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Journal of Medicinal
Plants Research
Volume 6 Number 12 30 March, 2012
ISSN 1996-0875

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Editors
Prof. Akah Peter Achunike
Editor-in-chief
Department of Pharmacology & Toxicology
University of Nigeria, Nsukka
Nigeria
Dr. Ugur Cakilcioglu
Elazıg Directorate of National Education
Turkey.
Dr. Jianxin Chen
Information Center,
Beijing University of Chinese Medicine,
Beijing, China
100029,
China.
Dr. Hassan Sher
Department of Botany and Microbiology,
College of Science,
King Saud University, Riyadh
Kingdom of Saudi Arabia.
Dr. Jin Tao
Professor and Dong-Wu Scholar,
Department of Neurobiology,
Medical College of Soochow University,
199 Ren-Ai Road, Dushu Lake Campus,
Suzhou Industrial Park,
Suzhou 215123,
P.R.China.
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Department of Biological Science,
Faculty of Science,
Ubon Ratchathani University,
Ubon Ratchathani 34190,
Thailand.
Prof. Parveen Bansal
Department of Biochemistry
Postgraduate Institute of Medical Education and
Research
Chandigarh
India.
Dr. Ravichandran Veerasamy
AIMST University
Faculty of Pharmacy, AIMST University, Semeling –
08100,
Kedah, Malaysia.
Dr. Sayeed Ahmad
Herbal Medicine Laboratory, Department of
Pharmacognosy and Phytochemistry,
Faculty of Pharmacy, Jamia Hamdard (Hamdard
University), Hamdard Nagar, New Delhi, 110062,
India.
Dr. Cheng Tan
Department of Dermatology, first Affiliated Hospital
of Nanjing Univeristy of
Traditional Chinese Medicine.
155 Hanzhong Road, Nanjing, Jiangsu Province,
China. 210029
Dr. Naseem Ahmad
Young Scientist (DST, FAST TRACK Scheme)
Plant Biotechnology Laboratory
Department of Botany
Aligarh Muslim University
Aligarh- 202 002,(UP)
India.
Dr. Isiaka A. Ogunwande
Dept. Of Chemistry,
Lagos State University, Ojo, Lagos,
Nigeria.

Editorial Board
Prof Hatil Hashim EL-Kamali
Omdurman Islamic University, Botany Department,
Sudan.
Prof. Dr. Muradiye Nacak
Department of Pharmacology, Faculty of Medicine,
Gaziantep University,
Turkey.
Dr. Sadiq Azam
Department of Biotechnology,
Abdul Wali Khan University Mardan,
Pakistan.
Kongyun Wu
Department of Biology and Environment Engineering,
Guiyang College,
China.
Prof Swati Sen Mandi
Division of plant Biology,
Bose Institute
India.
Dr.Wafaa Ibrahim Rasheed
Professor of Medical Biochemistry National Research
Center Cairo
Egypt.
Dr. Arash Kheradmand
Lorestan University,
Iran.
Prof Dr Cemşit Karakurt
Pediatrics and Pediatric Cardiology
Inonu University Faculty of Medicine,
Turkey.
Samuel Adelani Babarinde
Department of Crop and Environmental Protection,
Ladoke Akintola University of Technology,
Ogbomoso
Nigeria.
Dr. Ujjwal Kumar De
Indian Vetreinary Research Institute,
Izatnagar, Bareilly, UP-243122
Veterinary Medicine,
India.

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Journal of Medicinal Plants Research
International Journal of Medicine and Medical Sciences
Table of Contents: Volume 6 Number 12 30 March, 2012
nces
Review
ARTICLES
Research development on volatile oil from chuanxiong rhizoma 2240
Jipeng Hou and Xin He
Macroelements nutrition (NPK) of medicinal plants: A review 2249
Naser Boroomand and Mohammad Sadat Hosseini Grouh
Research Articles
Chemical composition and antimicrobial activities of Urena lobata L.
(Malvaceae) 2256
E. D. Fagbohun, R. R. Asare and A. O. Egbebi
Screening of Brazilian plants for antiviral activity against animal
herpesviruses 2261
M. J. B. Fernandes, A. V. Barros, M. S. Melo and I. C. Simoni
Anti-ulcer activity of Swietenia mahagoni leaf extract in ethanol-induced
gastric mucosal damage in rats 2266
Salmah Al-Radahe, Khaled Abdul-Aziz Ahmed, Suzy Salama, Mahmood
Ameen Abdulla, Zahra A. Amin, Saad Al-Jassabi and Harita Hashim
Analysis on the main active components of Lycium barbarum fruits and
related environmental factors 2276
Jing Z. Dong, Shu H. Wang, Linyao Zhu and Y. Wang

Table of Contents: Volume 6 Number 12 30 March, 2012
nces
ARTICLES
In vitro antiplasmodial activity of seven plants commonly used against
malaria in Burkina Faso 2285
Kumulungui Brice Serge, Ondo-Azi Alain Serges, Mintsa Ndong Armel,
Fumoux Francis and Traore Alfred
Phytochemicaland proximate analyses and thin layer chromatography
fingerprinting of the aerial part of Chenopodium ambrosioides Linn.
(Chenopodiaceae) 2289
Okhale Samuel Ehiabhi, Egharevba Henry Omoregie, Ona Eneyi Comfort and
Kunle Oluyemisi Folashade
Antifungal activity of coptidis rhizoma against Candida species 2295
Jae Young Kim, Yongsub Yi and Yoongho Lim
Evaluation and comparison of antifungal activities of Terminalia
catappa and Terminalia mantaly (Combretaceae) on the in
vitrogrowth of Aspergillus fumigatus 2299
ZIRIHI Guédé Noël, N’GUESSAN Koffi, KASSY N’dja Justin,
COULIBALY Kiyinlma and DJAMAN Allico Joseph
Optimization of callus induction medium for Hymenocallis littoralis
(Melong kecil) using root and bulb explants 2309
Rosli Noormi, Vikneswaran Murugaiyah and Sreeramanan Subramaniam
Effects of pomegranate seed extract on liver paraoxonase and bcl-xL
activities in rats treated with cisplatin 2317
Serap YILDIRIM, Fikrullah KISA, Ali KARADENIZ, Abdulkadir YILDIRIM,
Akar KARAKOC, Ismail CAN5, Adem KARA and Nejdet SIMSEK
Medicinal plants from an old Bulgarian medical book 2324
Anely Nedelcheva

Table of Contents: Volume 6 Number 12 30 March, 2012
ences
ARTICLES
Chemical composition, antioxidant activity and toxicity evaluation of
essential oil of Tulbaghia violacea Harv. 2340
O. S Olorunnisola, G. Bradley and A. J Afolayan
Optimum extraction conditions for arbutin from Asian pear peel by
supercritical fluid extraction (SFE) using Box-Behnken design 2348
Byung-Doo Lee and Jong-Bang Eun
A comparison of volatile components of flower, leaf and peel ofCitrus
reticulata Blanco (Citrus nobilis Lour var. deliciosa swingle) 2365
Behzad Babazadeh Darjazi
Optimization of total flavonoids extraction from mulberry leaf using an
ethanol-based solvent system 2373
Kai Nie, Zhonghai Tang, Xu Wu, Xiaona Xu, Yizeng Liang, Huang Li
and Liqun Rao
Evaluation of the antimicrobial activity in species of a Portuguese
“Montado” ecosystem against multidrug resistant pathogens 2381
B. Lai, G. Teixeira, I. Moreira, A. I. Correia, A. Duarte and A. M. Madureira
Genetic diversity and population structure of Dactylorhiza hatagirea
(Orchidaceae) in cold desert Ladakh region of India. 2388
Ashish R. Warghat, Prabodh K. Bajpai, Ashutosh A. Murkute, Hemant Sood,
Om P. Chaurasia and Ravi B. Srivastava
In vitro antioxidant capacities of rice residue hydrolysates from fermented
broth of five mold strains 2396
Wei Tian, Qinlu Lin and Gao-Qiang Liu

Table of Contents: Volume 6 Number 12 30 March, 2012
ences
ARTICLES
Determination of scavenging effect of Chinese medicinal herbs on
hydroxyl radical using a new chemiluminescence system 2402
Cai Zhuo, Qiu Xia-lin, Liang Xin-yuan, Mo Li-shu and Jiang Cai-ying
A survey of medicinal plants in mangrove and beach forests from sating
Phra Peninsula, Songkhla Province, Thailand 2421
Oratai Neamsuvan, Patcharin Singdam, Kornkanok Yingcharoen and
Narumon Sengnon
High-performance liquid chromatography (HPLC) determination of five
active ingredients in the calyces of Physalis Alkekengi L. var.franchetii
(mast.) Mskino 2438
Baoli Xu, Chengguo Ju, Huijie Guan, Liang Xu, Nan Xu and Bing Wang
Betalains from stem callus cultures of Zaleya decandra L. N. Burm. f. – A
medicinal herb 2443
M. Radfar, M. S. Sudarshana and M. H. Niranjan
Anti-Candida activity of ethanolic extracts of Iranian endemic medicinal
herbs against Candida albicanss 2448
H.A. Rohi Boroujeni, A. Ghasemi Pirbalouti, B. Hamedi, R. Abdizadeh and F.
Malekpoor
Acute toxicity of Clarias gariepinus exposed to Datura innoxia leaf extract 2453
Ayuba V. O., Ofojekwu P. C. and Musa S. O.
Dietary supplementation of green tea by-products on growth performance,
meat quality, blood parameters and immunity in finishing pigs 2458
Md. Elias Hossain, Seok Young Ko and Chul Ju Yang

Table of Contents: Volume 6 Number 12 30 March, 2012
ences
ARTICLES
Dietary supplementation of green tea by-products on growth performance,
meat quality, blood parameters and immunity in finishing pigs 2468
H. Abbaspour, H. Afshari1 and M. A. Abdel-Wahhab
Influence of salt stress on growth, pigments, soluble sugars and ion
accumulation in three pistachio cultivars 2468
H. Abbaspour, H. Afshari1 and M. A. Abdel-Wahhab
Study on salicin content correlation between taxilli herba and their
willow host plants 2474
Dong Lu, Benwei Su, Yonghua Li, Kaixin Zhu, Hehuan Pei, Minghui Zhao
and Jing Li
Antimicrobial activity and chemical composition of essential oils of four
Hypericum from Khorasan, Iran 2478
Motavalizadehkakhky Alireza
The antitumor activities of Lentinula edodes C91-3 mycelia fermentation
protein on S180 (Mouse sarcoma cell) in vivo and in vitro 2488
Mintao Zhong, Ben Liu, Yingli Liu, Xiaoli Wang, Xingyun Li, Lei Liu,
Anhong Ning, Jing Cao and Min Huang
Effect of plant density on phenology and oil yield of safflower herb
under irrigated and rainfed planting systems 2493
Roghayeh Shakeri Amoghein, Ahmad Tobeh and Shahzad Jamaati-e-Somarin
Astragalus polysaccharides induced gene expression profiling of
intraepithelial lymphocytes in immune-suppressed mice 2504
Lu Cheng, He Xiaojuan, Xiao Cheng, Guo Yuming, Zha Qinglin, Liu Yuanyan,
Liu Zhenli, Chen Shilin and Lu Aiping

Table of Contents: Volume 6 Number 12 30 March, 2012
ences
ARTICLES
Palatability perception of herbal teas: Impact of extraction time and
saccharose 2520
Yang Xie, Zongbao Ding, Wenjun Duan and Qingsheng Ye
Isolation and purification of terpenoids from Celastrus aculeatus Merr.
by high-speed counter-current chromatography 2520
Yang Xie, Zongbao Ding, Wenjun Duan and Qingsheng Ye

Journal of Medicinal Plants Research Vol. 6(12), pp. 2240-2248, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR10.208
ISSN 1996-0875 ©2012 Academic Journals
Review
Research development on volatile oil from chuanxiong
rhizoma
Jipeng Hou 1 and Xin He 1,2 *
1 College of Chinese Materia Medica, Tianjin University of Traditional Chinese Medicine, Tianjin, 300193, China.
2 Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin, China.
Accepted 15 July, 2010
Chuanxiong Rhizoma, as an authentic herbal medicine of Sichuan province, is one of the main plant
sources of Umbelliferae rhizoma which has been used to treat headache, rheumatic arthralgia and
coronary heart diseases. The current review covers its chemical constituents, pharmacological
activities, pharmacokinetic and combination therapy. The phytochemistry of Chuanxiong Rhizoma has
been studied extensively in recent decades with many investigations that focused on
tetramethylpyrazine and ferulic acid that is considered as active components of Chuanxiong Rhizoma.
However, the quantities of tetramethylpyrazine and ferulic acid in Chuanxiong Rhizoma are lower when
compared with volatile oil accounting for a considerable proportion of it. Senkyunolide A and ligustilide
belong to volatile oil and have been reported as main active components of Chuanxiong Rhizoma.
Pharmacokinetic studies are important for the application to understand the therapeutic and toxicity of
the Chinese traditional medicine, although numerous animal studies have demonstrated the
pharmacokinetic process of volatile oil; few clinical trials are conducted. Therefore, large randomized
clinical trials and further scientific researches to determine its mechanism of actions will be necessary
to ensure the safety, effect and better understanding of its action.
Key words: Chuanxiong Rhizoma, volatile oil, chemical study, pharmacology, pharmacokinetics.
INTRODUCTION
Chuanxiong Rhizoma, like most traditional Chinese
medicine (TCM) herbs, has been used clinically over
thousand years in China. The first recorded is in the
Divine Husbandmans Classic of the Materia Medica
(Shen Nong Ben Cao Jing). It belongs to the Umbelliferae
family, Ligustrum genus. Chuanxiong Rhizoma is warm in
property and pungent in flavor according to TCM theory
and is used for the treatment of headache, rheumatic
arthralgia and coronary heart diseases (China
Pharmacopoeia Committee, 2005). The volatile oil
compounds in this medicinal plant are recognized as
important part for its pharmacological activities mentioned
earlier. In addition to single-herb preparations, various
Chuanxiong Rhizoma-based proprietary products are
also used in clinical practice. For example, Quick-Acting
Heart-Saving Pill (Chinese name: Suxiao Jiuxin Wan), a
*Corresponding author. E-mail: hexintn@yahoo.com. Tel: 86-22-
59596231. Fax: 86-22-59596153.
contains Chuanxiong Rhizoma essential oil extract as the
primary ingredient for the treatment of cardiovascular
disorders and all kinds of pain (Sun et al., 2002).
In view of its large market in Asian countries as well as
keen interest in the use and modernization of herbal
products throughout the world, this article provides an
overview of chemistry, pharmacology and pharmacokinetics
of Chuanxiong Rhizoma.
PLANT STUDIES
Chuanxiong Rhizoma (Figure 1) is the dried root of
Ligusticum chuanxiong Hort. (Figure 2) which enjoys the
warm and moist environment, fears the high temperature
but can endure severe cold weather and get through the
winter in the farmland. As a naturally occurring medicinal
herb, the geographical region of its growth and the
season of its harvest vary in the active components of
Chuanxiong Rhizoma. Sichuan province, China, has
been traditionally recognized as the authentic and superior

Figure 1. Chuanxiong Rhizoma
(http://www.huilinnatural.com/cn/productshow.asp?keyno=3).
Figure 2. Plant of L. chuanxiong Hort.
plant source for the herbal Chuanxiong Rhizoma with a
suitable harvest time from April to the end of May
depending upon the commercial interest of herbal
producers or herbal pharmaceutical manufacturers (Li et
al., 2006).
CHEMICAL STUDIES
Main chemical constituents
The main constituents of Chuanxiong Rhizoma oil are
phthalides and terpenes. Phthalides such as Z-ligustilide,
senkyunolide, neocnidilide, 3-n-butylphthalide,
butylidenephthalide and E-ligustilide. Terpenes include pcymene,
g-terpinene, terpinolene, terpinen-4-ol, αcedrene,
β-selinene, α-selinene and spathulenol. These
principal compounds from Chuanxiong Rhizoma oil were
Hou and He 2241
identified and quantified under supercritical fluid
extraction in gas chromatography-mass spectrometry
(GC-MS) (Zhang et al., 2006). For a long time, the aerial
parts of L. chuanxiong Hort. were discarded as waste
when harvested. In the aerial parts of L. chuanxiong
Hort., forty-six components which make up 85.82% of the
total oil were identified by means of GC-MS and GC
under steam distillation extraction (Guo et al., 1993).
There was no difference in the composition of
Chuanxiong Rhizoma oil except the proportion of it, so
the aerial parts of L. chuanxiong Hort. have a good
prospect.
The different packing and storage life will affect the
content of active components in Chuanxiong Rhizoma,
guiding the moist, volatile oil and ferulic acid in
Chuanxiong Rhizoma from two good agriculture practice
base by the related determination method in the
supplement of Chinese Pharmacopeia (2005). The
content of total alkaloids was determined by acid dye
colorimetry. The loss of active component in Chuanxiong
Rhizoma was the least when in vacuum packing, sack
and weave packet. During storage, the content of moist
and volatile oil decreased; the content of ferulic acid
increased; the content variety of total alkaloids had no
regulation. Therefore, the packing of Chuanxiong
Rhizoma should choose the sack and weave packet. If
the quantity is small, vacuum packing should be used.
Combined with the changes of Chuanxiong Rhizoma
active component and the phenomenon of mildew and
worm eaten during storage, it should not be stored for a
long time (Jiang et al., 2005). The distilled fluid of
Chuanxiong Rhizoma should be preserved at low
temperature under shady place, kept at pH of 5 to secure
its quality and clinical effects (Tian et al., 2003). The
Chuanxiong Rhizoma oil content of processed products
has been reduced in different degrees when compared
with raw drug, the content of volatile oil: raw drug >
alcohol-broiled product > vinegar processed product > fry
yellow product > sample baked with boiled wine (Zhang
et al., 1998). As the special smell oily and unstable
substance, it can be included in β-cyclodextrin to improve
the stability in normal situation, avoiding the loss of active
ingredient (Li et al., 2005).
Comparison of different extraction process of
Chuanxiong Rhizoma oil
The extractions of Chuanxiong Rhizoma oil include steam
distillation extraction, supercritical fluid extraction (SFE),
organic solvent extraction and the new method,
headspace solid phase micro-extraction (HS-SPME).
Different extractions will vary the yield of the volatile oil of
Chuanxiong Rhizoma. Steam distillation extraction is a
conventional method which has been used frequently to
extract the volatile oil from Chuanxiong Rhizoma in the
past. But it requires lot of time and the yield of volatile oil

2242 J. Med. Plants Res.
Figure 3. Total ion current chromatogram of essential oil in Chuanxiong Rhizoma.
et al. (2002) identified about 44 compounds from
Chuanxiong Rhizoma and the yield of volatile oil is 4.16%
(v/w). In contrast, 30 compounds were determined by
steam distillation extraction and the yield of volatile oil by
steam distillation extraction is 0.8% (v/w) (Hong et al.,
2002). Although, both techniques including steam
distillation extraction and supercritical fluid extraction,
could extract the main components of volatile oil. The
contained components of samples extracted by
supercritical fluid CO2 extraction were most and the
whole operation process toke short time and had high
efficiency (Wang et al., 2009). They thought, since SFE is
operated at high pressure and lower temperature,
compounds with thermo unstable or high volatility would
probably have better solubility in supercritical fluid.
Wan et al. (2003) used steam distillation extraction,
supercritical fluid extraction and organic solvent
extraction to extract essential oil of Chuanxiong Rhizoma.
The three methods are able to extract main lactones of
volatile oil. Organic solvent extraction can extract fatty
acids and fatty acid ester which accounted for 30% of its
extracted substance, except for lactone and terpene type,
because neutral ethanol is one of the polar organic
solvent. Oleic acid can only be obtained by organic
solvent extraction (Wan et al., 2003).
HS-SPME was the first high concentration capacityheadspace
sampling technique to appear. It was
introduced by Zhang and Pawliszyn (1993) as an
extension of SPME, which had been developed by Arthur
and Pawliszyn (1990) to overcome some drawbacks of
solid phase extraction in sampling organic pollutants from
water. In recent years, HS-SPME has gained wide
acceptance as an effective extraction technique for a
wide variety of samples. This method followed by GC-MS
is described for the analysis of volatile compounds in the
dry rhizome of L. chuanxiong Hort.. As a result, 73
compounds were determined and identified by the HS-
SPME-GC-MS method with at least 20 more compounds
than those in the methods available. Using much less
sample amount, shorter extraction time and simpler
procedure, HS-SPME method can achieve similar results
to those by steam distillation. The HS-SPME method is
simple, rapid and effective and can be used for the
analysis of volatile compounds in medicinal plants (Zhang
et al., 2007).
Chemical fingerprint
As the complexity of Chinese medicine herbs, many
factors can influence the bioactive ingredients of the
plants which will vary the therapeutic outcome. Therefore,
the quality control is vital to Chinese medicine herbs. The
characteristic fingerprint (Figure 3 and Table 1) of
Chuanxiong Rhizoma volatile oil has been established to
scientifically evaluate and effectively control the inner
quality of Chuanxiong Rhizoma, and the quality
information has been provided by GC-MS method, using
the twelfth (ligusticum) peak as reference (Shi et al.,
2007).
PHARMACOLOGICAL EFFECTS
Anti-fibrosis effects
Hepatic fibrosis is due to diseases, such as chronic
hepatitis and alcoholic liver disease (Hui and Friedman,
2003). Compounds that promote apoptosis in hepatic
stellate cells which play a central role in both fibrogenesis
and fibrolysis may have anti-fibrotic potential.
Chuanxiong Rhizoma has been found to have the ability
of anti-proliferative and pro-apoptotic effects on hepatic
stellate cells (HSC), and the pathways mediated by Fas
and Bcl2 were involved in herb-induced apoptosis in
HSC-T6 (Chor et al., 2005). Two isomeric compounds,

Table 1. Identification of main peaks in essential oil of Chuanxiong Rhizoma.
Hou and He 2243
Peak No. Name Relative peak area Relative retention time
1 terpilenol 0.0637 0.4758
2 2-methoxyl-4-vinylphenol 0.0255 0.6167
3 1-pheny-1-pentanone) 0.0095 0.6650
4 2,2-dimethyl-1-phenyl,3-butene-1-ketone 0.0051 0.6746
5 spainulenol 0.0423 0.9053
6 butylphthalide 0.1975 0.9409
7 butylidenephthalide 0.2435 0.9592
8 unindentified 0.0566 0.9642
9 cnidilide 0.0421 0.9674
10 east ligustilide 0.3287 0.9862
11 neocindilide 0.1073 0.9914
12 ligusticum 1.0000 1.0000
13 isomer of ligusticum 0.0134 1.0320
ZZ-6,8',7,3'-diligustilide and levistolide A which are the
main compounds of volatile oil were identified to be active
components against hepatic fibrosis, and they did not
decrease the viability after 48 h incubation in both HSC-
T6 and LI-90 cells. The mechanism of the two
compounds is to inhibit platelet derived growth factoractivated
HSC proliferation, possibly through cell cycle
inhibition and apoptosis (Lee et al., 2007).
Vasorelaxing effects
Ligustilide and senkyunolide A, two main constituents of
Chuanxiong Rhizoma oil, had similar relaxation potencies
against contractions to 9,11-dideoxy-9α, 11αmethanoepoxyprostaglandin
F2α, phenylephrine, 5hydroxytryptamine
and KCl. Their vasorelaxation effects
were not affected by endothelium removal, the adenylate
cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6amine,
the soluble guanylate cyclase inhibitor 1H-
[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one or the nonselective
K + channel blocker tetraethylammonium (Chan
et al., 2006). By using cell membrane chromatography
(CMC) and GC-MS, Liang et al. (2005) found out that
ligustilide and butylidenephthalide significantly inhibited
the vasoconstrictions induced by norepinephrine (NE)
bitartrate and calcium chloride (CaCl2) in a concentrationdependent
manner (Liang et al., 2005). The mechanism
of ligustilide is to induce vasodilatation in rat mesenteric
artery by inhibiting the voltage-dependent calcium
channel and receptor-operated calcium channel, and
receptor-mediated Ca 2+ influx and release (Cao et al.,
2006). Butylidenephthalide-mediated vasorelaxation
comprises of both endothelium-dependent and
independent components, and it is acting through an
inhibitory mechanism downstream to L-type voltageoperated
and prostanoid TP receptor-operated Ca 2+
channels operating late in the contractile pathway (Chan
et al., 2006). Butylidenephthalide has also been found as
a synergism with NO donor sodium nitroprusside (SNP)
in relaxing rat isolated aorta. The interaction is related to
an enhancement of the effectiveness of SNP in producing
relaxation under tone induced mainly by Ca 2+
sensitization (Chan et al., 2009). This finding may serve
as the rationale behind the frequent use of a Chuanxiong
Rhizoma-NO donor combination in China.
Protective effects
The extract of Fo Shou San including Chuanxiong
Rhizoma and Angelicae Sinensis Radix dosedependently
and time-dependently protected human
umbilical vein endothelial cells against hydrogen peroxide
damage and suppressed the production of reactive
oxygen species (Hou et al., 2004). Essential oil of
Chuanxiong Rhizoma is able to inhibit DNA damage
induced by ultraviolet B via their antioxidant activities
through the methods of 1,1dipheny1-2-picryl hydrazyl
(DPPH) and 2,2-azinobis (3-ethylbenzothiozoline-6sulfonic
acid) diammonium salt (ABTS) scavenging
assay. In this assay, the essential oils showed very high
activity in the DPPH test with an IC50 value of 6.79 mg/ml
in Chuanxiong Rhizoma. In addition, the essential oils in
ABTS test exhibited an IC50 value of 1.58 mg/ml in
Chuanxiong Rhizoma. The mechanism for antioxidants is
to remove free radical that involves the donation of
hydrogen to a free radical, and hence, its reduction to an
unreactive species through removing the odd electron
feature which is responsible for radical reactivity (Jeong
et al., 2009).
Z-Ligustilide has significant neuroprotective effects on
transient forebrain ischemia in mice; focal cerebral
ischemia and chronic cerebral hypoperfusion in rats may
through antioxidant, improved cholinergic activity and
antiapoptotic mechanisms (Kuang et al., 2006; Peng et

2244 J. Med. Plants Res.
Table 2. Pharmacokinetic parameters of senkyunolide A in rats after i.v., i.p. and p.o. administration (n =5, Mean ± S.D.).
Pharmacokinetic
parameter
Route of administration
i.v. i.p. p.o.
Dose (mg/kg) 20 (pure) 7.65 (ext) † 50 100 100
Tmax (h) - - 0.04 ± 0.01 0.04 ± 0.01 0.21 ± 0.08***
Cmax (mg/L) 19.67 ± 3.48 4.86 ± 0.45 17.60 ± 3.35 31.01 ± 4.26 1.66 ± 0.23***
t1/2 (h) 0.65 ± 0.06 0.69 ± 0.31 0.67 ± 0.10 0.99 ± 0.23 0.52 ± 0.03
AUC0-∞ (mg·h/L) 2.81 ± 0.19 0.95 ± 0.16 5.29 ± 0.94 10.65 ± 0.98 1.11 6 ± 10***
Vd/F (l/kg) 6.74 ± 0.73 7.12 ± 2.16 9.98 ± 1.66 13.78 ± 3.30 68.97 ± 4.64***
CL/F (l/h/kg) 7.20 ± 0.48 9.17 ± 1.81 10.92 ± 2.10 9.72 ± 0.91 94.81 ± 12.06***
F (%) - 45.7 75.3 75.8 7.9***
***P < 0.001 compared with the same intraperitoneal doses. † The herbal extract containing 7.65% of senkyunolide A was given at a doses
of 100 mg/kg.
al., 2007; Kuang et al., 2008). It was also pronounced
that Z-Ligustilide has aprotective effect against H2O2induced
cytotoxicity, at least partly through improving
cellular antioxidant defense and inhibiting the
mitochondrial apoptotic pathway (Yu et al., 2008).
Antipyretic effects
The activity of body temperature regulation was involved
by central monoamine neurotransmitters. The theory of
monoamine of body temperature recognized that the
quantity dynamic balance of 5-hydroxytryptamine (5-HT)
and norepinephrine (NE) can keep a relative constancy of
body temperature. The report showed that the essential
oil of Chuanxiong Rhizoma have the ability of antipyretic
effect and make the proportion of 5-HT and NE in
hypothalamus have a significant change, so it was
considered as one of the mechanism of Chuanxiong
Rhizoma to antipyretic effect (Li et al., 2003b).
Anti-trichophyton effects
At present, trichophyton are frequently treated by oral
administration of ketoconazole or the development drug.
However, ketoconazole have unpleasant side effects and
the other drug has relatively low bioavailability after oral
administration and adverse effects. Ligustilide and
butylidenephthalide, which are major oil components
comprising of the volatile oil of Chuanxiong Rhizoma,
have the synergistic anti-fungal effects in combination
with antifungal drugs by checkerboard microtiter and
microdilution tests. With this synergistic anti-fungal effect,
the use of lower concentrations of ketoconazole may
facilitate and minimize its potential side effects. The
improvement of anti-trichophyton activity of itraconazole
administered in combination with ligustilide or
butylidenephthalide could provide alternative therapies to
overcome current limitations in the use of itraconazole for
treatment of trichophyton infections (Sim and Shin, 2008).
PHARMACOKINETICS
In recent years, with the development of analytical
technique, many investigations have been reported the
pharmacokinetics of Chuanxiong Rhizoma oil. However,
these reports were concentrated on senkyunolide A,
ligustilide and butylidenephthalide, the other compounds
of essential oil of Chuanxiong Rhizoma are very limited.
The main pharmacokinetics of these three active
components is summarized as the following.
Senkyunolide A
The pharmacokinetic parameters of senkyunolide A after
three different ways of administration, including
intravenous (i.v.), intraperitoneal (i.p.) and per os (p.o.)
are as shown in Table 2. After i.v. administration,
senkyunolide A was extensively distributed [apparent
volume of distribution based on the terminal phase (Vd/F):
6.74 ± 0.73 l/kg] and rapidly eliminated from the plasma
[apparent plasma clearance (CL/F): 7.20 ± 0.48 l/h/kg
and biological half life (t1/2): 0.65 ± 0.06 h]. Hepatic
metabolism was suggested as the major route of
senkyunolide A elimination as indicated by the results of
in vitro S9 fraction study. After i.p. administration,
senkyunolide A exhibited dose-independent
pharmacokinetics. The absorption after i.p. administration
was rapid [maximum concentration (Tmax): 0.04 ± 0.01 h],
and the absolute bioavailability (F) was 75%. After p.o.
administration, senkyunolide A was also absorbed rapidly
(Tmax: 0.21 ± 0.08 h); however, its oral bioavailability was
low (~8%). The contributing factors were determined to
be instability in the gastrointestinal tract (accounting for
67% of the loss) and hepatic first-pass metabolism
(accounting for another 25%). Pharmacokinetics of
senkyunolide A were unaltered when Chuanxiong
Rhizoma extract was administered, which suggests that
components in the extract have insignificant effects on

Table 3. Pharmacokinetic parameters of ligustilide in rats after i.v., i.p. and p.o. administration (n=5, Mean ± S.D.).
Pharmacokinetic
parameter
Route of administration
Hou and He 2245
i.v. i.p. p.o.
Dose (mg/kg) 15.6 14.9 a 26 52 500
Tmax (h) - - 0.05 ± 0.02 0.08 ± 0.01 0.36 ± 0.19
Cmax (mg/L) 13.19 ± 0.84 6.93 ± 0.60*** 7.48 ± 1.10*** 20.75 ± 2.55 ### 0.66 ± 0.23***
t1/2 (h) 0.31 ± 0.12 0.22 ± 0.07 0.36 ± 0.05 0.44 ± 0.08 # 3.43 ± 1.01***
AUC0-∞(mg·h/L) b 1.81 ± 0.24 0.79 ± 0.10** 0.93 ± 0.07* 1.77 ± 0.23 # 0.047 ± 0.012**
Vd/F (l/kg) c 3.76 ± 1.23 5.62 ± 1.19 6.54 ± 1.56 6.32 ± 1.81 16 41.9 ± 121.6***
CL/F (l/h/kg) c 9.14 ± 1.27 20.35 ± 3.05** 16.9 ± 1.21** 9.26 ± 1. 04 ## 411.1 ± 145.7***
MRT (h) 0.3 ± 0.07 0.19 ± 0.03 0.3 ± 0.05 0.41 ± 0 .03 5.14 ± 1.56***
F (%) - 45.7d 51.7 97.7 2.6
*P < 0.05, **P < 0.01, ***P < 0.001, compared with i.v. dosing of the isolated ligustilide. # P < 0.05, ## P < 0.01, ### P < 0.001, compared with the lower
i.p. dose of the isolated ligustilide. a Dose of ligustilide in 100 mg/kg of Chuanxiong Rhizoma extract. b Normalized with dose. c Data represent Vd and
CL in the case of i.v. dosing of the isolated ligustilide. d Relative bioavailability compared with that of i.v. dosing of the isolated ligustilide.
senkyunolide A pharmacokinetics (Yan et al., 2007).
Ligustilide
The pharmacokinetic parameters of ligustilide after three
different administrations, including i.v., i.p. and p.o. are as
shown in Table 3. After i.v. administration of pure
ligustilide, it was distributed extensively (Vd/F: 3.76 ± 1.23
l/kg) and eliminated rapidly (t1/2: 0.31 ± 0.12 h). The i.v.
CL/F of ligustilide after Chuanxiong extract administration
was significantly higher than that dosed in its pure form
[CL/F: 20.35 ± 3.05 versus 9.14 ± 1.27 l/h/kg, P Cspleen > Ckidney. The concentration of
ligustilide in tissues in different time is as shown in Figure
4 (Qian et al., 2008). There is a difference in the
metabolism of the main active constituent monomer and
the active constituent group. High-performance liquid
chromatography with diode array detection was used to
analyze the metabolites in the urine, feces and bile of rats
dosed with the essential oil or ligustilide. A clear
difference in the metabolism of the essential oil and
ligustilide indicating that the multiple components of
essential oil influence the types of metabolites produced
and the relative ratios of the main active components.
The possible pathway for the metabolism of ligustilide in
rats is as shown in Figure 5 (Ding et al., 2008).
Butylidenephthalide
Butylidenephthalide quickly permeate into peripheral
circulation system without accumulation in the skin and
then distribute into lung, liver, bile and kidney after dermal
application by the whole body autoradiogram and liquid
scintillation analysis, and excretion mainly into urine. In
the case of i.v. administration, 80% of the administered
butylidenephthalide was excreted into urine within 24 h,
while only 5% was excreted into feces within 24 h. The
metabolite in urine was determined to be a cysteine
conjugate by LC-MS/MS method (Sekiya et al., 2000).
TOXICITY STUDIES
L. chuanxiong Hort., which is contained in China plant
map database is one of the poisonous plant. The acute
toxicity (LD50) was 328 mg/kg in mice when the essential
oil of radix and stem of L. chuanxiong Hort. was
administered by intraperitoneal injection resulting in death
within 4 to 5 h.
Using toxic effect method to detect pharmacokinetic
parameter of Chuanxiong Rhizoma oil, LD50 was 517.51 ±
90.01 mg/kg and 2982.37 ± 345.12 mg/kg after the
administration to mice by i.p. and i.g., respectively (Pan
et al., 1999).
CONCLUSION
L. chuanxiong Hort., as one of the TCM herbs has been
used clinically for thousands of years in China. In the

2246 J. Med. Plants Res.
Figure 4. Concentration of ligustilide in tissues in different time (n=12, Mean ± Standard
deviation).
Ligustilide
Figure 5. Proposed metabolic pathway of ligustilide in the rat. Cly, glycine;
Cys, cysteine; Clu, glutamate.

active component study, many investigations suggested
that alkaloids (tetramethylpyrazine), phenolic acids
(ferulic acid) and volatile oils (ligustilide and senkyunolide
A), especially tetramethylpyrazine are considered as the
main factor to the effect of blood circulation. However, the
quantities of tetramethylpyrazine and ferulic acid in raw
Chuanxiong Rhizoma were lower (

2248 J. Med. Plants Res.
Sim Y, Shin SG (2008). Combinatorial anti-trichophyton effects of
Ligusticum chuanxiong essential oil components with antibiotics.
Arch. Pharm. Res., 31: 497-502.
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pharmacokinetics, pharmacodynamics and clinical research of
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Tian YQ, Wang XX, Gao LH (2003). On the stability of volatile oil of
Rhizoma Chuanxiong. Shanghai J. Trad. Chin. Med., 37: 49-52.
Wang Y, Fang CZ, Bi YX (2009). Effect of different extraction methods
on the chemical components of volatile oil from Ligusticum
chuanxiong. J. Anhui Agr. Sci., 37: 6425-6426.
Wan Q, Zhang Y, Hu YY, He XX (2003). The effect of different
preparative methods to the component of Chuanxiong Rhizome
volatile oil. China J. Chin. Mater. Med., 28: 572-574.
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metabolism of ligustilide, a major bioactive component in Rhizoma
Chuanxiong, in the Rat. Drug Metab. Dispos., 36: 400-408.
Yan R, Lin G, Ko NL, Tam YK (2007). Low oral bioavailability and
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Yan R, Li SL, Chung HS, Tam YK, Lin G (2005). Simultaneous
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2249-2255, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPRx11.019
ISSN 1996-0875 ©2012 Academic Journals
Review
Macroelements nutrition (NPK) of medicinal plants: A
review
Naser Boroomand and Mohammad Sadat Hosseini Grouh*
Department of Plant Science, Faculty of Agriculture, University of Jiroft, Jiroft, Iran.
Accepted 01 November, 2011
The use of medicinal plants to treat diseases since ancient times has been applied. Nowadays, the
number of the plants used for medicinal purpose is about 35,000 species. In this study, the effect of
macro elements (nitrogen, phosphorus and potassium) on the properties of medicinal plants was
reviewed. Investigations carried out showed that with adequate supply of N, P and K utilization by
various spice crops are enhanced. Macro elements caused increase the number of traits such as plant
height, leaf area, yield seed, and oil content. Some of traits of medicinal plants such as basil, turmeric,
black pepper, cardamom, fennel, fenugreek, and Aloe vera with used potassium were changed.
Key words: NPK fertilizer, medicinal plants, soil, nutrition.
INTRODUCTION
Plants have been used in treating human diseases for
thousands of years. Some 60,000 years ago, it appears
that Neanderthal man valued herbs as medicinal agents,
this conclusion is based on a grave in Iran in which pollen
grains of eight medicinal plants were found (Solecki and
Shanidar, 1975). Plant materials are used throughout
developed and developing countries as home remedies,
over-the-counter drug products and raw materials for the
pharmaceutical industry, and represent a substantial
proportion of the global drug market (Ross, 2005). The
interest in Nature as a source of potential
chemotherapeutic agents continues. Natural products
and their derivatives represent more than 50% of all the
drugs in clinical use in the world. Higher plants contribute
no less than 25% of the total (Ameenah, 2006). There are
some 240,000 species of higher plants (medicinal and
non medicinal) and not all of those species will have the
same mineral needs, at the same scale. Some will
require a specific element in much higher concentration
than others, and others will be able to tolerate a much
higher concentration of an essential element that would,
be toxic, to a different species. Such variability is inherent
*Corresponding author. E-mail: hosseinim@alumni.ut.ac.ir,
msadat.1983@gmail.com. Tel: +98-348-3260062. Fax: +98-
348-3260065.
in biology, and for this reason most generalities, such as
the definition of an essential nutrient, need to have some
wiggle room in interpretation. Nutrient deficiencies in
plants are often made most evident by plant physiological
responses (Clarkson and Hanson, 1980). Nutrient
deficiency symptoms tend to occur in three major
patterns: localized to the younger tissues, localized to the
more mature tissues, or widely distributed across the
plant. In each case, the distribution of the symptoms can
help a person determine the nature of the deficiency
experienced by the plant or, if the deficient nutrient is
already known, make an inference about the role the
nutrient plays in the plant body (Wiedenhoeft, 2006).
Fourteen mineral nutrients are classified as either
macronutrients or micronutrients based on their plant
requirements. There are six macronutrients: Nitrogen (N),
phosphorus (P), potassium (K), calcium (Ca), magnesium
(Mg), and sulfur (S). The macronutrients, N, P, and K, are
often classified as ‘primary’ macronutrients, because
deficiencies of N, P and K are more common than the
‘secondary’ macronutrients, Ca, Mg, and S. The
micronutrients include boron (B), chlorine (Cl), copper
(Cu), iron (Fe), manganese (Mn), molybdenum (Mo),
nickel (Ni) and zinc (Zn). Most of the macronutrients
represent 0.1 to 5%, or 100 to 5000 parts per million
(ppm), of dry plant tissue, whereas the micronutrients
generally comprise less than 0.025%, or 250 ppm, of dry
plant tissue (Wiedenhoeft, 2006).

2250 J. Med. Plants Res.
PHOSPHORUS (P)
Phosphorus is frequently a limiting nutrient, particularly in
tropical regions, where the soil chemistry differs from
temperate soils, or in highly weathered soils, where
phosphorus has long since leached away. Phosphorus is
one of the three main elements in commercial lawn
fertilizers, though there is mounting evidence that many
lawns and green areas already have ample phosphorus,
and thus it is being phased out of some commercial
fertilizers. The ultimate source of virtually all terrestrial
phosphorus is from the weathering of minerals and soils
in the Earth’s crust. Phosphorus is generally available as
phosphate, an anion that is not bindable by the cation
exchange complex and thus can be easily leached from
the soil by rain or runoff (Wiedenhoeft, 2006)
Role of phosphorus in plant growth
Phosphorus has many important functions in plants and
medicinal plants, the primary one being the storage and
transfer of energy through the plant. Adenosine
diphosphate (ADP) and adenosine triphosphate (ATP)
are high-energy phosphate compounds that control most
processes in plants including photosynthesis, respiration,
protein and nucleic acid synthesis, and nutrient transport
through the plant’s cells (Sharpley et al., 1996).
Role of phosphorus in medicinal plants
Plant height
Boroomand et al. (2011a) found that application of 100 kg
P2O5 ha -1 increased leaf number and leaf length in Aloe
Vera. Boroomand et al. (2011b) reported significantly
higher plant height of Basil at 150 kg P2O5 ha -1 (78.96 cm
in 2009) which was superior over all other P levels. One
of the earliest and most pronounced responses to
phosphorus deficiency is reduction in shoot growth,
specifically reduction in leaf number and leaf size (Lynch
et al., 1991). Shoot development requires the production
of leaves by shoot apical meristem and their subsequent
expansion. Decreased number of leaves with phosphorus
deficiency implies changes in leaf initiation and activity of
shoot apical meristem. Plant height of Phaseolus trilobus
was increased due to increased phosphorus fertilization
at 80 kg P2O5 ha -1 (Kumar et al., 2002).
Anilkumar et al. (2001) reported significantly higher
plant height of mustard at 37.35 kg P2O5 ha -1 (209.4 and
208.0 cm during 1998 and 1999, respectively) which was
superior over all other P levels. Panwar and Singh (2003)
recorded significantly higher plant height of groundnut at
60 kg P2O5 ha -1 (49.80 cm) over control (46.55 cm). Field
investigation carried out by Pareek et al. (2002), to study
the effect of P2O5 levels on growth of Panchpatri
(Ipomoea pestigrides), showed that maximum plant
height per plant was obtained by application of 25 kg
P2O5 per ha. But increased levels of P2O5 over 25 kg
P2O5 decreased this parameter. Harendra and Yadav
(2007) reported that plant height of mustard increased
significantly with each increment in the dose of
phosphorus up to 13.1 kg P2O5 ha -1 (165.7 cm). However,
the differences in plant height due to further increase in
the dose of phosphorus to 39.3 kg P2O5 ha -1 were not
significant.
Number of branches in a plant
Pareek et al. (2002) reported significantly higher number
of branches of Panchpatri at 25 kg P2O5 ha -1 . Harenda
and Yadav (2007), reported significant increase in the
number of primary branches of mustard with an increase
in the phosphorus levels from 0 to 13.1 kg P2O5 ha -1
which was 5.3 and 6.2 plant -1 , respectively. But, there
was no significant increase in the number of primary
branches with further increase in the P level at 39.3 kg
P2O5 ha -1 (6.5 plant -1 ).
Saraf et al. (2002), studied the effect of two levels of
P2O5 (25 and 50 kg ha -1 ) on the yield of Danti
(Baliospermum montanum) crop. The effect of
phosphorus was significant and the highest plant height,
number of leaves per plant, branches per plant and plant
spread were observed with the application of 50 kg P2O5
per ha as compared to 25 kg P2O5 per ha -1 .
Leaf area
Plants ultimately depend on green leaf area for dry matter
accumulation as the leaves intercept solar radiation and
produce photosynthates through photosynthesis. The
production, expansion and survival of green leaves are
the important determinants of crop productivity. The
primary symptoms of nutrient deficiency are reduction in
leaf expansion. Boroomand et al. (2011b), in a study on
Basil reported that increased level of P2O5 from 50 to 150
kgh increased leaf area from 18.13 to 23.25, respectively.
In Aloe Vera application, 150 kgh P2O5 cause increased
leaf area (Boroomand et al., 2011a).
Seed yield
Though the magnitude of the responses of oilseed crops
to applied phosphorus is less than that of nitrogen,
results of several experiments do indicate that in situation
where soil phosphorus level is low, significant responses
can be obtained. Kumar et al. (1987), studied the
responses of fenugreek to phosphorus application 50 kgh
caused increased seed yield. Application of phosphorus
at 0, 17 and 34 kg ha -1 recorded seed yield of 963, 1178

and 1185 kg ha -1 in the first site and 1038, 1435 and
1494 kg ha -1 in the second site. Esskaf and Aljaro (1982)
recorded no significant effect on the growth and yield of
Garlic with the application of 41.11 kg P ha -1 during
bulbing and root growth. Minard (1978), on the other
hand reported 114.75 kg P ha -1 as optimum for increased
final bulb yield among other parameters. Bandopadhyay
and Samul (1999) studied the effect of varied levels of
phosphorus on seed yield of groundnut and concluded
that there was significant response to phosphorus up to
100 kg ha -1 (1625.20 kg ha -1 ). Bhari et al. (2000)
recorded significantly higher seed yield of mustard at 45
kg P2O5 ha -1 (2.36 q ha -1 ) which was superior over all
other P levels including control. Farahani and Khalvati
(2011) reported significantly higher seed yield of
coriander at 70 kg ha -1 P2O5 which was superior over all
other P levels.
Oil content
Application of 100 kg P2O5 h to Basil resulted in
significantly higher oil content (2.40%) which was
superior over all other P levels (Boroomand et al.,
2011b). Harendra and Yadav (2007) reported significant
increase in the oil content of mustard with increase in P
level from 0 to 39.3 kg P2O5 ha -1 .30 per cent of oil content
was recorded at 39.3 kg P2O5 ha -1 which was significant
over all other levels including control. Lewis et al. (1987)
examined the response of oilseed rape to phosphorus
fertilization at 21 sites and significant increase in oil
content to the tune of 0.7 per cent was recorded in only
one site. Boroomand et al. (2011a) recorded significantly
higher gel content of Aloe Vera at 150 kgh P2O5 which
was superior over all other P levels including control.
Response of various crops to phosphorus application
indicated that yield and growth attributing parameters
increased with increase in levels of P2O5 and the
response varied from 25 to 80 kg P2O5 per ha -1 .
NITROGEN (N)
The largest natural source of nitrogen is the Earth’s
atmosphere, which is roughly 78% gaseous nitrogen, an
inert and essentially biologically unavailable form of the
element. Its biological unavailability is because the two
nitrogen atoms form an extremely stable bond, which is
not easily broken. Apart from human industrial processes
that fix nitrogen gas to solid or liquid forms, the primary
means of nitrogen fixation are through the high
temperature and energy of lightning strikes and biological
nitrogen fixation by bacteria. These processes produce
nitrogen in three main forms, each of which are available
to plants: nitrate, nitrite, and ammonium (Wiedenhoeft,
2006).
Role of nitrogen in plant growth
Boroomand and Grouh 2251
Within the plant, nitrogen serves in the same ways as it
does in other organisms as a component of amino acids
and nucleic acids. Nitrogen also plays a critical role in the
structure of chlorophyll, the primary light harvesting
compound of photosynthesis. This, along with its
structural role in amino acids, explains why plants require
large amounts of nitrogen, and thus why it is often the
limiting nutrient for plant growth.
Role of nitrogen in medicinal plants
Plant height
Moradkhani et al. (2010) reported in Mellissa officinalis L
plant height (61.63 cm) was obtained under application of
90 kg N ha -1 . Suresh (1980) revealed that, application of
nitrogen at 100 kg per ha was optimum in increasing
stem diameter, while nitrogen at 150 kg per ha was found
to be optimum in increasing plant height of Catharanthus
roseus (79.69 cm).
Garnayak et al. (2000) studied the effect of varied
levels of nitrogen on plant height of mustard and reported
significant increase in the plant height with an increase in
the level of nitrogen application from 0 to 120 kg N ha -1 .
The highest was recorded in the treatment supplied with
120 kg N ha -1 which was 200 cm. Okut and Yidirmi (2005)
concluded that application of 90 kg N ha -1 in coriander
resulted in significantly higher plant height (37.10 during
1998/1999). Attoe and Osodeke (2009) reported
significantly higher plant height of Ginger (Zingiber
officinale roscoe) at 200 kg N ha -1 (54.50 cm) than other
level of nitrogen.
Number of branches in a plant
Number of branches plant, the parameter related to yield
and dry matter production of crops, is positively affected
by nitrogen. Mekawey et al. (2010) reported that
application of 150 kg N ha -1 in coriander recorded
significantly higher number of secondary branches (8.37
plant -1 ) over control (6.05 plant -1 ) and was on par with
100 kg N ha -1 (6.57 plant -1 ) and 200 kg N ha -1 (6.91 plant -
1 ). Amit and Sundeep (2007) reported significantly higher
number of primary branches of mustard at 140 kg N ha -1
(7.9 plant -1 ) which was superior over all other N levels but
was on par with 120 kg N ha -1 (7.9 plant -1 ). Attoe and
Osodeke (2009) studied the effect of nitrogen level on
number branch in ginger. They understood that treatment
200 kg of nitrogen produces greatest number of branches
Seed yield
Sadanandan and Shashidharan (1979) studied the effect

2252 J. Med. Plants Res.
of six levels of N on yield of ginger with (0, 25, 50, 75,
100 and 125 kg ha -1 ). The effect of nitrogen was
significant and the highest yield (8595 kg ha -1 ) was
obtained from the treatment receiving 50 kg N per ha and
the treatment with zero N recorded the lowest yield (2995
kg ha -1 ). Lalitha and Gopala (2004) reported significantly
higher seed yield of groundnut at 80 kg N ha -1 (2532 kg
ha -1 ) over all other N levels, but was significantly lower at
120 kg N ha -1 (2383 kg ha -1 ). Buwalda (1986) studied the
effect of nitrogen level between 0 and 240 kgh on yield in
late California cultivar Garlic. He reported that the rate of
120 kgh of N had the best relationship between yield and
quality. To obtain high yield, it is necessary that
continuous nitrogen assimilation be maintained until the
maturation stage. According to Sarma (1985),
significantly higher seed yield of castor was recorded at
80 kg N ha -1 with 90 kg nitrogen as basal dose and 40 kg
as top dressing (1426 kg ha -1 ) which was superior over all
other N levels including control.
Oil content
Katarzyna and Jarosz (2006) reported significantly higher
essential oil of Satureja hortensis L. at 0,8 g dm -3 N which
was superior over all other N levels. Hocking et al. (1997)
assessed the influence of nitrogen on oil content of
canola in a two year field experiment and reported that
nitrogen application up to 75 kg ha -1 increased the oil
content in both years with the maximum increment of 1%.
Application of 120 kg N ha -1 recorded significantly higher
oil content of mustard (39.8 and 39.0% during 1998 and
1999, respectively) which was superior over all other N
levels (Anilkumar et al., 2001). Shishu and Vinay (2005)
recorded significantly higher oil content of mustard at 80
kg N ha -1 (37.61%) which was superior over all other N
levels including control.
POTASSIUM (K)
Potassium is a soil exchangeable cation and is actively
absorbed by plant roots. It is a major component of many
soils and is ultimately derived from the weathering of soil
parent materials such as potassium-aluminum-silicates in
the soil. Potassium though a part of the cation exchange
complex, is only weakly held to the soil particles and is
highly leachable. Due to plants and other organisms
holding potassium as free ions in their cells, once an
organism dies, its potassium quickly reenters the soil
solution. If other organisms do not quickly take up
potassium, it is easily lost from the soil due to leaching
and runoff. A loss of potassium is a common result of
forest fires, clear-cut harvest methods, and other major
disturbances that cause runoff and erosion (Wiedenhoeft,
2006).
Role of potassium in plant growth
Potassium is the primary osmolyte and ion involved in
plant cell membrane dynamics, including the regulation of
stomata and the maintenance of turgor and osmotic
equilibrium. It also plays important roles in the activation
and regulation of enzyme activity (Wiedenhoeft, 2006).
Role of potassium in medicinal plants
Plant height
Borromand et al. (2011a) studied the effect of varied
levels of potassium on leaf length of Aloe Vera and
reported significant increase in the leaf length with an
increase in the level of potassium application from 0 to
150 kg K ha -1 . Balashanmugam and Subramanian (1991)
recorded maximum plant height (152.80 cm), number of
leaves (15.80), and number of suckers per clump (11.32)
in turmeric with the application of potassium at 90 kg per
ha -1 .
Yield
Muralidharan (1973) and Muralidharan et al. (1976)
reported a slight increase in the yield of fresh ginger
rhizome when potassium was raised from 100 to 150 kg
per ha -1 . Further increase in K2O levels showed a decline
in the yield. However, the differences were not significant.
A field experiment conducted with two levels of
potassium at 25 and 50 kg K2O per ha -1 revealed that the
plant characters studied did not show any significant
differences due to the application of K2O to Danti crop
(Saraf et al., 2002). Ashokan et al. (1984) reported that
application of 60 kg K2O per ha -1 produced maximum
marketable tuber yield of sweet potato (13.8 t ha -1 ), dry
matter content in vine (11.9 %) and tuber (30.7 %) as
compared to 30 kg K2O per ha -1 .
Some medicinal plant responses to potassium
Cardamom (Elettaria cardamomum)
Vasantha and Mohanakumaran (1989) reported that for
cardamom, potassium content of leaves and pseudo
stem was maximum at the flower bud growth and peak
flowering stages respectively, and thereafter K
concentration of rhizome, roots and panicles showed
peak values at capsule maturity stage. Sadanandan et al.
(1993) reported that 150 kg K2O ha –1 year is optimum,
whereas Korikanthimath (1994) reported that 240 kg K2O
ha –1 year is needed under high-density trench system of
planting. Cardamom being a perennial crop and steady
bsorption and utilization of K take place throughout its life
cycle and it is a heavy feeder of K (Kulkarni et al., 1971).

Table 1. Recommendation for use fertilizer in medicinal plants on soil analysis.
Boroomand and Grouh 2253
Level of available (NPK) in soil Quantity Probability response to NPK fertilizer
< 0.2 (% O.C*0.5 (%O.C>2) Low
Pav (mgkg -1 )
Kav (mgkg -1 )
*O.C= Organic Carbon, AV= Available.
Ginger (Zingiber officinale)
It was reported that, for optimum productivity, NPK 75,
50, 50 kg together with 25 tonnes green leaf and 20
tonnes farmyard manure (FYM) is required (Sadanandan
et al., 1988). Sadanandan and Rohini (1986) reported
that application of two tonns neem cake together with 75,
50, 50 kg NPK has increased ginger yield by 32.8% and
restricted rhizome rot disease incidence to 4.7%
compared to 14.5% in FYM treated plots.
Pepper (Piper nigrum L)
Integrated nutrient and disease management studies in a
mixed cropping system with black pepper conducted in
the farmer’s fields for four years showed that application
of FYM, Neem cake and bone meal 5, 1 and half kg vine
year together with NPK fertilizer at a subdued level of
100, 40, 140 g vine year increased soil available K status
by 45%, leaf K status by 13% and pepper yield by 172%
(Sadanandan et al., 1993b). For pepper, Pillai et al.
(1987) reported that 200 g K2O vine as optimum whereas
Sadanandan (2000) reported up to 270 g K2O vine as
optimum dose by studying response function. Waard and
De (1969) reported critical level of K up to 2% in pepper
leaf. For bush pepper growing in pots, application of NPK
1, 0.5, 2g pot (10 kg soil) was optimum. Further K2SO4
was a better source than KNO3, KCl and wood ash for
bush pepper (Sadanandan and Hamza, unpublished).
Leaf nutrient norms for black pepper using diagnostic
recommendation integration system (DRIS) Sadanandan
et al. (1996) reported that in pepper leaves potassium
concentration of 0.33% is deficient, 0.33 to 17% as low,
1.18 to 2.84% as optimum, 2.85 to 3.68% as high and
more than 3.68% as excessive level.
15 Low
200 Low
Turmeric (Curcuma longa)
Rethinavel (1983) reported significant increase in plant
height, tiller production number of leaves, number of
mother, primary and secondary rhizomes and ultimately
yield of turmeric due to potassium application.
Sadanandan and Hamza (1996b, 1998) reported that
NPK 60, 50, 120 kg ha –1 with micronutrients were
optimum for varieties Suvarna, Suguna and Alleppey,
whereas NPK 50, 40, 100 kg ha –1 with micronutrients was
optimum for Sudarshana.
Seed spices: Coriander (Coriandrum sativam), Cumin
(Cuminum cyminum), Fennel (Foeniculum vulgare) and
Fenugreek (Trigonella foenum graecum). Fertilizer
response studies for seed spices were conducted by
several workers. Fageria et al. (1972) reported good
response to K2O up to 80 kg ha –1 for cumin. Pillai and
Boominathan (1975) reported response of coriander to
potassium up to 20 kg ha –1 . Afridi et al. (1983) reported
response of potash to fennel up to 90 kg ha –1 . Studies
conducted by “all India coordinated work project on
Spices” at their centers in major seed spices growing
belts of Rajasthan and Gujarat showed that for coriander
20 kg K ha –1 was optimum (Rethinam and Sadanandan,
1994). The requirement of K for cumin is 30 to 45 kg
depending upon K status of the soil. For a crop of
fenugreek 50 kg K2O ha –1 was adequate (Sadanandan
and Hamza, 1998). Our general recommendations for the
use of fertilizer N, P and K on the medicinal plants are
found in Table1.
CONCLUSION
Depending on crop, the response to N, P and K

2254 J. Med. Plants Res.
application varied. Medicinal crops like C. roseus showed
response to as low as 20 kg K2O per hectare and on the
other hand, ginger yield could be raised by applying 150
kg K2O per ha -1 . Studies indicates that N, P and K
nutrients applied in some definite combinations caused
increase in yields rather than supplying either of the
major nutrients alone.
ACKNOWLEDGEMENTS
We thank the University of Jiroft for financial supports
and Dr. Hamdollah Ravand is also acknowledged for
kindly editing the manuscript.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2256-2260, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR10.233
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Chemical composition and antimicrobial activities of
Urena lobata L. (Malvaceae)
E. D. Fagbohun 1 , R. R. Asare 2 and A. O. Egbebi 3
1 Department of Microbiology, University of Ado-Ekiti, P.M.B. 5363, Ado-Ekiti, Nigeria.
2 Department of Science Laboratory Technology, Faculty of Science, University of Ado-Ekiti, Nigeria.
3 Department of Food Technology, The Federal Polytechnic, P.M.B. 5351, Ado-Ekiti, Nigeria.
Accepted 21 June, 2011
The chemical composition and antimicrobial activity of the leaves of Urena lobata L. were investigated.
The proximate analysis showed that the leaves contained moisture (7.21%), crude fibre (6.31%),
carbohydrate (47.53%), crude protein (19.79%), fat (10.21%) and ash (8.95%). The mineral analysis in
mg/100 g indicated that the leaves contained calcium (42.09), copper (0.31), iron (9.35), magnesium
(35.38), manganese (0.81), phosphorus (24.91), potassium (35.38), sodium (29.48) and zinc (51.55). The
phytochemicals detected in the leaves of U. lobata L. were alkaloids, cardiac glycoside, tannins,
terpenoid and saponin; while flaonoid, phlobatanin and steroid were not detected. Antibacterial activities
of the leaf extract showed that Escherichia coli was sensitive to the methanolic extract of the plant and
had a zone of inhibition that varied from 1 - 4 mm with concentration that varied from 6.25 - 50 mg/ml.
Staphylococcus aureus had zone of inhibition that varied from 1.0 - 3.0 mm, Enterococcus spp. had zone
of inhibition that varied form 1.0 - 2.0 mm with concentration that varied between 25 - 50 mg/ml
respectively. Klebsiella spp. had a zone of inhibition of 2.0 mm at 50 mg/ml concentration, while
Pseudomonas aeruginosa was resistant to the extract at all the concentrations tested. The effect of
methanolic extract of U. lobata L on radial mycelial growth of the test fungi revealed that Botryodiplodia
theobromae had a percentage inhibition which varied from 20 - 50% with concentration of 6.25 - 50 mg/ml,
however, B. theobromae was not sensitive at 3.13 mg/ml. However, Rhizopus spp. had a percentage
inhibition that varied from 20 - 50% with concentration that varied from 12.5 - 50 mg/ml.
Key words: Chemical composition, phytochemical analysis, antimicrobial activities, Urena lobata L.
INTRODUCTION
Medicinal plants are plants which contain substances that
could be used for therapeutic purposes or which are
precursors for the synthesis of useful drugs (Sofowora,
2008; Gill, 1992). In recent years, there has been a
gradual revival of interest in the use of medicinal plants in
developing countries because herbal medicines have
been reported to be safe and without any adverse side
effect especially when compared with synthetic drugs and
because of low income of the majority of the populace.
Thus, a search for new drugs with better and cheaper
substitute of plant origin is a natural choice. The
importance of higher plants cut across all aspects of
*Corresponding author. E-mail: fagbohundayo@yahoo.com.
Tel:(+234)8035070548.
life and economy of man. Compounds from plant materials
have been reported to possess in vitro activities against
pathogenic microorganisms (Bringman et al., 1999) and
reduce or cure infections of microbial origin (Ekwuete,
1992; Kim et al., 2002). Medicinal plants have been
formulated into powders, concoctions, decortions, soap
and ointment. Various parts of plants are used which
include the leaves, stem, roots, stem bark and fruits (Gbile
and Adeshina, 1999).
Urena lobata L. is a member of the Malvaceae. The
mallow family are found all over the world with a primary
concentration in the tropics. There are about 110 genera
and over 2,300 species divided into five tribes. The plant
has pink flowers like a miniature hollyhocks. It grows to
about 2 m high stellate trichomes (star-shaped plant hairs)
which gives the leaves a grayish colour and raspy feel
(Dalziel, 1937). The origin of the plant is not well known

and it has been traced to South America, Florida and
Australia but taxonomist believed it evolved in Africa. It is
clutivated in Brazil and Congo for its fibres which are tough,
flexible and used for making sack and twine (Wunderlin,
1998). The Nigerian local names are: akeri, ilasa-agbunrin,
ilasa-omode (Yoruba), rama-rama (Hausa),Oronhon
(Benin), ridiri (Efik) and Ebe-izili (Edo). The leaves and the
whole plant are used as an emollient and expectorant. In
Edo North, the herbalists use the juice of the leaves for
treatment of dysentery. The plant is reputed for its
antimicrobial properties (Gill, 1992). Phytochemical study
of the aerial parts of the plant reported the presence of
mangeferin and quercetin while imperation has been
isolated from the roots (Keshab, 1976).
The aim of this work was to investigate the chemical
composition, phytochemical properties and antimicrobial
activities of the methanolic extract of the leaves of U.
lobata L.
MATERIALS AND METHODS
Collection of plant materials
The fresh leaves of U. lobata L. (Malvaceae) were collected from a
local farm in Ado-Ekiti, Ekiti-State, Nigeria. Identification and
authetication were carried out in the herbarium section of the
Department of Plant Sciences, University of Ado-Ekiti, Ekiti-State,
Nigeria.
Processing of plant materials
The leaves of the plants were air-dried at room temperature for 30
days. This was grinded into fine powder using mortar and pestle. The
powdered leaves materials were stored in a cool dry container until
use.
Determination of antimicrobial activity
Source of microorganisms
The test bacteria used were Staphylococcus aureus, Pseudomonas
aeruginosa, Klebsiella spp. and Enterococcus spp. while the fungi
were Botryodiplodia theobromae and Rhizopus spp. They were
obtained from the Department of Microbiology, University of
Ado-Ekiti. The bacteria were maintained on Nutrient Agar (NA) and
the fungi on Potato Dextrose Agar (PDA) and stored at 4°C until
ready for use.
Standardizaion of inocula
The test bacteria were grown (in separate tubes) at 37°C in Mueller-
Hilton (Oxoid)broth McFarland standard) at optical activity of 625 nm
with Mueller- Hilton (Oxoid) broth and stored at 4°C t o arrest further
bacteria growth/multiplication (Bauer et al., 1966).
Antibacterial testing
This was determined using agar diffusion method (Gnanamanickam
and Smith, 1980; Odeyemi and Fagbohun, 2005). About 0.2 ml of
standardized 24 h old culture of each of the test organisms
Fagbohun et al. 2257
(containing 10 5 cfu/ml) was aseptically transferred to seed the agar
plate. A sterile cork borer (8 mm diameter) was used to cut six wells
on the agar plate. The wells in each plate were filled with various
concentrations of the extracts 50, 25, 12.5, 6.25 and 3.13 mg/ml
while sixth hole in centre was filled with the extracting solvent
(methanol) as control. The tests were set up in duplicates. The
dishes were incubated at 28°C for 18 – 24 h and zone s of inhibition
were measured in millimeter.
Antifungal testing
Radial mycelial growth assay technique of Smith (1978) and
Odeyemi and Fagbohun (2005) were used whereby sterile plant
extracts of the following concentration 50, 25, 12.5, 6.25 and 3.13
mg/ml were introduced aseptically into sterile petri-dishes. About 18
ml of sterilized PDA was added to each of the dishes containing the
various concentration of the plant extracts. The plates were swirled
carefully to ensure proper mixing and allowed to set. Mycelial discs
(6 mm diameter) taken from the advancing edges of 3 – 5 days old
culture of each of the test fungi on PDA were placed centrally on the
cooled seeded agar plates, incubated at 28°C. The ra dial mycelial
growth was measured every 24 h for 5 days. All the plates were in
duplicate and the test carried out twice. Control plates were treated
as described above using the extracting solvent (methanol) only.
Phytochemical analysis of U. lobata L.
Quantitative phytochemical screenings to determine the presence of
alkaloids, tannins, saponins steroids, phlobatannin, terpenoids,
flavonoid and cardiac glycosides using standard methods as
described by Trease and Evans (1985), Harbone (1984) and
Sofowora (2008) were carried out.
Proximate analysis
The proximate analyses of the sample for moisture, ash, fibre and fat
were done by the method of AOAC (2005). The nitrogen was
determined by micro-Kjeldahl method as described by Pearson
(1976) and the percentage nitrogen was converted to crude protein
by multiplying with 6.25. Carbohydrate was determined by difference.
All determinations were performed in duplicates.
Mineral analysis
The mineral was analyzed by using a flame photometer (Model 405
Corning, UK), using NaCl and KCl to prepare the standards.
Phosphorus was determined colometrically using Spectronic 20
(Gallenkap, UK) as described by Pearson (1976) with KH2PO4 as
standard.
All other metals were determined by atomic absorption
spectrophotometer (Pekin-Elmar Model 403, Norwalk CT, USA). All
determinations were done in duplicates. All chemicals used were
analytical grade (BDH, London). Earlier, the detection limit of the
metals was determined according to Techtron (1975). The optimum
analytical range was 0.1 - 0.5 absorbance unit with a coeffient of
variation of 0.87 - 2.20%. All the proximate values were reported as
percentage while the minerals were reported as milligram/100
grams.
RESULTS AND DISCUSSION
The results of proximate analysis of U. lobata L. leaves are

2258 J. Med. Plants Res.
Table 1. Results of proximate analysis of U. lobata L.
(Malvaceae) (%).
Test Percentage of dry sample
Ash content 8.95
Carbohydrate 47.53
Crude protein 19.79
Fat 10.21
Crude fibre 6.31
Moisture content 7.21
Table 2. Results of mineral analysis of U. lobata L.
(Malvaceae) (mg/100 g).
Test Results (mg/100 g)
Calcium 42.09
Copper 0.31
Iron 9.35
Magnessium 35.38
Manganese 0.81
Phosphorus 24.91
Sodium 29.48
Zinc 51.55
shown in Table 1. The plant contained higher amount of
carbohydrate 47.53%. This was similar to the findings of
Abolaji et al. (2007) who reported that Blighia sapida
contained 44.09% carbohydrate but is higher than that of
Senna obtusfolia 23.70% and Amaranthus incurvatus
39.05% (Faruq et al., 2002). It is however, lower than the
value for Corchorus tridens 75.0% and sweet potato
leaves 82.8% (Asibey-Berko and Taiye., 1999).
Carbohydrates are essential for the maintenance of life in
both plants and animals and also provide raw materials for
many industries (Ebun-Oluwa et al., 2007). The plant is a
good source of carbohydrate when consumed because it
meets the recommended dietary allowance (RDA) values
(FND, 2002).
The plant contained crude protein value of 19.79%
which is higher than the value reported for Momordica
balsania L. 11.29% and Lesianthera africanas 13.1%
(Isong and Idiong, 1997), but lower than those value
reported for Piper guineeses 29.78% and Talinum.
triangulare 31.00% (Etuk et al., 1998; Akindahunsi and
Salawu, 2005). However, it compared favourably with the
value reported for Gnetum africana 17.50% and
Leptadenia hastata by Ekop (2007). The plant is
considered a good source of protein because it provides
more than 12% of caloric value from protein (Pearson,
1976).The leaves contained 10.21% of crude fat which is a
moderate amount when compared to those of T.
triangulare 5.9%, Baseila alba 8.71% and Amaranthus
hybridus 4.80% (Akindahunsi and Salawu, 2005). Dietary
fat increases the palatability of food by absorbing and
retaining flavours (Antia et al., 2006). A diet providing 1 -
2% of its caloric of energy as fat is said to be sufficient for
human being as excess fat consumption is implicated in
certain cardiovascular disorders (Antia et al., 2006).The
moisture content value for the leaves of U. lobata L. was
7.21% which is relatively low, therefore it would hinder the
growth of spoilage microorganisms and enhance the shelf
life.
The ash content of U. lobata L. leaves was 8.95% which
is lower than the value reported for the leaves of T.
triangulare 20.05% (Ifon and Bassir, 1980; Ladan et al.,
1996), Ipomea batatas 11.10%, Vernonia colorate 15.86%
and Moringa oleiifera 15.09% (Lockett et al., 2000; Antia et
al., 2006). It is however higher than that of some Nigerian
leafy vegetables such as Ocimum gratissium 8.0%
(Akindahunsi and Salawu, 2005).
The crude fibre of U. lobata was 6.31%. This value
compares favourably with those reported for I. batatas
7.20%, T. triangulare 6.20% and P. guineeses 6.40%
(Akindahunsi and Salawu, 2005).
The mineral composition of U. lobata L. leaves in
mg/100 g were shown in Table 2. It contained sodium
29.45 mg/100 g and potassium 35.38 mg/100 g. The ratio
of sodium to potassium is less than 1(0.8); therefore
consumption of the plants would reduce high blood
pressure disease because Na:K is less than one as
recommended by FND (2002).
The value of calcium and phosphorus in the leaves of U.
lobata L. were 42.09 mg/100 g and 24.91 mg/100 g
respectively. Calcium and phosphorus are associated with
each other for growth and maintenance of bones, teeth
and muscles (Okaka et al., 2006). The phosphorus content
of the leaves 24.91 mg/100 g compared favourably with
that of I. batatas 37.28 mg/100 g (Antia et al., 2006). For
good calcium and phosphorus intestinal absorption,
calcium and phosphorus ratio should be close to 1 or unity
(Gull-Guerrero et al., 1998) hence, calcium and
phosphorus would be well absorbed in the intestine
because the ratio or Ca:P in leaves was (0.6) close to
unity.
Magnessium content of the leaves of U. lobata L. was
found to be 35.38% mg/100 g. This value is high when
compared to that of Xylopia aethiopica 24 mg/100 g
(Abolaji et al., 2007). Magnessium is a component of
chlorophyll and it is an important mineral element in
connection with ischemic heart disease and calcium
metabolism in bones (Ishida et al., 2000).
The values of copper and manganese in the leaves of U.
lobata L. were 0.31 mg/100 g and 0.81 mg/100 g
respectively. This suggests that the leaves of U. lobata L.
does not contribute or rather cannot be used as a
substitute for blood forming leafy vegetables because it fell
far below RDA values (Bogert et al., 1973).
Iron content of the leaves of U. lobata L. was 9.35
mg/100 g. This value compared favourably with the value
reported for I. batatas 16.00 mg/100 g by Antia et al.

Table 3. Results of phytochemical analysis of U.
lobata L. (Malvaceae).
Test Results
Alkaloid +ve
Cardiaglycoside +ve
Flavonoid -ve
Phlobatanin -ve
Tannins +ve
Terpenoid +ve
Saponin +ve
Steroid -ve
+ve = Presence of constituents; -ve = Absence of
constituents.
Fagbohun et al. 2259
Table 4. Antibacterial activity of methanolic extract of U. lobata L. (Malvaceae) on some selected bacterial using punch agar method (Zone of Inhibition
in mm).
Concentrations
50 mg/ml 25 mg/ml 12.5 mg/ml 6.25 mg/ml 3.13 mg/ml Control
Escherichia coli 4.0 4.0 2.0 1.0 0.0 0.0
Staphylococcus aureus 3.0 1.0 0.0 0.0 0.0 0.0
Klebsiella species 2.0 0.0 0.0 0.0 0.0 0.0
Enterococcus species 2.0 1.0 0.0 0.0 0.0 0.0
Pseudomonas aeruginosa 0.0 0.0 0.0 0.0 0.0 0.0
(2006) but low when compared to the vaules of other
green leafy vegetables (Ibrahim et al., 2001). Iron is an
essential element for haemoglobin formation, normal
functioning of central nervous system and oxidation of
carbohydrate, protein and fats (Adeyeye and Otokili, 1999).
The leaves are a good source of iron because it meets the
RDA value of 11.2 mg (Bogert et al., 1973).
The zinc value for the leaves of U. lobata L. was 51.55
mg/100 g. This is the most abundant mineral found in the
leaves in this work. Zinc is involved in normal function of
immune system and is a component of over 50 enzymes in
the body (Okaka et al., 2006). The leaves are a good
source of zinc because it is far above 6.23 mg
recommended by RDA (Borgert et al., 1973).
The result of phytochemical analysis of leaves of U.
lobata L. are shown in Table 3. The plant contained
alkaloid, saponin, tannins, terpenoid and cardiac glycoside
while flavonoid, steroid and phlobatanin were absent.
Alkaloids has been found to have microbiocidal effect and
the major anti-diarrheal effect is probably due to their
effects on small intestine and antihypertensive effect
(Trease and Evans, 1978) Some alkaloids are useful
against HIV infection as well as intestinal infection
associated with AIDS (McDevitt et al., 1976). Saponin was
detected in the leaves of U. lobata L. This compound has
been reported to have antihyper-cholesterol, hypotensive
and cardiac depressant properties (Trease and Evans,
1985; Ghoshal, 1996). The plant also showed the
presence of tannins. The presence of tannin in U. lobata L.
suggests the ability of the plant to play a major role as anti
diarrheaic and anti-haemorrhagic agent (Asquith and
Butler, 1986). The leaves of U. lobata L. showed a positive
result for cardiac glycoside, which have been used for two
centuries as stimulants in treatment of cardiac failure and
diseases (Trease and Evans, 1985; Olayinka et al., 1992).
This perhaps justifies its use by local herbalists for
treatment and management of hypertension (Cowan,
1999). The plant also showed presence of terpenoids. The
anti-bacterial and anti-protozoan properties of this
compound have been reported by Ghoshal et al. (1996);
and Mendoza et al. (1997).The result of antibacterial
activities of methnolic extract of leaves of U. lobata L. are
shown on Table 4. E. coli was sensitive to the methanolic
extract with zones of inhibition that varied from 1.00 to 4.00
mm at concentration that varied from 6.25 – 50 mg/ml
while S. aureus had zone of inhibition that varied from 1.0 -
3.0 mm. Enterococcus sp. had zone of inhibition that
varied from 1.0 - 2.0 mm at concentration that varied
between 25 - 50 mg/ml. However, P. aeruginosa was not
sensitive to the extract at all the concentrations tested. The
antifungal activities of the methanolic extract of U. lobata L.
on the radial mycelia growth of the test fungi are shown
shown in Table 5. B. theobromae had a percentage of
inhibition that varied from 20 - 50% at concentration of
6.25 - 50 mg/ml while Rhizopus sp. had a percentage of
inhibition that varied from 20 - 50% at concentration of

2260 J. Med. Plants Res.
Table 5. Effects of the methanolic extract of U. lobata L. (Malvaceae) on the radial mycelial growth on the test fungi (mm).
Concentration of
extract (mg/ml)
Botrydiplodia theobromae Rhizopus species
Control Test % inhibition Control Test % inhibition
50 34 17.0 50 39 19.0 50
25 34 21.0 40 39 26.0 30
12.5 34 24.0 30 39 32.0 20
6.25 34 29.0 20 39 22 40
3.13 34 34.0 0 39 23.0 40
12.5 - 50.0 mg/ml. From the above results U. lobata L. may
serve as a constituent of human diet supplying the body
with minerals, protein and energy. The use of the plant by
traditional practitioners to treat bacterial and fungal
diseases is therefore justified.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2261-2265, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR10.040
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Screening of Brazilian plants for antiviral activity
against animal herpesviruses
M. J. B. Fernandes*, A. V. Barros, M. S. Melo and I. C. Simoni
Centro de P&D de Sanidade Animal, Instituto Biológico, São Paulo, Brazil.
Accepted 22 June, 2010
In a screening of Brazilian plants, extracts from 27 species were assayed in vitro for antiviral activity
against bovine and suid herpesviruses type 1. The plants considered promising as source of antiviral
substances were those that presented viral inhibition index equal or more than 1.5 meaning a difference
of viral titers between treated and untreated infected cells. Out of the 27 plants tested, extracts of
Bumelia sertorum, Coffea arabica, Endopleura uchi, Leandra purpurescens, Psidium cattleianum and
Uncaria tomentosa showed antiviral activity for both viruses. Extracts of Prunus myrtifolia and
Symphyopappus compressus were active only against bovine herpesvirus while those of Bauhinia
blakeana, Origanum vulgare, Ricinus communis and Tibouchina mutabilis inhibited only suid
herpesvirus. Most of these plants are part of Brazilian folk medicine warranting the ethnopharmacology
as an efficient strategy for selecting the plants for antiviral studies. Plants that presented activity
against both animal herpesviruses are promising for further studies as antiviral components source.
Key words: Brazilian plants, cytotoxicity, suid and bovine herpesviruses type 1, antiviral activity.
INTRODUCTION
Researches on natural products have significantly
progressed over the last decades, mainly on plants
corroborating their importance to the discovery of new
biological and medicinal agents (Rates, 2001; Newman et
al., 2003; Calixto, 2000). Treatment of viral infections with
synthetic substances is often unsatisfactory and limited
due a narrow spectrum of activity, limited therapeutic
usefulness, toxicity and resistant viral strains (Martim and
Ernest, 2003; Chattopadhyay and Naif, 2007). Many
plants have been reported to have antiviral activity and
may serve as promising sources of novel viral prototypes
(Cowan, 1999; Jassim and Naji, 2003; Martim and
Ernest, 2003; Chattopadhyay and Naif, 2007).
In the veterinary area, the bovine (BoHV-1) and the
suid (SuHV-1) herpesviruses are important pathogens,
because of the significant economic losses incurred by
diseases and trading restrictions. BoHV-1 is a major
pathogen of cattle, causing infection bovine
*Corresponding author. E-mail: judite@biologico.sp.gov.br. Tel:
+551150871714. Fax: +551150871791.
rhinotracheitis (IBR) and abortions (Muylkens et al.,
2007). SuHV-1 causes the Aujeszky’s disease (ADV) or
pseudorabies (Nauwynck, 1997; Groff et al., 2005). The
aim of the present work is to assess the in vitro antiviral
activity of 27 Brazilian plant extracts against these two
animal herpesviruses together with studies of cytotoxicity,
because Brazil has the largest tropical forest in the world,
where medicinal plants are most abundant.
MATERIALS AND METHODS
Plants
The 27 plant species used in this study are as shown in Table 1.
Majority of the plants were collected in Serra do Itapeti Municipal
Natural Park, Mogi das Cruzes, São Paulo (SP) State, Brazil during
2004 to 2005. Only Coffea arabica, Ligustrum lucidum, Morus nigra,
Potomorphe umbellata and Ricinus communis were from the park
to the Instituto Biológico, São Paulo/SP. The plant specimens were
identified and authenticated by botanist Dra. Inês Cordeiro, of the
Instituto de Botânica de São Paulo, São Paulo by comparison with
exsiccates deposited at this herbarium. The crude aqueous extracts
(CAE) were obtained from dried leaves which were powdered,
dissolved in sterile distilled water (10%, w/v) and maintained
overnight at 4°C. Then, CAE were filtered on filter paper and were

2262 J. Med. Plants Res.
Table 1. Species of Brazilian plants with their correspondent families and popular names.
Species Family Popular name
Bauhinia blakeana Dunn Caesalpiniaceae Pata de vaca
Begonia fruticosa A.DC. Begoniaceae Begônia
Bumelia sartorum Mart. Sapotaceae Quixaba
Cassia ferruginea Schrad Caesalpiniaceae Canafístula
Chenopodium ambrosioides L. Chenopodiaceae Erva de Santa Maria
Citrus limon (L.) Burm. f. Rutaceae Limão
Coffea arabica L. Rubiaceae Café
Endopleura uchi (Huber) Cuatrec. Humiriaceae Uxi amarelo
Ficus enormis Miq Moraceae Figueira
Impatiens walleriana Hook. f. Balsaminaceae Maria sem vergonha
Leandra purpurescens Cogn. Melastomataceae Pixirica
Ligustrum lucidum W.T. Aiton Oleaceae Ligustro
Morus nigra L. Moraceae Amora
Origanum vulgare L. Lamiaceae Orégano
Pittosporum undulatum Vent. Pittosporaceae Pau – incenso
Plectranthus barbatus Andrews Lamiaceae Boldo brasileiro
Pothomorphe umbellata (L.) Miq. Piperaceae Pariparoba
Prunus myrtifolia (L.) Urban Rosaceae Pessegueiro bravo
Psidium cattleianum Sabine Myrtaceae Araçá
Rhynchelytrum repens (Willd.) C.E.Hubb. Poaceae Capim favorito
Ricinus communis (L.) Euphhorbiaceae Mamona
Solidago chinesis (Osbeck) Merr. Asteraceae Arnica
Symphyopappus compressus (Gardner) B.L.Rob. Asteraceae not known
Tabebuia impetiginosa (Mart. ex DC.) Standl Bignoniaceae Ipê roxo
Tibouchina granulosa Cogn. Melastomataceae Quaresmeira
Tibouchina mutabilis Cogn. Melastomataceae Manacá da serra
Uncaria tomentosa (Willd. ex Roem. & Schult.) DC. Rubiaceae Unha de gato
lyophilized. For the assays, the lyophilized extracts were dissolved
in equal parts of distilled water and Eagle's minimum essential
medium (MEM) (Cultilab®, Campinas/SP, Brazil) for final
concentration of 4,000 µg/ml.
Virus and cell line
Strains Nova Prata of SuHV-1 and Los Angeles of BoHV-1 were
propagated in cell line MDBK (ATCC-CCL 22), and were
maintained in MEM with 10% fetal calf serum.
Cytotoxicity assays
The cytotoxicity of the plant extracts was based on cellular
morphologic alterations using 96-well plates with 30,000 cells/well
(Simoni et al., 2007; Gomes et al., 2008). Briefly, MDBK cells were
exposed to concentrations of extracts, ranging from 31.25 to 2.000
µg/ml, in triplicate. Daily, the cell morphologies were checked to
determine the maximum non-cytoxic concentration (MNCC) of each
extract used in the antiviral tests. Cells incubated only with MEM
were used as controls.
Antiviral assays
The antiviral activity of the extracts was determined by the reduction
of virus titers that was calculated by the method of Reed and
Muench (1938) and expressed in 50% tissue culture infective dose
(TCID50). The difference of viral titer between treated and untreated
control cultures is expressed as viral inhibition index (VII) and was
considered significant when the value is greater or equal to 1.5
(Simoni et al., 2007; Silveira et al., 2009). Briefly, cell monolayers
were treated with the extracts at their respective MNCC for I h and
were inoculated with the logarithmic dilutions (10 -1 to 10 -7 ) of each
correspondent virus. Controls consists of untreated infected (virus
titer), treated non-infected (cytotoxicity control) and untreated noninfected
(cell control) cells.
RESULTS
The 27 plant species screened in this study and their
respective family and popular name are listed in Table 1.
They covered 19 families and 26 genera.

(a) (b) (c)
Figure 1. (a) Normal cells MDBK; (b) infected cells with SuHV-1; (c) infected cells with BoHV-1; (×200).
The appearance of normal cells MDBK is as shown in
Figure 1a. Figure 1b and c shows the cytopathic effect of
suid and bovine herpesviruses type 1, respectively. It is
characterized by cell rounding and ballooning. The
cytotoxic and antiviral activities of these plant extracts
against BoHV-1 and SuHV-1 are as shown in Table 2.
Most of them showed low cytotoxicity to cells with MNCC
ranging from 1.000 to 125 µg/ml, but some of them were
more cytotoxic with MNCC between 62.5 and 7.8 µg/ml.
The extracts of Bumelia sertorum, C. arabica, Endopleura
uchi, Leandra purpurescens, Psidium cattleianum and
Uncaria tomentosa presented VII greater or equal to 1.5
for both viruses. The extracts of Prunus myrtifolia and
Symphyopappus compressus were positive for BoHV-1,
while the extracts of Bauhinia blakeana, Origanum
vulgare, R. communis and Tibouchina mutabilis were
positive for SuHV-1.
DISCUSSION
Although, the collect of the plants was based on random
study of natural flora of the region/country; majority of the
plants after identification were found to be medicinal and
some of them are also part of human feeding (Cordeiro et
al., 2002). Considering the plants with antiviral effect for
at least one of the viruses, all of them have use in
Brazilian folk medicine except S. compressus. There is
no information about it in literature. These data
corroborated the importance of ethnopharmacology as an
efficient strategy for selecting a plant for drug
development and antiviral studies (Vlietinck and Vanden
Berghe, 1991; Rates, 2001; Jassim and Naji, 2003; Cos
et al., 2006).
Plant extracts should be firstly assayed for toxicity
evaluation since the safety of a therapeutic agent is of
paramount importance (Harbell et al., 1997; Rates, 2001;
Veiga et al., 2005). Cytotoxicity tests based on the cell
Fernandes et al. 2263
morphological alterations, although qualitative and more
subjective can initially be used in in vitro antiviral
screening programs when evaluating many plants.
Furthermore, with the use of the extracts in their
respective MNCC in the antiviral tests can distinguish the
antiviral effects of the possible toxic effects of the
extracts.
Studies with some of these extracts have already
demonstrated their antiviral activity as E. uchi
(Plantamed, 2009). Simões et al. (1999) reported the
antiviral effect of P. cattleianum against herpes simplex
(HSV) type 1 and 2, but using hydroalcoholic extracts
from leaves. Our extract is aqueous and then probably
the active compound could probably be a polar chemical
substance since both preparations presented activity.
Barks and roots of U. tomentosa have a broad
therapeutic potential including treatment of viral infections
(Aquino et al., 1989; Willians, 2001). Otherwise, among
the diverse properties and uses of C. arabica, L.
purpurescens and P. cattleianum, an antibacterial activity
is also included (Almeida et al., 2006; Coelho de Souza
et al., 2004; Plantamed, 2009). However, in the studies of
Mccutcheon et al. (1995), P. myrtifolia did not present
antiviral activity against other viruses including HSV type
1. Kudi and Myint (1999) showed the antiviral activity of
B. blakeana against other viruses as equine herpesvirus;
the essential oils from O, vulgare presented antibacterial
and antifungicidal activities among others (Bozin et al.,
2006). Semple et al. (1998) and Kudi and Myint (1999)
described the antiviral activities of Pittosporum undulatum
and Cassia ferruginea, respectively, against a range of
viruses, although these plants were not active in this
study for the two herpesviruses.
Researches for antiviral effects of plant extracts against
these two herpesviruses have continuously been made
(Ahmad et al., 1996; Simoni et al., 1996; Summerfield et
al., 1997; Barrio and Parra, 2000; Felipe et al., 2006;
Simoni et al., 2007). The positive plants for the BoHV-1

2264 J. Med. Plants Res.
Table 2. Cytotoxic and antiviral activity of Brazilian plant extracts against bovine (BoHV-1) and
suid (SuHV-1) herpesviruses type 1.
Species
MNCC
(g/ml)
Antiviral activity
SuHV-1 BoHV-1
Bauhinia blakeana 1000 + -
Begonia fruticosa 250 - -
Bumelia sertorum 250 + +
Cassia ferruginea 31.25 - -
Chenopodium ambrosioides 250 - -
Citrus limon 250 - -
Coffea arabica 250 + +
Endopleura uchi 250 + +
Ficus enormis 250 - -
Impatiens walleriana 62.5 - -
Leandra purpurensis 125 + +
Ligustrum lucidum 500 - -
Morus nigra 500 - -
Origanum vulgare 62.5 + -
Pittosporum undulatum 31.2 - -
Plectranthus barbatus 125 - -
Potomorphe umbellata 500 - -
Prunus myrtifolia 1000 - +
Psidium cattleyanum 62.5 + +
Rhynchelytrum repens 62.5 -
Ricinus communis 250 + -
Solidago chenesis 7.8 - -
Symphyopappus compresses 1000 - +
Tabebuia impetiginosa 250 - -
Tibouchina granulosa 15.62 - -
Tibouchina mutabilis 500 + -
Uncaria tomentosa 500 + +
MNCC = Maximal non cytotoxic concentration. += viral inhibition index 1.5.
and SuHV-1, and other viruses of the Herpesviridae
family may have a spectrum of action directed for this
family. On the other hand, plants that presented activity
for only one of viruses and/or were negative for the other
family members would have a mechanism more specific
for each virus. Plants that presented activity against both
animal herpesviruses are promising as antiviral
components source.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2266-2275, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR10.409
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Anti-ulcer activity of Swietenia mahagoni leaf extract in
ethanol-induced gastric mucosal damage in rats
Salmah Al-Radahe 1 , Khaled Abdul-Aziz Ahmed 2,3 *, Suzy Salama 2 , Mahmood Ameen Abdulla 2 ,
Zahra A. Amin 2 , Saad Al-Jassabi 1 and Harita Hashim 4
1 Institute of Biological Sciences, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia.
2 Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia.
3 Department of Medical Sciences, Faculty of Dentistry, Ibb University, P. O. Box 70627, Ibb, Yemen.
4 Department of Biology, Faculty of Applied Science, University Teknologi MARA 40450 Shah Alam, Malaysia.
Accepted 13 April, 2011
Swietenia mahagoni (West Indian mahogany) has been reported to have medicinal uses, such as
treatment for hypertension, cancer, amoebiasis, chest pains and intestinal parasitism. The present
study was performed to evaluate the acute toxicity and anti-ulcer activity of S. mahagoni ethanol leaf
extract against ethanol-induced gastric ulcer. 24 rats included in this study were divided into 4 groups
with 6 rats each group. Group 1 rats (ulcer control group) were pre-treated with vehicle (Carboxyl
methyl cellulose). Group 2 (reference group) was orally pretreated with 20 mg/kg omeprazole. Group 3
and 4 (experimental groups) were orally pre-treated with S. mahagoni ethanol leaf extract at 250 and 500
mg/kg doses, respectively. After one hour later, all groups received absolute ethanol to generate gastric
mucosal injury. After an additional hour, all the rats were sacrificed and the ulcer areas of the gastric
walls were determined. Grossly, the ulcer control group exhibited severe mucosal injury, whereas pretreatment
with either omeprazole or plant extract resulted in significantly protection of gastric mucosal
injury and increase in mucus production. Flattening of gastric mucosal folds was also observed in rats
pretreated with S. mahagoni leaf extract. Histological studies of the gastric wall of ulcer control group
revealed severe damage of gastric mucosa, along with edema and leucocytes infiltration of the
submucosal layer compared to rats received either omeprazole or S. mahagoni leaf extract where there
was marked gastric protection along with reduction or absence of edema and leucocytes infiltration of
the submucosal layer. Acute toxicity study with a higher dose of plant extract at 5 g/kg did not reveal
any toxicological signs in rats. In conclusions, the present findings suggest that S. mahagoni ethanol
leaf extract exhibit an anti-ulcer activity against ethanol-induced gastric ulcer in experimental animals.
Key words: Swietenia mahagoni leaf extract, gastric ulcer, histology.
INTRODUCTION
Various parts of Swietenia mahagoni have been used as
folk medicine for the treatment of hypertension, malaria,
cancer, amoebiasis, chest pains, fever, anemia diarrhea,
dysentery, depurative and intestinal parasitism
(Nagalakshmi et al., 2001; Maiti et al., 2007). Other
studies have showed other medicinal values of S.
mahagoni plant like antibacterial (Majid et al., 2004) and
*Corresponding author. E-mail: khaaah@gmail.com.
antioxidant activities (Sahgal et al., 2009a) as well as
diabetes therapy (Li et al., 2005). The biologically active
ingredients, tetranortriterpenoids and fatty acids, are
considered to be responsible for these therapeutic effects
(Bacsal et al., 1997).
Previous phytochmical investigations on S. mahogani
have led to the isolation of more than 45 limonoids
belonging to the structural types of andirobin, gendunin,
mexicanolide, phragmalin, triterpens, tetranortriterpenes,
and chlorgenic acid (Abdelgaleil et al., 2006; Chen et al.,
2007). From S. Mahagoni, eighteen tetranotriterpenoids
Wer isolated (Kadota et al., 1990) and the presence of

known fatty acids and terpenoids were reported (Saad et
al., 2003). There are no data available regarding antiulcerogenic
property of S. mahagoni in rats. Therefore,
the present study was undertaken to evaluate the
antiulcerogenic activity of S. mahagoni ethanol leaf
extract against ethanol-induced gastric mucosal damage
in experimental rats.
MATERIALS AND METHODS
Omeprazole
Omeprazole, a proton pump inhibitor, has been widely used as an
acid inhibitor agent for the treatment of disorders related to gastric
acid secretion for about 15 years (Li et al., 2004). In this study,
Omeprazole was used as the reference anti-ulcer drug, and was
obtained from the University of Malaya Medical Centre (UMMC)
Pharmacy. The drug was dissolved in carboxylmethyl cellulose
(CMC) and administered orally to the rats in concentrations of 20
mg/kg body weight (5 ml/kg) (Pedernera et al., 2006).
Plant material
S. mahagoni leaves were obtained from Ethno Resources
Company (Selangor Malaysia) and identified by comparison with
the Voucher specimen deposited at the Herbarium of Rimba Ilmu,
Institute of Biological Sciences, University of Malaya, Malaysia.
Preparation of plant extract
S. mahagoni leaves were shade-dried for 7 to 10 days and were
then powdered using electrical blender. 100 g of fine powder were
soaked in 500 ml of 95% ethanol in conical flask for 3 days. After 3
days the mixture was filtered using a fine muslin cloth followed by
paper filtration (Whatman No. 1) and distilled under reduced
pressure in an Eyela rotary evaporator (Sigma-Aldrich, USA). The
dry extract was then dissolved in CMC (0.25% w/v) and
administered orally to rats in concentrations of 250 and 500 mg/kg
body weight (5 ml/kg body weight) (De Pasquale et al., 1995).
Acute toxicity test
Experimental animals
Adult healthy male and female Sprague Dawley rats (6 to 8 weeks
old) were obtained from the Animal House, Faculty of Medicine,
University of Malaya, Kuala Lumpur. The rats weighed between 150
to 180 g. The animals were given standard pellets and tap water.
The acute toxicity study was used to determine the safe dose for
the plant extract. Thirty six Sprague Dawley rats (18 males and 18
females) were assigned equally into 3 groups.
The first group was labeled as vehicle (CMC, 0.25% w/v, 5 ml/kg)
while the second and third groups of animals were pretreated with 2
and 5 g/kg of S. mahagoni leaf extract, respectively. The animals
were fasted overnight (food but not water) prior dosing. Food was
withheld for a further 3 to 4 h after dosing. The animals were
observed for 30 min and 2, 4, 8, 24 and 48 h after extract
administration for the onset of clinical and/or toxicological
symptoms. The animals were sacrificed on the 14 th day and
histological, hematological and serum biochemical parameters were
Al-Radahe et al. 2267
determined following standard methods (Bergmeyer and Horder,
1980; Tietz et al., 1983).
Behavioral observation and mortality
Throughout the study period, all animals were observed for
behavioral signs of toxicity, morbidity and mortality. Mortality checks
were made twice daily and determination of behavioral signs was
observed daily for all animals. Detailed observations of the
individual animals were made weekly in comparison with the vehicle
treated animals.
Observations included gross evaluations of the skin, any signs of
respiration (dyspnea), salivation, exophthalmia, convulsion and any
changes in locomotion such as whether the animals tend to stay
quietly or actively moving in their cage.
Hematological and biochemistry analysis
The animals were fasted overnight prior to necropsy and blood was
collected. Blood samples were drawn from jugular vein under
diethyl ether anesthesia. Blood samples were collected into EDTA
tubes for total and differential white blood cell (WBC) count. For
serum biochemistry analysis, blood was collected into
anticoagulant-free tubes.
Biochemical parameters include aspartate aminotransferase
(AST), alanine aminotransferase (ALT), total protein, albumin,
globulin, total bilirubin, conjugated bilirubin, alkaline phosphatase,
gamma glutamyl transferase (GGT), urea, creatinine, anion gap and
serum electrolytes (CO2, Potassium, Sodium and Chloride). All
samples were sent immediately to the Clinical Diagnostic
Laboratory at University of Malaya Medical Centre for liver and
renal function tests. The results were compared to that of the rats’
respective control groups.
Gross necropsy and histopathology
At scheduled termination, all surviving animals were anesthetized
by diethyl ether inhalation and quickly sacrificed by exsanguinations
of jugular vein for blood sample collection. Gross postmortem
examinations were performed on all terminated animals. Liver and
kidney from each animal were routinely processed and embedded
in paraffin. After sectioning and staining with Haematoxylin and
Eosin (H and E) stain method, all slides were observed under
microscope to observe for any pathological changes.
Anti-ulcer activity studies
Experimental animals
Sprague Dawley healthy adult male rats were obtained from the
Experimental Animal House, Faculty of Medicine, University of
Malaya. The animals were kept at room temperature in humidity
rooms on a standard light/dark cycle (12 h light; 12 h dark cycle).
The rats were divided randomly into 4 groups of 6 rats each. Each
rat that weighted between 200 to 225 g was placed individually in
separate cage (one rat per cage) with wide-mesh wire bottoms to
prevent coprophagia during the experiment. The animals were
maintained on standard pellet diet and tap water.
Gastric ulcer induction by ethanol and tissue sample collection
The rats were fasted for 48 h before the experiment (Garg et al.,

2268 J. Med. Plants Res.
1993), but were allowed free access drinking water up to 2 h before
the experiment. Gastric ulcer in Sprague Dawley was induced by
orogastric intubation of absolute Ethanol (5 ml/kg) according to the
method described by De Pasquale et al. (1995). Ulcer control group
was orally administered with vehicle (CMC, 0.25% w/v, 5 ml/kg).
The reference group was received oral doses of 20 mg/kg
Omeprazole in CMC (5 ml/kg) as positive controls.
Experimental groups (groups 3 and 4) were orally administered
250 and 500 mg/kg of plant extract dissolved in CMC solution (5
ml/kg), respectively. One hour after this pre-treatment; all groups of
rats were gavaged with absolute ethanol (5 ml/kg) in order to
induce gastric ulcers (Hollander et al., 1985). After one hour, the
rats were sacrificed by cervical dislocation under overdose of
diethyl ether anesthesia (Paiva et al., 1998). The stomachs were
immediately excised and rapidly immersed in 10% buffered formalin
solution.
Measurement of acid of gastric juice and mucus production
Each stomach was opened along the greater curvature. Samples of
gastric contents were analyzed for hydrogen ion concentration by
pH-meter titration with 0.1 N NaOH solutions using digital pH meter.
The acid content was expressed as mEq/l based on the method of
Tan et al. (2002). Gastric mucus production was measured in the
rats that were subjected to absolute ethanol-induced gastric
mucosal injury. The gastric mucosa of each rat was gently scraped
using a glass slide and the mucus obtained was weighed using a
precision electronic balance (Tan et al., 2002).
Gross gastric lesions evaluation
Any ulcers would be found in the gastric mucosa, appearing as
elongated bands of hemorrhagic lesions parallel to the long axis of
the stomach. Each gastric mucosa was thus examined for damage.
The length (mm) and width (mm) of the ulcer on the gastric mucosa
were measured by a planimeter (10 × 10 mm 2 = ulcer area) under
dissecting microscope (1.8 x).
The area of each ulcer lesion was measured by counting the
number of small squares, 2 × 2 mm, covering the length and width
of each ulcer band. The sum of the areas of all lesions for each
stomach was applied in the calculation of the ulcer area (UA) where
the sum of small squares × 4 × 1.8 = UA mm 2 as described by
Kauffman and Grossman (1978) with slight modification. The
inhibition percentage (I %) was calculated by the following formula
as described by Njar et al. (1995):
(I %) = [(UAcontrol − UAtreated) UAcontrol] × 100.
Histological evaluation of gastric lesions
Specimens of the gastric walls from each rat were fixed in 10%
buffered formalin and processed in a paraffin tissue processing
machine. After embedding, sections of the stomach were made at a
thickness of 5 µm and stained with Hematoxylin and Eosin for
histological evaluation.
Ethics
The study was approved by the ethics Committee for animal
experimentation, Faculty of Medicine, University of Malaya,
Malaysia (Ethics No. PM 07/10/2009 MAA (a)(R). Throughout the
experiments, all animals received human care according to the
criteria outlined in the “Guide for the Care and Use of Laboratory
Animals” prepared by the National Academy of Sciences and
published by the National Institute of Health.
Statistical analysis
All values were expressed as mean ±S.E.M. The statistical
significance of differences between groups was assessed using
one-way analysis of variance (ANOVA). The Mann-Whitney U test
was used to compare the difference between two groups. A value
of p

Table 1. Renal function tests of rats in acute toxicity study of S. mahagoni leaf extract.
Dose
Sodium
(mmol/L)
Pottasium
(mmol/L)
Chloride
(mmol/L)
CO2
(mmol/L)
Anion gap
(mmol/L)
Urea
(mmol/L)
Al-Radahe et al. 2269
Creatinine
(µmol/L)
Vehicle (CMC, 0.25% w/v) 137.17 ± 0.47 5.17±0.2 103.02 ± 0.17 23.11 ± 0.86 18.17 ±0.75 5.61 ± 0.44 50.33 ± 1.74
S. mahagoni extract (2 g/kg b.wt) 137.67±0.42 5.32±0.16 101.69±1.23 20.84 ±0.77 19.17 ± 1.56 4.86 ± 0.46 49.67 ± 0.84
S. mahagoni extract (5 g/kg b.wt) 138.01 ± 0.50 4.88 ± 0.17 102.83 ± 0.79 21.9 ±0.88 17.76 ±0.61 5.72±0.38 48.74±1.88
Values are expressed as mean ± S.E.M. There are no significant differences among all groups.
Table 2. Liver function tests of rats in acute toxicity study of S. mahagoni leaf extract.
Dose Total protein (g/L) Albumin (g/L) Globulin (g/L) TB (µmol/L) CB (µmol/L) AP (IU/L) ALT (IU/L) AST (IU/L) GGT (IU/L)
Vehicle (CMC, 0.25% w/v) 71.2 + 1.7 11.33 + 0.71 59.86 + 1.70 1.81 + 0.17 0.86 + 0.17 134.5 + 17.9 53.05 + 3.17 152.9 + 8.3 4.87 + 0.92
Plant extract (2 g/kg b.wt) 71.2 + 0.5 10.87 + 0.33 59.62 + 0.34 2.07 + 0.17 1.00 + 0.00 133.7 + 8.9 50.83 + 1.37 152.7 + 3.6 5.01 + 1.21
Plant extract (5 g/kg b.wt)t) 71.8 + 1.0 11.63 + 0.6 60.00 + 0.93 1.77 + 0.22 1.00 + 0.00 135.1 + 6.9 52.65 + 3.27 154.0 + 11.3 5.13 + 1.09
Values are expressed as mean ± S.E.M. There are no significant differences among all groups. TB: Total bilirubin; CB: Conjugated bilirubin; AP: Alkaline phosphatase; ALT: Alanine aminotransferase; ST:
Aspartate aminotransferase; GGT: G-Glutamyl Transferase.
Table 3. Effect of leaf extract of S. mahagoni on the ulcer area pre-treated with different preparations.
Animal group Pre-treatment (5 ml/kg dose) Mucus production pH of gastric content Ulcer area (mm) 2 Inhibition (%)
1 Ulcer control (CMC) 0.34 + 0.009 a 4.0 + 0.09 a 920.00 ± 10.72 a -
2 Omeprazole (20 mg/kg b.wt) (positive control) 0.57 + 0.006 b 6.85 + 0.15 b 205.00 + 7.64 b 77.72
3 S. mahagoni leaf extract (250 mg/kg b.wt) 0.89 + 0.08 c 7.0 + 0.13 b,c 185.33 ± 6.77 b 79.86
4 S. mahagoni leaf extract (500 mg/kg b.wt) 1.05 + 0.02 d 7.25 + 0.07 c 45.50 + 5.16 c 95.05
All values are expressed as mean ± S.E.M of six rats per group. Mean values with different superscripts are significantly different. The mean difference is significant at P value of

2270 J. Med. Plants Res.
Figure 1.
Gastric lesions
mmmmmmmmmmmmmmmmmmmmmmmmmm
Figure 1. Macroscopic appearance of the gastric mucosa in a rat pre-treated with
5 ml/kg of CMC (ulcer control). Severe injuries were seen in the gastric mucosa.
Gastric lesions
Figure 2. Macroscopic appearance of the gastric mucosa in a rat pre-treated with
hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh
5 ml/kg of Omeprazole (20 mg/kg). Injuries to the gastric mucosa were milder
compared to the injuries seen in the ulcer control rat.

Al-Radahe et al. 2271
Figure 3.nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
re 4
Figure 3. Macroscopic appearance of the gastric mucosa in a rat pre-treated with 5
ml/kg of S. mahagoni extract (500 mg/kg). Gastric mucosa showed flattening of
mucosal fold, and no prominent injuries were observed.
nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
Edema with
leucocytes
Mucosal
lesions
Figure 4. Histological section of gastric mucosa in a rat pre-treated with 5 ml/kg of CMC (ulcer
control). There was severe disruption to the surface epithelium, and edema of the submucosal
layer with leucocytes infiltration (H&E stain 10x magnification).
hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh

2272 J. Med. Plants Res.
Figure 5.nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
Mucosal
lesions
Edema with
leucocytes
Figure 5. Histological section of gastric mucosa in a rat pre-treated with 5 ml/kg of
Omeprazole (20 mg/kg). There was mild disruption to the surface epithelium with mild
edema and leucocytes infiltration of the submucosal layer (H&E stain 10x).
Figure 6.mmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmm
Intact
mucosa
Figure 6. Histological section of gastric mucosa in a rat pre-treated with 5 ml/kg of
Swietenia mahagoni (500 mg/kg). There was very mild disruption to the surface epithelium
with no edema and no leucocytes infiltration of the submucosal layer (H&E stain 10x).
mmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmmm
the result of an imbalance between aggressive
factors and maintenance of the mucosal integrity through
the endogenous defense mechanism (Piper and Stiel,
1986). It is known that gastric lesions produced by
ethanol administration appeared as multiple-hemorrhagic
red bands of different size along the glandular stomach.
Absolute ethanol is commonly used for inducing ulcer
in experimental rats and lead to intense gastric mucosal
damage. Studies suggest that the ethanol damage to the
gastrointestinal mucosa starts with microvascular injury,

namely disruption of the vascular endothelium resulting in
increased vascular permeability, edema formation and
epithelial lifting (Szabo et al., 1995). Furthermore, ethanol
produces necrotic lesions in the gastric mucosa by its
direct toxic effect, reducing the secretion of bicarbonates
and production of mucus (Marhuenda et al., 1993).
The results of the present study show that the S.
mahagoni leaf extract is capable of inhibiting gastric
lesions formed by ethanol. The accompanying significant
increase in mucus production suggests that the gastric
mucosal strengthening mechanism contributes to the
anti-irritant potential of the S. mahagoni. It is evident that
increased mucus productions have largely contributed to
preventive effect of the extract. The gastric wall mucus is
thought to play an important role as a defensive factor
against gastrointestinal damage (Davenport, 1968).
Pre-treatment of animals with leaf extract of S.
mahagoni significantly increased gastric mucus content
which suggests that gastroprotective effect of this plant is
mediated partly by preservation of gastric mucus
secretion.
In the present study, we observed flattening of the
mucosal folds which in turn suggests that gastroprotective
effect of S. mahagoni leaf extract might be due
to a decrease in gastric motility. It is previously reported
that the changes in the gastric motility may play a role in
the development and prevention of experimental gastric
lesions (Garrick et al., 1986; Takeuchi et al., 1987).
Relaxation of circular muscles will increase the mucosal
area to be exposed to necrotizing agents and reduce the
volume of the gastric irritants on rugal crest leading to
protection of the gastric mucosa against damage
(Takeuch and Nobuhara, 1985).
Our findings have revealed protection of gastric
mucosa and inhibition of leucocytes infiltration of gastric
wall in rats pretreated with S. mahagoni leaf extract.
Kobayashi et al. (2001) reported that teprenone exerts a
protective effect against mucosal lesions through
inhibition of neutrophil infiltration in the ulcerated gastric
tissue while Shimizu et al. (2000) demonstrated that the
reduction of neutrophil infiltration into ulcerated gastric
tissue promotes the healing of gastric ulcers in rats.
Absolute alcohol would extensively damage the gastric
mucosa leading to increased neutrophil infiltration into the
gastric mucosa. Oxygen free radicals derived from
infiltrated neutrophils in ulcerated gastric tissues have
inhibitory effect on gastric ulcers healing in rats (Suzuki et
al., 1998). Neutrophils are a major source of inflammatory
mediators and can release potent reactive oxygen
species such as superoxide, hydrogen peroxide and
myeloperoxidase derived oxidants. These reactive
oxygen species are highly cytotoxic and can induce
tissue damage (Cheng and Koo, 2007). Therefore,
suppression of neutrophil infiltration during inflammation
was found to enhance gastric ulcer healing (Tsukimi et
al., 1996). S. mahagoni leaf extract has been shown to
contain anti-inflammatory activity (Ghosh et al., 2009)
Al-Radahe et al. 2273
and it is speculated that the gastro-protective exerted by
this plant could be attributed to its anti-inflammatory
property. Swarnakar et al. (2005) have reported that the
anti-inflammatory activity of curcumin could be a key
factor in the prevention of gastric ulcer.
Oxidative stress plays an important role in the
pathogenesis of various diseases including gastric ulcer,
with antioxidants being reported to play a significant role
in the protection of gastric mucosa against various
necrotic agents (Trivedi and Rawal, 2001).
Antioxidants could help to protect cells from damage
caused by oxidative stress while enhancing the body’s
defense systems against degenerative diseases.
Administration of antioxidants inhibits ethanol-induced
gastric injury in rat (Ligumsky et al., 1995).
S. mahagoni have been reported to contain flavonoids
(Sahgal et al., 2009a, b) and it could be conceivable that
the anti-ulcer activity of S. mahagoni leaf extract could be
linked to the flavonoids since flavonoids are reported to
protect the mucosa by preventing the formation of lesions
by various necrotic agents (Saurez et al., 1996). It is well
known that many flavonoids display anti-secretory and
cytoprotective properties in different experimental models
of gastric ulcer (Zayachkivska et al., 2005). Flavonoids
possess anti-oxidant properties in addition to
strengthening the mucosal defense system through
stimulation of gastric mucus secretion (Martin et al.,
1994).
The acute toxicity profile of S. mahagoni leaf extract
could be considered favorable judging from the absence
of adverse clinical manifestations in experimental animals
after 14 days of observation. Liver and kidney of the
treated rats showed no significant changes as compared
to the control group. Hematology and clinical
biochemistry values were within the range of the control
animals tested and similar to some of the control
reference values published by other researchers (Ghosh
et al., 2009; Radhamani et al., 2009).
The highest dose of ethanol extract of S. mahagoni
which did not cause any toxicity was 5 g/kg body weight,
suggesting that the S. mahagoni is relatively non-toxic
since in acute toxicity studies, the product is considered
non-toxic if no deaths are registered after 14 days of
observation and no clinical signs of toxicity are observed
at doses at or below 5 g/kg. These results indicate that
the extract is quite safe even at these higher doses and
has no any acute toxicity.
In conclusion, S. mahagoni leaf extract could
significantly protect the gastric mucosa against ethanolinduced
damage. Such protection was shown to be dose
dependent as ascertained by the reduction of ulcer areas
in the gastric wall, reduction or inhibition of edema, and
leucocytes infiltration of submucosal layers.
ACKNOWLEDGEMENTS
The authors express gratitude to the staff of the Faculty

2274 J. Med. Plants Res.
of Medicine Animal House for the care and supply of rats,
and to the University of Malaya for the financial support
(P0102/2009C).
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2276-2283, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR10.780
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Analysis on the main active components of Lycium
barbarum fruits and related environmental factors
Jing Z. Dong 1,2 , Shu H. Wang 1 , Linyao Zhu 3 and Y. Wang 1 *
1 Key Laboratory of Pant Germplasm Enhancement and Speciality Agriculture, Wuhan Botanical Garden, Chinese
Academy of Sciences, Wuhan 430074, China.
2 School of Biological Science and Technology, Hubei University for Nationalities, Enshi, 445000, China.
3 Wuhan Vegetable Extension Station, Wuhan, 430012, China.
Accepted 29 February, 2012
Fruits of Lycium barbarum L. (wolfberry or “Gouqi” in Chinese) are widely used as traditional Chinese
medicine and functional food in China, South-east Asia, Europe and North America. Polyphenols,
polysaccharides and carotenoids are the main active compounds in L. barbarum fruits. Based on a
rapid sample preparation with ultrasonic-assisted extraction (UAE) established, contents of
polyphenols, polysaccharides and carotenoids of L. barbarum fruits from 8 main producing areas of
western China were analyzed and the related environmental factors were investigated. The results are:
L. barbarum fruits from Zhongning showed the highest polyphenols contents (2.9749%) and the highest
antioxidant activity (RSA = 56.82%), the highest content of polysaccharides (8.9041%) was from Jinghe,
the highest content of carotenoids (0.1642%) was from Julu; the highest fruit weight and pulp weight
were from Numhon. Contents of polysaccharides, polyphenols and carotenoids were significantly
affected by environmental factors: the principle factor for polyphenols was soil organic matter (SOM) (r
= 0.964), the principle factors for carotenoids were temperature-sunshine (r = 0.826), polysaccharides
were mainly affected by soil available phosphorus (r = 0.75), fruit weight or pulp weight were negatively
correlated with temperature (r = 0.953, 0.963). Each of the three active components has its own genuine
producing area rather than Zhongning as the only genuine producing area authenticated.
Key words: L. barbarum fruits, polyphenols, antioxidant activity, polysaccharides, carotenoids, genuineness.
INTRODUCTION
Lycium barbarum L. is one of the important traditional
Chinese medicinal plant species. It has been cultivated in
Northwest China and used as daily functional food in
China, Southeast Asia and many European countries. In
the Chinese medicinal monographs
“shennongbencaojing”, “ben cao gang mu” and “ben cao
hui yan”, L. barbarum fruits were recorded as nourishing
liver and kidney, enhancing eyesight, enriching blood,
invigorating sex, reducing rheumatism” and so on. More
functions were recently reported as immunity
improvement (Lin et al., 2008), anti-oxidation (Lu et al.,
2008), anti-radiation (Qian et al., 2004), anticancer (Chao
et al., 2006), enhancing hemopoiesis (Hsu et al., 1999),
anti-aging and enhancing sex (Yu et al., 2005).
*Corresponding author. E-mail: djz22cn@yahoo.com.cn. Tel:
86-27- 87510771.
Polyphenols, polysaccharides and carotenoids are the
important active compounds in L. barbarum fruits. Since
L. barbarum fruits have become one of the most popular
functional foods, L. barbarum has been cultivated in
many areas of western China, which leads to the
subsequent quality problems of L. barbarum fruits.
Environmental factors of L. barbarum fruits are important
for quality control on L. barbarum fruits. In order to make
a systematic evaluation on the quality of L. barbarum
fruits, the main active components of L. barbarum fruits
from different areas were analyzed, the related ecological
factors were investigated.
MATERIALS AND METHODS
Reagents and materials
Standard rutin were purchased from Chinese Authenticating
Institute of Material Medica and Biological Products (Beijing, China).

β-carotene were purchased from Sigma Company, USA, 1diphenyl-2-picrylhydrazyl
(DPPH) was purchased from Wako Pure
Chemical Industries, Ltd. (Japan), all the other chemicals were of
analytical grade. Ripe fruits of the same cultivated variety of L.
barbarum L. were collected from eight main producing areas in
Northwest China. In every producing area, 5 locations (N1 - N5)
were sampled as replicates. The fruits were dried with silica gel,
and then frozenly milled with liquid nitrogen to quickly go through
60-mesh screen. Then the collected powders were dried with silica
gel until constant weight.
Equipments
The following facilities were used: an ultrasonic cleaning bath
(Desktop NC, Xi'an Taikang Biotechnology Co., Ltd., China, 40 kHz,
400 W) equipped with an automatic temperature regulation system,
UV-VIS spectrophotometer Lambda 450 (PerkinElmer, Inc., USA),
Agilent 1100 HPLC system consisted of Agilent 1100 ChemStation
Rev.A.10.02, G1313A autosampler, G1311A quarternary pump,
G1314A variable wavelength ultraviolet (UV) detector (VWD), and
reverse phase ZORBAX SB-C8 column (5 µm, 4.6 × 250 mm,
Agilent Technologies, USA).
Extraction and determination of polyphenols
0.2 g of the dried powders were placed in soxhlet extractor and
refluxed with ether at 50°C water bath to remove oils for 2 h. After
the ether was volatilized, the deoiled powders were put in an
airproof box filled with silica gel for polyphenols analysis.
Ultrasonic-assisted extraction (UAE) is being used widely in
analytical chemistry, facilitating different steps in the analytical
process, particularly in sample preparation (Cabredo-Pinillos et al.,
2006; Wang et al., 2006), expeditious, inexpensive and efficient
alternative to traditional extraction techniques and microwaveassisted
extraction (Jalbani et al., 2006). In order to make rapid and
efficient extraction of polyphenols from L. barbarum fruits, UAE
method was employed for sample preparation. Factors as
extraction temperature, duration, concentration of ethanol and ratio
of liquid to solid were optimized.
Methanol was regarded as not in compliance with good
manufacturing practice (GMP) due to its high toxicity (Hemwimol et
al., 2006), so in this study only ethanol was used for extraction of
polyphenols. The deoiled powders were mixed with the appropriate
extraction solvent in a 100 ml conical flask that was immersed in
water of the ultrasonic cleaning bath. The bottom of the flask was
approximately 2 cm above that of the bath and the liquid level in the
flask was about 5 mm below the water surface in the bath. The
extracts were filtered through 0.22 µm microporous membranes
and the filtrates were collected for quantitation of polyphenols.
Optimization on UAE parameters was conducted by orthogonal
design and test (Table 1).The results were analyzed with SPSS
16.0.
Polyphenols in the extracts were determined according to the
established method (Dong et al., 2009), namely, polyphenols of the
extracts were directly determined at 258 nm with rutin as
equivalents. Recovery was obtained by adding rutin into the
extracts to investigate the accuracy of the determination on
polyphenols of the samples prepared by the UAE method.
Polyphenols contents and radical-scavenging activity
The capacity of polyphenols of L. barbarum fruits to remove 1, 1diphenyl-2-picrylhydrazyl
radical was determined according to the
method by Chon et al. (2009), namely, 1 ml of polyphenols extract
and 5 ml of freshly prepared 0.1 mM DPPH methanol solution were
Dong et al. 2277
thoroughly mixed and kept in the dark for 60 min. The absorbance
of the reaction mixture at 520 nm was measured with the UV
spectrophotometer. The blank was prepared by replacing the
extract with methanol. The percentage of free radical scavenging
activity was calculated as follows:
Scavenging activity (%) = [1 − (A520 nm, sample/A520 nm, blank)] × 100
Analysis on carotenoids of L. barbarum fruits
Ultrasonic cannot only destroy the wall of plant cells but enhance
mass transfer rates (Zhang et al., 2009; Dong et al., 2010), hence
increase extraction rate. The optimized UAE process for L.
barbarum fruit samples was also used for the extraction of
polyphenols, carotenoids and polysaccharides from L. barbarum
fruit samples. Six kinds of commonly used extraction solvents were
used in carotenoids extraction to find out the proper extraction
solvent. The same amount of dried fruit samples were weighted and
extracted with the same amount of solvents under the extraction
process modified from the UAE method established earlier, namely,
extraction temperature of 40°C, extraction time of 30 min, solid to
liquid of 3.0 mg/ml. The extraction was performed in three cycles.
Then the extracts were scanned within 350 to 600 nm. In Figure 2,
these extracts showed the special UV-visible absorption of βcarotene
(Fraser and Bramley, 2004). Absolute ethanol extract
showed the highest absorbance which indicated the highest
extraction yield of carotenoids. So, absolute ethanol was used as
the proper solvent for carotenoids extraction. The carotenoids
extract by absolute ethanol was separated by HPLC method to
detect possible interference (Figure 3). The HPLC conditions:
acetonitrile (solvent A, 60%) and dichloromethane (solvent B, 40%),
deaerated ultrasonically for 30 min, respectively, in advance were
used as the mobile phase with a flow rate of 1.0 ml/min. The
column temperature was set at 25°C and the sample volume
injected was 10.0 µl. The detector was set at 453 nm.
β-carotene was used as standard for determination of
carotenoids. Carotenoids concentrations were calculated according
to the calibration, with β-carotene as standard. A good linear
relationship was obtained within the range of 0.5 to 4.0 µg/ml, and
the regression equation is: y = 0.176 x + 0.0058, r = 0.9998, where
y is the absorbance at 453 nm, x is the concentration of β-carotene
(µg/ml), r is coefficient correlation.
Determination of polysaccharides
The prepared dried fruit powders were weighed and packed with
filter paper. The packed powders were placed in soxhlet extractor
and refluxed with ether at 60°C water bath for 4 h to remove lipids.
And the deoiled samples were further refluxed with 80% (v/v)
ethanol at 80°C for 4 h to remove polyphenols and flavonoids.
Finally, the samples were dried at 60°C for 4 h and then
polysaccharides of the samples were extracted with UAE method
established in this article. The extraction was performed in three
cycles.
Phenol-sulfuric method (Masuko et al., 2005) was used for
determination of polysaccharides. Polysaccharides concentrations
were calculated according to the calibration, as glucose standard. A
good linear relationship was obtained within the range of 8.5 to 38.5
µg/ml, and the regression equation is: y = 0.0069x + 0.0035, r =
0.9998, where y is the absorbance at 490 nm, x is the concentration
of glucose (µg/ml), r is coefficient correlation.
Analysis on environmental factors related with the main active
components and fruit weights
Potential influential environmental factors were collected and

Table 1. Orthogonal test and results (L16, 4 4 ).
Test no. Ethanol (%)
Extraction
temperature(°C)
Liquid/ solid (ml/mg)
Extraction time
(min)
Mean yield
(%, n = 3)
1 50 30 1 30 2.65
2 50 50 1.5 40 3.05
3 50 60 2 50 2.51
4 50 70 2.5 60 2.54
5 60 30 1.5 50 3.08
6 60 50 1 60 3.25
7 60 60 2.5 30 3.15
8 60 70 2 40 2.82
9 70 30 2 60 2.46
10 70 50 2.5 50 3.38
11 70 60 1 40 2.91
12 70 70 1.5 30 2.65
13 80 30 2.5 40 2.28
14 80 50 2 30 2.24
15 80 60 1.5 60 2.12
16 80 70 1 50 1.93
K1 2.69 2.62 2.69 2.67
K2 3.08 2.98 2.73 2.77
K3 2.85 2.67 2.51 2.73
K4 2.14 2.49 2.84 2.59
R 0.94 0.49 0.33 0.18
P value 0.002 ** 0.011 * 0.037 * 0.170
* Significant (P < 0.05); ** very significant (P < 0.01).
analyzed. These factors are: sunshine (kh/y), precipitation (mm/y),
cumulative temperature (≥10°C/y), mean annual temperature
(°C/y), frost-free period (d/y), and altitude (m) were collected from
local weather records of producing areas, respectively. Soil factors
as soluble salinity (%), SOM (soil organic matter, %), Soil available
nitrogen, phosphorus and potassium were analyzed according to
the method of Täumer et al. (2005). The factors as sunshine,
precipitation, cumulative temperature, mean annual temperature,
frost-free period, altitude, soluble salinity and SOM were analyzed
by principle factors analysis with SPSS 16.0 to investigate
correlations between these environmental factors and the main
active components, because these are environmental factors
deciding the genuineness of L. barbarum fruits. While soil available
nitrogen, phosphorus and potassium can be easily changed by
human farming, so the available nitrogen, phosphorus and
potassium were analyzed by linear regression independently.
RESULTS
Preparation of the samples
A four-level OAD with an OA16 (4 4 ) matrix was chosen to
optimize the UAE parameters (Table 1). Based on singlefactor
experiments (Figure 1), the levels are chosen as:
ethanol concentration of 50/60/70/80%, extraction
temperature of 30/50/60/70°C, ratio of liquid to solid of
1/1.5/2/2.5, extraction time of 30/40/50/60 min. The
results of orthogonal test and extreme difference analysis
are presented in Table 1. The P value analysis indicated
that the influential order of the four factors on the
extraction yield of polyphenols is ethanol concentration >
extraction temperature > ratio of liquor to solid >
ultrasonication time (Table 1). According to variance
analysis, the contributions of ethanol concentration,
extraction temperature and ratio of liquor to solid for the
extraction yield of polyphenols are significant (P < 0.05),
whereas extraction time was not significant factor which
means UAE can significantly shorten extraction time.
According to extreme difference analysis, the optimum
extraction condition of polyphenols was deduced as:
ethanol of 70%, liquid/solid of 1/2.5 (ml/mg), extraction
temperature of 50°C, extraction time of 30 min.
Accuracy of determination on polyphenols from UAE
extracts
The linearity range of standard rutin was determined as
4.0 to 15.0 µg/ml (R 2 = 0.9998). The equation was
obtained by linear regression: y = 0.0419x - 0.1015 (data
not shown), where y is absorbance, x is concentration of
rutin (µg/ml). Recovery was obtained by adding standard
rutin to extract solution to obtain total polyphenols. Final
concentrations were 6.0, 11.0 and 15.0 µg/ml. The assay

Figure 1. Effect of ethanol concentration, temperature, liquid/solid and extraction time on polyphenols yields.
Dong et al. 2279
Figure 2. UV-Visible chromatography of carotenoids extracted by different solvents (1 = absolute ethanol, 2 = chloroform, 3 = aether,
4 = acetone/aether (1/1), 5 = acetone, 6 = absolute methanol).

mAU
300
250
200
150
100
50
0
0 5 10 15 20 25
mAU
300
250
200
150
100
50
0
0 5 10 15 20 25
Figure 3. HPLC chromatography of caratenoids from L. barbarum fruit extract by absolute ethanol (1. carotenoids extract; 2.β-carotene).
Table 2. a Three active components levels of L. barabrum fruits from different producing areas and radical scavenging
activity of polyphenols
Producing areas PS (%) C (%) PP (%) FW PW RSA(%) R
ZN 5.3682 C 0.1005 B 2.9749 A 12.182 C 10.4231 C 56.82
SH 6.9639
0.968
B 0.1535 A 2.7081 B 13.738 C 11.3863 C 49.71
NM 7.2293 B 0.0511 D 2.54056 C 19.713 A 16.9451 A 46.25
GM 7.3586 B 0.0512 D 2.64098 C 18.531 A 16.3494 A DZ 7.4153
48.80
B 0.0570 D 2.63 C 17.98 B 15.4195 B 47.32
QT 7.3190 B 0.0907 B 2.40148 D 7.984 D 7.0674 D 45.18
JH 8.9041 A 0.0656 C 2.46116 D 13.313 C 11.8399 C 43.43
JL 7.0099 B 0.1642 E 1.594 E 8.946 D 7.1368 D 30.82
a : Each value is the mean of five replicates(RSD

Table 3. Climatic and soil factors of 8 producing areas of L. barbarum.
Dong et al. 2281
* S Pr C M F A D SOM N P K
ZN 2.972 250 3.349 9.2 153 1190 0.44 2.91 51.53 76.77 312.37
SH 3.200 171.6 3.447 8.5 157 1055 0.60 2.75 45.49 53.73 423.87
NH 3.090 38.9 1.921 4.4 112 2812 0.48 2.29 66.32 66.43 438.03
GM 3.150 45.8 2.014 4.2 140 3250 0.40 2.56 47.43 73.59 443.33
DZ 3.065 450 2.228 7.4 120 3667 0.37 1.90 53.29 65.37 535.75
QT 3.076 176 3.107 4.7 156 792 1.80 4.39 46.87 59.76 357.43
JH 2.800 111.6 3.582 7.2 175 366 0.03 0.52 25.36 18.92 121.10
JL 2.773 532 4.663 13.1 207 18 0.13 10.85 35.75 32.72 327.40
* Producing area codes are the same as those of Table 3. S = Sunshine (Kh/y); Pr = precipitation (mm/y); C = cumulative temperature
(≥10°C/y); M = mean annual temperature(°C/y); F = frost-free period (d); A = altitude (m); D = dissoluble salinity (%); SOM = soil organic
matter (%); N = available nitrogen (mg/kg); P = available phosphorus (mg/kg); K = available potassium (mg/kg).
Table 4. Linear correlations of L. barbarum fruits with climatic and soil factors producing areas.
Variable PS (%) C (%) PP (%) FW PW
S* +0.6402 -0.2260 -0.2323 +0.4738 +0.4685
Pr* -0.5563 +0.5080 -0.2268 -0.3932 -0.4391
C* -0.6166 +0.8300 -0.0650 -0.8309 -0.8545
M* -0. 5641 +0.7931 -0.2919 -0.5150 -0.5684
F* -0.6943 +0.7248 +0.0803 -0.7905 -0.7983
A* +0.4678 -0.6950 -0.0294 +0.8699 +0.8717
D* +0.1841 -0.0050 -0.1456 -0.3969 -0.3706
SOM* -0.8454 +0.7022 -0.2667 -0.5866 -0.6273
N* +0.4458 -0.3448 -0.5004 +0.5435 +0.5205
P* +0.4644 -0.3338 -0.7500 +0.4180 +0.4159
K* +0.2121 -0.1335 -0.3309 +0.5080 +0.4707
*: The codes are the same as those of Table 3 and 4.
Determination on contents of polysaccharides and
carotenoids
Recovery was obtained by adding standard glucose to
extracts to obtain polysaccharides. Final concentrations
were 9.0, 25.0, and 38.0 µg/ml. The assay was
conducted with 5 replicates. Recovery was within 98.7
to105.1% (data not shown). As shown in Table 2, the
order of polysaccharides contents is Jinghe (8.9041%) >
Qitai (7.3190%), Dazi (7.4153%), Goldmud (7.3586%),
Numhon (7.2293%), Shahai (6.9639%), Julu (7.0099%) >
Zhongning (5.3682%). So, Jinghe can be considered as
the genuine area for polysaccharides of L. barbarum
fruits.
β-carotene was added into the carotenoids extracts to
obtain recovery, final concentration 1.0, 2.5, 4.0 µg/ml.
The recovery was 97.45 to 109.3% (data not shown). As
is shown in Table 2, the order of carotenoids contents is
Julu (0.1642%) > Shahai (0.1535%) > Zhongning
(0.1005%), Qitai (0.0907%) > Jinghe (0.0656%) >
Numhon (0.0511%), Goldmud (0.0512%), Dazi (0.0570%).
Julu proved to be the genuine area for carotenoids of L.
barbarum fruits.
Correlation between environmental factors and the
main active components of L. barbarum fruits
Environmental factors of eight producing areas were
collected in Table 3. In Table 4, linear correlation analysis
showed that polyphenols contents was strongly
negatively correlated with SOM (r = 0.8454), which
indicated that soil containing relatively low SOM is proper
for polyphenols accumulation; carotenoids were positively
correlated with effective cumulative temperature (r =
0.83), mean annual temperature (r = 0.7931) and frostfree
period (r = 0.7248), meaning that areas of west
China with relatively low temperature are not the best for
carotenoids accumulation; fruit weight and pulp weight
were strongly positively correlated with altitude (r
=0.8699, 0.8717, respectively), strongly negatively
correlated with cumulative temperature and frost-free

Dong et al. 2282
Table 5. Correlation of main active components and fruit weights with eigenvectors of principal
factors by rotation and multiple regression.
Characters P a Extracted principle factors and equations b
Polyphenols (%) 0.024 Y = +0.714 (X1) 2 , r = 0.964
Carrotenoids (%) 0.000 Y = +1.641 (X2) 3 , r = 0.826
Weight/100 fruits (g) 0.000 Y = -1.791(X2) 3 , r = 0.953
Pulp/100 fruits (g) 0.000 Y = -1.848 (X2) 3 , r = -0.963
Polysaccharides (%)* Y = -0.819 (X3) 3 , r =- 0.750
a : P value of the model by tests of KMO and Barteltt; b : X1 = SOM factor (%); X2 = accumulated
temperature, frost-free days each year and altitude; X3 = available phosphorus; *: analyzed by linear
regression independently.
period (r = -0.8309, -0.7905, -0.8545, -0.7983,
respectively), at the relatively high altitude, strong
sunshine and high temperature variety between day and
night are good for photosynthesis and accumulation of
dried matters; polysaccharides was not significantly
affected by other environmental factors but negatively
correlated with soil available phosphorus (r = 0.75).
Based on rotation and multiple regression analysis (Table
5), SOM was the principle factor for polyphenols contents
(r = 0.964), effective cumulative temperature, frost-free
period and altitude were the principle factors significantly
affecting carotenoids contents, fruit weight and pulp
weight (r = 0.826, 0.953 and 0.963, respectively). Altitude
mainly leads to temperature-sunshine changes; so,
effective cumulative temperature, frost-free period and
altitude can be summed up as temperature-sunshine
factor, namely, temperature-sunshine is the principle
factor for carotenoids contents, fruit weight and pulp
weight.
DISCUSSION
Genuineness of L. barbarum fruits and medicinal
values
In ancient traditional Chinese medicine system,
Zhongning had been authenticated as the genuine
producing area where the L. barbarum fruits were of the
highest medicinal quality. In pharmacopoeia of China
(2005), polysaccharides were authenticated as the main
active components of L. barbarum fruits. In this study, L.
barbarum fruits from Zhongning showed the highest
content of polyphenols (2.9749%) and the highest
antioxidant activity (56.82%) with high correlation (r =
0.968), which indicated that content of polyphenols in L.
barbarum fruits can accurately reflect the anti-oxidant
ability and confirmed that Zhongning is the genuine
producing area for polyphenols. It was reported that more
than 100 human diseases are correlated with oxidation
caused by free radicals in the body (Gutteridge, 1993). L.
barbarum fruits from Zhongning presented the highest
content of polyphenols and the highest antioxidant
activity; it may be the main reason for the genuineness of
L. barbarum of Zhongning.
L. barbarum fruits produced in different areas had
different essential active components. Among the 8
producing areas, Zhongning proved to be the genuine
area for polyphenols production, while polysaccharides
genuine producing area was Jinghe, carotenoids genuine
producing area was Julu, the highest fruit weight was
from Numhon. Fruits from Zhongning contained the
highest content of polyphenols, but the lowest content of
polysaccharides in the 8 areas. So, polyphenols may be
the really main active component rather than
polysaccharides as for L. barbarum fruits from
Zhongning.
Since L. barbarum fruits of different areas showed
different contents of the main active components, their
main medicinal use should be consequently different.
Take for example, vitamin A deficiency was a worldwide
serious health problem, especially in Africa, South
America, Central America, and Southeast Asia (Sommer
et al., 1995). L. barbarum fruits of Julu should be the best
choice for vitamin A supplementation because L.
barbarum fruits of Julu have the significantly highest
content of β-carotene which was the precursor in vitamin
A synthesis in human body. While L. barbarum fruits of
Jinghe should be the best choice for polysaccharides
supplementation and extraction (Dong et al., 2008).
Principle factors affecting the genuineness of L.
barbarum fruits
The optimal environmental factors for each active
component are not the same, meaning that each active
component has its own genuine producing area.
Relatively low SOM is good for polyphenols
accumulation; relatively high temperature and low
ultraviolet in the sunshine are proper for carotenoids
synthesis and accumulation, while low temperature is
good for dried matter accumulation hence leading to
higher pulp weight. The climatic factors, SOM and
dissoluble salinity are not related with polysaccharides.
Only soil available phosphorus significantly affected the

content of polysaccharides. Phosphate is mainly stored in
membrane system in plants; deficiency of phosphate may
lead to the membrane phosphate transferred to growth
center, which resulted in degradation of phospholipids,
while galactose acts as substitute for phospholipids to
maintain the normal structure and functions of membrane
(Kochlan et al., 2004). Sun et al. (1997) reported that
69% of polysaccharides composition in L. barbarum fruits
was galactose, which confirmed the mechanism that
deficiency of phosphorus leads to polysaccharides
accumulation.
Conclusion
Extraction with UAE proved to be the proper sample
preparation for L. barbarum fruits. β-carotene is the main
component in carotenoids of L. barbarum fruits, which
can be a good source for VAD functional food. Absolute
ethanol is the best solvent for carotenoids extraction
concerning safety and efficiency. A direct UV
determination of carotenoids from absolute ethanol
extracts of L. barbarum fruits is convenient and accurate.
Contents of the main active components of L. barbarum
fruits from the 8 areas were significantly different. So,
medicinal values of L. barbarum fruits from different
producing areas might not be the same.
Polyphenols of L. barbarum fruits from Zhongning were
the main active components rather than polysaccharides.
L. barbarum fruits of Julu should be the best selection for
carotenoids supplementation because of highest βcarotene
content, while L. barbarum fruits from Jinghe
containing the highest content of polysaccharides should
be the best source for polysaccharides supplementation
or extraction.
Environmental factors have significant effects on
genuineness and the main active components of L.
barbarum fruits. The principle factors of polysaccharides,
carotenoids and polyphenols are soil available
phosphorus, temperature-sunshine and SOM,
respectively. These principle factors provide a basis for
scientific division of producing areas and quality control
for L. barbarum.
ACKNOWLEDGEMENTS
This research was partially funded by CAS/SAFEA
International Partnership Program for Creative Research
Teams Project and Doctor Research Project (498012) of
Hubei University for Nationalities, and Major Project of
Wuhan Municipal Bureau of Agriculture (200720322099).
We are grateful to anonymous reviewers and scientific
editor for their critical review and valuable suggestions.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2284-2288, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR10.806
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
In vitro antiplasmodial activity of seven plants
commonly used against malaria in Burkina Faso
Kumulungui Brice Serge 1,2 *, Ondo-Azi Alain Serges 2 , Mintsa Ndong Armel 3 , Fumoux Francis 4
and Traore Alfred 1
1 Laboratoire de Pharmacologie et Biochimie Clinique du CRSBAN, UFR-SVT, Université de Ouagadougou,
Ouagadougou, Burkina Faso.
2 Institut National Supérieur d’Agronomie et de Biotechnologies (I.N.S.A.B), Université des Sciences et Techniques de
Masuku (U.S.T.M.) Franceville, Gabon.
3 Laboratoire National, Ministère de la Santé. Libreville, Gabon.
4 Pharmacogénétique des Maladies Parasitaires, EA 864, IFR 48, Faculté de Pharmacie, Université de la Méditerranée,
Marseille cedex 5, France.
Accepted 21 December, 2010
We collected seven medicinal plants currently used for malaria treatment in Burkina Faso. The paper
reports the in vitro antiplasmodial activity of 26 crude extracts (alkaloids and tannins) from leaves and
roots. After scientific experimentation, we discovered that Alkaloids from Mitragyna inermis leaves and
roots and from Anogeissus leiocarpus leaves have a strong antiplasmodial activity on laboratory
strains and on Plasmodium falciparum strains isolated from infected patients (IC50

Serge et al. 2285
Table 1. Relative content of alkaloids and tannins on roots and leaves of the seven selected plants (nd: not
determined).
Medicinal plants
(number of extractions)
Roots Leaves
Alkaloids (%) Tannins (%) Alkaloids (%) Tannins (%)
A. leiocarpus (n = 3) 0.02 13 0.1 20
B. rufescens (n = 2) 0.03 13 0.04 4
C. sieberiana (n = 2) 0.01 11 0.02 14
M. inermis (n = 6) 0.2 5 0.6 7
N. latifolia (n = 3) 0.3 2 nd nd
P.biglobosa (n = 2) 0.06 12 0.04 10
T. roka (n = 2) 0.1 5 0.2 6
Benth.)), two Rubiaceae (Mitragyna inermis (Willd., O.Ktze) and
Nauclea latifolia Sm).
The plants were collected during the wet season around the town
of Ouagadougou. They were identified by the Department of
Botany, Ouagadougou University, where voucher specimens have
been deposited.
Preparation of crude extracts
The air dried plants were powdered using a semi-industrial crusher.
Alkaloids were extracted according to Lannuzel et al. (2002),
modified as follows: After humidification with NH4OH (12.5%),
alkaloids were extracted using petroleum ether and treated with
H2SO4 (5%). The aqueous acid solution was washed with hexane,
alkalised with Na2CO3 (10%), and extracted with chloroform. The
organic solution was washed with water, dried over Na2SO4, and
evaporated. Alkaloid bases were dissolved in acetone with
Hydrochloric acid (HCl) vapour. The presence of hydrochlorate
alkaloid residue was confirmed using the Mayer and Draggendorf
test.
The powder was delipided and tannins were extracted using
acetone. After elimination of pigments, the acetone was evaporated
and the tannin precipitates were characterised by FeCl3 in
accordance with CEE recommendations (Bruneton, 1993).
Quantitative studies were conducted using gravimetric analysis. For
in vitro antiplasmodial activity, stock solutions of alkaloids and
tannins at 5.000 µg/ml were prepared after filtration using 0.22 mm
Millipore membrane. A chloroquine stock was prepared at 1.000
µg/ml.
Antiplasmodial activity
P. falciparum strains were isolated from infected children (4 to 7
years) at Barogo village, north of Ouagadougou. Thin blood films
were prepared and stained with Giemsa. The selected subjects
showed a blood parasite density ranging from 300 to 10.000
infected red blood cells per µl. Before treatment, venous blood was
collected in ethylenediametetraacetic acid (EDTA) Vacutainer®
tubes and immediately transported to the laboratory. Seven isolates
were used for antiplasmodial assay. Standard protocols of P.
falciparum in vitro cultures (Trager and Jensen, 1976) were used
(RPMI 1640 with 10% human serum, human erythrocytes O+,
incubated at 37°C with 5% CO2).
Continuous in vitro culture of the 3D7 chloroquine sensitive strain
and W2 chloroquine resistant strain was maintained in the
laboratory. Quantitative assessment of in vitro antiplasmodial
activity was determined using four concentrations of each extract.
The initial highest concentration of each plant extract was 1250
µg/ml, and the lowest was 1.25 µg/ml. All tests were made in
duplicate for 24 and 48 h, in a 96 well flat-bottom culture plate
(Costar, UK). Chloroquine was used as a positive control and drugfree
medium as a negative control. After incubation, thin blood films
were prepared and stained with Giemsa, for each well; the
percentage of infected red blood cells was determined
independently by two readers. In case of discrepancy a third
reading was made. Percentage inhibition curves = f (-log [plants
extract]) following a two order polynomial regression. IC50 were
classified according to Deharo et al. (2001): If it is less than 5 µg/ml,
the extract was considered highly active, 5 to10 µg/ml moderately
active, and over 10 µg/ml inactive. Each assay was run in duplicate
and the values given are averages of the two independent assays.
All field isolates were chloroquine sensitive (IC50 < 15 ng/ml).
Haemolytic activity
We determined the in vitro haemolytic activity of extracts using
human red blood cells O+; haemoglobin rate was determined at
550 nm (Tietz and Fiereck, 1973).
RESULTS AND DISCUSSION
Twenty-six extracts were prepared from the seven plants.
A very unequal distribution of alkaloids and tannins in
leaves and roots of the plants was observed. Qualitative
analysis showed that leaves and roots of A. leiocarpus,
C. sieberiana and P. biglobosa are rich in tannins and
poor in alkaloids. Conversely, M. inermis and N. latifolia
contain many alkaloids but few tannins. These raw
results were confirmed by quantitative study using
gravimetric analysis. A. leiocarpus, C. sieberiana, and P.
biglobosa contain 13, 11 and 12% tannins respectively in
the roots, and 20, 14 and 10% in the leaves (Table 1). M.
inermis, T. roka and N. latifolia contain 0.2, 0.1 and 0.3%
alkaloids, respectively in the roots and 0.6, 0.2 (M.
inermis and T. roka) in the leaves.
In preliminary studies, chloroquine and all 26 extracts
(alkaloids and tannins) were screened at four
concentrations on 3D7, W2 and on the seven strains
isolated from infected patients. Only a few extracts
showed an IC50 inferior to 10 µg/ml. Considerable

2286 J. Med. Plants Res.
Table 2. Number of P. falciparum strains (schizontes) per µl of blood after 24 h incubation with various concentrations of alkaloids from A. leiocarpus and M. inermis. % of inhibition is
reported as IC50 values.
A. leiocarpus
(Leaves)
M. inermis
(Leaves)
M. inermis
(Roots)
Alkaloids extracts
concentration in µg/ml
P. falciparum strains number / µl before Alkaloids using
Isolate 1 Isolate 2 Isolate 3 Isolate 4 Isolate 5 Isolate 6 Isolate 7 Inhibition IC50
540 1480 336 2780 1060 320 9940 % µg/ml
P. falciparum strains number / µl after Alkaloids using
1250 0 0 0 0 0 0 0 100
125 4 40 0 0 0 0 32 99
12.5 160 400 96 64 20 16 9080 78 2.59
1.25 168 768 320 2176 1020 380 9800 32
1250 0 0 0 0 0 0 0 100
125 8 0 0 0 0 0 0 100
12.5 144 620 160 1320 64 0 6040 66 2.61
1.25 152 1060 352 1340 1820 264 8200 31
1250 0 0 0 0 0 0 0 100
125 0 0 16 0 8 0 8 100
12.6 164 820 176 1240 840 56 4280 67 2.35
1.25 264 1068 420 1280 1060 320 11360 30
Chloroquine
IC50 for each 11.45 20.19 11.57 17.14 29.27 18.28 7.09
Isolate (nM)
IC50 of chloroquine (CQ) on isolates and reference strains of Plasmodium falciparum (Pf). W2 and 3D7 are expressed in nM to validate the sensitivity of P. falciparum (IC50100 µg/ml, and

Percentage inhibition
% of inhibition
Percentage inhibition
% of inhibition
(A) (A)
120
(B)
120
100
100
100
99
120
100
100
99
80
78100
80
78
60
80
60
40
32
Serge et al. 2287
20
0
20
0
0
0 1 2
0
3
1 2 3
0
-log[alkaloids
4
extract] µg/ml
0 1
4
2
0
3
1 2 3
-log[alkaloids
4
extract] µg/m
(C)
120
100
80
60
40
100
-log[alkaloids extract] µg/ml
100
% of inhibition
40
20
(A) (A)
(B)
(C)
(C)
120
100
6760
100
20
0
0
0 1 2 3 4
0 1 2 3 -log[alkaloids 4 extract] µg/ml
30
% of inhibition
60
40
100
(B)
66 60
-log[alkaloids extract] µg/ml
Figure 1. Antimalarial -log[alkaloids activity of extract] alkaloids µg/ml of A. leiocarpus and M. inermis: A/Alkaloids from A. leiocarpus leaves. B
/Alkaloids from M. inermis leaves. C/ Alkaloids from M. inermis roots. Each point represents the average of the seven
isolates after incubation for 24 h at 37°C.
no more studies of these extracts were performed.
However, our study showed considerable antiplasmodial
activity for alkaloids from M. inermis and A. leiocarpus
leaves, and for alkaloids from M. inermis roots (IC50

2288 J. Med. Plants Res.
Scalbert (1991) and Malmberg et al. (1980). Their results
concerning the toxicity of tannins were similar to our
results. For example, they showed that tannins such as
lapachol or gossipol show an antiplasmodial activity on P.
falciparum strains, but that their toxicity limited their use,
as in our results.
Most of the plant extracts commonly used as medicinal
plants in Burkina Faso did not show activity in the malaria
assays used in this study. However, this does not mean
that extracts of the plants are not active, as they may
work via other mechanisms as pro-drugs or febrifuges.
The activity of some plant extracts may be mediated by
active metabolites formed in vivo. In vivo investigation of
these plants, for example on animal models, will be
necessary before we can draw any conclusion
concerning their efficacy against human malaria. Finally,
some extracts of the seven plants, in particular alkaloids
extracted from M. inermis and A. leiocarpus, showed
considerable antiplasmodial activity. After toxicological
studies, these plants may be of use, particularly in rural
communities where conventional drugs are unavailable
and health facilities are insufficient.
IC50 of chloroquine (CQ) on isolates and reference
strains of P. falciparum (Pf), W2 and 3D7 are expressed
in nM to validate the sensitivity of P. falciparum (IC50

Journal of Medicinal Plants Research Vol. 6(12), pp. 2289-2294, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.201
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Phytochemicaland proximate analyses and thin layer
chromatography fingerprinting of the aerial part of
Chenopodium ambrosioides Linn. (Chenopodiaceae)
Okhale, Samuel Ehiabhi 1 *, Egharevba, Henry Omoregie 1 , Ona, Eneyi Comfort 1,2 and
Kunle, Oluyemisi Folashade 1,3
1 Department of Medicinal Plant Research and Traditional Medicine, National Institute for Pharmaceutical Research and
Development (NIPRD), Idu Industrial Area, Idu, P. M. B. 21 Garki, Abuja, Nigeria.
2 Department of Chemistry, Ahmadu Bello University, Zaria, Nigeria.
3 Department of Pharmacognosy, Faculty of Pharmacy, University of Jos, Nigeria.
Accepted 1 February, 2012
The aerial part of Chenopodium ambrosioides L., reputable for the treatment of malaria and diabetes in
Nigeria, was qualitatively screened for the presence of secondary metabolites using standard methods.
Some proximate parameters were also determined. The result of the phytochemical screening revealed
the presence of alkaloids, tannins, saponins, flavonoids, terpenes, sterols, cardenolide aglycone,
volatile oils and carbohydrates. The proximate analysis revealed moisture content of 10.90%, total ash
value of 14.65%, acid-insoluble ash value of 3.05%, water-soluble ash of 6.25%, water-soluble extractive
value of 3.18% and alcohol-soluble extractive value of 13.20%. The thin layer chromatography
fingerprinting and phytochemical screening revealed several chemical components, which could be
isolated from the plant. This study shows that C. ambrosioides is a potential drug plant considering the
rich phytochemical and proximate pharmacognostic profile of the aerial part, hence its folkloric uses.
The result of this study is the first of its kind on the Nigerian species of this plant drug, and is
informative for standardization and monograph development of this herbal plant.
Key words: Chenopodium ambrosioides, secondary metabolites, thin layer chromatography, pharmacognostic
analysis, antimalaria, Mexican tea, ash value.
INTRODUCTION
Chenopodium ambrosioides L. also known as epazote,
Mexican tea, and wormseed, belongs to the family
Chenopodiaceae. The species is wide-spread and
originates from tropical America (Munz, 1975). C.
ambrosioides is an annual or short-lived perennial herb
that grows to over 1 m high, with aromatic glandular
hairs. It has a strong rank smell when bruised and the
leaves have a pungent smell (Burkill, 1985).
Chenopodium species have a wide variety of medicinal
properties (Burkill, 1985; Tapondjou et al., 2002). In
*Corresponding author. E-mail: samokhale@yahoo.com.
Spain, the dry or fresh aerial part of C. ambrosioides is
boiled in water and drunk after meals as digestifs,
stomachics, and in some cases as hemostatic, stimulant,
laxative and antidiarrhoeic. It is also used to reduce blood
pressure and to treat colds and fibroids (Filipoy, 1994). It
has been used for centuries as condiment, traditional
antihelmintic and antimalarial (Ruffa et al., 2002). The
plant is sometimes cultivated principally for medicinal
uses in West Africa where the leaves are added as
flavorings to soup, pounded leaves are applied to sores,
or to swellings on the body and to areas of pain. The
aromatic smell is inhaled for headache. The plant is used
for the treatment of diabetes. In Nigeria, the whole plant
is pounded and eaten as a laxative, and its infusion is

2290 J. Med. Plants Res.
used as febrifuge, as well as to treat coughs and
tuberculosis (Burkill, 1985). A decoction of the aerial part
is used in Northern Nigeria as an antimalarial.
In view of the enormous bioactivity profile and the
documented ethnomedicinal uses of C. ambrosioides,
this study aims to establish its phytochemical,
pharmacognostic and thin layer chromatographic
characteristics that could be useful for standardization
and monograph development of this potential drug plant.
MATERIALS AND METHODS
All chemicals used were of analytical reagent grade (Sigma) and
used as supplied.
Plant materials and processing
Plant material was collected by Mallam Muazzam Ibrahim from
Chaza in Suleja, Niger state, Nigeria. The plant was identified and
authenticated at the Herbarium of the National Institute for
Pharmaceutical Research and Development, Abuja, Nigeria. The
aerial part of the plant was air-dried for three weeks at room
temperature, pulverized and used immediately for analyses.
Phytochemical screening
The phytochemical and proximate analyses of the powdered aerial
part of the plant were performed using the methods described by
Harborne (1998), MHFW (1999), Evans (2002) and Sofowora
(2008). The analyses were carried out as thus explained.
Test for cardenolide aglycone
0.5 g of the powdered sample was boiled with 10 ml of 95% alcohol
for 2 min. The mixture was filtered; the filtrate was allowed to cool
and diluted with water to 10 ml. Three drops of a saturated solution
of lead sub-acetate were added and filtered thoroughly. The filtrate
was treated with 1 ml of 2% solution of 3, 5-dintrobenzoic acid in
95% alcohol and the solution basified with 5% sodium hydroxide. A
purple-blue colour indicates the presence of free or combined
cardenolide aglycone (Sofowora, 2008).
Test for terpenes and sterols
5 g of the powdered sample was extracted by maceration with 50
ml of chloroform. The extract was filtered and evaporated to
dryness on water bath. The residue was dissolved in 10 ml of
anhydrous chloroform and filtered. The filtrate was divided into two
equal portions and used for the following tests:
Lieberman-Burchard test for terpenes: To the first portion of the
chloroform solution was added to 1 ml of acetic anhydride and
shaken. Then 1 ml of concentrated sulphuric acid was added down
the wall of the test tube to form a layer underneath. The formation
of a reddish-violet colour at the lower layer indicates the presence
of terpenes (Sofowora, 2008).
Salkowski’s test for sterols: The second portion of the chloroform
solution was carefully mixed with 2 ml of concentrated sulphuric
acid so that the sulphuric acid formed a lower layer. A reddish-
brown colour at the interface indicates the presence of a steroidal
ring.
Test for saponins
0.5 g of the powdered plant material was added to 5 ml of 95%
ethanol and boiled for 2 min; filtered into a test tube and diluted to
10 ml with distilled water. The test tube was shaken vigorously for 1
min. The formation of a persisting honey comb indicates the
presence of saponins.
Test for tannins and phlobatannins
Ferric chloride test for tannins: 10 ml of distilled water was added
to 1 g of the powdered sample in a test tube and boiled for 3 min in
a water bath. The mixture was allowed to cool and then filtered with
Whatman No.1 filter paper. 1 ml of the filtrate was diluted with 4 ml
of distilled water and few drops of 10% ferric chloride were added.
Instant formation of blue-black or green coloured solution indicates
the presence of tannins (Evans, 2002).
Test for Phlobatannins: 10 ml of distilled water was added to 1g of
the powdered sample in a test tube and boiled for 3 min in a water
bath. The mixture was allowed to cool and then filtered with
Whatman No. 1 filter paper. 1 ml of the filtrate was boiled with equal
volume of 1% v/v hydrochloric acid. The formation of a red
precipitate indicates the presence of phlobatannins (Evans, 2002).
Test for anthraquinone (Borntrager’s tests)
0.5 g of the powdered sample was poured into a dried test tube and
10 ml of chloroform added. The mixture was shaken for 5 min and
filtered. The filtrate was shaken with 10% w/v potassium hydroxide
solution. A bright pink-red colour in the upper aqueous layer
indicates the presence of free anthraquinones (Evans, 2002).
Test for balsams
1 g of the powdered sample in a test tube was extracted with 10 ml
of 90% v/v ethanol and filtered. Two drops of 10% (w/v) alcoholic
ferric chloride solution was added to 5 ml 5 of the filtrate. Formation
of a dark green coloured solution indicates the presence of
balsams.
Tests for resins
0.5 g of the pulverized plant was extracted with 15 ml petroleum
ether and filtered. 5 ml of the filtrate was dispensed into a test tube
and shaken vigorously with equal volume of copper acetate solution
TS. The mixture was allowed to stand for a few minutes. Formation
of a green coloured solution indicates the presence of resins.
Test for alkaloids
3 g of the powdered sample was macerated with 50 ml of methanol
at room temperature and filtrate evaporated to dryness on a hot
water bath without overheating. 10 ml of 1% v/v HCL was added to
the residue. The solution was divided equally into five test tubes,
and two drops of the following reagents were added to the
respective test tubes: Mayer’s reagent – (potassium mercuric iodide

Table 1. Result of phytochemical screening of the aerial
part of C. ambrosioides.
Phytochemical constituents Result
Cardenolide aglycone +
Terpenes +
Sterols +
Saponins +
Tannins +
Anthraquinones ND
Balsams ND
Resins ND
Alkaloids +
Phlobatannins ND
Flavonoids +
Phenols
+
Volatile oil +
+: Detected; ND: not detected.
solution); Dragendorff’s reagent - (potassium bismuth iodide
solution); Wagner’s reagent – (solution of iodine in potassium
iodide); Hager’s reagent – (a saturated solution of picric acid); and
10% tannic acid solution. The formation of amorphous or crystalline
precipitates or coloured precipitate in at least 3 or all of these tests
indicates the presence of alkaloids.
Test for flavonoids
Lead acetate test: 5 g of the powdered sample was detanned by
wetting it with acetone, and the acetone was completely evaporated
on a hot water bath. The residue was extracted with 20 ml of warm
distilled water and filtered. 5 ml of the filtrate in a test tube was
added two drops of 10% (w/v) lead acetate solution. Formation of a
coloured precipitate indicates the presence of flavonoids.
Sodium hydroxide test: To 5 ml of the filtrate from above equal
volume of 10% (w/v) sodium hydroxide solution was added.
Formation of yellow coloured solution indicates the presence of
flavonoids.
Test for volatile oil
0.5 g of powdered sample was shaken with 1 ml of 0.1 M sodium
hydroxide solution and 1% aqueous hydrochloric acid. The
formation of a white precipitate indicates the presence of volatile oil.
Determination of water-soluble ash
Water soluble ash was determined as reported in MHFW (1999),
with slight modification. Briefly, total ash was determined. The total
ash was boiled for 5 min with 25 ml of distilled water; the insoluble
matter was collect on an ashless filter paper, washed with hot
distilled water, and ignited for 15 minutes at a temperature not
exceeding 450°C. The weight of the insoluble matter was
subtracted from the weight of the total ash; the difference in weight
represents the water-soluble ash. The percentage of the watersoluble
ash was calculated with reference to the air-dried powdered
plant sample.
Okhale et al. 2291
Extraction and thin layer chromatographic fingerprinting
Successive extraction of 1 g powdered aerial part was carried out
with n-hexane (10 ml × 2); ethyl acetate (10 ml × 2); and methanol
(10 ml × 2) at room temperature (27 to 30°C) for 24 h. The extract
from each solvent was vacuum filtered with Whatman No. 1 filter
paper, and the filtrates evaporated to dryness in the fume hood at
room temperature. The yield for each extract was determined. Then
0.05 g each of the extracts was reconstituted in 5 ml of their
respective solvent of extraction and spotted on glass TLC plate
precoated with silica gel 60. The plate was previously activated at
105°C for 2 h. The plates were developed using a mobile phase
comprising n-hexane and ethyl acetate (3: 2). The plates were
observed in daylight and under UV at 365 nm. Visible spot were
marked. The plates were then sprayed with a solution of 1% (w/v)
vanillin in sulphuric acid and heated in oven at 110°C for 3 min. The
coloured spots revealed after spraying were marked. The
retardation factors (Rf) of all components detected were then
computed.
RESULTS AND DISCUSSION
Phytochemical screening
The results of the phytochemical screening of the aerial
part of Chenopodium ambrosioides collected from
Northern Nigeria are presented in Table 1. Nine major
classes of secondary metabolites were detected, namely
cardenolide aglycone, terpenes, sterols, saponins,
tannins, alkaloids, flavonoids, phenols and volatile oil.
C. ambrosioides has been shown to exhibit antifungal,
antihelminthic, anticatarrhal, antibacterial, antiviral,
insecticidal, nematicidal and allelopathic activities (Verma
et al., 1983; Dubeyand Kishore, 1987; Peterson et al.,
1989; Begum et al., 1993; Kishore et al., 1993; Hegazy
and Farrag, 2007; Valery et al., 2008). The antihelminthic
and anticatarrhal activities have been attributed to the
presence of some bioactive compounds shown in Figure
1 such as ascaridole (1), isoascaridole (2), α-terpinene
(3) and ascaridole glycol (4) (Valery et al., 2008).
Ascaridole has been reported to possess sedative, painrelieving
and antifungal properties (Okuyama et al., 1993;
Pare et al., 1993). It has also been reported to exhibit
antimalarial and antiparasitic activities. Pollack et al.
(1990) reported its inhibition of the in vitro development of
Plasmodium falciparum, while its activities against
Trypanosoma cruzi and Leishmania amazonensis were
reported by Kiuchi et al. (2002) and Monzote et al.
(2006), respectively. Ascaridole was also reported to
exhibit in vitro activity against different tumor cell lines
(CCRF-CEM, HL60, and MDA-MB-231) (Valery et al.,
2008).
Our finding is in agreement with previously reported
work of Hegazy and Farrag, (2007), who reported the
presence of sterols and terpenes in the Egyptian species,
and Onocha et al. (1999), who reported the presence of
essential oil in Chenopodium ambrosioides collected from
Western Nigeria. Several other secondary metabolites
have been previously isolated from Chenopodium

2292 J. Med. Plants Res.
ambrosioides, such as alkaloids (Haseeb et al., 1978),
saponins (Gupta and Behari, 1972) and flavonoids (Jain
et al., 1990).
Proximate analysis
The proximate analysis (Table 2) revealed moisture
content of 10.90%, which is within the acceptable limits of
about 6 to 15% for most vegetable drugs (Kunle, 2000).
Low moisture content reduces errors in the estimation of
the actual weight of drug material, reduces components
hydrolysis by reducing the activities of hydrolytic
1
3
O
O
4
O
2
O
O
OH
OH
Figure 1. Chemical structures of some bioactive
compounds of Chenopodium ambrosioides.
Table 2. Result of proximate analysis of the aerial part of C.
ambrosioides.
Parameter Values (%)*
Moisture content 10.90 ± 0.05
Total ash value 14.65 ± 0.3
Acid-insoluble ash value 3.05 ± 0.05
Water-soluble ash value 6.25 ± 0.02
Alcohol-soluble extractive value 13.20 ± 0.06
Water-soluble extractive value 3.18 ± 0.03
* Each value in the table was obtained by calculating the average of
three experiments ± SEM.
enzymes which may destroy the active components, and
also reduces the proliferation of microbial colonies and
therefore minimize the chance of spoilage due to
microbial attack (Shellard, 1958). Total ash value of
14.65%, though high, is within range given for some
official drugs such as citrus leaf (7.0%), neem leaf
(11.6%) and atropa leaf (16%) (Kunle, 2000). The total
ash value is a diagnostic purity index. It represents the
physiological ash and non-physiological ash.
Physiological ash is the ash inherent in the plant due to
biochemical processes and the non-physiological is
contaminants from the environment. These may be
carbonates, phosphates, nitrates, sulphates, chlorides

Table 3. Result of thin layer chromatography fingerprint of nhexane
extract of aerial part of C. ambrosioides.
Spot Rf value Color/visualization
1 0.15 Pink/vanillin spray
2 0.31 Purple/vanillin spray
3 0.49 Purple/vanillin spray
4 0.65 Pink/vanillin spray
5 0.70 Light green/daylight; pink/UV 365 nm
6 0.81 Green/daylight; pink/UV 365 nm
7 0.89 Deep pink/vanillin spray
8 0.93 Deep pink/vanillin spray
Table 4. Thin layer chromatography fingerprint of ethyl acetate
extract of aerial part of C. ambrosioides with Rf values.
Spot Rf value Color/visualization
1 0.05 Green/daylight
2 0.50 Purple/vanillin spray
3 0.56 Purple/vanillin spray
4 0.65 Pink/vanillin spray
5 0.70 Light green/daylight; pink/UV 365 nm
6 0.81 Green/daylight; pink/UV 365 nm
7 0.93 Deep pink/vanillin spray
and silicates of various metals which were taken up from
the soil (Kunle, 2000). The non-physiological ash
component of the total ash could be reduced by rinsing
the fresh plant material several times in clean water
before drying and processing for medicinal uses. The
acid-insoluble ash value measures the amount of silica,
especially siliceous earth, present in the drug plant
(Kunle, 2000). The physiological ash gets dissolved in the
dilute acid while some of the non-physiological ash
remains undissolved (Shellard, 1958). The value of
3.05% which was obtained in this work is within range
reported for some official vegetable drugs like cajanus
seed (not more than 0.15%), capsicum fruits (not more
than 1.5%), euphorbia whole herb (3%) and atropa leaf
(5%) (Kunle, 2000). The value obtained indicates that
about 11.6% of the total ash will be physiologically
available when the plant drug is ingested (Shellard,
1958). The water extractive value of 3.18%, which is less
than the alcohol extractive value of 13.20%, implies that
alcohol will be a better solvent for the extraction of the
plant constituents.
Thin layer chromatography fingerprinting
The TLC Rf values of the hexane and ethyl acetate
extracts are shown in Tables 3 and 4, respectively. The
Okhale et al. 2293
chromatogram showed eight spots and seven spots for
the hexane and ethyl acetate extracts, respectively.
Conclusion
Nigerian species of C. ambrosioides is rich in
phytochemicals, some of which may be attributed to its
ethnomedicinal uses for management of coughs,
tuberculosis, diabetes, and as antimalarial. The presence
of some secondary metabolites like alkaloids, flavonoids,
saponins, tannins, phenols, terpenes and sterols, all of
which have been reported to exhibit physiological
activities in man, animals and microorganisms, suggests
that the plant may be use as a potent vegetable drug.
Some phytochemicals are used in the pharmaceutical
industry for the production of various drugs. Examples
include the quinine analogues, nicotine, taxol, artemisinin
and cocaine (Evans, 2002). Flavonoids and saponins
have been reported to possess anti-oxidants, antiinflammatory
and hypoglycemic activities, and are used
as anti-microbial, anti-cancer and anti-allergic remedies.
Saponins and cardiac glycosides have also been
reported as antifungal as well as cardiotonics (Kunle and
Egharevba, 2009). Some tannins had been reported as
anti-viral and anti-tumor agents as well as diuretics.
Terpenes like the mono-, sesqui- and triterpenes, and
sterols had been reported to exhibit various biological
activities in animals and microorganisms. Some of which
include, anti-inflamatory, anti-microbial and hormonal
activities. Some steroidal compounds have been reported
to exhibited anti-diabetic properties (Evans, 2002).
ACKNOWLEDGEMENT
The authors wish to thank Muazzam Ibrahim Wudil for
providing ethnomedicinal uses of C. ambrosioides in
Northern Nigeria.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2295-2298, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR011.272
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research paper
Antifungal activity of coptidis rhizoma against Candida
species
Jae Young Kim 2 , Yongsub Yi 1,2 * and Yoongho Lim 3
1 Department of Herbal Cosmetic Science, Hoseo University, Asan 336-795, South Korea.
2 Department of Biochemistry, Hoseo University, Asan 336-795, South Korea.
3 Department of Bioscience and Biotechnology, Konkuk University, Seoul 143-701, South Korea.
Accepted 12 March, 2012
The extract of Coptidis Rhizoma was determined antifungal activity against five different Candida
species. The extract showed antifungal activity against Candida albicans at 200 μg/ml, and C. tropicalis
and C. glabrata at 50 μg/ml. A compound 1 was separated by thin layer chromatography (TLC) analysis
and confirmed as berberine by high performance liquid chromatography (HPLC) analysis. The minimum
inhibitory concentration (MIC) values of the compound 1 and berberine were 24 and 48 μg/ml against C.
tropicalis and C. glabrata, and 30 and 50 μg/ml against C. tropicalis and C. glabrata. The IC50 values of
the compound 1 were 58 μM at 24 h and 40 μM at 48 h by MTT assay of cell viability using HaCaT cell
line. The above results indicated that berberine was a good candidate to inhibit the propagation of C.
tropicalis and C. glabrata, even in human skin.
Key words: Anticandidial activity, coptidis rhizoma, MTT assay, HaCaT cell line.
INTRODUCTION
Fungal infections have shown increased rate at the
numerous surveys over the past decades. The most
notable infections were due to Candida spp., which were
found to be the fourth most common cause of
nosocomical blood stream infection among hospitals
during the 1980s (Pfaller, 1995; Abi-Said et al., 1997;
Richardson and Kokki, 1998; Verduyn et al., 1993). This
trend has continued into the 2000s. Notably, these
infections were increased by species of Candida other
than C. albicans.
The emergence of species other than C. albicans is
clearly a concern. Urinary infection is the frequent fungal
infection in patients with HIV infection and in people with
long term antibiotics and steroid use. Fungal infections
were generally treated by antibiotics such as
amphotericin B (Amp B) and the zoles (Blumberg and
Reboli, 1996; Girmenia and Martino, 1998). Data have
reported that the resistance of Candida species to Amp B
and the zoles has been increasing (Fonos and Cataldi,
2000; Gallis et al., 2000).
*Corresponding author. E-mail: yongsub@hoseo.edu. Tel: 82-
41-540-5979. Fax: 82-41-541-5979.
MATERIALS AND METHODS
The dried Coptidis Rhizoma was crushed with a pulverizer and
extracted two times with a 70% ethanol at room temperature for 24
h. The extracts were evaporated to dryness using a rotary
evaporator (Eyela, Japan). The extract powders were stored at -
20°C until use. Candida species, C. albicans, C. tropicalis, C.
glabrata, C. parapsilensis, and C. utils were purchased from Korea
Gene Bank (Daejeon, Korea). Candida species were culture on YM
(Difco, USA) media with the extract for antifungal activity test. The
isolated compounds separated by Thin Layer Chromatography on
silica gel plates 60F254 (Merck, Darmstadt, Germany) were
analyzed using HPLC (Varian, Walnut Creek, CA) equipped with
PDA detector, and a Varian polar C18 reversed-phase column (4.6
× 250 mm, 0.45 μm). The mobile phase was composed of a
phosphate buffer-acetonitrile solution containing 0.1% formic acid.
The elution program for the mobile phase was as follows; 10%
acetonitrile at 0 min, 30% acetonitrile at 10 min, 80% acetonitrile at
20 min, 80% acetonitrile at 25 min, 10% acetonitrile at 30 min. The
flow rate was 1 ml/min and UV detection was dually performed at
290 nm and 340 nm. For cell viability assay, HaCaT cells line was
obtained from Korean cell Line Bank (Seoul, Korea). Cells were
cultured in DMEM containing FBS (10%), penicillin (100 U/ml),
streptomycin (0.1 mg/ml) at 37°C in a humidified atmosphere of 5%
CO2. Cells were harvested after incubation for 24 h. Viability of
cultured cells was determined by MTT method (Mosmann, 1983).
Cells were added to each well in 96-well plates, and cultured for 24
h. After samples treatment, MTT (5 mg/ml in PBS) was added 100
µL to each well. Cells were incubated at 37°C for 30 min, and

2296 J. Med. Plants Res.
Table 1. Antifungal activity of extract of Coptidis Rhizoma against Candida species.
Species
Clear zone (mm) contents of extract (μg/ml)
10 50 100 150 200
C. albicans - - - - 3
C. tropicalis - 2 3 6 9
C. glabrata - 3 4 4 5
C. parapsilensis - - - - -
C. utils - - - - -
-: Not detected.
DMSO was added to dissolve the formazan crystals. The
absorbance was measured at 560 nm with a spectrophotometer.
RESULTS AND DISCUSSION
Anticandidial activity of Coptidis Rhizoma extract was
determined against five different Candida sp. by agar
diffusion assay. The extract of Coptidis Rhizoma showed
antifungal activity against Candida albicans at 200 μg/ml,
C. tropicalis and C. glabrata at 50 μg/ml. The extract
showed best antifungal activity against C. glabrata at 50
μg/ml (Table 1). A compound 1 was separated by TLC
analysis from Coptidis Rhizoma extract, and the above
compound 1 was confirmed as berberine by HPLC
analysis (Figure 1). The MIC (minimum inhibition
concentration) of the compound 1 and berberine were
Figure 1. HPLC analysis of compound 1 isolated from coptidis rhizoma.
determined and compared. The MIC values of the
compound 1 and berberine were 24 and 48 μg/ml against
C. tropicalis and C. glabrata, and 30 and 50 μg/ml against
C. tropicalis and C. glabrata (Table 2). The difference of
MIC values of both compounds was supposed an error of
weighting materials. The IC50 values of the compound 1
were 58 μM at 24 h and 40 μM at 48 h by MTT assay of
cell viability using HaCaT cell line (Figure 2). The above
results indicated that the compound 1, berberine, was a
good candidate to inhibit the propagation of C. tropicalis
and C. glabrata, even in human skin.
Candidiasis has been the most common fungal
infection in HIV infected individuals and patients in
hospital. Although Candida spp. resistant to antifungal
agents have been a rarity until three decades ago,
resistance of Candida spp. was spread in a serious
infection disease of the hospital environment. Although

Table 2. MIC of a compound 1 from coptidis rhizoma.
Species Compound 1 (μg/ml) Berberine (μg/ml) Amphotericin B (μg/ml)
C. albicans - - 0.2
C. tropicalis 24 30 0.4
C. glabrata 48 50 0.4
C. parapsilensis - - 0.4
C. utils - - 0.4
- : Not detected.
Cell viability ( IC 50 value(M) )
120
100
80
60
40
20
0
24 h 48 h
Amphotericin B 24hr 48hr
Berberin
Figure 2. IC50 values for viability on HaCaT cells. Growth inhibition of HaCaT cell
after treatment with berberine and Amphotericin B. Cells were plated at 1x10 4
cell/well per 96 well plate, and incubated for 24 h. The cells treated with variable
concentrations of berberine for 24 and 48 h, and amphotericin B for 24 h and the
viability were measured by MTT assay. The data represent means ±SD of five
independent experiments.
uncommon, this outbreak should serve as a warning that
multi-resistance of Candida strains may develop and
spread within the hospital environment (Bodey, 1988;
Schaberg et al., 1991; Masia and Gutierrez, 2002).
In this work, we did seek what a kind of the extract from
Coptidis Rhizoma was responsible for the antifungal
effect. It is suggested that the compound 1, berberine,
can be applied as a phytomedicine for treatment of the
Candidal infection (Williamson, 2001; Han and Lee, 2005).
ACKNOWLEDGEMENT
This research was supported by the Academic Research
Fund of Hoseo University in 2010 (2010-0131).
REFERENCES
Kim et al. 2297
Abi-Said D, Anaissie E, Uzun O, Raad I, Pinzcowski H, Vartivarian S
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Bodey GP (1988). The emergence of fungi as major hospital pathogens.
J. Hosp. Infect., 11: 411-426.
Fonos V, Cataldi L (2000). Amphotericin B-induced nephrotoxicity. J.
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epidemiology of candidemia. Clin. Infect Dis., 27: 234.
Han Y, Lee JH (2005). Berberine synergy with amphotericin B against
disseminated candidiasis in mice. Biol. Pharm. Bull., 28: 541-544.
Masia CM, Gutierrez RF (2002). Antifungal drug resistance to azoles

2298 J. Med. Plants Res.
and polyenes. Lancet Infect. Dis., 2: 550-563.
Mosmann T (1983). Rapid colorimetric assay for cellular growth and
survial: application to proliferation and cytotoxicity assays. J. Immunol.
Methods, 65: 55-63.
Pfaller MA (1995). Nosocomical candidiasis: Epidermiology of
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Richardson MD, Kokki MH (1998). Diagnosis and prevention of fungal
infection in the immunocompromized patient. Blood Rev., 12: 241-
244.
Schaberg DR, Culver DH, Gaynes RP (1991). Major trends in the
microbial etiology of nosocomial infection. Am. J. Med., 91: 72S-75S.
Verduyn LFM, Meis JF, Voss A (1993). Nosocomial fungal infections:
candidemia. Diagn. Microbiol. Infect. Dis., 34: 213-220.
Williamson EM (2001). Synergy and other interactions in
phytomedicines. Phytomedicine, 8: 401-409.

Journal of Medicinal Plants Research Vol. 6(12), pp. 2299-2308, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11-411
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Evaluation and comparison of antifungal activities of
Terminalia catappa and Terminalia mantaly
(Combretaceae) on the in vitro growth of Aspergillus
fumigatus
ZIRIHI Guédé Noël 1 *, N’GUESSAN Koffi 1 , KASSY N’dja Justin 1 , COULIBALY Kiyinlma 1,3 and
DJAMAN Allico Joseph 2
1 University of Cocody-Abidjan, Unité de Formation et de Recherche (U.F.R.) Biosciences, Laboratoire de Botanique, 22
BP 582 Abidjan 22, Côte-d’Ivoire.
2 University of Cocody-Abidjan, Unité de Formation et de Recherche (U.F.R.) Biosciences, Laboratoire de
Pharmacodynamie Biochimique, 22 BP 582 Abidjan 22, Côte-d’Ivoire.
3 Unité des Antibiotiques des Substances naturelles et de Surveillance de la Résistance des Micro-organismes aux Antiinfectieux,
Institut Pasteur de Côte d’Ivoire, 01 BP 490 Abidjan 01, Côte-d’Ivoire.
Accepted 21 December, 2011
Many surveys performed around the world have mentioned that among the plant species belonging to
Combretaceae family, Terminalia catappa is the most requested medicinal plant. In recent decades,
traditional healers in southern region of Côte d’Ivoire prefer to use the bark of Terminalia mantaly
instead of those of T. catappa. The purpose of this study is to compare the pharmacological activities of
these plants. The results of the anti-fungal activities of aqueous, hydroalcoholic and residual extracts
on the growth of Aspergillus fumigates, a fungal pathogen, show that Terminalia mantaly water extract
is 64 times more active than T. catappa water extract; hydroalcoholic extract of T. mantaly is 2 times
more active than hydroalcoholic extract of T. catappa and residual extract of T. mantaly is 128 times
more active than residual extract of T. catappa. Analysis of these results shows clearly that T. mantaly
extracts are more active than extracts of T. catappa. The choice of T. mantaly and abandon of T.
catappa by traditional healers in making medicines against skin infections is due to abundance of this
plant in all areas and settlements and its excellent activities on many pathogens.
Key words: Côte d’Ivoire, Terminalia catappa, Terminalia mantaly, antifungal activity.
INTRODUCTION
Ethnopharmacological surveys performed around the
world have mentioned that among the plant species
belonging to Combretaceae family, Terminalia catappa is
the most requested medicinal plant (N’guessan, 2008). In
Côte d’Ivoire, the roots bark decoction is used as antipyretic
(Zirihi, 1991; N’guessan, 1995). In Nigeria, the
decoction of leaves is used as medicine against malaria
and abdominal pains (Amenoudji, 1990). In Togo and
Benin root barks decoction is used in the treatment of
*Corresponding author. E-mail: noel_zirihi @yahoo.fr. Tel:
0022548042873.
various dermatosis (Amenoudji, 1990; Batawila et al.,
2005; Baba-moussa, 1999; Baba-moussa et al., 1999). In
Phillipines, leaves extract is used against leprosies
(Burkill, 1997). In recent decades, traditional healers in
southern region of Côte d’Ivoire prefer to use the bark of
Terminalia mantaly instead of those of T. catappa (Zirihi,
1991). According to Coulibaly (Coulibaly, 2006), roots of
T. catappa and leaves of Terminalia mantaly are used
against the loss of voice. Indeed, unlike T. catappa, T.
mantaly abounds in this part of Côte d’Ivoire, this plant is
found along roads side, streets and even in the
settlements (Aké-Assi, 1984). The purpose of this study
is to compare the pharmacological activities of T. catappa
a medicinal plant well known and well studied to those of

2300 J. Med. Plants Res.
Figure 1. Situation of the study site; Geographical situation of Côte-d’Ivoire in West Africa; Geo graphical situation of
the department of Tiassalé, in Côte-d’Ivoire; (CEDA, 2001, modified by Coulibaly).
T. mantaly introduced in 1972 in Côte d’Ivoire. We
present here the results of the anti-fungal activities of
aqueous, ethanolic and residual extracts in the in vitro
growth of Aspergillus fumigatus a fungal pathogen that
causes very severe and irreversible lung infections in
Human immunodeficiency virus (HIV) positive patients.
MATERIALS AND METHODS
Study site
Our investigations took place in Tiassalé Department (Figure 1),
Located at 130 km of Abidjan. Tiassalé is part of the Southern
forest of Côte-d’Ivoire (West Africa), in the guinea field of the

Zirihi et al. 2301
Figure 2. Terminalia mantaly H. Perrier (Combretaceae): Portion of stem bark. Picture by Coulibaly K., Tiassalé, 06/12/08.
mesophilic sector, characterized by dense moist semi-deciduous
forest. Currently, the original vegetation has been degraded by
human activities (Chevalier, 1948). Annual average pluviometry is
about 1708.73 mm of water. Its climate, warm and humid, is
characterized by two seasons: a dry season from December to
February with two dry month, January and February, and a long
rainy season from March to November, with the largest recorded in
June, the other in October; between the two month, there is a
period of less rainfall during August.
Vegetal material
T. mantaly
T. mantaly grows 10 to 20 m with an erect stem and layered
branches. Bark is pale grey and smooth. Leaves are smooth and
bright green when young and in terminal rosettes; they are 5 to 7
cm long with short stem, apex broadly rounded and base tapred
(Figures 2 to 5). Flowers small, greenish in erect spikes. Fruit small
oval, about 1.5 cm long
T. catappa
T. catappa is a mean tree, a Mesophanerophyte from 25 to 30 m of
height and of 1.60 m of diameter. The trunk of T. catappa, without
footing, comprises a rhytidom split lengthwise (Figures 1 and 3); the
branches are staged. The simple leaves, bound without peduncles,
are in subvertilled tufts with. Flowers without petals, gathered in
terminal tufts are bisexual.
Preparation of extracts
The drugs (stems barks) of T. catappa coded « CTA » and
Terminalia mantaly coded « MTA», used in this study, were
collected in the region of Tiassalé, South of Côte d’Ivoire. The
collected drugs, were washed up, cut in small dices and dried at the
laboratory at the ambient temperature (25 to 27°C) during three
weeks and crushed out fine powder. Powders were extracted
separately with a blinder, according to the method of Zirihi and Kra
(2003), as follows: 100 g of powder of barks from each plant were
extracted hot (decoction) with one liter of distilled water using a
Mixer (Blinder). After three cycles of extraction, the solution
obtained was filtered, then the solvent of extraction (water) was
eliminated using a rotary evaporator; the paste obtained was
freeze-dried; it constituted the total water extracts codified ( CTAwat
and MTAwat). 30 g of each total water extract were treated by 300
ml of hydroalcoholic solution (70% Ethanol and 30% distilled water)
using a separating funnel. Two phases were obtained (the alcoholic
upper phase (CTA0, MTA0) and a residue named (CTA1, MTA1).
CTA0, MTA0, CTA1 and MTA1 were freeze-dried. All extracts
obtained were kept in glass sterilized bowls at -20°C. The six
extracts of CTAwat, MTAwat, CTA0, MTA0, CTA1 and MTA1 were
tested on the in vitro growth of A. fumigatus.
Microbial stock and realization of in vitro tests
The stock of A. fumigatus on which we worked was provided to us
by the Laboratory of Mycology of the U.F.R of Medical Sciences of
the University of Cocody-Abidjan (Côte d’Ivoire). The incorporation
of different extracts of CTA and MTA at the Sabouraud agar was

2302 J. Med. Plants Res.
Figure 3. Terminalia catappa Linn. (Combretaceae): Portion of stem bark. Picture by Coulibaly K.,
Tiassalé, 10/01/09.
Figure 4. Terminalia mantaly H. Perrier (Combretaceae): Young leaves and fruits. Picture by Coulibaly K.
Tiassalé, 06/12/08.

Figure 5. T. catappa Linn. (Combretaceae): Leaves and fruits. Picture by Coulibaly K.,
Tiassalé, 10/01/09.
done according to the double dilution method, in angle sloping
tubes. The extracts were tested separately. For the T. mantaly
extract, each set comprises of 10 test tubes for the water extract
and 12 test tubes for the ethanolic and residual extract in which we
have, respectively eight and ten test tubes containing vegetal
extracts, and two testimony tubes including one without vegetal
extract serving to control the growth of germs, the other one without
germs and without the extract, serving to control the sterility of the
field of cultivation. The concentrations of the extracts vary from
3.125 mg/ml to 24 µg/ml for the eight test tubes and from 12.5
mg/ml to 24 µg/ml for the ten test tubes (according to a geometric
line of ½ reasons). For the T. catappa extract the set representing
the water extract and the residual extract comprises of 15 test
tubes, including 13 test tubes, and 02 testimony tubes.
The concentrations of the extracts from the 13 test tubes vary
from 100 mg/ml to 24 µg/ml. Concerning the ethanolic extract we
have 14 test tubes, including 12 test tubes and 02 testimony tubes.
The concentrations of the extracts vary from 50 mg/ml to 24 µg/ml.
After the incorporation of the extracts, all the tubes of each set are
sterilized with an autoclave at 121°C, during 15 min and then
inclined at room temperature, to allow the agar-agar to quench itself
and to solidify itself (Ajello et al., 1963; Holt, 1975; Guede-Guina et
al., 1995). For each set of the different extracts, the antifungal tests
were carried out by the cultivation of 1000 cells of A. fumigatus on
the fields previously prepared. All the cultivations were incubated at
30°C during 72 h. The colony of A. fumigatus was numbered and
the growth in experimental tubes of each set was assessed in
percentage of survival, calculated in proportion to 100% of survival
in the testimony tube of control of the growth (Ajello et al., 1963;
Holt, 1975; Guede et al., 1995).
RESULTS
Antimicrobic tests
Zirihi et al. 2303
After 48 h of incubation at 30°C, we observe
comparatively to the testimony bowls, a progressive
reduction of colonies number with an enhancement of
plant extract concentrations in experimental tubes. This is
observed for all the sets (Figures 6 to 11). The
experimental data expressed in the form of curves of
sensibilities are summarized in Figure 12. The values of
the antifungal parameters, MFC (Minimal Fungicid
Concentration) and CI50 (Concentration for 50%
Inhibition) of the 06 extracts, are recorded in the Table 1.
All the six curves representing the evolution of the activity
of each extract, presenting a deceasing speed, with
slopes of more or less strong.
DISCUSSION
The botanical study of these two plants shows great
differences; stems, barks, leaves and fruits are different.
The analysis of the results of the microbiological tests
shows that A. fumigatus is sensitive to all the extracts
(CTAwat, MTAwat, CTA0, MTA0, CTA1 and MTA1), according

2304 J. Med. Plants Res.
Figure 6. A. fumigatus: Aspect of culture in presence of water extracts of T. mantaly, at different concentrations.
Figure 7. A. fumigatus: Aspect of culture in presence of water extracts of T. catappa at different concentrations.
to a relation dosis-dependent. In all experiments we
noticed effective inhibition of A. fumigatus growth after 48
h of incubation and at 30°C. The comparison of all
antifungal parameters shows that the six plants extracts
are active on A. fumigatus growth. T. mantaly extracts
activities comparisons shows the following results:
MFCMTAwat / MFC MTA1 = 1.56 / 0.78 = 2; MFCMTAwat /MFCMTA0= 1.56 / 1.56 = 1; MFCMTA0 / MFCMTA1 = 1.56 / 0.78 =
2.

Figure 8. A. fumigatus: Aspect of culture in presence of 70 % ethanolic extracts of T. mantaly, at different
concentrations.
Figure 9. A. fumigatus: Aspect of culture in presence of 70% ethanolic extracts of T. catappa, at different
concentrations.
According to these results residual extract (MTA1) is twice
more active than the water extract of T. mantaly (MTAwat).
Activities of water extract (MTAwat) and hydroalcoholic
Zirihi et al. 2305
extract (MTA0) are the same. T. catappa extracts
activities comparisons shows the following results:
MFCCTAwat / MFCCTA1 = 100 /100 =1 MFCCTA1 / MFCCTA0= 100 / 3.125 = 32; MFCCTAwat / MFCCTA0 = 100 /3.125 =
32.

2306 J. Med. Plants Res.
Figure 10. A. fumigatus: Aspect of culture in presence of residual extracts of T. mantaly, at different
concentrations.
Figure 11. A. fumigatus: Aspect of culture in presence of residual extracts of T. catappa, at different
concentrations.
The comparisons of these results show that
hydroalcoholic extract (CTA0 ) of T. catappa is 32 times
more active than water (CTAwat ) and residual extract
(CTA1).
In order to know the plant which is more active, water
extracts, hydroalcoholic extracts and residual extracts are
compared separately. -water extracts: MFCCTAwat
/MFCMTAwat =100/ 1.56 = 64; this ratio show that T.
mantaly water extract is 64 times more active than T.
catappa water extract. Hydroalcoholic extracts: MFCCTA0/
MFCMTA0 = 3.125/1.56 =2; According to this proportion T.
mantaly hydroalcoholic extract is 2 times more active
than hydroalcoholic extract of T. catappa. -residual
extracts: MFCCTA1 / MFCMTA1 = 100/ 0.78 = 128; this ratio
proves that T. mantaly residual extract is 128 times more
active than residual extract of T. catappa. Analysis of
these results shows clearly that T. mantaly extracts are
more active than extracts of T. catappa. Ackah (2004)
and Ouattara (2005), tested, respectively the 96%
alcoholic residue (MISCA-F3) of Mitracarpus scaber
(MFC = 18.750 mg/ml) and the 70% alcoholic residue
(MISCA-F2) of Mitracarpus scaber (MFC = 50.000 mg/ml)

Figure 12. Sensitivity of A. fumigatus to the extracts of T. mantaly and T. catappa (MTAwat, CTAwat,
MTA0, CTA0, MTA1 and CTA1).
Table 1. Antifungal parameters Values of the six extracts of the MTA and the CTA at 48 h of
incubation and at 30°C.
Type of extract
Water extracts
Hydroalcoholics extracts
Residual extracts
on the same germ and in the same experimental
conditions, we can say that Terminalia mantaly, MFC =
0.78 mg/ml (MFCMTA1); present a better activity than M.
scaber.
CONCLUSION AND PERSPECTIVES
According to all these results we can make the following
observations: water, hydroalcoholic and residual extracts
of T mantaly and T catappa inhibits the in vitro growth of
A. fumigatus, hydroalcoholic extract of T. mantaly is 2
times more active than hydroalcoholic extract of T.
catappa, residual extract of T. mantaly is 128 times more
active than residual extract of T. catappa. Analysis of
these results shows clearly that T. mantaly extracts are
more active than extracts of T. catappa. T. mantaly
extracts are more active than those of M. scaber on the in
Antifungal parameters
CI50 (mg/ml) CMF (mg/ml)
MTAwat 0.098 1.56
CTAwat 0.84 100
MTA0 0.10 1.56
CTA0 0.197 3.125
MTA1 0.043 0.78
CTA1 1.58 100
Zirihi et al. 2307
vitro growth of A. fumigatus. The choice of T. mantaly
and renunciation of T. catappa by traditional healers in
making medicine against skin infections is due to
abundance of this plant in all areas and settlements and
its excellent activities on many pathogens. Phytochemical
analysis associated to antifungal tests is needed to
isolate the active compound.
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in vitro de Candida albicans, Cryptococcus neoformans, Aspergillus
fumigatus, Aspergillus flavus, Trichophyton rubrum, Trichophyton
mentagrophytes. Master of Biotechnology and Plant Production
Improvement, Option Pharmacology of Natural Substances,
University of Cocody-Abidjan, Dept. Biosci., Côte d'Ivoire, pp. 20-24.
Amenoudji AD (1989). Contribution to the knowledge of the flora oust
Africa: systematic and ethnobotany of some species of angiosperms,
Internship Report, National Center for Flora, Univ. The Ivory Coast, p.
86.

2308 J. Med. Plants Res.
Ajello L, Georg LK, Kaplan W, Kaufman L (1963). Laboratory manual
for medical mycology, 2nd. Ed., John Wiley. and sons, Inc. New-
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seven West African Combretaceae used in traditional medicine. J.
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Chevalier A (1948). Biogeography of the dense rainforest of Ivory
Coast. Rev. Bot. Appl. Agric. Trop., 28: 305-306, 101-115.
Coulibaly K (2006). Evaluation of the antifungal activity of extracts of
bark of commercial species, category P1 the forest of Mopri, Tiassalé
(Southern Ivory Coast).Memory Master in Tropical Ecology, Plant
Option, University of Cocody-Abidjan, Dept. Biosci., pp. 23-25.
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activity of a plant extract against the opportunistic pathogens in AIDS.
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Holt R (1975). Laboratory test of antifungal drug. J. Clin. Pathol., 18:
767-774.
N’Guessan K (1995). Contribution to the ethnobotanical study in
Krobou countries (Republic of Ivory Coast). PhD Thesis of 3rd cycle,
FAST, Laboratory of Botany, Natl. Univ. Ivory Coast, pp. 180-182.
N’Guessan K (2008). Medicinal plants and traditional medical practices
among the peoples and Abbey Krobou Department Agboville (Ivory
Coast). PhD Thesis State in Natural Sciences, Department
Biosciences, Lab. Bot, Univ. Cocody-Abidjan, 27- 56.
Ouattara S (2005). Spectrum anti-infective MISC-F2 on in vitro growth
of Candida albicans, Cryptococcusneoformans, Aspergillus
fumigatus, Aspergillusflavus, Trichophyton rubrum, Trichophyton
mentagrophytes. Master of Biotechnology and Plant Production
Improvement, Option Pharmacology of Natural Substances,
University of Cocody-Abidjan, Dept. Biosci., Côte d'Ivoire, pp. 20- 23.
Zirihi GN (1991). Contribution to the census, identification and
knowledge of several plant species in traditional medicine in
cattle Department Issia.PhD thesis of third cycle, F.A.S.T, Natl.
Univ. Cote d'Ivoire, pp. 76-78.
Zirihi GN, Kra AKM (2003). Evaluation of the antifungal activity
ofmicroglossia pyrifolia (Lam.) O. Ktze(Asteraceae) PYMI''''on the in
vitro growth ofCandida albicans. Rev. med. Pharm. Afr., 17 : 1-19.

Journal of Medicinal Plants Research Vol. 6(12), pp. 2309 -2316, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.739
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Optimization of callus induction medium for
Hymenocallis littoralis (Melong kecil) using root and
bulb explants
Rosli Noormi 1,3 , Vikneswaran Murugaiyah 2 and Sreeramanan Subramaniam 1 *
1 School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia.
2 School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, Malaysia.
3 Faculty of Applied Sciences, Universiti Teknologi MARA, Negeri Sembilan Branch, Kuala Pilah Campus,
Negeri Sembilan, Malaysia.
Accepted 1 March, 2012
Callus was initiated using bulb and root explants of Hymenocallis littoralis on the Murashige and Skoog
(MS) basal media supplemented with various auxins, namely 2,4-dichlorophenoxyacetic acid (2,4-D),
dicamba, picloram, napthaleneacetic acid (NAA) and indole-3-acetic acid (IAA) at various
concentrations. Degree of callus formation based on weight was found highest (+++++) in NAA
treatment at 2.0 mg/L (93.33%). Dicamba produced the lowest (+) number of callus formation at 3.0 mg/L
(13.33%). In terms of physical appearance, callus induced from NAA at 2.0 mg/L produced white
yellowish and globular callus and Dicamba at 3.0 mg/L produced white and hard callus. Callus
induction from bulb is possible except with IAA. Bulb explants grown on 2.0 mg/L 2,4-D and Dicamba
formed callus after 11 days, followed by 3.0 mg/L picloram, as well but only 9 days for 2.0 mg/L NAA.
Degree of callus formation was found to be the highest (+++++) in 2,4-D at 2.0 mg/L (93.33%). Picloram
and NAA produced the lowest (++) callus formation at 3.0 and 2.0 mg/L (40.0% each). In terms of
physical appearance, callus induced from 2, 4-D at 2.0 mg/L showed yellowish, and soft globular callus.
Picloram at 3.0 mg/L produced white yellowish and soft callus; meanwhile, NAA at 3.0 mg/L produced
small callus appearance of white and yellowish colour. The callus induction protocol developed in this
study provides a fundamental investigation of bioactive constituents from the H. littoralis medicinal
plant.
Key words: Hymenocallis littoralis plants, callus induction, auxins, bulb explants, root explants.
INTRODUCTION
Plants are a valuable source of a vast array of chemical
compounds, and they synthesize and accumulate
extractable organic substances in quantities sufficient to
be economically useful as raw materials for various
commercial applications (Rashida and Rabia, 2007).
Plant cell and tissue cultures provide an alternative
approach to plants which are difficult to cultivate, or has a
long cultivation period, or has a low yield, product yield by
cell culture may significantly produce a higher yield than
those obtained from the parents (Hippolyte et al., 1992;
*Corresponding author. E-mail: sreeramanan@gmail.com. Tel:
+06-046533528. Fax: +06-046565125.
Zhong et al., 1994). Plant cell cultures are generally more
desirable than a solid medium because of higher growth
rates resulting from high medium to tissue contact
(Rashida and Rabia, 2007). However, plant cell cultures
have been used for producing valuable biochemicals,
such as drugs, flavourings, pesticides and fragrances
(Nagamori et al., 2001). Cultured plant cells and tissues
are widely recognized as promising alternatives for the
production of valuable secondary metabolites (Wu et al.,
2003; Rosli et al., 2009; Maziah et al., 2010).
Hymenocallis littoralis (Melong kecil) commonly known as
‘spider lily’ is a bulbous, herbaceous plant from the family
of Amaryllidaceae (Rafael and Michael, 2009). The plant
is distributed by the sea and in swamps in tropical, subtropical,
and temperate regions throughout the world

2310 J. Med. Plants Res.
(Ji and Merow, 1985). Throughout the history of H.
littoralis, several alkaloids had been discovered from its
bulb. The first alkaloid lycorine was proven to have
antineoplastic, cytotoxic and antiviral properties (Renard-
Noiaki et al., 1989, Lin et al., 1995, Abou-Donia et al.,
2008).
The present investigation was carried out to study
callus induction from the bulb and the root explants of H.
littoralis. The objectives of present study were to test
various concentrations of phytohormone for callus
induction of root and bulb of H. littoralis wild plant.
MATERIALS AND METHODS
Plant source
H. littoralis plants used in this study was obtained from Penang
Botanical Gardens, Penang, Malaysia.
Sterilization of explants
The explants collected from H. littoralis plants were washed under
running tap water for 45 min. The explants then were place into the
laminar flow and were soaked in the 75% ethanol within five
minutes. Then 75% ethanol used was discarded and three drops of
Tween-20 (Sigma) were added as a wetting agent into 15% (v/v) of
commercial Clorox solution (5.2% HgCl2) and the explants were
soaked in the sterile solution for 20 min. The solution used then
was discarded. The explants then were soaked in the 75% ethanol
within five minutes, and then the explants were then rinsed for 5,
10, and 20 min with 200 ml of sterile distilled water. The sterilized
explants were then cut into 1.5 × 0.2 cm for roots and 0.6 cm × 0.7
cm × 0.8 cm for bulbs.
Initiation of callus culture
The sterilized explants, namely bulb and root of H. littoralis, were
cultured in the vial containing 10 ml of MS on the (Murashige and
Skoog, 1962) basal media containing vitamins, 3% (w/v) sucrose
and 0.25% (w/v) Gelrite supplemented with various auxins, namely
2,4-dichlorophenoxyacetic acid (2,4-D), dicamba, picloram, NAA
and IAA at concentrations of 1.0, 2.0, 3.0, 4.0 and 5.0 mg/L. The pH
of the medium was adjusted to 5.7 to 5.8, prior autoclaving at 106
kPa, 121°C. Cultures were incubated at 25 ± 2°C in the dark for 4
weeks to determine the best condition for the initiation and
maximum production of callus culture using bulb and root explants.
The cultures were observed daily for one month to determine the
time, amount of initiation and formation of the callus. The
experiment was conducted in ten replicates.
Statistical analysis
The data were compared by one-way ANOVA following by a Tukey
to compare means of the sample.
RESULTS AND DISCUSSION
In the first part of the study, various concentrations of
phytohormones were used to select the suitable hormone
for induction and development of callus cultures. Different
parts of young explant such as root and bulb were
cultured in basal MS medium supplemented with auxins.
Callus was first initiated along the cut edges of culture,
depending on the different auxin concentrations in the
culture medium.
Tables 1 and 2 showed the duration for callus formation
based on number of days, percentage of callus induction
(Cip), nature of response and degree of callus formation
of callus induced from root and bulb explants in the
induction media supplemented with different auxins. For
root explants, results obtained revealed that all of the five
auxins, the first callus could induce callus within 8 days
grown on 1.0 mg/L of 2,4-D, followed by 9 days for
picloram (2.0 and 3.0 mg/L), NAA (1.0, 2.0, 3.0 and 4.0
mg/L) and IAA (3.0 mg/L). Similar observation was
reported on callus induction from root explants of the
Eurycoma longifolia plants within 8 days (Maziah et al.,
2010). However, degree of callus formation based on
weight was found to be the highest (+++++) in NAA
treatments at 2.0 mg/L (93.33%) followed by 2,4-D (+++)
at 2.0 mg/L (70.0%), picloram and IAA (++) at 3.0 mg/L
(70.0 and 73.33%).
On the other hand, dicamba produced the lowest (+)
number of callus formation at 3.0 mg/L (13.33%). This
result showed a significant increase in the induction of
the callus (Table 1). In addition, no callus was formed
from the treatment without any hormone. In terms of
physical appearance (Figure 1), callus induced from NAA
at 2.0 mg/L produced white yellowish, and globular
callus, followed by 2, 4-D at 1.0 mg/L with white and soft
callus. Meanwhile, Dicamba at 3.0 mg/L produced white
and hard callus. Additional roots were formed in medium
containing IAA at 3.0, 4.0 and 5.0 mg/L.
Meanwhile, callus induction for bulb results obtained
revealed that four auxin (except IAA) could produced
callus (Table 2). Bulb explants grown on 1.0, 2.0, 3.0 and
5.0 mg/L 2,4-D and 1.0, 2.0, 3.0 and 4.0 mg/L dicamba
formed callus after 11 days of culture followed by 2.0 and
3.0 mg/L picloram. Degree of callus formation was found
to be the highest (+++++) in 2, 4-D at 2.0 mg/L (93.33%)
followed by dicamba (+++) at 2.0 mg/L (63.33%).
Picloram and NAA produced the lowest (++) at 3.0 and
2.0 mg/L (40.0% each) callus formation. Similar
observations were reported on callus induction in
Tabernaemontana pandacaqui (Sierra et al., 1991),
Miscanthus x ogiformis Honda ‘Giganteus’ (Holme and
Petersen, 1996), Withania somnifera (Manickam et al.
2000), maize (Zacchini et al., 2000), Genista plants
(Luczkiewicz and Glod, 2003), and Asiatic salsola
species (Stefaniak et al., 2003).
No callus was formed from the treatment of Murashige
and Skoog (1962) basal media supplemented without
phytohormone. In addition, the bulb explants turned to
green hard structure and shoots were formed on media
without auxin. In terms of physical appearance (Figure 2),
callus induced from 2,4-D at 2.0 mg/L produced
yellowish, and soft globular callus, followed by dicamba

Noormi et al. 2311
Table 1. Callus Induction from root explants of Hymenocallis littoralis in MS basal medium + MS vitamins, 3% (w/v) sucrose, 0.25% (w/v) Gelrite agar supplemented with
different hormones concentration (0, 1, 2, 3, 4, 5 mg/L).
Hormones and
concentration (mg/L)
2,4-D
Dicamba
Picloram
NAA
IAA
Period to form
callus (day)
% of callus
induction (Cip)
Nature of response
Degree of callus
formation
0.0 NoC NoC Explant turned to brown -
1.0 8 40.0 ab White soft callus form +++
2.0 9 70.0 ab White soft callus form +++
3.0 9 56.67 b White soft callus form, little roots were grown +++
4.0 9 46.67 ab White soft callus form +++
5.0 10 3.33 ab White yellowish, nodular callus ++
0.0 NoC NoC Explant turned to green and hard structured, shoots formed -
1.0 NoC NoC ab Explant turned to brown -
2.0 NoC NoC ab Explant turned to brown -
3.0 11 13.33 b White hard callus form. Explant turned to brownish +
4.0 NoC NoC ab Explant turned to brown -
5.0 NoC NoC ab Explant turned to brown -
0.0 NoC NoC Explant turned to green and hard structured, shoots formed -
1.0 11 6.67 ab Explant turned to brown. Small white soft callus formed +
2.0 9 63.33 ab Explant turned to small soft white callus +++
3.0 9 70.0 b Explant turned to small soft white callus ++
4.0 10 50.0 ab Explant turned to small soft white callus ++
5.0 9 46.67 ab Explant turned to small soft white callus ++
0.0 NoC NoC Explant turned to brown -
1.0 9 33.33 ab Explant turned to green ++
2.0 9 93.33 ab White yellowish globular callus +++++
3.0 9 73.33 b White yellowish globular callus +++++
4.0 9 73.33 ab White yellowish soft callus +++++
5.0 10 53.33 ab White yellowish soft watery ++++
5.0 10 16.67 ab White hard callus formed +
0.0 - NoC Explant turned to green and hard structured, shoots form -
1.0 10 30.0 ab Small soft callus, Explant turn to white and green ++
2.0 10 36.67 ab Small soft callus, Explant turn light brown ++

2312 J. Med. Plants Res.
Table 1. Contd.
3.0 9 73.33 b Small white hard callus. Explant turns to white hard structure +
4.0 10 20.0 ab Explant turns to brown. Large and long root formed +
5.0 10 16.67 ab White hard callus formed +
- = no callus formed, + = very few callus formation, ++ = minor callus formation, +++ = slight callus formation, ++++ = moderate callus formation, +++++ = profuse callus formation,
Means within the column having the same letter were not significantly different by the Turkey HSD test (p > 0.05), NoC=No callus.
Table 2. Callus induction from bulb explants of Hymenocallis littoralis in MS basal medium + MS vitamins, 3% (w/v) sucrose, 0.25% (w/v) Gelrite agar supplemented with
different hormones concentration (0, 1, 2, 3, 4, 5 mg/L).
Hormones and
concentration (mg/L)
2,4-D
Dicamba
Picloram
NAA
Period to form
callus (day)
% of callus
induction(Cip)
Nature of response
Degree of callus
formation
0.0 NoC NoC Explant turned to green and hard structured, shoots formed -
1.0 11 23.33 ab White soft callus, shoot formed ++
2.0 11 93.33 b Yellowish, soft globular callus +++++
3.0 11 73.33 ab Yellowish, soft globular callus +++
4.0 15 23.33 ab White- yellowish, nodular callus ++
5.0 11 20.00 ab White -yellowish, nodular callus ++
0.0 NoC NoC Explant turned to green and hard structured, shoots formed -
1.0 11 10.00 ab White-yellowish, small globular callus +
2.0 11 63.33 b Yellowish-white, soft and compact callus, torpedo shape +++
3.0 11 40.00 ab Yellowish-white compact callus ++
4.0 11 46.67 ab Yellowish, soft and compact callus ++
5.0 13 10.00 ab White yellowish, not all callus produced from the explant +
0.0 NoC NoC Explant turned to green and hard structured, shoots formed -
1.0 13 NoC ab Explant turned to expand hard white and red in colour -
2.0 11 10.00 b Explant turned to small globular callus +
3.0 11 40.00 ab White yellowish callus, soft callus ++
4.0 13 13.33 ab Small white callus +
5.0 11 10.00 ab White soft callus +
0.0 NoC NoC Explant turned to green and hard structured, shoots formed -
1.0 10 10.00 ab White soft, watery callus +
2.0 9 40.00 b White, yellow small callus ++
3.0 9 23.33 ab White soft watery and friable +

Table 2. Contd.
NoC=No callus.
4.0 13 10.00 ab White soft watery and friable +
5.0 13 NoC ab Explant turned to hard white structure -
5.0 NoC NoC ab Explant turn to white and green hard structure -
Figure 1. Induction of Hymenocallis littoralis callus cultures derived from root explants in MS basal medium + B5 vitamins, 3%
(w/v) sucrose, 0.25% (w/v) Gelrite agar supplemented with different hormones concentration (0, 1, 2, 3, 4, 5 mg/L) after 30 days
of culture. A (2,4-D), B (dicamba), C (picloram), D (NAA), E (IAA). The scale (1 cm = 0.5 cm) representing the explants above.
Noormi et al. 2313

2314 J. Med. Plants Res.
Figure 1. Contd.
Figure 2. Induction of Hymenocallis littoralis callus cultures derived from bulb explants in MS basal medium + B5 vitamins, 3%
(w/v) sucrose, 0.25% (w/v) Gelrite agar supplemented with different hormones concentration (0, 1, 2, 3, 4, 5 mg/L) after 30 days
of culture. A (2,4-D), B (dicamba), C (picloram), D (NAA), E (IAA). The scale (1 cm = 0.5 cm) representing the explants above.

Figure 2. Contd.
at 2.0 mg/L yellowish-white, soft, compact callus and
torpedo in shape. Meanwhile, picloram at 3.0 mg/L
produced white yellowish callus and soft callus
appearance. NAA at 3.0 mg/L produced small callus
appearance of white and yellowish in colour.
No response was observed in the medium containing
IAA, even the cultures were kept for prolonged eriod. A
similar result was obtained by Das et al. (1995). The
percentages of callus induction using various types of
explants were found to be increased significantly by using
selected types of auxin in Eurycoma longifolia plants.
Conclusion
The best auxin used for callus induction from root
explants of H. littoralis was NAA at 2.0 mg/L followed by
2,4-D at 2.0 mg/L, picloram at 2.0 mg/L, IAA at 2.0 mg/L,
and dicamba at 3.0 mg/L. Meanwhile, the best auxin
used for callus induction from bulb explants of H. littoralis
(Melong kecil) was 2,4-D at 2.0 mg/L, followed by
Dicamba at 2.0 mg/L, picloram at 3.0 mg/L, and NAA at
2.0 mg/L.
ACKNOWLEDGEMENTS
This research was supported by Universiti Sains
Malaysia, Malaysian Ministry of Higher Education
(MOHE) and Universiti Teknologi MARA (UiTM)
scholarship.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2317-2323, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR011.788
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Effects of pomegranate seed extract on liver
paraoxonase and bcl-xL activities in rats treated with
cisplatin
Serap YILDIRIM 1 *, Fikrullah KISA 2 , Ali KARADENIZ 3 , Abdulkadir YILDIRIM 4 , Akar KARAKOC 4 ,
Ismail CAN 5 , Adem KARA 5 and Nejdet SIMSEK 5
1 Department of Physiology, Faculty of Medicine, Atatürk University, Erzurum, Turkey.
2 Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Atatürk University, Erzurum, Turkey.
3 Department of Physiology, Faculty of Veterinary Medicine, Atatürk University, Erzurum, Turkey.
4 Department of Biochemistry, Faculty of Medicine, Atatürk University, Erzurum, Turkey.
5 Department of Histology and Embryology, Faculty of Veterinary Medicine, Atatürk University, Erzurum, Turkey.
Accepted 4 July, 2011
The aim of the present study was to investigate the protective effect of pomegranate seed extract (PSE)
on the changes caused by cisplatin (CP) in rat liver paraoxonase and bcl-xL activities. Twenty-four
Sprague-Dawley rats were randomly divided into four groups of six animals: (1) Control; (2) PSE:
Treated for 15 consecutive days by gavage with PSE (300 mg/kg/day); (3) CP: Injected intraperitoneally
with cisplatin (7 mg/kg body weight, single dose); and (4) PSE+CP: Treated by gavage with PSE 15 days
after a single injection of CP. Blood and liver tissue samples were taken from each animal after
experimental procedures. PON-1 paraoxonase and arylesterase activities and malondialdehyde (MDA)
levels were estimated from liver homogenates; the liver tissue was also immunohistochemically
examined for anti-apoptotic activity (bcl-xL). The reduction caused by cisplatin in paraoxonase and
arylesterase activities could be prevented at a significant level in the rats given a pre-treatment with
PSE before the injection of cisplatin. Also, PSE significantly attenuated the cisplatin-induced structural
alterations in the liver tissue, and increased anti-apoptotic hepatocyte numbers in the interlobular area
and around the central vein of liver, but they were not observed in CP treated rats. PSE may be used as
a preventive agent against the cisplatin-induced hepatotoxicity in patients receiving chemotherapy
medications.
Key words: Arylesterase, cisplatin, paraoxonase, pomegranate, anti-apoptosis.
INTRODUCTION
Pomegranate (Punica granatum) fruit has been
consumed extensively in the form of fresh fruit,
concentrate juice and pomegranate sour in salads in
Turkey and the Mediterranean region) (Tezcan et al.,
2009). The pomegranates are found to be a rich source
of polyphenolic compounds that include flavonoids
(anthocyanins, catechins and other complex flavonoids)
and hydrolyzable tannins (punicalin, pedunculagin,
punicalagin, gallagic acid and ellagic acid esters of
*Corresponding author. E-mail: serapyildirim10@hotmail.com.
Tel: +90 442 2316617. Fax: +90 442 2361054.
glucose) which account for 92% of their antioxidant
activities (Zahin et al., 2010). In recent years, many
research results have been published about the
beneficial effects of pomegranate fruit. For example, it
has been reported that consumption of pomegranate
juice may be helpful against coronary heart disease
(Sumner et al., 2005) and Alzheimer’s disease (Singh et
al., 2008). Also, pomegranate extract significantly
improve arteriogenic erectile dysfunction (Zhang et al.,
2011) and sperm quality in male patients (Turk et al.,
2008b). In addition, the more remarkable scientific
articles are available on cancer chemoprevention by
pomeganate extracts and active compounds in vitro, as
well as in experimental animal models (Adhami et al.,

2318 J. Med. Plants Res.
2009). Cisplatin (cis-diamine-dichloroplatinum) is one of
the principal drugs used to treat various types of cancers,
including lung cancer, ovarian cancer and testicular
cancer (Gottfried et al., 2008; Turk et al., 2008a).
However, the use of high-dose cisplatin can lead to
serious side effects. The toxic effects which occur
primarily in the liver and other organs restrict the clinical
use of cisplatin (Jiang and Dong, 2008; Liao et al.,
2008). Cisplatin toxicity may occur with different
mechanisms. Studies have shown that cisplatin leads to
the formation of reactive oxygen species, lipid
peroxidation and DNA damage (Cayir et al., 2009).
Therefore, an important point that should be considered
during treatment with cisplatin is that consumption of
dietary antioxidants may be useful against free radical
damage.
Serum paraoxonase-1 (PON-1) is a calcium-dependent
ester hydrolyse which has got both paraoxonase and
arylesterase activities. It is one of the three members of
the paraoxonase family (PON-1, PON-2, PON-3). Serum
PON-1 is synthesized mainly in the liver and secreted
into the blood circulation (Draganov et al., 2005;
Rosenblat et al., 2006). PON-1 has anti-oxidative
properties, which are associated with the enzyme’s
capability to protect LDL and HDL from oxidation, to
decrease the lipid peroxidation caused by the free
radicals on the cell membranes and lipoproteins and to
slow the development of atherosclerosis (Costa et al.,
2005; Ferretti et al., 2003; Toker et al., 2009; Yildirim et
al., 2007). Aviram et al. (2000) reported that the
consumption of pomegranate juice by healthy human
subjects for 2 weeks significantly reduced the oxidation of
both LDL and HDL and increased the activity of serum
paraoxonase. In addition, it has been shown in a recent
study that PON-1 expression in hepatocytes is
upregulated by pomegranate polyphenols (Khateeb et al.,
2010).
The consumption of dietary antioxidants such as
pomegranate juice, blueberry juice and green tea inhibit
oxidative stress and prevent free radical injury (Zhang et
al., 2011). Thus, we envisage that pomegranate seed
may indicate protective antioxidant activity which might
have promising therapeutic potential against the oxidative
organ damage caused by cisplatin. For this purpose, the
antioxidant effects of pomegranate seed extract have
been analysed with the PON-1 paraoxonase and
arylesterase enzyme activities measured in the liver
tissue samples. Further, the hepatotoxicity associated
with cisplatin treatment and the protective effects of
pomegranate extract were examined histologically.
MATERIALS AND METHODS
Animals and experimental procedure
After obtaining approval from the local ethics committee of animal
experiments in Ataturk University, we used 24 female adult
Sprague-Dawley rats weighing approximately 200 g. Before starting
the experimental protocols, the rats were randomly divided into four
groups of six animals: Control group (Group C), the group that took
pomegranate seed extract (Group PSE), the group that took
cisplatin (Group CP) and the group that took pomegranate seed
extract+cisplatin (Group PSE+CP). The animals were kept in the
metal cages with a temperature of 22 to 24°C and a 12-h light / dark
cycle during the study. The rats in the control group were fed with
standard rat food and tap water, while the PSE group rats were
given 300 mg/kg/day pomegranate seed extract (Balen
Pomegranate Seed Extract Capsule, Arı Mühendislik Company,
Ankara, Turkey) through the oragrastric tube for 15 days. Group CP
rats were given a single dose of cisplatin (7 mg kg −1 body weight
i.p.). Group PSE+CP rats were given PSE 300 mg/kg/day through
oragrastic way for 15 days and then a single dose of cisplatin (7 mg
kg −1 , i.p.). The doses of cisplatin and PSE used in this study were
selected in accordance with the previous studies (Al-Majed, 2007;
Cayir et al., 2011).
Taking of the tissue and blood samples and biochemical
measurements
Twenty-four hours after the end of the study, the animals in the
control and experimental groups were anaesthetized with an i.p.
injection of 60 mg sodium pentobarbitone per kg of body weight.
Then the rats were killed by taking blood samples from the heart,
and liver tissues were immediately removed. Blood samples were
centrifuged for 5 min. at 3500 g and their serum parts were
separated and stored at -80°C until they were analyzed. Tissue
samples were cleaned by the solution of isotonic NaCl at ice cold
for the removal of bloody spots, and then they were dried with
blotting paper.
About 300 mg liver tissue was weighed for each rat and
homogenized within the tampon solution at ice cold buffer (50 mM
Tris-HCl, pH 8.0, containing 2 mM CaCl2) (OMNI TH homogenizer,
Warrenton, VA, USA). Tissue homogenates were centrifuged at
15,000 g for 15 min. (4°C) and the supernatant part was kept for
biochemical analysis at -80°C. Tissue protein levels were
determined with Bradford method (Bradford, 1976) and the serum
ALT level was determined with Roche Cobas biochemical
autoanalyzer by using a commercial kit (Roche Diagnostics,
Mannheim, Germany).
Measurement of PON-1 paraoxonase and arylesterase (ARE)
activities
PON-1 and ARE activities were assessed by methods described
previously (Beltowski et al., 2005; Eckerson et al., 1983), with some
modifications. The original method was made semi-automatized
with the adaptation of 96-well microplate and thus in a short time
the analysis of a multitude of samples was made possible. The
kinetic measurements were conducted with the use of
spectrophotometric microplate reader (PowerWave XS, Bio-Tek
Instruments, Inc.) and its software program (KC Junior software,
Bio-Tek Inc.).
PON-1 measurement was realized at 405 nm and ARE at 270
nm. As ARE measurements were made at ultraviolet wavelength
(270 nm), the 96-well ultraviolet plate was used for assays. For the
measurements of PON-1 and ARE activities, diethyl-p-nitrophenyl
phosphate (Sigma Co, UK) and phenyl acetate (Sigma Co, UK)
were used as the substrates, respectively. Molar absorption
coefficients were used in the calculation of PON-1 and ARE
activities (17100 and 1310 M −1 cm −1 , respectively). One unit for
PON-1 activity was defined as 1 nmol 4-nitrofenol/mL serum/min
and that for ARE activity was defined as 1 mmol fenol/mL
serum/min. Tissue-specific activities of PON-1 and ARE in liver
were calculated and results are expressed as U/mg protein.

Table 1. Biochemical parameters in the study groups (values are mean ± SD).
Parameter
C (n=6)
Groups
PSE (n=6) CP (n=6) PSE+CP (n=6)
Serum ALT activity (U/L) 32.8 ± 4.6 33.2 ± 4.1 50.5 ± 5.7 b 45.9 ± 8.7 a
Liver MDA (nmol/g protein) 36.9 ± 8.1 34.1 ± 9.7 59.8 ± 16.3 c
50.2 ± 13.7 d
Paraoxonase (U/mg protein) 8.1 ± 1.6 10.2 ± 1.8 5.2 ± 1.1 a
6.8 ± 1.3 e
Arylesterase (U/mg protein) 6.6 ± 0.5 8.7 ± 1.2 a
4.2 ± 0.7 b
5.6 ± 1.1 f
a , p

2320 J. Med. Plants Res.
Histological results
A comparison of anti-apoptotic activity of the groups is
shown in Figures 1 to 4. Based on the histological
evaluation, it could be concluded that although few
numbers of bcl-xL immune positive cells were observed
around the central vein in the control groups (Figure 1),
these reactions were mildly observed at both the
interlobular area and around the central vein in the PSE
group (Figure 2). On the other hand, in the CP treated
group, histological alterations were characterized with
sinusoidal dilatation, hepatocellular degeneration,
necrosis, and bcl-xL immun negative hepatocyte (Figure
3). In the PSE+CP group, interestingly decreases in
cytoplasmic alteration of the hepatocytes were observed
Figure 1. Bcl-xL reactions in interlobular area (a) and around central
vein (b) in control group. Streptavidin-biotin peroxidase staining.
Figure 2. Bcl-xL reactions in interlobular area (a) and around central
vein (b) in pomegranate seed extract group. Streptavidin-biotin
peroxidase staining.
when compared to the CP treated group. Anti-apoptotic
activities were strongly observed in hepatocytes placed
radially around the central vein in the liver tissue of
PSE+CP group (Figure 4) compared with CP group.
Semiquantitative analysis of bcl-xL reactivity (antiapoptotic)
in liver was determined as follows: Control
group, mild (+); PSE group, severe (+++); CP group,
none or light (-/+); PSE+CP group, very strong (++++)
staining immune positive reactions.
DISCUSSION
Free-radical damage and increased oxidative stress have
been proposed as a mechanism of cisplatin-induced

Figure 3. Bcl-xL reactions in interlobular area (a) and around central
vein (b) in cisplatin control group. Streptavidin-biotin peroxidase
staining.
Figure 4. Bcl-xL reactions in interlobular area (a) and around central vein
(b) in pomegranate seed extract+cisplatin group. Streptavidin-biotin
peroxidase staining.
toxicity (Cayir et al., 2009; Cepeda et al., 2007; Martins et
al., 2008). Kart et al. (2010) reported that some
antioxidant molecules that are influential as scavenger or
that prevent the formation of reactive oxygen species
eliminate the hepatotoxicity caused by cisplatin.
Therefore, we investigated whether pomegranate seed
extract possesses the protective effect against the toxicity
of cisplatin. For this purpose, we made both the
biochemical and histopathological examination by
creating an experimental rat model. In the biochemical
examination, we measured serum ALT levels, PON-1
Yildirim et al. 2321
paraoxonase and arylesterase activities and MDA levels
in liver tissue samples.
Cisplatin is a drug commonly used in the treatment of
cancer (Gottfried et al., 2008; Turk et al., 2008a).
However, especially when used at high doses, cisplatin
can lead to serious side effects such as nephrotoxicity
and hepatotoxicity (Jiang and Dong, 2008; Liao et al.,
2008). In the animal experiment studies, it has been
shown that cisplatin at a single dose of 7 mg/kg (i.p.)
leads to hepatotoxicity in rats (Al-Majed, 2007).
Therefore, we also have created hepatotoxicity with a

2322 J. Med. Plants Res.
single dose injection of cisplatin (7 mg/kg, i.p.).
Hepatotoxicity was shown by measuring serum ALT
levels and the histological examination of the liver tissue
samples after administration of cisplatin in the rats. In the
present study, cisplatin led to a significant increase in the
serum ALT levels when compared with the control group,
and the obvious histopathological changes such as
mononuclear cell infiltration, congestion and
hepatocellular degeneration were observed in liver
tissues of the rats. These results were consistent with
those of the previous published studies of cisplatininduced
hepatotoxicity (Cayir et al., 2009; Kart et al.,
2010; Yuce et al., 2007). In addition, in our study, MDA
levels were increased significantly in the liver tissues
from cisplatin-treated animals. This result seems to be
confirmative of the literature information that increased
free radical levels and resulting oxidative stress may play
an important role in cisplatin-induced toxicity (Cayir et al.,
2009; Martins et al., 2008).
PON-1 is a calcium-dependent esterase synthesized
primarily in the liver and possesses both paraoxonase
and arylesterase activities (Draganov et al., 2005). In
many experimental and clinical studies, it has been
revealed that PON-1 is an antioxidant enzyme and also
protects HDL and LDL from oxidation (Rosenblat et al.,
2006; Costa et al., 2005; Ferretti et al., 2003). In the
present study, it was observed that the administration of
cisplatin to rats caused a significant decrease in hepatic
PON-1 paraoxonase and arylesterase activities. The
decreases of these enzyme activities may be due to a
direct toxic effect of cisplatin on hepatocytes or the
increased free radical production (Martins et al., 2008),
and namely, in cases of increased oxidative stress, the
excessive amounts of free radicals can disrupt the
functions of proteins by attacking specific chemical
groups of proteins (Yildirim et al., 2003). PON-1 enzyme
protein has three cysteine (Cys) residues in positions 42,
284 and 353, with a disulfide bond between Cys-42 and-
353 and a Cys-284 as a free thiol (-SH) (Costa et al.,
2005). Although Cys-284 is not essential for the
hydrolytic activity of enzyme, this reactive sulfhydryl (-SH)
group at position 284 is required for the tertiary structure
to maintain the active-site residues in their optimal spatial
arrangement (Mackness et al., 1998). Also, the free Cys-
284 residue is necessary for PON-1 to be protective
against LDL oxidation (Costa et al., 2005). It thus
appears likely that the formation of free radicals caused
by cisplatin may inhibit enzyme activity by interacting with
sulfhydryl groups on PON-1 protein.
Certain antioxidants may increase PON-1 activity by
preventing its oxidative inactivation. Khateeb et al. (2010)
reported that pomegranate juice polyphenols stimulate
PON-1 hepatic expression via PPAR signalling cascade
which result in an increased secretion of a biologically
active PON-1 from the hepatocytes. In another study,
whether there are beneficial effects of pomegranate juice
consumption by mice on their serum PON-1 activity and
macrophage PON-2 expression has been investigated
(Rosenblat et al., 2010). Their data showed that
pomegranate juice consumption led to a significant
increase in serum PON-1 catalytic activities (36%) and
macrophage PON-2 expression. Similar to the results of
Khateeb et al. (2010), we observed that the consumption
of pomegranate seed extract stimulates PON-1
paraoxonase and arylesterase activities in the liver tissue
of rats treated with pomegranate extract. Moreover, the
administration of pomegranate seed extract was
observed to significantly prevent the decrease in liver
paraoxonase enzyme activities caused by cisplatin. This
effect is probably related to the antioxidant characteristic
of pomegranate.
As we stated previously, increased free radical levels
[that is, the superoxide (O2 .- ), hydroxyl (HO . ), perhydroxyl
.-
radicals (HO2 )] and/or decreased enzymatic antioxidant
defense system in the cell play important roles in cisplatin
toxicity (Liao et al., 2008; Cayir et al., 2009; Cepeda et
al., 2007). In contrast, the pomegranates are found to be
a rich source of polyphenolic compounds (flavonoids and
tannins) which account for 92% of their antioxidant
activities (Zahin et al., 2010), and has scavenging activity
against HO . .-
and O2 (Noda et al., 2002). Therefore, it is
probable that pomegranate polyphenolic compounds
inactivate free oxygen radicals such as HO . .-
and O2
which are indeed very reactive (Yildirim et al., 2003), thus
protecting the PON-1 paraoxonase and arylesterase from
the harmful effects of free radicals. Cisplatin has been
found to have an apoptotic effect and mitochondrial
damage on liver tissue (Wetzel et al., 2001). Apoptosis is
characterized by membrane budding, cell shrinkage,
chromatin condensation. The caspase enzymes (initiator
caspases e.g. 2, 8, 9, 10 and 12 and effector caspases
e.g. 3, 6 and 7) triggers the apoptotic process, whereas
bcl-xL is generally acts anti-apoptotic, which
overexpression are delay or inhibit apoptosis as well as
provide a true survival advantage in cell treated with
chemotherapeutic agents (Walker et al., 1997; Zhan et
al., 1999).
In our previous study, CP has been found to have an
apoptotic effect on proximal tubules and loop of henle in
the kidney and around of the central vein in the liver
(Cayir et al., 2011). In this study, anti-apoptotic cells in
the liver tissue were investigated with bcl-xL antibody.
Anti-apoptotic reactions were observed throughout of the
lobules and around of the central vein in the only PSE
treated group and PSE pretreatment before CP injection
in liver tissue, whereas bcl-xL activity was not observed in
the CP group.
Consequently, the administration of pomegranate seed
extract was observed to significantly prevent the
decrease in rat liver paraoxonase enzyme activities
caused by cisplatin, possibly because of the antioxidant
characteristic of pomegranate. Also, cisplatin-induced
apoptosis may be prevented as a result of
overexpression pattern of the bcl-xL by PSE.

ACKNOWLEDGMENTS
The authors would like to express their gratitude to the
Center of Experimental Research and Practice at the
Ataturk University for providing the animals.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2324-2339, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.831
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Medicinal plants from an old Bulgarian medical book
Anely Nedelcheva
Sofia University “St. Kliment Ohridski”, Faculty of Biology, Department of Botany, Sofia, Bulgaria.
E-mail: aneli_nedelcheva@yahoo.com.
Accepted 18 July, 2011
The aim of this study was to conduct an ethnobotanical research of old written sources which give
information about medicinal plants and preparation of folk remedies for a particular historical period.
The object of the present study is “Canon Prayer to St. Ivan Rilski and Medicinal Text” (1845) - a part of
the Bulgarian early printed literature heritage. The 92 submitted recipes cover a wide range of illnesses
and symptoms ranging from antiseptic to cures for neurological diseases. High species diversity of
medicinal plants is represented in the book - most of them are vascular plants from 36 families
(Leguminosae, Umbeliferae, Compositae, Zingiberaceae, Piperaceae, Myristicaceae, Lauraceae,
Labiatae, Liliaceae, etc.) and 65 genera. The main components in written folk remedies are medicinal
plants (more than 69), followed by the animals and animal products (20) such as honey, eggs, leeches,
blood, musk, etc., mineral elements (sulphur (S), mercury (Hg), Au, gold (Au), iron (Fe)) and other
organic and inorganic compounds (30). The significant participation of spices such as clove, cinnamon,
mastic and ginger in folk remedies sheds new light on the list of species that are traditionally used in
the folk medicine. The ethnobotanical study on this book, support the thesis that it was founded on
authentic recipes from the healing activity of St. Ivan Rilski, which has increased its historical value a
lot.
Key words: Ethnobotany, folk remedies, medicinal plants, old book, St. Ivan Rilski.
INTRODUCTION
Because of the recent trends to search for new medicinal
plants and the rediscovery of alternative methods to treat
diseases, the interest to all sources of popular knowledge
concerning the folk medicine is expected and logical.
This trend in scientific studies is quite visible in many
contemporary documents of world health organization
(WHO) (Bodeker et al., 2005). In the recent years, a lot of
ethnobotanical investigations were aimed at collecting,
analyzing and systematizing the accumulated traditional
folk knowledge (Hatfield, 2004). The methods applied
mainly by conducting interviews in different regions of the
world are followed by modern quantitative and numerical
analysis. The number of these studies has increased in
Europe and in particular in the Balkan region (Redzic,
2009; Santayana et al., 2010; Dogan et al., 2011).
Bulgarians have been using herbal medicine to treat
some common diseases for centuries. The empirical data
of medicinal plants and traditional herbal drugs is passed
on from one generation to another as oral folklore and
only little part of it can be found in written texts -
manuscripts or herbal books. Most of them are well
preserved and recorded with regard to the responsibility
to keep the national traditional knowledge (Balan, 1909;
Pogorelov, 1923; Stoyanov, 1957-1959; Petkanova,
2003).
Written historical records are documentary sources with
greater degree of reliability of the information. They
provide data which summarizes the folklore experience of
many generations. A good example in that regard is the
study of the works of Cervantes with references to plants,
plant communities, and products (Santayana et al.,
2006). Almanacs, orthodox books, books with herbal
recipes and books of domestic medicine abundant during
the XIX century were a mix of officinal and folk medicine.
Some ethnobotanical and ethnopharmacological studies
have focused on written documents - historical
documents, herbal books, literature, etc. (Richmond et
al., 2003; Santayana et al., 2006; Quave et al., 2008;

Figure 1. The first page of the book.
Leonti et al., 2009). The Bulgarian ethnobotanical
literature generally neglected the oldest text documents
related to Bulgarian herbal history (Ahtarov et al., 1939;
Stojanov and Kitanov, 1960; Vakarelski, 1977, Petkov,
1982; Ivancheva and Stancheva, 2000; Leporatti and
Ivancheva, 2003; Ploetz and Orr, 2004; Nedelcheva and
Dogan, 2009).
The aim of this study was to conduct an ethnobotanical
research of old written sources which give information
about medicinal plants and preparation of folk remedies
for a particular historical period. The research was
focused on the medicinal plant identification, the
determination of species‟ richness and diversity, and the
analysis of the level of herbal folk knowledge. One of its
purposes was to show features of historical printed
sources as well as to reveal the possibilities of analysis
that the collected information provides.
MATERIALS AND METHODS
Nedelcheva 2325
The object of the present study is “Canon Prayer to St. Ivan Rilski
and Medicinal Text” (1845), (P. Sapunov Publ., Bucharest, 65 pp).
Figure 1 - a part of the Bulgarian early printed literature heritage
(Balan, 1909; Pogorelov, 1923; Stoyanov, 1957-1959). Written in
Old Church Slavonic language by an anonymous author, the book
contains three main parts: 1) “Canon-prayer”, 2) “Prayer to St. Ivan
Rilski” and 3) Folk remedies. According to Balan (1909), the
presumed author of the manuscript from which he made the printed
edition is Neofit Rilski (a 19th-century Bulgarian monk, teacher and
artist, and an important figure of the Bulgarian National
Rennaisance). The printed edition of this book is stored in the fund
for old, rare and valuable books on St. St. Cyril and Methodius
National Library, Sofia.
Why this book?
The book is one of the oldest written documental sources with
traditional herbal remedies. “Lekarstvenik” (in Bulgarian) means
book with collection of folk recipes and home remedies (medicinal
text) (Figure 1). The texts that it contains are much more than a
catalog of natural cures.
The book is dedicated to St. Ivan Rilski (876 - circa 946) - the first
Bulgarian hermit. Today he is honoured as the patron of the
Bulgarian people and one of the most important saints of the
Bulgarian Orthodox Church. St. Ivan Rilski is also legendary known
to have performed a multitude of miracles in order to help people
heal of illnesses and infirmities (Duichev, 1947; Pulos, 1992;
Bayramova, 1997). The few data about herbs, remedies, etc. that
he used has been found until now (Stranski, 1953; Nedelcheva,
2009).
The book chosen for the subject of this study relates to several
specific time periods: 1) The period of the creation and the
implementation of the recipes - the most active years of St. Ivan
Rilski during the reign of Tsar Peter I (927-969); 2) The period of
the storage and transmission (it continued about 900 years); 3) The
period of creating a written document (around 1827) (the activity of
N. Rilski as a scholar in Rila Monastery); 4) The period of printed
book (1845).
The book is one of the oldest catalogues of natural cures in
Bulgaria and consists of 92 folk remedies, used in wide spectra of
health problems. The feature for each remedy describes the kind of
disorders it is intended for, its ingredients, the order of preparation
and some instructions for use (Figure 2).
The herbs are mentioned with their vernacular names. The
identification of the plants on these folk names is a major problem
because nowadays most of them are old and unknown or with very
limited use. The plants were identified according to their scientific
(Latin) names in the medicinal and botanical books of that time
(Pirovo, 1854), classical Bulgarian ethnobotanical sources (Ahtarov
et al., 1939; Stojanov and Kitanov, 1960; Stranski, 1963) and
modern ethnobotanical databases and glossaries (Katzer, 2011).
The information set out in any written source is directly connected
to a region characterized by geographical, ethnic and social
features.
Study area
Bulgaria is a country in the Balkans in south-eastern Europe. It
borders five other states: Romania to the north (mostly along the
River Danube), Serbia and the Republic of Macedonia to the west,

2326 J. Med. Plants Res.
and Greece and Turkey to the south. The Black Sea defines the
extent of the country to the east (42 41′0″N and 23°19′0″E).
Phytogeographically, Bulgaria straddles the Illyrian and Euxinian
provinces of the Circumboreal region within the Boreal kingdom.
The territory of Bulgaria can be subdivided into two main
ecoregions: the Balkan mixed forests and Rhodope Mountain mixed
forests. Small parts of other four ecoregions are also present in the
Bulgarian territory. Bulgarian flora comprises 159 families, 906
genera and 3900 species, of which 12.8% are endemics, 750
medicinal plants, 300 medicinal plants gathered yearly and 200 in
active use (Petrova 2005; Petrova and Vladimirov, 2009).
Bulgaria's population consists mainly of ethnic Bulgarians
(83.9%), with two sizable minorities, Turks (9.4%) and Roma
(4.7%), (NSI, 2011). The official language is Bulgarian (written in
Cyrillic alphabet), a member of the Slavic linguistic group.
The plant nomenclature is given according to Flora Europaea
(Tutin, 1964-1993).
RESULTS
The third part of the printed edition of the book (3 Folk
remedies) contains 92 recipes which are not arranged
thematically. The recipes are graphically separated by a
plant ornamental frieze (Figure 2).
Structure of the recipes
Each recipe is numbered with Arabic numerals. The titles
Figure 2. The recipe № 10 demonstrated the structure of the folk
remedies description.
of the recipes are a little long. They are often very
descriptive and pictorial and incorporate sounds, events
and feelings associated with certain symptoms. At the
beginning of the recipe the people can find the
ingredients and their quantity, the order in which they
should be placed, and a clear indication of storage and
intake well as the amount and the duration (Figure 2).
Unit
The units are usually expressed in grams, and measures
as “oka” and “denk” (water, honey) are used. Oka is an
Ottoman measure of mass of weight equal to 1.28 kg.
Denk means „number of fruits„(7 red peppers). In very
rare cases the book indicates the amount of money for
which one should buy the component (take chickpea for
10 “pará” (old coins).
Mode of preparation
The author gives instructions for the temperature (testing
the temperature of the water using the finger) and
detailed and coherent explanation of the process of
mixing the ingredients. Techniques such as cooking to
reduce the amount of the fluid on half, boiling so that the
consistency can become mushy, filtration through a cloth,

Figure 3. The most widely included in book system disorders according to the number of recipes.
sieving, shaking, thickening with flour, frying, “frying” in
wine, etc. are used. For internal use in most recipes are
prepared pills. The most commonly used fillers are flour
or batter and it is indicated that the pills should be with
the size of a chickpeas.
A lot of the remedies contain honey as a base and
healing ingredient. These are mainly herbal pastes for
internal administration, but there are some for external
use as well. Honey is first heated and then added to the
rest of the ingredients. This description is in accordance
with the modern researches on honey and its properties
during thermal processing (Turhan et al., 2008). In many
cases it is recommended to add "margarit" (pearls) to
facilitate the mixing to homogenization. Some instructions
are given for the storage of the medicine: to stand for
several days before use, to be kept tightly closed in a
cool, dark place, etc.
Mode of use
Nedelcheva 2327
In several places in the book an accompanying diet is
recommended which prescribes abstinence from salty,
sour and spicy food and alcohol in cases of skin diseases
and an infectious disease (jaundice). Vegetarian diet is
recommended too. Each recipe ends with a detailed
indication of when and how to apply the medicine.
Disorders
The 92 submitted recipes cover a wide range of illnesses
and symptoms ranging from antiseptic to cures for
neurological diseases (Figure 3).
In many of the titles of the recipes a symptom called
“stitch” is noted, which is a popular name for a pain of

2328 J. Med. Plants Res.
uncertain origin. Its intensity is graded - for example, one
of the grades is"… cannot be tolerated". On this basis in
the study a group of pain relievers (15) has been
separated. This significant group of recipes (15) specifies
descriptive symptoms that may be associated with the
occurrence of different types of pain caused by various
reasons. It is therefore logical and explicable that they
represent 16.3% of all prescriptions. The largest numbers
of recipes are those concerning Nerve disorders (15)
such as headache (6), disorders associated with
dizziness and procrastination of the head. Others are
focused on getting seizures and treating insomnia and
fear. Prescriptions for headache were based mainly on
herbs, and most of them are for external use - acting as a
local analgesic. One of them is a herbal paste taken
internally and another - of unknown composition - is
snorted as snuff. Two consecutive recipes in the text
refer to occurrence of seizures with different frequency.
The first is addressed to seizures once a month and the
second - “when a man collapses rarely in the year, but
not every month”. In the first case it is recommended to
be treated burned swallowtail chapter (of the same sex
as the patient). The ash is sanctified repeatedly and is
then placed into the drink water from a spring and Myron
(holy oil) is added. This is the only recipe in which a ritual
is included. In the latter case, the brain of a badger is
mixed with butter and pills are produced.
“When humans are frightened” and “when a man is
frightened in a dream” are two recipes related to the
treatment of fear. The description of the first is unclear,
because of damage to the page of the book, but
apparently it involves more inorganic substances. The
second consists entirely of herbal pasta and is combined
with swallowing a fulminatory bullet. “Sleep Balm” is
made mainly of herbs. In it and in several other remedies
opium poppy (seeds) is included which has a sedative
effect.
Analysis of the prescriptions associated with nerve
disorders is important because the “Lives of St. Ivan
Rilski” mainly focus on treatment of fear, madness,
stuttering and alike (Duichev, 1947; Pulos, 1992;
Bayramova, 1997). Some of the famous paintings of the
Saint focus exactly on his talent of a healer (Figure 4).
The fact that these recipes occupy a large proportion of
the book and with their rational nature of formulation and
implementation are the main arguments in support of the
presumption that the text is drawn after the recipes
associated with the healing activity of the Saint. The large
proportion of the cures of disorders is for the digestive
system and intestinal-digestive disorders (9) - these are
mainly stomachache, diarrhea and constipation. Other
prescriptions concern the treatment of diseases with
symptoms that impede the daily life of the people and are
assumed to be common - skin disorders (7), ophthalmic
disorders (7), respiratory system disorders (6), dental
disorders (6), and urinary system disorders (5). Ways to
alleviate the symptoms of cardiovascular disorders (4),
gynecological disorders (1), ear disorders (1), liver-spleen
disorders (1), throat disorders (4), hemorrhoids (2) and
cough symptoms (3) are also included. Several of the
recipes are compound recipes (for more than one
disease or symptoms). Some of the remedies and
procedures described in them are focusing on antiinflammatory
(antiseptic) (3), antipyretic (4) and insects‟
repellent (1) action. Three of the recipes are oriented
generally on strengthening the body after a serious
illness or fatigue - herbal pastes with a general
strengthening effect (immune supporter) (3). In the book
there are two recipes related to the treatment of jaundice
(2) showing the importance of this disease, as the period
during which the recipes were created and the time at
which the book was published, nearly 10 centuries ago,
was manifestly plagued by significant for the society
outbreaks of jaundice. The special significance of the
horse in the life of the people as transport and power is
evident from both veterinary prescriptions (horse skin and
urinary diseases) (2).
Ingredients
Constituents (with repetitions) of the recipes are totally
480. The average number of the components of a recipe
is 5-6, with the minimum being 1 and the maximum - 17.
Eighty-five of all prescriptions contain plants in its
composition. Herbal ingredients are marked a total of 309
times. One of them contain only one plant, the average
number of plant ingredients is 3-4 and mostly up to 5 in
the final product. The remedies with general
strengthening action on the basis of honey have the
highest number of herbal ingredients.
The main components in written folk remedies are
medicinal plants (146), followed by the animals and
animal products (20) such as honey, eggs, leeches,
blood, musk, etc., mineral elements (sulphur (S), mercury
(Hg), gold (Au), iron (Fe)) and other organic and
inorganic compounds (30).
The basis for the preparation of remedies is often
honey and sheep fat, as well as eggs, blood, musk, etc.
Interestingly, along with the use of wild animals such as
eagle, swallow, jackdaw, magpie, turtle, trout, leeches,
crabs, tadpoles, there is a single mention of use of bone
marrow from tibia of horse, bovine testes and bile from a
goat ling. The use of animals (meat from their bodies)
made it clear that the women should be treated with
female animals and men - with male animals. Very often
in folk remedies there are mineral elements such as
sulfur, mercury, gold, ferrum and some appear as the
main constituent. Some common compounds are silver
nitrate (AgNO3), alum, naphtha, cinder, asphalt,

Figure 4. Lithographic print “St. Ivan Rilski and his Monastery” 1866.
gunpowder, glaze, citric acid and plant and animal
products such as amber, wax, coal, mastic (plant resin),
pearls, Gummi Arabica, treacle, egg albumen and yolk,
brandy, red wine, sugar, yogurt, vinegar, salt, butter and
camphor. From the first to the last prescription the
ingredients are of the same “list” and are found in various
St. Ivan Rilski heal furious.
St. Ivan Rilski heal person with
rabies.
Nedelcheva 2329
combinations, thus showing the unity of the source.
Plants
High species diversity of medicinal plants is represented

2330 J. Med. Plants Res.
6
5
4
3
2
1
0
Anacardiaceae
Caprifoliaceae
Compositae
Crucuferae
Cucurbitaceae
Geraniaceae
Gramineae
Labiatae
Lauraceae
Leguminosae
Liliaceae
Malvaceae
Myristicaceae
Piperaceae
Polygonaceae
Ranunculaceae
Rosaceae
Umbeliferae
Urticaceae
Zingiberaceae
Figure 5. The systematic structure of medicinal plants represented in the book.
in the book - most of them are vascular plants from 36
families and 65 genera. The predominant numbers of
species (and the most notable ones) are from
Leguminosae, Umbeliferae, Compositae, Zingiberaceae,
Piperaceae, Myristicaceae, Lauraceae, Labiatae,
Liliaceae, etc. families (Figure 5). Thirty-three plant
species mentioned in the text are representatives of the
Bulgarian flora, others were subject to cultivation during
the historical period, but no small part were spices and
raw materials imported from Middle Eastern markets. In
Table 1, sixty-nine species are included which are clearly
identified in the studied text.
DISCUSSION
Most of the plants are naturally distributed species in the
Bulgarian flora (50.7%). Large part of them is known by
the population as food and 20.3% are cultivated. The
second largest group is the species used as spices
(24.6%). Many plants have been cultivated as ornamental
in the courtyards of buildings (10.1%), but there are also
weed and ruderal species included (6%) (Figure 6). It is
noteworthy to avoid poisonous plants. Even when using
the fruits of the elder, they should be ripe. This
corresponds to the phytochemical data showing the
presence of toxic substances in the unripe fruits of this
plant. It is well known that the green berries are toxic.
Descriptions rarely specify what part of the plant should
be used (in less than 10%). The explanation for this may
be the widespread use of herbs i.e. perceived as
Number of species
something that does not call into question which part of
the plant is used. Contrary to this, “roots of nettles” is
clearly specified, because seeds may be used too; “roots
of elder”, because its flowers and fruit are the more
oftenly used parts, melon seed (commonly known as fruit
for food) and others.
Chickpea (Cicer arietinum) is mentioned in four recipes.
In some cases it is difficult to assess whether it is the
basis of a drug or it just adds healing properties when
combined with other herbs. The constant comparing of
the size of the pills to the size of chickpeas shows that
people knew well this plant and that it was widely used in
everyday life. “Nahut” is the standard measure for the
preparation of the pills. Chickpea was studied for its
appearance in arhaeobotanical records from this region
by Marinova and Popova (2008) and Marinova (2009).
According to the results the occurrence of the chickpea
only for a short period of time around the end of the
Bulgarian early Neolith and at the beginning of the middle
Neolith, as well as during the late Chalcolith, supports the
suggestion that the chickpea was mostly a weed. It might
have been imported with other crops during more intense
contacts with Anatolia during these periods.
Although rock rose (Cistus landaniferus) is associated
with religious rituals in the Orthodox Christianity, here it is
mentioned only as an integral part of biologically active
action. This plant is widely used in herbal medicine
around the world as anti-diarrheal, antiacid and
antispasmodic as visible from many ethnobotanical
works. Essential oil been shown to exhibit antifungal and
antibacterial effect (Aziz et al., 2006).

Table 1. List of plants included in folk remedies from the book
Nedelcheva 2331
Scientific name Family Local name Type Part used Mode of administration
Cotinus coggygria Scop. Anacardiaceae tekla W shoot 7: diarrhea; crushed and boiled in water
19: pain in the gums; boil in vinegar; liquid used for gargling
Pistacia lentiscus L. Anacardiaceae bial sakaz I plant resin 14: difficulty urinating; mix and boil in water
25: purgative; herbal pasta based on honey
38: in fart a lot; herbal pasta based on honey. Diet.
43: in jaundice; herbal pasta based on honey
45: for problems in heart, eyes and ears
77: wound healing; ointment based on butter
80: in distress; herbal pasta based on honey
82: for wounds; ointment based on olive oil and butter
92: accidental urinating; compress
Cocos nucifera L. Arecaceae
izistan zhevizi S, I nut 1: general strengthening; herbal pasta based on honey
15: cough with bloody secretions; based on red wine
38: in fart a lot; herbal pasta based on honey. Diet.
40: back pain; compress of herbs and cooked meat from eagle
43: in jaundice; herbal pasta based on honey
79: in difficult sleeping; mixture based on brandy
Alkanna tinctoria (L.) Tausch Boraginaceae aivazhiva W root 77: wound healing; ointment based on butter
Sambucus nigra L. Caprifoliaceae svirchov lek W flower
root
fruit
9: fever; based on sugar syrup
21 a: scabies skin infection; boil in water
38: in fart a lot; herbal pasta based on honey. Diet.
38: in fart a lot; herbal pasta based on honey. Diet.
Sambucus ebulus L. Caprifoliaceae bazei W maturated
fruit
Cistus landaniferus L. Cistaceae tamian R, I leaf, oil 25: purgative; herbal pasta based on honey
38: in fart a lot; herbal pasta based on honey. Diet.
60: in eye secretion; ointment based on olive oil, honey and flour
67: in blocked ear; water based liquid for ear lavage. Diet.
82: for wounds; ointment based on olive oil and butter
84: strong headache; compress based on rose oil
Artemisia spp. Compositae pelin W aerial part 11: general strengthening; mixture based on vinegar. The same recipe is
recommended for making beverages “pelinash”, which uses red wine a
basis.
72: headache; compress based on brandy
Artemisia alba Turra Compositae bozhie darvo W aerial part 11: general strengthening; mixture based on vinegar
72: headache; compress based on brandy

2332 J. Med. Plants Res.
Table 1. Contd.
Tanacetum balsamita L. Compositae kalofer C leaf 11: general strengthening; mixture based on vinegar
Tanacetum vulgare L. Compositae vratiga W corymb 11: general strengthening; mixture based on vinegar
Sinapis nigra L. Cruciferae
cheren sinap W seed 12: pain (stitch one the body); mix with honey and prepare compress. The
place in advance is smeared with olive oil. Stand for 24 hours.
13: joint pain; the seed is mixed with strained yogurt and prepare com
Stand for 24 hours.
72: headache; crushed herbs and mixed with brandy; compress on the fore
74 b: general strengthening, in the beginning of the cold symptoms; m
based on olive oil and vinegar
Citrullus lanatus (Thunb.) Matsum. Cucurbitaceae libenitsa, dinia F, C fruit 35: fever; watermelon is baked in an oven. Crust is smeared all over bo
& Nakai.
sweating.
48: in eye edem; compress for a night on the shaved head
Cucumis melo L. Cucurbitaceae pipon F, C seed 55: difficulty urinating; sugar syrup
Quercus spp. Fagaceae shikalki W gallae 38: in fart a lot; herbal pasta based on honey. Diet.
Geranium spp. Geraniaceae zdravets W root 72: headache; compress based on brandy
Pelargonium roseum Willd. Geraniaceae indrishak W leaf 22: heart arrhythmia; in red wine
Cynodon dactylon (L.) Pers. Gramineae troskot W rhizome 39: in edem; pasta based on butter and treacle; compress is changing
2 hour.
Hordeum vulgare L. Gramineae echemik F, C grain 14: difficulty urinating; mix and boil in water
33: spider bites; barley flour mixed with egg yolk to the dough; ointment.
Aesculus hippocastanum L. Hippocastanaceae at kestene W seed 43: in jaundice; herbal pasta based on honey
Crocus sativus L. Iridaceae shafran W, I anther 34: for heart problems and stomach ache; balsam based on brandy
pollen 85: eye pain and secretion; compress based on alcohol
Juglans regia L. Juglandaceae oreh W seed 10: cough; crushed and mixed; shaped as pills
21 b: scabies skin infection; fresh chopped; ointment. Diet.
Mentha spicata L. Labiatae nane, dzhodzhen,
C leaf 11: general strengthening; mixture based on vinegar
giuzum
52: a swollen lips; compress with honey based on leaf of cabbage
Ocimum basilicum L. Labiatae bosilek W aerial part 11: general strengthening; mixture based on vinegar
Salvia officinalis L. Labiatae kakule W leaf 23: headache; herbal pasta based on honey
Cinnamomum camphora L. Lauraceae kamphor S, I oil 4: cough; mixture based on water

Table 1. Contd.
Nedelcheva 2333
Cinnamomum verum J. Presl. Lauraceae kanela S, I inner bark 1: general strengthening; herbal pasta based on honey
4: cough; mixture based on water
27: to improve voice; boil in red wine and sweetened with honey
28: pain in heart area; mix based on brandy. Diet.
38: in fart a lot; herbal pasta based on honey. Diet.
40: back pain; compress of herbs and cooked meat from eagle
66: cracking of skin, wounds; mixture based on olive oil
72: headache; crushed herbs and mixed with brandy; compress on the fore
Laurus nobilis L. Lauraceae dafinov list S, I leaf 34: for heart problems and stomach ache; balsam based on brandy
Cassia acutifolia Delile Leguminosae silimakia I root
leaf
fruit
14: difficulty urinating; mix and boil in water
25: purgative; herbal pasta based on honey
38: in fart a lot; herbal pasta based on honey. Diet.
42: in the cold of feed; brandy compress based on rabbit skin
Astragalus spp. Leguminosae klinavche W seed 38: in fart a lot; herbal pasta based on honey. Diet.
Cicer arietinum L. Leguminosae leblebia, nahut F, I seed 36: back pain; boil in water; compress for 24 hours
46: chest pain; herbal pills based on dough. Diet.
42: in the cold of back; herbal pills based on dough
84: strong headache; compress based on rose oil
Glycyrrhiza glabra L. Leguminosae sladak koren W root 4: cough; mixture based on water
5: cough; herbal pasta based on honey
71: bleeding in the eye; compress on neck area
Phaseolus vulgaris L. Leguminosae bob F, C seed 36: back pain; boil in water; compress for 24 hours
48: in eye edem; compress for a night on the shaved head
Vicia faba L. Leguminosae bakla F, C seed 36: back pain; boil in water; compress for 24 hours
Aloe spp. Liliaceae
leaf 1: general strengthening; herbal pasta based on honey
sari sabur
O
2: stomach complaints and loss of appetite; pills based on dough
17: eye ache; based on red wine. Used as eye drops. Diet.
24: scabies skin infection; pills based on dough. Diet.
34: for heart problems and stomach ache; balsam based on brandy;
46: chest pain; herbal pills based on dough. Diet.
86: eye pain; compress based on rose oil
Allium cepa L. Liliaceae luk F,C bulb 21 b: scabies skin infection; fresh chopped; ointment. Diet.
68: gallbladder pain; fresh juice in water (red onion)
69: scabies skin infection; fresh chopped with sulfur; ointment

2334 J. Med. Plants Res.
Table 1. Contd.
Allium sativum L. Liliaceae chesnov luk F, C bulb 11: general strengthening; mixture based on vinegar
21b: scabies skin infection; fresh chopped; ointment. Diet.
Althaea officinalis L. Malvaceae Biala ruzha O, W root 14: difficulty urinating; mix and boil in water
Malva sylvestris Malvaceae slez O, W leaf 67: in blocked ear; water based liquid for ear lavage. Diet.
Ficus carica L. Moraceae inzhirki F, I fruit 10: cough; crushed and mixed; shaped as pills
18: jaundice; based on vinegar. Eat macerated fruits.
Myristica fragrans Houtt. Myristicaceae pespase S, I nut 1: general strengthening; herbal pasta based on honey
23: headache; herbal pasta based on honey
Syzigium aromaticum (L.) Merr. &
Perry
Myristicaceae karamfil S, I flower bud 1: general strengthening; herbal pasta based on honey
4: cough; mixture based on water
6: fever; crushed and boiled in water
20: general strengthening; boil in water and sweetened with honey
23: headache; herbal pasta based on honey
52: a swollen lips; compress with honey based on leaf of cabbage
74a: general strengthening, in the beginning of the cold symptoms; mix
based on olive oil and vinegar
76: body pain; ointment based on honey
79: in difficult sleeping; mixture based on brandy
89: headache and toothache; compress, ointment
90: mouth sores; boil in vinegar; liquid used for gargling
Papaver somniferum L. Papaveraceae afion O seed 79: in difficult sleeping; mixture based on brandy
80: in distress; herbal pasta based on honey
Pinus sylvestris L. Pinaceae
bial bor W sawdust 90: mouth sores; boil in vinegar; liquid used for gargling
plant resin 91:bleeding and pain in the gums; boil in water; liquid used for gargling
Phytolacca decandra L. Phytolaccaceae karmaza O root
77: wound healing; ointment based on butter
82: for wounds; ointment based on olive oil and butter
92: accidental urinating; compress
26: in distress; mixture based on water
fruit 31: general strengthening for baby; based on sheep fat; ointment
Piper cubeba L. Piperaceae kebabie S, I fruit 1: general strengthening; herbal pasta based on honey
Piper nigrum L. Piperaceae cher piper S, I fruit 4: cough; mixture based on water
31: general strengthening for baby; based on sheep fat; ointment
42: in the cold of feed; compress based on rabbit skin
76: body pain; ointment based on honey
89: headache and toothache; compress, ointment

Table 1. Contd.
Nedelcheva 2335
Rheum palmatum L. Polygonaceae revent rosiiski I root 1: general strengthening; herbal pasta based on honey
2: stomach complaints and loss of appetite; pills based on dough
37: in vomiting; pasta based on honey
38: in fart a lot; herbal pasta based on honey
59: in jaundice; herbal pasta based on honey
Rheum rhaponticum L. revent W root 9: fever; based on sugar syrup
27: to improve voice; boil in red wine and sweetened with honey
34: for heart problems and stomach ache; balsam based on brandy
43: in jaundice; herbal pasta based on honey
80: in distress; herbal pasta based on honey
Polypodium vulgare L. Polypodiaceae sladka paprat W rhizome 27: to improve voice; boil in red wine and sweetened with honey
Clematis vitalba L. Ranunculaceae povet W flower 55: difficulty urinating; sugar syrup
Nigella sativa L. Ranunculaceae chere otu S, W seed 25: purgative; herbal pasta based on honey
53: in vomiting; boil in water
89: headache and toothache; compress, ointment
Cydonia oblonga Mill. Rosaceae diulia F, W seed 27: to improve voice; boil in red wine and sweetened with honey
Rosa damascena Mill. Rosaceae giul O, C rose oil 31: general strengthening for baby; based on sheep fat; ointment
40: back pain; compress of herbs and cooked meat from eagle
Citrus × limon (L.) Burm.f Rutaceae limon F, O, I fruit 79: in difficult sleeping; mixture based on brandy
Digitalis lanata Ehrh. Scrophulariaceae nezhitniche W root 45: for problems in heart, eyes and ears
Verbascum spp. Scrophulariaceae ribe bile W leaf 24: scabies skin infection; pills based on dough. Diet.
Capsicum spp. Solanaceae cherven piper,
chushki
F, S, C fruits 10: cough; crushed and mixed; shaped as pills
Camellia sinensis (L.) Kuntze Theaceae chai B, I leaf 11: general strengthening; mixture based on vinegar
Angelica officinalis Hoffm. Umbeliferae angelika W root 1: general strengthening; herbal pasta based on honey
Anethum graveolens L. Umbeliferae kopar S, C fruit 61: to increase milk during lactation; herbal pasta based on honey and sug
Foeniculum vulgare Mill. Umbeliferae horezene S, C fruit 25: purgative; herbal pasta based on honey
Petroselinum crispum (P.Mill.)
Nyman ex A.W. Hill
Umbeliferae magdanoz S, C root 14: difficulty urinating; mix and boil in water
Pimpinella anisum L. Umbeliferae anason S, W fruit 1: general strengthening; herbal pasta based on honey
14: difficulty urinating; mix and boil in water
25: purgative; herbal pasta based on honey
72: headache; crushed herbs and mixed with brandy; compress on the fore
89: headache and toothache; compress, ointment
Urtica dioica L. Urticaceae kopriva F, W root 14: difficulty urinating; mix and boil in water
seed 23: headache; herbal pasta based on honey

2336 J. Med. Plants Res.
Table 1. Contd.
Urtica urens L. Urticaceae kopriva grachka W herba 9: fever; based on sugar syrup
Curcuma zedoaria L. Zingiberaceae zulumbat S, I rhizome 1: general strengthening; herbal pasta based on honey
2: stomach complaints and loss of appetite; pills based on dough
23: headache; herbal pasta based on honey
34: for heart problems and stomach ache; balsam based on brandy
Zingiber officinale Rosc. Zingiberaceae isiot, dzhindzhifil,
darifilfil
S, I rhizome 23: headache; herbal pasta based on honey
25: purgative; herbal pasta based on honey
38: in fart a lot; herbal pasta based on honey. Diet.
42: in the cold of feed; compress based on rabbit skin
45: for problems in heart, eyes and ears
62: itchy skin on horse; mixture based on vinegar
76: body pain; ointment based on honey
80: in distress; herbal pasta based on honey
89: headache and toothache; compress, ointment
90: mouth sores; boil in vinegar; liquid used for gargling
W, Wild; S, spice; C, cultivated in Bulgaria; O, ornamental; F, food, I, import plant or plant product; R, plant with religious importance; B, beverage. Mode of administration (recipe number: symptoms;
preparation; use, etc.).
Both species Rheum palmatum and Rheum
raponticum (localized in Rila Mt.) are included in
present recipes. The folk use of the second one is
derived from the healing experience of the Rila
Monastery monks and St. Ivan Rilski is the
founder of Rila Monastery (Stranski, 1953;
Nedelcheva, 2009). Plants used as spices are
mainly biologically active components, but also
determine the taste, aroma and colour of the final
product. Among them are the most commonly
used components in the folk remedies: clove
(Syzigium aromaticum) - which has a very strong
and distinct taste and flavor; mastic (Pistacia
lentiscus) - specifically strong, slightly smoky,
resin aroma, bitter and spicy taste, yellow colour;
cinnamon (Cinnamomum verum) specifically
aroma and spicy taste; ginger (Zingiber officinale)
with warming, sharp flavor and sharp taste, etc.
All of them are well known in folk medicine in
Europe and widely studied for their medicinal
effect (Kwang-Geun and Takayuki, 2001; Dogan
et al., 2004; Lev, 2006; Ayoola et al., 2008), as a
natural dye sources (Dogan et al., 2003) and etc.
Relatively less folk remedies contain onion (3) and
garlic (2), which give sweet sour taste and strong
specific flavor. This seems a bit strange if we bear
in mind the significant role of these two plants in
the diet of Bulgarians and their traditional use in
the folk medicine. Peppermint is also not among
the preferred flavors and ingredients. From the
representatives of this genus only mint (2) is
mentioned here, which is a widely used spice in
the Bulgarian culinary traditions.
Some researches show that the links between
the taste perceptions and the medicinal uses of
herbal drugs may be understood as bio-cultural
phenomena rooted in the human physiology, and
dependent on the individual experiences and
culture (Pieroni and Torry, 2007). The established
species that determine the taste of the medicines
and are noted have been registered and
investigated by the same authors. On the other
hand, these results show that the relationships
based on a source, referring to a particular
historical period should be considered in its light.
In the studied documentary source, traces of
mysticism and superstition, which are an integral
part of the folk medicine in the western countries
(Europe) from this period, are not present. The
made of amulets is not mentioned. As far as any
visible impact here, it is from Arab and

14
7
1
15
1
35
17
16
Imported plants
Spices
Wild
Religious
Cultivated Bg
Ornamental
Food
Bevarage Beverage
Figure 6. The plant biodiversity in folk remedies according to their general usage, distribution
and economical importance.
Byzantine medicine. This confirms the opinion of many
authors for the original and rational nature of the folk
remedies from this period.
Conclusion
This study demonstrates how the old written sources can
be used to collect information for: new medicinal plants
and traditional folk remedies, historical information about
the level of trade contacts and some socio-cultural
processes in the society. The obtained data are analyzed
by taking into account the historical fact regarding the
period of the application of the treatment and the time of
the creation and the printing of the text. Reading such a
source is slow and requires multi-layered knowledge -
botany, history and linguistics. The correct interpretation
of the text requires knowledge of the relevant literature of
the period such as “Lives of St. Ivan Rilski”, herbal books,
sources about trade contacts and plant cultivation,
linguistic literature, language dictionaries (Turkish,
Russian, and Serbian), etc.
This study contributes to the ethnobotanical studies in
Bulgaria and on the Balkans, and presents data derived
from a written source. The book “Cannon…” was first
being studied to enlarge the knowledge of species used
in the traditional medicine in the investigated period.
The wide variety of folk remedies including mainly
plants, mineral elements and other organic and inorganic
Nedelcheva 2337
ingredients found by the study is consistent with the
results of other authors about the rich traditional
knowledge and practice in relation to medicinal plants
and folk medicine. The use of many wild species of the
Bulgarian flora in the folk remedies is well known by all
previous studies.
The significant participation of spices such as clove,
cinnamon, mastic and ginger in folk remedies sheds new
light on the list of species that are traditionally used in the
folk medicine. The importance of these species, together
with the presence of many organic and inorganic
compounds, showed greater significance than previously
suspected. The traditional use of medicinal plants is more
precise and more oriented to the “East plants“than it was
believed until now.
Last but not the least, the ethnobotanical study on this
book and the presented results support the thesis that it
was founded on authentic recipes from the healing
activity of St. Ivan Rilski, which has increased its
historical value a lot. Some data that can be used as a
proof for the link between the text and the knowledge of
St. Ivan Rilski (in hagiography) is as follows:
C. arietinum is the only plant that has been mentioned
in various “Saint‟s Lives” as one used by the hermit. The
same plant is a very common ingredient in recipes and is
also used as a standard measure for the preparation of
pills.
Both species R. palmatum and R. raponticum (localized
in Rila Mt.) are included in present recipes. The folk use

2338 J. Med. Plants Res.
of the second one is related to the healing experience of
the Rila Monastery monks. St. Ivan Rilski is the founder
of Rila Monastery.
St. Ivan Rilski is well known as a nerve disorders‟
healer - a fact that corresponds to the large number of
remedies in text, aimed at curing this illness.
The ethnobotanical data is in accordance with the
modern hagiographic concept that St. Ivan Rilski is a
highly educated person (Pulos, 1992), which contradicts
to traditional beliefs.
ACKNOWLEDGEMENT
The author would like to say thanks to Dr. Stefan
Draganov (Sofia University St. Kliment Ohridski”, Sofia,
Bulgaria) and Dr. Yunus Dogan (Dokuz Eylul University,
Izmir, Turkey) for help in plant folk names identification,
their useful recommendations, for critical reading and
correction of the manuscript. The author would like to
express his sincere gratitude to Dr. Stefan Draganov and
his family who provided original lithographic print “St. Ivan
Rilski and his Monastery” (1866) for this study.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2340-2347, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.843
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Chemical composition, antioxidant activity and toxicity
evaluation of essential oil of Tulbaghia violacea Harv.
O. S Olorunnisola, G. Bradley and A. J Afolayan*
School of Biological Sciences, University of Fort Hare, Alice 5700, South Africa.
Accepted 1 November, 2011
Essential oil from the rhizomes of Tulbaghia violacea of the family Alliaceae was obtained by hydrodistillation
using an all-glass Clevenger-type apparatus. In vitro antioxidant activities of the oil at
various concentrations were assessed using 2, 2-diphenyl-1-picryl hydrazyl (DPPH), nitric oxide
scavenging, reducing power and lipid peroxidation inhibition assay. The results were compared with
butylated hydroxytoluene (BHT) and ascorbic acid. Brine shrimp lethality test was used to determine
cytotoxicity of the oil. Gas chromatography/mass spectrophotometer (GC/MS) analyses of oil revealed 7
polysulfides with a pungent garlic-like odor. The principal constituents were dimethy disulfide, dimethy
trisulfide, (methyl methylthio) methyl, 2,4-dithiapentane (11.35%) and (methylthio) acetic acid, 2-
(methylthio) ethanol, 3-(methylthio)- and propanenitrile (7.20%). The essential oil demonstrated
moderate radical scavenging activities. Although, their EC50 value was lower than those of the BHT and
ascorbic acid, the value was close to those reported for other Alliaceae family. The LC50 value of 12.59
µg/ml obtained showed that the essential oil of T. violacea was toxic. The implication of the toxicity of
the oil is discussed.
Key words: Tulbaghia violacea, antioxidant, essential oils, gas chromatography/ gas chromatography-mass
spectrophotometer (GC/GC-MS), toxicity.
INTRODUCTION
Essential oils are made up of different volatile
compounds and aromatic oily liquid obtained from
different plant parts (Amal et al., 2010). The composition
of essential oils often varies between species (Mishra
and Dubey 1994) and it determines the organoleptic
properties and biological activity of the oil (Misharina et
al., 2009). The study of individual components of different
essential oils has shown that many terpenes containing
oils possess antiradical and antioxidant activity
(Misharina et al., 2009). When essential oil is isolated
from plants, they are not usually extracted as chemically
pure substances, but as mixtures of many compounds,
like monoterpenes and sesquiterpines which are mainly
hydrocarbon (Amal et al., 2010). Essential oils and
extracts have been used for thousands of years, especially
in food preservation, pharmaceuticals, alternative
*Corresponding author. E-mail: aafolayan@ufh.ac.za. Fax:
+27866282295.
medicine and natural therapies (Imelouane at al., 2009).
It has been well established that some plant essential oils
exhibit antimicrobial properties against bacterial pathogens
(Koba et al., 2009; Prabuseenivasan et al., 2006).
Many plants have been employed in the manage-ment
and treatment of diseases. One of such plants is
Tulbaghia violacea Harv. It was recently reported that the
extract of rhizomes of T. violacea is commonly employed
in the management of heart diseases in Nkonkobi
municipality Eastern Cape of South Africa (Olorunnisola
et al., 2011).
T. violacea is commonly known as wild garlic, wilde
knoffel (Afrikaans), isihaqa (Zulu) or itswele lomlambo
(Xhosa). It is indigenous to the Eastern Cape region of
South Africa and is widely used as an herbal remedy for
various ailments. Its leaves and bulbs are the most
commonly used. T. violacea has been reported to have
various medicinal applications which include treatment for
fever and colds, asthma, tuberculosis, stomach problems
and oesophageal cancer (Bungu et al., 2008).
The plant is also used as a snake repellent (Hutchings

et al., 1996; van Wyk and Gericke, 2000). In spite of the
traditional medicinal claims on the use of T. violacea,
there is dearth of information on the pharmacological
activity its essential oil. This study was designed to
determine the chemical constituents of the essential oil
from the rhizomes of T. violacea using hydro- distillation
techniques and also to assess it antioxidant activities and
toxicity using brine shrimp lethality test which is a bench
top bioassay for elementary cytotoxicity study.
MATERIALS AND METHODS
Plant materials
Fresh rhizome of T. violacea was collected in April from Alice,
Eastern Cape, South Africa, and authenticated by Prof. D.S
Grierson of Botany Department, University of Fort Hare. Voucher
specimen (Sin 2010/2) was deposited at the Giffen Herbarium.
Extraction of essential oils
Rhizomes were hydro-distilled for 3 h in a Clevenger-type
apparatus in accordance with the British pharmacopoeia
specifications (1980). The essential oil was collected and analyzed
immediately.
GC-MS analyses of the oil
GC-MS analyses of the oil was carried out using Hewlett-Packard
HP 5973 mass spectrometer interfaced with an HP-6890 gas
chromatograph with an HP5 column. The following conditions were
used: Initial temperature 70%, maximum temperature 325°C,
equilibration time 3 min, ramp 4°C/min, final temperature 240°C;
inlet: split less, initial temperature 220°C, pressure 8.27 psi, purge
flow 30 ml/min, purge time 0.02 min, gas type helium; column:
capillary, 30 m × 0.25 mm i.d., film thickness 0.25 µm, initial flow
0.7 ml/min, average velocity 32 cm/s; MS: EI method at 70 eV.
Identification of components
The individual constituents of the oil were identified by matching
their mass spectra and retention indices with those of Wiley 275
library (Wiley, New York) in computer library (Kovats, 1958; Adams,
1995; Joulain et al., 2001; Joulain and Konig, 1998). The yield of
each component was calculated per g of the plant material, while
the composition was calculated from the summation of the peak
areas of the total oil composition. The whole experiment was
replicated thrice.
Antioxidant activity
2, 2-diphenyl-1-picryl hydrazyl (DPPH) radical scavenging
activity
The method of Liyana-Pathiranan and Shahidi (2005) was used for
the determination of scavenging activity of free radical in the
essential oil. A solution of 0.135 mM 2, 2-diphenyl-1-picrylhydrazyl
radical (DPPH) in methanol was prepared. 1.0 ml of this solution
was mixed with 1.0 ml of oil prepared in methanol containing 0.1 to
0.5 mg/ml of the oil and standard drugs (BHT and ascorbic acid).
The reaction mixture was vortexed thoroughly and left in the dark at
room temperature for 30 min. The absorption of the mixture was
Olorunnisola et al. 2341
measured spectrophotometrically at 517 nm. The actual decrease
in absorption was measured against that of the control. All test and
analysis were run in triplicates and the results obtained were
averaged. The activities were also determined as a function of their
% Inhibition which was calculated using the formula;
% scavenging activity = [Ac - As / Ac] × 100
Ac = Absorbance of the control; As = Absorbance of the sample.
Nitric oxide-scavenging activity
Nitric oxide scavenging activity was measured
spectrophotometrically (Govindarajan et al., 2003). Sodium
nitroprusside (5 mM) in phosphate buffered saline was mixed with
different concentrations of the extract (100 to 500 μg/ml) was
prepared in methanol and incubated at 25°C for 30 min. A control
without the test compound but with an equivalent amount of
methanol was taken. After 30 min, 1.5 ml of the incubated solution
was removed and diluted with 1.5 ml of Griess reagent (1%
sulphanilamide, 2% phosphoric acid and 0.1% N-1-
naphthylethylenediamine dihydrochloride). The absorbance of the
chromophore formed during diazotization of the nitrite with
sulphanilamide and subsequent coupling with N-1-
naphthylethylene diamine dihydrochloride was measured at 546 nm
and percentage scavenging activity was measured with reference
to standard. BHT vitamin C was used as a positive control.
Lipid peroxidation and thiobarbituric acid reactions
A modified thiobarbituric acid reactive species (TBARS) assay
(Ohkowa et al., 1979) was used to measure the lipid peroxide
formed using egg yolk homogenate as lipid rich media (Ruberto et
al., 2000). Egg homogenate (0.5 ml of 10%, v/v) and 0.1 ml of the
oil extract were added to a test tube and made up to 1 ml with
distilled water; 0.05 ml of FeSO4 (0.07 M) was added to induce lipid
peroxidation and the mixture incubated for 30 min. Then, 1.5 ml of
20% acetic acid (pH 3.5) and 1.5 ml of 0.8% (w/v) thiobarbituric acid
in 1.1% sodium dodecyl sulphate were added and the resulting
mixture was vortexed and then heated at 95°C for 60 min. After
cooling, 5.0 ml of butan-1-ol were added to each tube and
centrifuged at 3000 rpm for 10 min. The absorbance of the organic
upper layer was measured at 532 nm. Inhibition of lipid peroxidation
percent by the oil extract was calculated as:
[(1-E)/C] × 100
Where C is the absorbance value of the fully oxidized control;
E is the absorbance in presence of extract.
Reducing power of the extract
The reducing power of the oil was determined according to the
method of Yen and Chen (Ebrahimzadeh et al., 2008a; Nabavi et
al., 2008a). 2.5 ml of extract (100 to 500 µg/ml) in water were mixed
with a phosphate buffer (2.5 ml, 0.2 M, pH 6.6) and potassium
ferricyanide [K3 Fe (CN)6] (2.5 ml, 1%). The mixture was incubated
at 50°C for 20 min. A portion (2.5 ml) of trichloroacetic acid (10%)
was added to the mixture to stop the reaction, which was then
centrifuged at 3000 rpm for 10 min. The upper layer of the solution
(2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3 (0.5 ml,
0.1%), and the absorbance was measured at 700 nm. Increased
absorbance of the reaction mixture indicated increased in reducing
power. Vitamin C was used as a positive control.

2342 J. Med. Plants Res.
Table 1. Compounds obtained from GC/GC-MS analysis of T. violacea rhizome essential oil.
S/N Retention time (min) Chemical composition % Area
1 21.94 Dimethy trisulfide, 0.57
2 26.77 Dimethy disulfide, methyl (methylthio) meth 2,4-dithiapentane 11.35
3 33.92 (Methylthio) acetic acid 2.58
4 38.00 (Methylthio) acetic acid, 2-(methylthiol) ethanol, propanitrile, 3-(methylthio)- 7.20
5 44.99 2,4-dithiapntane,bis-(methlythio), disulfide 0.78
Total (%) 22.48
Brine shrimp lethality test
Shrimp eggs were allowed to hatch and mature as nauplii in two
days in a hatching tank filled with seawater. The free-swimming
nauplii were attracted by a light to a compartment from which they
could be collected for the assay proper. Vials containing 2.5 to 20
µg ml -1 samples were prepared by dissolving the oils in dimethyl
sulfoxide (DMSO) and transferring the solution to each vial. The
solvent was evaporated at room temperature and seawater was
added to achieve the correct concentration. 15 shrimps were added
to three vials for each dose via a disposable pipette. The number of
deaths out of 15 shrimps per dose was recorded after 24 h and
LC50 values obtained from the best-fit line slope. The control
solution consisted of 15 nauplii in the artificial seawater. For
acceptable readings; the LC50 for the toxicant should fall within 27
to 35 µg ml -1 range (Sam et al., 1986).
Statistical analysis
Statistical analysis was carried out with Statistical Package for
Social Sciences (SPSS). The data was expressed as the mean ±
standard deviation and a probability of less than 0.05 (p

DPPH inhibition (%)
DPPH % inhibition
120
100
80
60
40
20
0
100 200 300 400 500
Concentration (µg/ml)
Figure 1. Scavenging effects of oil extracts from rhizomes of T. violacea, vitamin C and BHT on DPPH.
Petroselinum sativum (Parsley) herb oil (EC50 = 82.1
µg/ml), Cuminum cyminum (Cumin) oil (EC50 = 81.5
µg/ml) and Allium cepa L. (Onion) oil (EC50 = 80.0 µg/ml)
at the same concentrations (Shalaby et al., 2011).
However, the oil extract of rhizome of T. violacea
demonstrate lower EC50 value when compared with
vitamin C (EC50 = 52.0 µg/ml) and BHT (EC50 = 67.0
µg/ml) reference drugs. These values revealed that the
antioxidant activity of T. violacea rhizome oil was still less
active than butylated hydroxytoluene (BHT) and vitamin
C. It was observed that the antioxidant activity of the oil
extract is higher than for their individual components and
this might be as a result of synergetic effects of multi
component of oil (Misharina et al., 2009).
Nitric oxide scavenging activity
Nitric oxide has been implicated in inflammatory and
pathogenesis of various human diseases such as cancer
and cardiovascular diseases, Hence, nitric oxide
scavenging capacity of extracts may help to arrest the
chain of reactions initiated by excess generation of nitric
oxide (NO) that are detrimental to the human health
(Raushanar et al., 2009). In this study, we demonstrate
that the oil extract significantly inhibited NO production
from sodium nitroprusside in an aqueous solution at
physiological pH and reacts with oxygen in the reaction to
form nitrite. The extracts inhibit nitrite formation by
directly competing with oxygen in the reaction with nitric
oxide and other nitrogen oxides such as NO3, and N2O3
Olorunnisola et al. 2343
Oil oil Oil extracts
extract
% Inhibition inhibition of Vitamin vitamic vitamin C C (%)
% Inhibition inhibition of of BHT BHT (%)
(Osamuyimen et al., 2011). The extract showed strong
concentration dependant inhibitory activities with highest
percentage inhibition of nitric oxide at 0.5 mg/ml (Figure
2). Though, the oil extract demonstrated a significant
inhibitory activity against nitric oxide radical, its 50%
effective inhibition concentration (EC50 = 180 µg/ml) was
comparably lower than what was obtained for garlic oil
(IC50 = 50 µg/ml) (Reena and Kapil, 2011) and the
reference drugs BHT (EC = 115 µg/ml) and ascorbic acid
(EC = 134 µg/ml) (Table 2). The nitric oxide inhibiting
ability of the oil extract could support the use of the plant
in the treatment of oxidative induce ailments such as
cardiovascular diseases.
Lipid peroxidation
Reacting oxygen species are known to cross react with
lipid constituents of the cell membranes causing changes
in fluidity and permeability (Nigam and Schewe, 2000),
DNA mutation (Russo et al., 2001) and lipid peroxidation.
The effect of these delirious reactions is the cause of
most human diseases. The generation of lipid peroxidase
in egg yolk lipids when incubated in the presence of
ferrous sulphate with subsequent formation of
malonodialdehyde (MDA) and other aldehydes that form
pink chromogen (Kosugi et al., 1987) was strongly
inhibited in concentration dependant manner (Figure 3)
by oil extract of T. violacea with EC50 value of 192.3
μg/ml (Table 2). This value was much lower than what
was reported for methanol extract of Mucuna pruriens

2344 J. Med. Plants Res.
0 200 400 600
Concentration (µg/ml)
T. violacea oil
BHT
Vitamin C
Figure 2. Scavenging effects of oil extracts from rhizomes of T. violacea, vitamin C
and BHT on nitric oxide radical.
Table 2. Comparison of 50% effective inhibitory concentration for nitric oxide and lipid peroxidation of oil extract
of rhizomes of T. violacea.
Extract Nitric oxide inhibition EC50 (µg/ml) LP inhibition EC50 (µg/ml)
OTV 180.0 192.3
BHT 115.0 138
AA 134.0 100
OTV represent oil extract of Tulbaghia violacea. BHT represents, AA represent ascorbic acid. LP inhibition EC50
represents concentration of oil extract for 50% inhibition of lipid peroxidation.
Inhibition of lipid peroxidation (%)
Inhibition of nitric oxide radical (%)
T. violacea oil
Vitamin C
Figure 3. Scavenging effects of oil extracts from rhizomes of T. violacea, vitamin C and
BHT on lipid peroxidation.
Concentration (µg/ml)
(Yerra et al., 2005) however, the value is higher when results of the investigations revealed that oil extract of
compared with the standard (BHT 0 and AA) (Table 200 2). The 400 rhizome of 600 T. violacea had potent lipid peroxidation
BHT

% Mortality
Concentration (µg/ml)
T. violacea oil
BHT
Vitamin C
Figure 4. Ferric reducing power of oil extracts from rhizomes of T. violacea,
vitamin C and BHT.
120
100
80
60
40
20
0
-20
y = 5.681x - 18.29
R² = 0.968
0 0 5 200 10 15400 20 600 25
Log Conc. (µg/ml)
% Mortality
Linear (% Mortality)
Figure 5. Determination of LC50 of essential oil of rhizome of T. violaceae against
brine shrimps nauplii.
inhibition activity. Although, phytochemical evaluation
was not assessed in this study, other group of workers
who has work on other members of Alliaceae species
(onion and garlic) has reported that the observed
inhibition of lipid peroxidation and radical scavenging
activities might be due to the phenolic contents (Nuutila
et al., 2003).
Reducing power of the extract
Absorbance (700 nm)
Mortality (%)
The antioxidant potentials of the oil extract was estimated
from their ability to reduce Fe 3+ to Fe 2+ . This was
observed from yellow colour of the test solution that
changed to various shades of green and blue depending
on the concentration of the plant extracts. The reducing
power of the oil of T. violacea and the reference compounds
increased with increasing concentration (Figure
4). In addition, the reducing value of the oil extract was
significantly lower than that of BHT and ascorbic acids,
used as reference compounds in this study (Figure 4). As
the concentration of the oil extract increases, the reducing
Olorunnisola et al. 2345
power assay absorbance also increased. This observation
follow similar trend reported for garlic oil extracts
(Reena and Kapil, 2011). The reducing properties of the
oil might be due to the presence of reductones (Saha et
al., 2008). From the previous results and discussion it
can be concluded that the oil extract of rhizome of T.
violacea possesses the potent antioxidant substances
which may be responsible for its anti-inflammatory and
chemoprotective mechanism as well as justify the basis
of using this plant’s extract as folkloric remedies.
Cytotoxic activity in brine shrimps bioassay
Brine shrimp lethality assay is frequently used as model
system to measure cytotoxic effects of variety of toxic
substances and plant extracts against brine shrimps
nauplii (Morshed et al., 2011). The method provides a
simple and inexpensive screening test for cytotoxic compounds
and possesses the advantages of requiring only
small amounts (0.6 mg) of compounds for investigation.
The LC50 (12. 59 µg/ml) value (Figure 5) obtained in

2346 J. Med. Plants Res.
Table 3. Brime shrimp lethality test of the essential oil of T. violacea rhizome.
Concentration
(µg/ml)
Essential oil of T. violacea rhizome
Average no. of survivors Average no. of dead Mortality (%)
2.5 15.0 ±0.00 15.0 ± 0.00 0
5.0 13.0 ± 0.00 2.0 ± 0.00 13.3
10.0 11.0 ± 0.00 4.0.0 ± 0.00 26.6
20 0 0 100
Control 15 0 0
LC50 = 12.59 µg/ml
Data were expressed as mean ± SD.
these studies showed that the oil extract of rhizome of T.
violacea was cytotoxic and this toxicity is concentration
dependant (Table 3). It was observed that all the nauplii
survive at the lowest concentration (2.5 µg/ml). This
significant lethality of the oil extracts(LC50values less than
100 µg/ml) against brine shrimps nauplii might be due to
the presence of polysulfides which has be implicated as
cytotoxic agents with potential anticancer, antimicrobial
and antifungal activities (Münchberg et al., 2007; Anwar
et al., 2008).
The present study indicates that the essential oil of
rhizome of T. violacea exhibit interesting biological
activities such as antioxidant and cytototoxic effect and
may serve as alternative natural source of anticancer,
and antibiotic and antimicrobial agents.
ACKNOWLEDGEMENT
This study was supported with a grant from the National
Research Foundation of South Africa.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2348-2364, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.890
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Optimum extraction conditions for arbutin from Asian
pear peel by supercritical fluid extraction (SFE) using
Box-Behnken design
Byung-Doo Lee and Jong-Bang Eun*
Department of Food Science and Technology and and Functional Food Research Center Chonnam National University,
Gwangju, 500-757, South Korea.
Accepted 28 October, 2011
Asian pear (Pyrus pyrifolia cv. Niitaka) peel, a by-product that results from juice processing, is a good
source of arbutin, a polyphenol, which has a whitening effect on the skin. The objective of this study
was to investigate the optimum extraction conditions for arbutin from Asian pear peel by supercritical
fluid extraction (SFE) using response surface methodology (RSM) based on Box-Behnken experiment
design. Arbutin was extracted using co-solvent, methanol and ethanol. A three-level four-factor Box-
Behnken experiment design was performed to evaluate the combination effect of four independent
variances, co-solvent concentration (22 to 30%), extraction pressure (250 to 300 bar), extraction
temperature (30 to 60°C) and extraction time (30 to 60 min), coded for X1, X2, X3 and X4, respectively. The
coefficients of determination (R 2 ) of response surface regression equations were 0.89 (p

Table 1. Levels of independent variables for Box-Behnken experiment design in extraction of arbutin.
Independent variable
Levels
-1 0 1
Co-solvent concentration (%, X1) 22 26 30
Extraction pressure (bar, X2) 250 275 300
Extraction Temp (°C, X3) 32 45 60
Extraction Time (min, X4) 90 120 150
and Fukuda, 1991; Maeda and Fukuda, 1996) and as the
species that accumulate it survive extreme environmental
stresses, such as frost and drought, arbutin may
contribute to their stress hardiness.
The physiological role of arbutin in resurrection plants
is unknown, but it is believed that arbutin contributes to
the protection of membrane components in the dry state
(Escarpa and Gonzalez, 1999), as it has been shown to
be an antioxidant (Couteau and Coiffard, 2001) and also
to inhibit phospholipase A2 (PLA2) activity in mostly
dehydrated systems (Masse, 2001). Arbutin is a solute
accumulated to high concentration in drought and frost
resistant plant. This hydroquinone derivative composed
by glucose and a phenol moiety is isolated from the
leaves of the bearberry shrub, cranberry, blueberry and
most types of pears (Frias et al., 2006). Arbutin is a skin
care products and as a whitening agent, it can compete
with L-DOPA for receptor site on tyrosinase and hinders
the oxidation of L-DOPA, thrums can inhibit the formation
of eumelanin (Lin et al., 2007).
Unfortunately, few studies deal with method
development and validation using statistical designs and
response surface techniques to determine the optimum
operational conditions for the hydrolysis. The
conventional approach for the optimization of a
multivariable system is usually one variable at a time.
This can be very time-consuming and when interactions
exist between the variables, it is unlikely to find the true
optimum. RSM is a very useful tool for this purpose as it
provides statistical models that help in understanding the
interactions among the parameters that should be
optimized.
This aim of this study was to investigate the optimum
extraction conditions for arbutin from Asian pear peel by
SFE using RSM based on Box-Behnken experimental
design.
MATERIALS AND METHODS
Asian pear cultivars (P. pyrifolia cv. Niitaka), is grown in private
orchard in Naju city of South Korea was used for this study. In 2007
harvest season, the fruits were harvested carefully by hand at their
commercial maturity stage and transferred to the laboratory.
Supercritical fluid extraction (SFE) condition
Extractions were carried out for SFE (Insong) with a 100 g
Lee and Eun 2349
extraction cell. The extraction pressure was controlled by micro
metering valves and the carbon dioxide pump was from Bran-
Luebbe (Norderstedt, Germany). Fractionation was achieved in two
different vessels, with independent temperature and pressure
control, by a decrease in pressure.
The extraction cell was filled up with 60 g of ground laurel and 90
g of washed sea sand (Panreac, Barcelona, Spain). Dynamic
extraction was performed at the following experimental conditions:
extraction pressure, 250 bars; extraction temperature, 60°C; 4% of
ethanol as modifier; pressure of separator 1, 100 bar; temperature
of separator 1, 60°C; pressure of separator 2, 20 bar and
temperature of separator 2, 20°C. Extraction time was 75 min and
the addition of ethanol started when selected pressure was reached.
All extracts were kept under N2, at 20°C in the dark and ethanol
was eliminated at 35°C in a vacuum rotary evaporator.
Experimental design
Optimization of conditions for arbuitn from Asian pear was carried
out using RSM. Experiments with four independent variables, cosolvent
concentration (X1), extraction pressure (X2), extraction
temperature (X3) and extraction time (X4) were conducted following
the experimental design statistical analysis obtained by the Box–
Behnken experimental design.
This design was selected due to the small number of
experiments required to estimate complex response functions. For
the three-level four factorial Box–Behnken experimental were
design, a total of 27 experimental that were runs are necessary.
The uncoded and coded independent variables and experimental
design are listed in Tables 1 and 2.
Determination of arbutin
The arbutin (4-hydroxyphenyl-â-D-glucopyranoside) contents of
samples were determined by HPLC. The column used was waters
spherisorb ODS2 (25.0 × 0.46 cm, 5 µm). The mobile phase was
water/formic acid (19:1, v/v) and methanol at a flow rate of 0.9
mL/min. Eluates were detected at 280 nm (UV-975, Jasco, Japan).
Statistical analysis
The experimental data (Table 1) were analyzed by response
surface regression (RSREG) procedures using SAS software to fit
the following second-order polynomial Equation (1):
Where Y is the response (percent of molar conversion); b0 is a
constant, bi, bii and bij are coefficients; xi and xj are the uncoded
(1)

2350 J. Med. Plants Res.
Table 2. Box-Behnken experiment design setting in the original and coded form of the independent variables (X1, X2, X3 and X4).
No X1 X2 X3 X4
Co-solvent
concentration (%)
Extraction pressure
(bar)
Extraction temp
(℃)
Extraction time
(min)
1 -1 -1 0 0 22 250 45 120
2 0 -1 -1 0 26 250 32 120
3 0 -1 0 -1 26 250 45 90
4 0 -1 0 1 26 250 45 150
5 0 -1 1 0 26 250 60 120
6 1 -1 0 0 30 250 45 120
7 -1 0 -1 0 22 275 32 120
8 -1 0 0 -1 22 275 45 90
9 -1 0 0 1 22 275 45 150
10 -1 0 1 0 22 275 60 120
11 0 0 -1 -1 26 275 32 90
12 0 0 -1 1 26 275 32 150
13 0 0 0 0 26 275 45 120
14 0 0 0 0 26 275 45 120
15 0 0 0 0 26 275 45 120
16 0 0 1 -1 26 275 60 90
17 0 0 1 1 26 275 60 150
18 1 0 -1 0 30 275 32 120
19 1 0 0 -1 30 275 45 90
20 1 0 0 1 30 275 45 150
21 1 0 1 0 30 275 60 120
22 -1 1 0 0 22 300 45 120
23 0 1 -1 0 26 300 32 120
24 0 1 0 -1 26 300 45 90
25 0 1 0 1 26 300 45 150
26 0 1 1 0 26 300 60 120
27 1 1 0 0 30 300 45 120
independent variables. The options of RSREG SAS and RIDGE
MAX were employed to compute the estimated ridge of maximum
response for increasing radii from the center of the original design.
RESULTS AND DISCUSSION
Optimization of extraction conditions using methanol
Figure 1 shows the three dimensional plots of the effect
of the independent variables co-solvent concentration
and extraction pressure on the arbutin content with SFE
extraction. Response surface for the effect of extraction
temperature and co-solvent concentration on arbutin
content of arbutin extracted from Asian pear peel (Figure
2). Three dimensional plots of the effect of the
independent variables extraction time and co-solvent
concentration on the arbutin content with SFE extraction
(Figure 3). Figure 4 shows the three dimensional plots of
the effect of the independent variables of extraction
temperature and extraction pressure on the arbutin
content with SFE extraction.
Response surface for the effect of extraction time and
extraction pressure on arbutin content of arbutin
extracted from Asian pear peel (Figure 5). Three
dimensional plots of the effect of the independent
variables extraction time and extraction temperature on
the arbutin content with SFE extraction (Figure 6). In
order to obtain a model for CAPE synthesis, the results
from the 3-level-4-factor Box-Behnken design (Table 3)
were used and the RSREG procedure from SAS was
employed to fit the second-order polynomial Equations 1
and 2 was thus generated and is given as:
Y MeOH = 31.695451 + 0.442257X1 + 0.185411X2 + 0.105694X3 + 0.061407X4 - 0.007786X1 2 + 0.000083X2X1 -
0.000343X2 2 - 0.000764X3X1 - 0.000044X3X2 - 0.000815X3 2 - 0.000528X4X1 + 0.000067X4X2 - 0.000026X4X3 -
0.000717X4 2 (R 2 =0.89) (2)
Analysis of variance indicates that this second-order
polynomial model was highly significant and adequate to
represent the actual relationship between the response
(percent molar conversion) and the variables. The p-value

Arbutin (mg/g)
Co-solvent
Figure 1. Response surface for the effect of extraction pressure and co-solvent concentration on arbutin content of arbutin extracted from Asian pear peel.
Lee and Eun 2351

2352 J. Med. Plants Res.
Arbutin (mg/g)
Co-solvent
Figure 2. Response surface for the effect of extraction temperature and co-solvent concentration on arbutin content of arbutin extracted from Asian pear
peel.

Arbutin (mg/g)
Co-solvent
Figure 3. Response surface for the effect of extraction time and co-solvent concentration on arbutin content of arbutin extracted from Asian pear peel.
Lee and Eun 2353

2354 J. Med. Plants Res.
Arbutin (mg/g)
Extraction pressure (bar)
Figure 4. Response surface for the effect of extraction temperature and extraction pressure on arbutin content of arbutin extracted from Asian pear
peel.

Arbutin (mg/g)
Extraction pressure (bar)
Figure 5. Response surface for the effect of extraction time and extraction pressure on arbutin content of arbutin extracted from Asian pear peel.
Lee and Eun 2355

2356 J. Med. Plants Res.
Arbutin (mg/g)
Extraction temperature (°C)
Figure 6. Response surface for the effect of extraction time and extraction temperature on arbutin content of arbutin extracted from Asian pear peel.

Table 3. Arbuitn content of Asian pear peel after extracting with supercritical fluid (unit: mg/g).
No
Co-solvent
concentration (%)
Extraction pressure
(bar)
Extraction temp
(℃)
Extraction time
(min)
Lee and Eun 2357
Arbutin content (mg/g)
MeOH EtOH
1 22 250 45 120 3.22 ±0.01 2.45 ±0.01
2 26 250 32 120 3.18 ±0.02 2.53 ±0.02
3 26 250 45 90 3.28 ±0.02 2.45 ±0.01
4 26 250 45 150 3.27 ±0.03 2.57 ±0.01
5 26 250 60 120 3.18 ±0.02 2.46 ±0.01
6 30 250 45 120 3.34 ±0.03 2.56 ±0.03
7 22 275 32 120 3.13 ±0.02 2.44 ±0.01
8 22 275 45 90 3.27 ±0.03 2.56 ±0.01
9 22 275 45 150 3.35 ±0.02 2.54 ±0.03
10 22 275 60 120 3.35 ±0.02 2.54 ±0.03
11 26 275 32 90 3.35 ±0.02 2.54 ±0.03
12 26 275 32 150 3.28 ±0.01 2.56 ±0.03
13 26 275 45 120 3.16 ±0.01 2.54 ±0.02
14 26 275 45 120 3.13 ±0.02 2.52 ±0.02
15 26 275 45 120 3.10 ±0.01 2.54 ±0.03
16 26 275 60 90 3.28 ±0.03 2.45 ±0.01
17 26 275 60 150 3.18 ±0.02 2.55 ±0.04
18 30 275 32 120 2.31 ±0.02 1.76 ±0.02
19 30 275 45 90 2.44 ±0.03 1.94 ±0.03
20 30 275 45 150 2.58 ±0.02 2.10 ±0.02
21 30 275 60 120 2.64 ±0.03 2.13 ±0.03
22 22 300 45 120 2.59 ±0.01 2.13 ±0.01
23 26 300 32 120 2.58 ±0.02 2.11 ±0.02
24 26 300 45 90 2.48 ±0.02 2.04 ±0.02
25 26 300 45 150 2.46 ±0.01 2.10 ±0.01
26 26 300 60 120 2.45 ±0.01 2.04 ±0.04
27 30 300 45 120 2.52 ±0.02 2.03 ±0.02
was

2358 J. Med. Plants Res.
Arbutin (mg/g)
Co-solvent
Figure 7. Response surface for the effect of extraction pressure and co-solvent concentration on arbutin content of arbutin extracted from Asian pear peel.

Arbutin (mg/g)
Co-solvent
Figure 8. Response surface for the effect of extraction temperature and co-solvent concentration on arbutin content of arbutin extracted from Asian pear peel.
Lee and Eun 2359

2360 J. Med. Plants Res.
Arbutin (mg/g)
Co-solvent
Figure 9. Response surface for the effect of extraction time and co-solvent concentration on arbutin content of arbutin extracted from Asian pear peel.

Arbutin (mg/g)
Extraction pressure (atm)
Figure 10. Response surface for the effect of extraction temperature and extraction pressure on arbutin content of arbutin extracted from Asian pear peel.
Lee and Eun 2361

2362 J. Med. Plants Res.
Arbutin (mg/g)
Extraction pressure (atm)
Figure 11. Response surface for the effect of extraction time and extraction pressure on arbutin content of arbutin extracted from Asian pear peel.

Arbutin (mg/g)
Extraction temperature (°C)
Figure 12. Response surface for the effect of extraction time and extraction temperature on arbutin content of arbutin extracted from Asian pear peel.
Lee and Eun 2363

2364 J. Med. Plants Res.
was

Journal of Medicinal Plants Research Vol. 6(12), pp. 2365-2372, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.972
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
A comparison of volatile components of flower, leaf and
peel of Citrus reticulata Blanco (Citrus nobilis Lour var.
deliciosa swingle)
Behzad Babazadeh Darjazi
Department of Plant Production, Faculty of Agriculture, Roudehen Branch, Islamic Azad University (I. A. U),
Roudehen, Iran. E-mail: babazadeh@riau.ac.ir. Tel: +98 2133009743.
Accepted 30 November, 2011
The volatile flavor components of flower, leaf and peel of Citrus nobilis Lour var. deliciosa swingle were
investigated in this study. Flower, leaf and peel flavor components were extracted using water
distillation method and then eluted using n-hexane solvent. Then they were all analyzed by Gas
chromatography- flame ionization detector (GC-FID) and Gas chromatography–mass spectrometry (GC-
MS). 39 flower components, 39 leaf components and 25 peel components including: aldehydes,
alcohols, esters, ketons, monoterpenes and sesquiterpenes were identified and quantified. The major
flavor components were linalool, limonene, sabinene, a-pinene, β-myrcene, terpinene-4-0l, (E)-βocimene
and γ-terpinene. The flower oil showed the highest content of aldehydes and alcohols. Since
the aldehyde content of citrus oil is considered as one of the most important indicators of high quality,
organ apparently has a profound influence on citrus oil quality.
Key words: Local tangerine, Citrus reticulata Blanco, tangerine flower oil, tangerine leaf oil, tangerine pee oil,
flavor components, water-distillation.
INTRODUCTION
Citrus nobilis Lour var. deliciosa Swingle is a local
tangerine and is widely planted on a large scale in Iran. It
is probably a native of southern China, and is now found
in all warm countries. Its fruit is characterized by a loose
skin and pleasant flavor (Asgarpanah, 2002). In Citrus L.
species essential oils occur in special oil glands in
flowers, leaves, peel and juice. These valuable essential
oils are composed of many compounds including:
terpenes, sesquiterpenes, aldehydes, alcohols, esters
and sterols. They may also be described as mixtures of
hydrocarbons, oxygenated compounds and nonvolatile
residues. Essential oils of citrus are used commercially
for flavoring foods, beverages, perfumes, cosmetics,
medicines, etc. (Salem, 2003). Up to now, numerous
investigations have been performed aimed at identifying
the aroma volatiles in the mandarin flower (Babazadeh
Darjazi, 2011 a, b, c; Salem, 2003; Kharebava and
Tsertsvadze, 1986; Yoshikawa et al., 1996), leaf
(Babazadeh Darjazi, 2011 a, b; Salem, 2003; Lota et al.,
2000, 2001; Ekundayo et al., 1990), peel (Babazadeh
Darjazi, 2009; Babazadeh Darjazi et al., 2009; Lota et al.,
2000, 2001) and juice (Babazadeh Darjazi, 2009;
Babazadeh Darjazi et al., 2009; Yajima et al., 1979). The
quality of an essential oil may be calculated from the
quantity of oxygenated compounds present in the oil
(Babazadeh Darjazi et al., 2009). Branched aldehydes
and alcohols are important flavor compounds in many
food products (Salem, 2003). Various studies have
shown that the tangerine-like smell was also suggested
to be mainly based on carbonyl compounds, such as αsinensal,
β-sinensal, geranial, citronellal and decanal
(Babazadeh Darjazi, 2009; Salem, 2003; Asgarpanah,
2002). The quality of a honey may be calculated from the
amount of oxygenated components present in the honey
(Alissandrakis et al., 2003; Alistair et al., 1993) and
various flowers may influence the quality of volatile flavor
components present in the honey. It had been recognized
previously that oxygenated compounds are important
factor in deceiving and attracting the pollinators. These
results may have consequences for yield in agricultural
(Kite et al., 1991; Andrews et al., 2007). There have been
very few studies on the essential oils of C. nobilis Lour
var. deliciosa swingle, even though citrus oil
compositions have been investigated in many areas

2366 J. Med. Plants Res.
throughout the world. In this study, we compare the
volatile compounds isolated from fresh flowers, leaves
and peel of C. nobilis Lour var. deliciosa swingle with the
aim of determining whether the quantity of oxygenated
compounds was influenced by the organs.
MATERIALS AND METHODS
In 1989, C. nobilis Lour var. deliciosa swingle trees, grafted on Sour
orange, were planted at 84m with three replication at Ramsar
research station (Kotra) (Latitude 36°54’ N, Longitude 50°40’ E ;
Caspian Sea climate, average rainfall 970 mm per year and
average temperature 16.25°C; soil was classified as loam-clay, pH
range 6.9 to 7). In the early week of June 2007, about 300 g of
leaves and at least 200 g flower were collected from many parts of
the same trees, located in Ramsar research station (Kotra), early in
the morning (6 to 8 am) and only by dry weather. Also we collected
at least 10 mature fruit from these trees in the last week of
November 2007. In order to obtain the volatile compounds, about
200 g fresh mature peel, 300 g of fresh leaves and 200 g of fresh
flower were subjected to hydro distillation for 3 h using a Clavengertype
apparatus. N-Hexane was used to isolate the oil layer from the
aqueous phase. The hexane layer was dried over anhydrous
sodium sulphate and stored at -4°C until used.
GC and GC-MS
An Agilent 6890N GC equipped with a DB-5 (30 m 0.25 mm i.d;
film thickness = 0.25 m) fused silica capillary column (J and W
Scientific) and a FID was used. The column temperature was
programmed from 50°C (2 min) to 188°C (20 min) at a rate of 3°C/
min. The injector and detector temperatures were 220°C and
helium was used as the carrier gas at a flow rate of 1.0 ml/min and
a linear velocity of 22 cm/s. The linear retention indices (LRIs) were
calculated for all volatile components using a homologous series of
n-alkanes (C9 to C22) under the same GC conditions. The weight
percent of each peak was calculated according to the response
factor to the FID. GC-MS was used to identify the volatile
components. The analysis was carried out with a Varian Saturn
2000R. 3800 GC linked with a Varian Saturn 2000R MS. The oven
condition, injector and detector temperatures and column (DB-5)
were the same as those given above for the Agilent 6890 N GC.
Helium was the carrier gas at a flow rate of 1.1 ml/min and a linear
velocity of 38.7 cm/s. Injection volume was 1 l.
Identification of components
Components were identified by comparing their LRIs and matching
their mass spectra with those of reference compounds in the data
system of the Wiley library and National Institute of Standards and
Technology (NIST) Mass Spectral Search program (Chem. SW. Inc;
NIST 98 version database) connected to a Varian Saturn 2000R
MS. Identifications were also determined by comparing the
retention time of each compound with that of the known compounds
(Adams, 2001; McLafferty and Stauffer, 1991).
RESULTS
Flower components of the C. nobilis Lour var.
deliciosa swingle
GC-MS analysis of the flavor compounds extracted from
C. nobilis flower using water distillation allowed
identification of 39 volatile components (Table 1):19
oxygenated terpenes (4 aldehydes, 9 alcohols, 5 ester
and 1 ketone), and 20 non oxygenated terpenes (13
monoterpens and 7 sesqiterpens).
Leaf components of the C. nobilis Lour var. deliciosa
swingle
GC-MS analysis of the flavor compounds extracted from
C. nobilis leaf using water distillation allowed
identification of 39 volatile components (Table 1 and
Figure 1): 17 oxygenated terpenes (3 aldehydes, 14
alcohols), 22 non oxygenated terpenes (14 monoterpens,
8 sesqiterpens).
Peel components of the C. nobilis Lour var. deliciosa
swingle
GC-MS analysis of the flavor compounds extracted from
C. nobilis peel using water distillation allowed
identification of 25 volatile components (Table 1): 9
oxygenated terpenes (3 aldehydes, 3 alcohols, 2 esters,
1 ketones), 16 non oxygenated terpenes (12
monoterpens, 4 sesqiterpens).
Aldehydes
Seven aldehyde components that were identified in this
analysis were octanal, citronellal, decanal, neral,
geranial, -sinensal and -sinensal (Table 2). In
addition they were quantified (from 0.29 to 3.94%) that it
was determined and reported as relative amount of those
compounds in oil in this study. These findings were
similar to the previous study undertaken by Salem
(2003); Lota et al. (2001); Asgarpanah (2002). Tangerine
oil is easily distinguished from other citrus oils by its
content of various aliphatic aldehydes. Two main aliphatic
aldehydes were -sinensal and -sinensal. In addition,
tangerine oil also contained citronellal (Asgarpanah,
2002). -sinensal has a woody aroma (Sawamura et al.,
2004), and is considered as one of the major contributors
to tangerine flavor (Asgarpanah, 2002). Since the
aldehyde content of citrus oil is considered as one of the
most important indicators of high quality, organ
apparently has a profound influence on C. nobilis oil
quality. Among the three organs examined, flower
showed the highest content of aldehydes (Table 2).
Flower aldehyds were also compared to those of leaf and
peel in this study. Neral, -sinensal and -sinensal
were identified in flower and leaf oil, while they were not
detected in peel oil. Compared with peel, the flower
improved and increased aldehyde components about 13
times for C. nobilis Lour (Table 2).

Table 1. Chemical composition of essential oils of the flower, leaf and peel of C. nobilis Lour var. deliciosa Swingle.
Darjazi 2367
S/no Component Flower Leaf Peel KI S/no Component Flower Leaf Peel KI
1 α- thujene * * * 925 30 Tymol * 1270
2 α - Pinene * * * 933 31 Geranial * 1275
3 Sabinene * * * 974 32 Terpinyl acetate * 1313
4 β - Pinene * * * 977 33 δ -elemen * 1342
5 β -myrcene * * * 989 34 Citronellyl acetate * 1353
6 Octanal * 1003 35 Neryl acetate * * 1356
7 α - phellandrene * * * 1003 36 Geranyl acetate * * 1381
8 α - terpinene * * 1017 37 β - elemene * * * 1389
9 P-cymene * 1023 38 (Z)- β -caryophyllene * * 1428
10 Limonene * * * 1029 39 Geranyl acetone * 1434
11 (Z)- β - ocimene * * 1035 40 γ-elemen * 1437
12 (E)- β - ocimene * * * 1046 41 α -guaiene * 1450
13 γ- terpinene * * * 1062 42 Allo aromadendrene * 1456
14 (E)-sabinene hydrate * * 1068 43 (Z)- β - farnesene * 1457
15 (Z)- linalool oxide * 1072 44 Germacrene D * * * 1490
16 α -terpinolene * * * 1088 45 E,E, α - farnesene * * * 1505
17 Linalool * * * 1100 46 δ -cadinene * * 1522
18 Cis-p-menth-2-en-1-ol * * 1114 47 Elemol * 1557
19 Trans-p-Menth-2-en-1-ol * * 1127 48 (E) – nerolidol * * 1562
20 (Z)-limonene oxid * 1141 49 Spathulenol * * 1588
21 (E)-β -terpineol * 1144 50 Caryophyllene oxide * 1596
22 Citronellal * 1154 51 Globulol * 1605
23 Terpinen-4-ol * * * 1185 52 α -muurolol * 1650
24 α -terpineol * * * 1194 53 α -cadinol * 1665
25 Decanal * 1205 54 β -sinensal * * 1700
26 Thymol methyl ether * 1235 55 E,E-cis-farnesol * 1725
27 Neral * * 1242 56 α-sinensal * * 1728
28 Carvone * 1250 57 Phytol * 1949
29 Linalyl acetate * 1253
*There is in oil.
Alcohols
Sixteen alcohol components identified in this study were
linalool, cis-p-menth -2-en-1-ol, trans-p-menth -2-en-1-ol,
(E)- -terpineol, terpinene-4-ol, -terpineol, thymol
methyl ether, tymol, elemol, (E) nerolidol, spathulenol,
-muurolol, -cadinol, phytol, globulol and E,E-cisfarnesol
(Table 2).
The total amount of alcohols ranged from (0.70 to
39.11%) that it was determined and reported as relative
amount of those compounds in C. nobilis oil. Linalool was
the major component in this study and it was the most
abundant. Linalool, the most significant alcohol
compound of tangerine oil, is recognized as being very
important to good tangerine flavor (Babazadeh Darjazi,
2009; Salem, 2003). Linalool has a flowery (rose-like)
aroma (Sawamura et al., 2004) and its level is important
to flavor character in tangerine flower, leaf and peel
(Salem, 2003). Among the three organs examined, flower
showed the highest content of alcohols (Table 2). Flower
alcohols were also compared to those of leaf and peel in
this study. Cis-p-menth -2-en-1-ol, trans-p-menth -2-en-
1-ol, (E)-nerolidol and spathulenol were identified in
flower and leaf oil, while they were not detected in peel
oil. Compared with peel, the flower improved and
increased alcohol components about 56 times for C.
nobilis Lour (Table 2).
Esters
Five ester components identified in the analysis were
linalyl acetate, terpinyl acetate, citronellyl acetate, neryl
acetate and geranyl acetate. The total amount of esters
ranged (from 0.00 to 0.13%) in oil and linalyl acetate was
the most abundant. Among the three organs examined,
flower showed the highest content of esters in oil (Table
2).

2368 J. Med. Plants Res.
ketones
Figure 1. HRGC chromatogram of C. nobilis Lour var. deliciosa Swingle leaf oil.
Two ketone compounds identified in the analysis were
carvone and geranyl acetone. Among the three organs
examined flower showed the highest content of ketones
(Table 2). Flower ketones were also compared to those
of leaf and peel in this study. Geranyl acetone was
identified in flower oil, while it was not detected in leaf
and peel (Table 2).
Monoterpene hydrocarbons
The total amount of monoterpene hydrocarbons ranged
(from 51.87 to 95.62%). Sabinene was the major
component among the monoterpene hydrocarbons of C.
nobilis flower and leaf oil while limonene was the major
component among the monoterpene hydrocarbons of C.
nobilis peel oil. Limonene has a weak citrus-like aroma
(Sawamura et al., 2004) and is considered as one of the
major contributors to tangerine flavor (Salem, 2003).
Among the three organs examined, peel had the highest
monoterpenes hydrocarbons in oil (Table 2).
Sesquiterpene hydrocarbons
The total amount of sesquiterpene hydrocarbons ranged
(from 0.31 to 2.01%). -elemene, (Z)-β-caryophyllene
and E, E-α- farnesene were the major components
among the sesquiterpene hydrocarbons of C.nobilis
flower and leaf oil. Among the three organs examined,

Darjazi 2369
Table 2. Statistical analysis of variation in volatile components of flower, leaf and peel of Citrus nobilis Lour var. deliciosa Swingle.
Mean is average composition (%) in the different organs used with three replicates. St. err. = standard error. F value is acc ompanied by
its significance, indicated by: NS = not significant, * = significant at P = 0.05, ** = significant at P = 0.01.
S/No Compounds
Oxygenated compounds
Flower
Mean St.err
Leaf
Mean St.err
Peel
Mean St.err
a Aldehyds
1 Octanal 0.00 0.00 0.00 0.00 0.14 0.006
2 Citronellal 0.00 0.00 0.00 0.00 0.03 0.005
3 Decanal 0.00 0.00 0.00 0.00 0.12 0.005
4 Neral 0.07 0.00 0.07 0.001 0.00 0.00
5 Geranial 0.009 0.001 0.00 0.00 0.00 0.00
6 β-sinensal 2.08 0.16 1.78 0.08 0.00 0.00
7 α-sinensal 1.79 0.19 0.85 0.04 0.00 0.00
Total 3.94 0.35 2.70 0.12 0.29 0.01
b b) Alcohols
1 linalool 24.91 1.35 23.47 0.10 0.58 0.01 F**
2 Cis-p-menth-2-en-1-ol 0.26 0.11 0.31 0.01 0.00 0.00
3 Trans-p-menth-2-en-1-ol 0.29 0.03 0.10 0.01 0.00 0.00
4 (E)- β-terpineol 0.00 0.00 0.01 0.00 0.00 0.00
5 Terpinene-4-ol 4.00 0.20 4.28 0.02 0.02 0.005 F**
6 α-terpineol 0.80 0.10 0.50 0.05 0.10 0.006
7 Thymol methyl ether 0.00 0.00 1.10 0.01 0.00 0.00
8 Tymol 0.00 0.00 0.14 0.01 0.00 0.00
9 Elemol 0.00 0.00 0.20 0.001 0.00 0.00
10 (E)-nerolidol 7.51 0.29 0.10 0.01 0.00 0.00
11 Spathulenol 0.46 0.06 0.14 0.01 0.00 0.00
12 α-muurolol 0.00 0.00 0.04 0.005 0.00 0.00
13 α-cadinol 0.00 0.00 0.05 0.005 0.00 0.00
14 Phytol 0.00 0.00 0.12 0.005 0.00 0.00
15 Globulol 0.09 0.001 0.00 0.00 0.00 0.00
16 E,E-cis-farnesol 0.79 0.03 0.00 0.00 0.00 0.00
Total 39.11 2.17 30.56 0.24 0.70 0.02
C Esters
1 Linalyl acetate 0.09 0.01 0.00 0.00 0.00 0.00
2 Terpinyl acetate 0.01 0.00 0.00 0.00 0.00 0.00
3 Citronellyl acetate 0.01 0.005 0.00 0.00 0.00 0.00
4 Neryl acetate 0.009 0.00 0.00 0.00 0.03 0.005
5 Granyl acetate 0.02 0.005 0.00 0.00 0.04 0.005
Total 0.13 0.02 0.00 0.00 0.07 0.01
d Ketones
1 Carvone 0.00 0.00 0.00 0.00 0.01 0.00
2 Geranyl acetone 0.04 0.01 0.00 0.00 0.00 0.00
Total 0.04 0.01 0.00 0.00 0.01 0.00
Monoterpenes
1 α -thujene 0.007 0.00 0.54 0.004 0.008 0.00
2 α -pinene 0.93 0.16 2.04 0.03 0.46 0.01 F**
3 sabinene 36.33 1.02 38.91 0.23 0.19 0.006 F**
4 β- pinene 1.42 0.45 1.75 0.06 0. 30 0.02

2370 J. Med. Plants Res.
Table 2 cont.
5 β-myrcene 2.65 0.53 3.84 0.05 2.06 0.02 F**
6 α -phellandrene 0.24 0.01 0.43 0.05 0.04 0.006
7 α-terpinene 0.00 0.00 1.04 0.04 0.007 0.00
8 P-cymene 0.00 0.00 0. 30 0.02 0.00 0.00
9 limonene 7.46 0.35 3.20 0.03 87.45 0.49 F**
10 (Z)- β -ocimene 0.23 0.01 0.29 0.01 0.00 0.00
11 (E)- β -ocimene 1.34 0.24 7.44 0.02 0.30 0.02 F**
12 γ - terpinene 0.55 0.14 1.29 0.007 5.10 0.20 F**
13 (E) Sabinene hydrate 0.41 0.03 0.60 0.006 0.00 0.00
14 (Z)-linalool oxide 0.02 0.006 0.000 0.00 0.00 0.00
15 α-terpinolene 0.29 0.03 1.10 0.01 0. 20 0.00
16 Trans-limonene oxide 0.00 0.00 0.00 0.00 0.01 0.006
Total 51.87 2.97 62.48 0.56 95.62 0.77
Sesquiterpenes
1 δ-elemene 0.00 0.00 0.01 0.00 0.00 0.00
2 β-elemene 0.53 0.06 0.33 0.02 0.04 0.005
3 (Z)- β-caryophyllene 0.64 0.03 0.51 0.03 0.00 0.00
4 γ -elemene 0.00 0.00 0.01 0.00 0.00 0.00
5 (Z)- β-farnesene 0.00 0.00 0.23 0.05 0.00 0.00
6 δ -guaiene 0.07 0.009 0.00 0.00 0.00 0.00
7 allo aromadendrene 0.03 0.006 0.00 0.00 0.00 0.00
8 Germacrene D 0.09 0.001 0.01 0.00 0.17 0.01
9 E,E- α-farnesene 0.36 0.06 0.50 0.05 0.10 0.005
10 δ -cadinene 0.00 0.00 0.007 0.00 0.007 0.00
11 Caryophyllene oxide 0.29 0.10 0.00 0.00 0.00 0.00
Total 2.01 0.26 1.60 0.15 0.31 0.02
Total oxygenated compounds 43.22 2.55 33.26 0.36 1.07 0.04
Total 97.10 5.78 97.34 1.07 97 0.83
flower had the highest sesquiterpenes content (Table 2).
Result of correlation
Correlations of the main components were evaluated with
Pearson correlation analysis. Simple intercorrellations
between 8 components are presented in a correlation
matrix (Table 3). The highest positive values or r
(correlation coefficient) were between [sabinene and
terpinene-4-ol (1.00%)]; [sabinene and linalool (99%)];
[terpinene-4-ol and linalool (98%)].
The highest significant negative correlations were
between [limonene and linalool (99%)]; [limonene and
terpinene-4-ol (99%)]; [limonene and sabinene (99%)]; [γ-
terpinene and linalool (99%)]. When 8 components were
cluster analyzed, there was clustering of only 3
components into 2 two-compound factors above the 98%
level of function. These 2 factors resulted from the
clustering of highly positively interrelated compounds
such as [sabinene and terpinene-4-ol (1.00%)]; [sabinene
and linalool (99%)] (Table 3).
Statistical analysis
Differences for main components among organs were
analyzed by performing separate one-way analysis of
variance (ANOVA). The Duncan’s Multiple Range test
was used to separate the significant organs. Of the 8
individual oil components analyzed, all showed
statistically significant differences due to the influence of
individual organs. These differences on the 1% level
occurred in linalool, terpinene-4-ol, -pinene, sabinene,
-myrcene, limonene, (E)-β- ocimene and γ- terpinene
(Table 2).
DISCUSSION
Observations were made that changing organ has an
effect on some of the components of tangerine oil accord
with other observations (Babazadeh Darjazi, 2011 b;

Table 3. Correlation matrix (numbers in this table correspond with main components mentioned in Table 2).
Terpinene-4-ol
α-pinene
Sabinene
β-myrcene
Limonene
(E)- β -ocimene
γ – terpinene
Darjazi 2371
Linalool
0.98**
Terpinene-4-ol α-pinene Sabinene β-myrcene Limonene (E)- β -ocimene γ - terpinene
0.68* 0.76*
0.99** 1.00** 0.76*
0.69* 0.73* 0.90** 0.74*
-0.99** -0.99** -0.75* -0.99** -0.74*
0.57 0.65 0.97** 0.65 0.93** -0.64
-0.99** -0.97** -0.60 -0.97** -0.62 0.97** -0.48
*=significant at 0.05. **=significant at 0.01.
Salem, 2003; Asgarpanah, 2002; Lota et al., 2000). The
compositions of C. nobilis Lour obtained from flower, leaf
and peel were very similar. However, relative
concentration of compounds differed according to type of
materials. Compared with peel, the flower improved and
increased alcohol components about 56 times for C.
nobilis Lour. The amount of alcohol components obtained
from peel were low probably because of decrease in
endogenous enzymes activity [isopentenyl
pyrophosphate isomerase (IPI) and
geranylpyrophosphate synthase (GPS) (Hay and
Waterman, 1995) resulting in decreased of labile
compounds. Also the lower proportion of the detected
alcohol components in peel was probably due to
seasonal temperature (Sekiya et al., 1984) that it is the
most important environmental factor in the control of
endogenous enzymes.
According to our results, it appears that the relative
percentages of the identified compounds depend on the
plant part studied. However, it should be kept in mind
that the isolation method has an effect on some of the
components of oil (Babazadeh Darjazi, 2011c). The
pronounced enhancement in the amount of oxygenated
compounds, when flower was used as the organ, showed
that either the synthesis of geranyl pyrophosphate (GPP)
is enhanced or activities of both enzymes (IPI and GPS)
increased (Hay and Waterman, 1995). High positive
correlations between two terpens such as (sabinene and
terpinene-4-ol (1.00%)); (sabinene and linalool (99%));
[terpinene-4-ol and linalool (98%)) suggest a genetic
control (Scora et al., 1976). Whether such dependence
between two terpenes is due to their derivation of one
from another is not known. Similarly, high negative
correlations observed between (limonene and linalool
(99%)); (limonene and terpinene-4-ol (99%)); (limonene
and sabinene (99%)); (γ- terpinene and linalool (99%))
suggest that one of the two compounds is being
synthesized at the expense of the other or of its
precursor. Non-significant negative and positive
correlations can imply genetic and /or biosynthetic
independence. However, without a thorough knowledge
of the biosynthetic pathway leading to each terpenoid
compound, the true significance of these observed
correlations is not clear.
Conclusion
In the present study we found that the percent of flavor
compounds was significantly affected by organ. The
essential oil obtained from flower contained more
oxygenated compounds and fewer monoterpene
components than those isolated from leaf and peel. It is
easy to observe the significant variations among flower
and other organs, mainly in terms of the quantities of
oxygenated compounds. The essential oils and their
aroma compounds are very important and widely used in
hygienic products, aromatherapy, pharmacy, food
industries, cosmetics and other areas. Therefore, many
studies, such as this study is very crucial in order to
identify what type of chemical constituents existing in
the materials that we want to use, before the essential
oil can be utilized in those industries. Further research on
the relationship between organ and essential oil
(oxygenated terpenes) is necessary.
ACKNOWLEDGEMENTS
The author would like to express his gratitude to
Z.Kadkhoda from Institute of Medicinal Plants located at
Supa blvd-Km 55 of Tehran – Qazvin (Iran) for her help in
GC-MS and GC analysis.
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Adams RP (2001). Identification of essential oil components by gas
chromatography / mass spectrometry. Allured Publishing
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Alissandrakis E, Daferera D, Tarantilis PA, Polissiou M, Harizanis PC
(2003). Ultrasound assisted extraction of volatile compounds from
citrus flowers and citrus honey. Food. Chem., 82: 575-582.
Alistair LW, Yinrong LU, Seng-To Tan (1993). Extractives from New
Zealand honey 4.linalool derivatives and other components from
nodding thistle (Corduus nutans) honey. J.Agric.Food.Chem., 41(6):
873-878.
Andrews ES, Theis N, Alder LS (2007). Pollinator and herbivore
attraction to cucurbita floral volatiles. J. Chem. Ecol., 33: 1682-1691.

2372 J. Med. Plants Res.
Asgarpanah J (2002). Extraction, analysis and identification of essential
oil components of the peel and leaves of the Bam tangerine ”Citrus
nobilis loureior var deliciosa swingle”. Master of Science Thesis,
Islamic Azad University. Pharmaceutical sciences Branch.
Babazadeh Darjazi B (2009). The effects of rootstock on the volatile
flavor components of Page mandarin juice and peel. Ph.D of science
Thesis. Islamic Azad Univ. Sci. Res. Branch.
Babazadeh Darjazi B, Rustaiyan A, Talaei A, Khalighi A, Larijani
K,Golein B, Taghizad R (2009). The effects of rootstock on the
volatile flavor components of Page mandarin juice and peel.
Iran.J.Chem.Chem.Eng., 28(2): 99-111.
Babazadeh Darjazi B (2011a). The effects of rootstock on the volatile
flavour components of Page mandarin flower and leaf. Afr. J. Agric.
Res., 6(7): 1884-1896.
Babazadeh Darjazi B (2011 b). Comparison of volatile components of
lower, leaf, peel and juice of ‘Page’ mandarin. Afr. J. Biotechnol.,
10(51): 10437-10446.
Babazadeh Darjazi B (2011c). A comparison of volatile components of
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and hydrodistillation. J. Med. Plant. Res., 5(13): 2840-2847.
Ekundayo O, Bakare O, Adesomoju A, Stahl-Bisk up E (1990). Leaf
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Kharebava LG, Tsertsvadze VV (1986). Volatile compounds of flower of
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Lota ML, Serra D, Tomi F, Casanova J (2001). Chemical variability of
peel and leaf essential oils of 15 species of mandarins.
Biochem.Syst. Ecol., 29: 77-104.
Lota ML, Serra D, Tomi F, Casanova J (2000). Chemical variability of
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McLafferty FW, Stauffer DB (1991). The important peak index of the
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Salem A (2003). Extraction and identification of essential oil
components of the peel, leaf and flower of tangerine”Citrus nobilis
loureior var deliciosa swingle” cultivated at the north of Iran. Master of
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Sawamura M, Minh Tu NT, Onishi Y, Ogawa E, Choi HS (2004).
Characteristic odor components of Citrus .reticulata Blanco (Ponkan)
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Sekiya J, Kajiwara T, Hatanaka A (1984). Seasonal changes in
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Composition of the volatiles of peel oil and juice from Citrus unshiu.
Agric.Biol.Chem., 43: 259-264.

Journal of Medicinal Plants Research Vol. 6(12), pp. 2373-2380, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.993
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Optimization of total flavonoids extraction from
mulberry leaf using an ethanol-based solvent system
Kai Nie 1# , Zhonghai Tang 1,2# , Xu Wu 1 , Xiaona Xu 3 , Yizeng Liang 2 , Huang Li 1 and Liqun Rao 1 *
1 College of Bioscience and Biotechnology, Hunan Agricultural University, Changsha, Hunan 410128, P. R. China.
2 Research Center of Modernization of Chinese Traditional and Herbal Drug Modernization, College of Chemistry and
Chemical Engineering, Central South University, Changsha, Hunan 410083, P. R. China.
3 College of Chemistry and Chemical Engineering, University of South China, Hengyang, Hunan 421001, P. R. China.
Accepted 24 August, 2011
Flavonoids from mulberry leaf are a kind of physiological active materials that can powerfully combat
with biological oxidation. They are widely used in medicines and health care products. The present
research established the best conditions for flavonoid extraction using a circulation method of 80%
alcohol (solid-liquid ratio = 1:30, temperature = 80°C). The extraction was performed 5 times for 3 h.
Ultraviolet spectrophotometery and high-performance liquid chromatography were used to determine
the extraction rate of total flavonoids.
Key words: Mulberry leaf, total flavonoids, best process, determination.
INTRODUCTION
Mulberry is a widely distributed deciduous tree belonging
to the Moraceae family. Some studies indicate that the
mulberry leaf contains diverse chemical ingredients such
as flavonoids, polysaccharides, alkaloids, sterols, volatile
oil, amino acids, vitamins and other trace elements.
Flavonoids are C6-C3-C6 chemical compounds that
include flavone, flavonol, dihydroflavone, dihydroflavonol,
isoflavone, dihydroisoflavone, chalcone, anthocyanidin,
etc (Liu et al., 2008). The flavonoid content of mulberry
leaf, consisting of flavones, flavonol, dihydroisoflavonol
and anthocyanidin, is impressively high. Interestingly,
significant differences in the flavonoid content can be
observed in diverse organs, habitats, periods and
varieties (Liu and Jia, 1997). Modern medical research
has proven that mulberry leaf flavonoids enhance
cerebrovascular and coronary blood flows, regulate
arrhythmia, soften angiosclerosis, as well as decrease
sugar and fat. Japanese researchers have also found that
flavonoids from mulberry leaf extracts have anticancer
effects (Jia et al., 1996; Akiko et al., 2007). Undoubtedly,
using high and new technologies to extract flavonoid
*Corresponding author. E-mail: raoliqun@163.com.
#These authors contributed equally to this work.
compounds from mulberry leave has numerous potential
applications in pharmaceuticals, cosmetics, and food
preparation.
MATERIALS AND METHODS
Plant material
Fresh mulberry leaves (Morus cathayana Hemsl. var. Cathayana)
were collected in October 2010 from the Sericultural Research
Institute of Hunan. The samples were oven-dried at 45°C until
constant weight was reached, and were then milled into powders
(0.250 mm in particle size). Ethylether was used to decrease the
powder using a Soxhlet extractor.
Reagents and instrument
Ethanol (95%), sodium nitrite, chloroform, aluminium nitrate,
sodium hydroxide and methanoic acid were all analytical grade.
Rutin (99%) was purchased from J&K (Beijing, China). Liquid
chromatography grade acetonitrile was obtained from Merck
(Darmstadt, Germany). In all experiments, the water used was
obtained from a Milli-Q purification system (Millipore, Molsheim,
France).
The analyses were performed on an Agilent 1100 highperformance
liquid chromatography (HPLC) instrument (Agilent,
USA). The UV-9100 UV-Vis spectrophotometer used was from the
Rayleigh Analytical Instrument Co. (Beijing, China). The RE-52AA
rotary evaporator was from the Yarong Biochemical instrument Co.

2374 J. Med. Plants Res.
Figure 1. Standard curve of rutin. The absorbance versus concentration is described by the equation:
y = 0.0091 xs+0.0018 (R 2 = 0.9996), where, y is the absorbance and x is the content.
(Shanghai, China). The AL204 electronic balance was from Mettler-
Toledo Instruments Co., Ltd (Switzerland).
Preparation of the rutin standard sample
The present research used rutin as the contrast sample to
determine total flavonoids. About 0.0100 g of rutin was accurately
weighed and dissolved by appropriate amounts of 60% alcohol.
Ethanol (30%) was then added until the solution volume was 10
mL, then the rutin standard sample (1 mg/ml) was obtained (Sun et
al., 2004).
Fabrication of the standard curve
Seven 10 ml test tubes were prepared and numbered 0 to 6, to
which 0, 0.1, 0.2, 0.3, 0.4, and 0.5 ml rutin standard samples were
added. About 0.5 ml of 5% NaNO2 was added to all seven test
tubes, which were shaken for 5 min.
About 0.5 ml of 10% Al(NO3)3 was also added, and the tubes
were shaken for 5 min. Water and 4 ml of 4% NaOH were then
added, and the tubes were shaken for 15 min. Test tubes 0 was the
contrast, and the absorbance was determined at 510 nm (Wu and
Tan, 2006). The Microsoft Excel software was used to draw the
standard curve (Figure 1).
Extraction method and determination of total flavonoids from
mulberry leaf
Each extraction process was performed according to the following
procedure. Mulberry leaf powder was accurately weighed, and then
reflux-extracted with ethanol at 80°C. To our knowledge, 80°C is the
optimal extraction temperature for flavonoids in mulberry (Cai et al.,
2003; Gao et al., 2005; Li 2003; Chen and Chen, 2008). The
volume of the extracting solution was concentrated to almost half
using a rotary evaporator. The absorbance of 1 ml of the
concentrated extracting solution was determined according to the
standard curve.
Calculation of the extraction rate
The absorbance and volume of the extracting solution after
concentration were determined. The extraction rate of total
flavonoids was calculated via the following equation:
Where A is the absorbance, V is the volume of the extracting
solution after concentration, and M is the weight of the mulberry leaf
powder.
Precision test
The absorbance of the 50 µg/mL rutin standard sample was
determined for six times.
Stability test for the extracts
After extraction, the absorbance of the extracting solution made
from 1.00 g of mulberry powder was determined nine times every
hour.
Reappearance test for the extraction of total flavonoids
Six mulberry powder samples (1.00 g each) were prepared. The
,

Table 1. Precision test.
extraction was performed at 80°C for 3 h using 80% alcohol and a
solid-liquid ratio of 1:40. After the extracting solution was
concentrated, the absorbance was determined at 510 nm. The
extraction rate was then calculated.
Recovery rate test
About 6.00 g of mulberry leaf powder was weighed. The extraction
conditions were the same as those in the reappearance test. After
the extracting solution was concentrated, 10 mL of the extract and 1
mg/mL rutin standard sample were added into each of the 6 test
tubes. The recovery rate was then calculated.
Single factor experiments of hot reflux extractions with ethanol
According to the extracting method, the alcohol concentration,
solid-to-liquid ratio, number of extraction, and extracting period
were chosen for single factors experiments. About 5.00 g of
mulberry leaf powder was weighed before each extraction.
Influence of the alcoholic concentration on the extraction
Alcoholic concentrations of 40, 50, 60, 70 and 80% were used. The
extraction was performed once every 3 h at 80°C using a solidliquid
ratio of 1:30. The extraction rate was then calculated.
Influence of the solid-liquid ratio on the extraction
Solid-liquid ratios of 1:10, 1:20, 1:30, 1:40, and 1:50 were used. The
extraction was performed once every 3 h at 80°C using 60%
alcohol (v/v). The extraction rate was then calculated.
Influence of the number of extraction on the extraction
The numbers of extraction used were 1, 2, 3, 4, and 5. The
extraction was performed for 3 h at 80°C using 60% alcohol and a
solid-liquid ratio of 1:30. The extraction rate was then calculated.
Influence of the extracting period on the extraction
Extracting periods of 60, 90, 120, 150, and 180 min were used. The
extraction was performed at 80°C using 60% alcohol and a solidliquid
ratio of 1:30. The extraction rate was then calculated.
Orthogonal design of total flavonoid extraction from mulberry
leaf
The alcohol concentration, solid-liquid ratio, number of extraction,
Test No. Absorbance
1 0.455
2 0.453
3 0.450
4 0.455
5 0.454
6 0.452
Nie et al. 2375
and extracting period were chosen as the orthogonal factors. The
orthogonal design L9 (3 4 ) was adopted to optimize the extraction
conditions (Chen and Jiang, 2008). For each orthogonal test, 5.00 g
of mulberry leaf powder was weighed.
Determination of the optimal system using HPLC
A standard solution of rutin was prepared as follow. About 0.0049 g
of rutin was accurately weighed and dissolved in an ethanol
solution. Ethanol was added until volume of 25 ml was reached in a
25 ml volumetric flask. About 1, 2, 4, 6, and 8 ml of rutin standard
solution were diluted to 10 ml by ethanol. Rutin determination was
performed as described above. Using the concentration of the rutin
standard solution as the abscissa and the peak area as the ycoordinate,
the linear chart was constructed. The regression
equation was y = 507.15x+23.364, R 2 = 0.9998. The HPLC analysis
was carried out on an Agilent Series 1100 liquid chromatograph.
The UV detection system therein was connected to a reversedphase
column (Kromasil C18; 5 μm, 250 mm × 4.6 mm). The HPLC
mobile phase was a methanol-acetonitrile-1% acetic acid (13:18:69,
v/v/v) solution. The absorbance was measured at 360 nm for the
rutin detection. The flow rate was 0.5 ml/min, and the column
temperature was maintained at 30°C. The sample acquired from the
optimal system was injected at a volume of 10 μl after dilution. The
peak area was used for quantification. The extraction rate was then
measured and compared with the results from the UV
spectrophotometer.
RESULTS
Precision test
After the absorbance was determined, the results showed
that the relative standard deviation (RSD) was 0.43%
(RSD < 2.0%), as shown in Table 1.
Stability test for the extracts
Table 2 shows that the absorbance of flavonoids showed
minor differences within 3 to 8 h. The RSD was 1.3%
(RSD < 2.0%).
Reappearance test for the extraction of total
flavonoids
Table 3 compares the results of the six numbers of

2376 J. Med. Plants Res.
Table 4. Recovery rate test.
Test No.
Flavonoids sample
(ml)
Table 2. Stability test.
Time (h) Absorbance
0 0.165
1 0.160
2 0.163
3 0.166
4 0.164
5 0.166
6 0.165
7 0.164
8 0.166
9 0.168
Table 3. Reappearance test.
Time (h) Absorbance (%) Extraction rate of flavonoids
1 0.163 2.48
2 0.166 2.53
3 0.167 2.54
4 0.161 2.45
5 0.159 2.42
6 0.163 2.48
Content of flavonoids
(mg)
Rutin standard sample
added (mg)
Total flavonoids
(mg)
Recovery
rate (%)
1 10 13.31 10.0 23.02 98.7
2 10 13.31 15.0 27.94 98.6
3 10 13.31 20.0 33.45 100.4
4 10 13.31 25.0 38.04 99.2
5 10 13.31 30.0 42.86 98.9
6 10 13.31 35.0 47.89 99.1
extraction. The RSD was 1.85% (RSD < 2.0%).
Recovery rate test
Table 4 shows that the RSD of the recovery rate test
was 0.66% (RSD < 2.0%).
Single factor experiments of hot reflux extractions
with ethanol
Influence of the alcoholic concentration on the
extraction
Figure 2 shows that the total flavonoids content increased
with increased alcoholic concentration. At alcohol
concentrations beyond 60%, no significant increase in
flavonoids content occurred. Hence, the alcoholic
concentrations (60, 70 and 80%) was then chosen for the
orthogonal experiments.
Influence of the solid-liquid ratio on the extraction
Figure 3 shows that at solid-liquid ratios beyond 1:30, the
total flavonoids content decreased. Hence, the solid-liquid
ratios of (1:20, 1:30 and 1:40) were chosen for the
orthogonal experiments.
Influence of the number of extraction on the
extraction
Figure 4 shows that when the number of extraction is

Figure 2. Effect of different alcohol concentrations on flavonoids extraction.
Figure 3. Effect of different solid-liquid ratios on flavonoids extraction
Figure 4. Effect of different numbers of extraction on flavonoids extraction.
Nie et al. 2377

2378 J. Med. Plants Res.
Figure 5. Effect of different extraction period on flavonoids extraction.
Table 5. Results of the orthogonal test.
Test No.
Concentration of
alcohol (%)
The ratio of
solid to liquid
Extraction
times
Extraction
time (min)
Extraction rate of total
flavonoids (%)
1 60 1:20 3 120 1.37
2 60 1:30 4 150 1.97
3 60 1:40 5 180 1.84
4 70 1:20 4 180 1.73
5 70 1:30 5 120 2.22
6 70 1:40 3 150 1.66
7 80 1:20 5 150 2.41
8 80 1:30 3 180 2.57
9 80 1:40 4 120 2.47
K1 5.18 5.51 5.6 6.06
K2 5.61 6.76 6.17 6.04
K3 7.45 5.97 6.47 6.14
k1 1.727 1.837 1.867 2.020
k2 1.870 2.253 2.057 2.013
k3 2.483 1.990 2.157 2.047
R 0.756 0.416 0.290 0.034
more than 3, the flavonoid content did not significantly
increase. Hence, the numbers of extraction chosen for
the orthogonal experiments were 3, 4 and 5.
Influence of the extracting period on the extracting
process
Figure 5 shows that total flavonoids content increased
with increased extracting period. At extracting period
beyond 150 min, flavonoids content slightly increased.
Hence, the extracting periods (120, 150, and 180 min)
were chosen for the orthogonal experiments.
Analysis of extraction results using the orthogonal
test
Total flavonoids were adopted as the index for the
orthogonal test. Table 5 shows that influences on
flavonoids extraction followed the trend alcoholic
concentration > solid - liquid ratio > number of extraction

Table 6. Results of ANOVA.
Nie et al. 2379
Sources Sum of square of deviations Degree of freedom Mean square F value Prominence
Concentration of alcohol 0.969 2 0.4845 484.5 **
The ratio of solid to liquid 0.266 2 0.133 133.0 *
Extraction times 0.130 2 0.065 65.00 *
Error E (extraction time) 0.002 2 0.001 1.000
F0.05 (2,6) = 19.00, F0.01(2,6) = 99.00.
Figure 6. Standard peak of rutin.
Figure 7. Sample peak of extracting solution from mulberry leaves.
> extracting period. After an analysis of variance
(ANOVA), the results (Table 6) indicated that the
alcoholic concentration, solid-liquid ratio, as well as
number of extraction were significant, and the extraction
period was not. Summarily, the optimum extracting
condition at 80°C was 80% alcohol, 1:30 solid-liquid ratio,
5 numbers of extraction, and 180 min extraction period.
The extraction experiment was repeated three times. The
average extraction rate was 2.99%. The results showed
that the proposed method was the most feasible among
all previously tried methods.
Results of the HPLC analysis
After measuring the peak areas in Figures 6 and 7, the
extraction rate was determined as 2.65%. This value was
lower than the extraction rate measured by the UV

2380 J. Med. Plants Res.
spectrophotometer.
Conclusions
In the present study, alcohol was used to extract
flavonoids from mulberry leaves. Total flavonoids content
was then determined using spectrophotometry. Based on
single-factor and orthogonal tests, the best conditions for
extraction at 80°C were 80% alcohol, 1:30 solid-liquid
ratio, 5 numbers of extraction, and 180 min extraction
period. The extraction rate measured by HPLC was lower
than that measured by the UV spectrophotometer. This
phenomenon is attributed to the fact that mulberry has
other classes of flavonoids. The results showed that most
of the flavonoids in the present experimental sample were
rutin. In conclusion, the method used in the present study
was easy to use, convenient, and highly accurate.
ACKNOWLEDGMENTS
We thank Yin Gong and Huang Li for correcting the
English and Xu Wu for providing mulberry leaf. This work
is financially supported by the International Cooperation
Project on Traditional Chinese Medicines of Ministry of
Science and Technology of China (no. 2007DFA40680),
the Technological Item of Hunan
Province(2011NK3062), the Youth Grant of Hunan
Agriculture University (10QN20). The innovation for
research projects of graduate in Hunan Province (no.
CX2011B294).
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Chen LS, Chen ZB (2008). Extracting and determinantion on flavone
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Sci., 32(4): 138-141.

Journal of Medicinal Plants Research Vol. 6(12), pp. 2381-2387, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Evaluation of the antimicrobial activity in species of a
Portuguese “Montado” ecosystem against multidrug
resistant pathogens
B. Lai 1 , G. Teixeira 2 , I. Moreira 1 , A. I. Correia 3 , A. Duarte 1 and A. M. Madureira 1 *
1 Universidade de Lisboa, Faculdade de Farmácia de Lisboa, iMed-UL, Avenida Prof. Gama Pinto, 1649-003, Lisboa,
Portugal.
2 Universidade de Lisboa, Faculdade de Farmácia de Lisboa, Centro de Biologia Ambiental, Avenida Prof. Gama Pinto,
1649-003, Lisboa, Portugal.
3 Universidade de Lisboa, Faculdade de Ciências de Lisboa, Centro de Biologia Ambiental, C2, Campo Grande, 1749-
016 Lisboa, Portugal.
Accepted 12 October, 2011
Forty polar and non polar extracts from six “Montado” species (Adenocarpus anisochilus., Erica
lusitanica, Lavandula stoechas subsp. luisieri, Paeonia broteroi, Quercus faginea subsp. broteroi and
Rosmarinus officinalis) were evaluated for their antimicrobial activity against a broad panel of
microorganisms that include standard and resistant strains of Gram-positive, Gram-negative bacteria,
Mycobacterium smegmatis and the yeast Candida albicans. From the forty tested extracts, 87%
inhibited the development of Gram-positive bacteria. Interesting results were obtained with the most
polar extracts of P. broteroi (leaves) displaying the best MIC (3.1 to 1.9 µg/ml) when tested against
Staphylococcus aureus standard, Vancomycin-Resistant S. aureus VRSA and meticillin-resistant S.
aureus (MRSA) strains. The extracts of E. lusitanica and P. broteroi displayed the broadest
antimicrobial activity spectra with the lowest minimum inhibitory concentration (MIC) values seeming
very promising and are worthy for further phytochemical studies.
Key words: “Montado” flora, antibacterial activity, MIC determination, phytochemical screening.
INTRODUCTION
Infectious diseases and global antibiotic resistant
pathogens are an increasing public health problem. The
lack of development of new antimicrobial agents in the
last decades, associated with their misuse, led to the
emergence of multiresistant microorganisms. These
multiresistant strains are a threat to disease management
and greatly increased the treatment costs. This may
result in a dark scenario where common infections
become untreatable and even lethal (Norrby et al., 2005).
Examples include meticillin-resistant Staphylococcus
aureus (MRSA) which is a major cause of serious
hospital-acquired infections (Appelbaum, 2006). This
justifies the search for new antimicrobial drugs.
*Correspondence author. E-mail: afernand@ff.ul.pt. Tel: (+
351)217946400. Fax: (+ 351)217946470.
Plants used for traditional medicine are known to be
effective in a wide range of diseases. The role of the
natural products is particularly relevant in the infectious
diseases area, where over 60% of the antimicrobial
agents used in therapy are from natural origin (Newman
and Cragg, 2007; Newman et al., 2003). With only 5 to
15% of the approximately 250 000 species of higher
plants systematically investigated, natural products
remain an important source of new molecules and
scaffolds which can lead to the development of new
effective drugs against multiresistant microorganisms
(Newman and Cragg, 2007).
The Mediterranean region is rich in medicinal and
aromatic plants. Southwestern Iberia is mainly covered
by cork-oak woodland, common speaking as “Montado”.
This is a unique Mediterranean ecosystem, whose
importance is related to its huge biodiversity, allied to a
traditional and sustainable management of the ecosystem.

2382 J. Med. Plants Res.
Table 1. Botanical data of the species used in the present study.
Family Taxa Plant part studied Voucher number
Leguminosae Adenocarpus anisochilus Boiss. Aerial parts and fruits LISU 221390
Ericaceae Erica lusitanica Rudolphi Leaves LISU 223635
Labiatae Lavandula stoechas subsp. luisieri (Rozeira) Rozeira Aerial parts LISU 223640
Paeoniaceae Paeonia broteroi Boiss. & Reut. Leaves and fruits LISU 221345
Fagaceae Quercus faginea subsp. broteroi (Cout.) A. Camus Leaves LISI 503/ 2011
Labiatae Rosmarinus officinalis L. Aerial parts LISI 2001/ 2009
Its unique characteristics were considered by the EU
(Santos-Reis and Correia, 1999) as a priority habitat for
conservation. Its botanical biodiversity is well adapted to
the harsh meteorological conditions characteristic of
Mediterranean ecosystems (inconsistent rainfall patterns,
dry summers and cool winters). These “montado” species
have developed various functional and structural
characteristics that allow them to survive. In those
extreme conditions several chemical adaptations occur,
as can be exemplified by the presence of a large
combination of metabolites, often present in glandular
structures (Figueiredo et al., 2007).
It is known that some of those species contain a variety
of biologically active compounds, such as terpenoids and
tannin (Harborne, 1997), which are believed to provide
protection against potential pathogenic microbes
(Hammer et al., 1997; Lis-Balchin et al., 1998). The
exploration of the antimicrobial properties of the
Portuguese “montado” flora is very limited and restricted
to a few botanical families, in particular to the Labiatae,
the mint family, being their biological activities mainly
attributed to the essential oils (Matos et al., 2009;
Rodrigues et al., 2008, 2006).
The aim of the present study was to evaluate the
antimicrobial activity of extracts of chosen indigenous
species from the Portuguese “montado”, against selected
human pathogens, Gram-positive and Gram-negative,
standard and resistant bacteria, Mycobacterium
smegmatis and against the yeast Candida albicans. The
preliminary phytochemical composition of each obtained
extract was also evaluated by chromatographic methods.
MATERIALS AND METHODS
Plant material and preparation of the extracts
The plant materials used in this study were six different Portuguese
“montado” species: Adenocarpus anisochilus Boiss. (Boiss.)
Franco, Erica lusitanica Rudolphi, Lavandula stoechas subsp.
luisieri (Rozeira) Rozeira, Paeonia broteroi Boiss. and Reut.,
Quercus faginea subsp. broteroi (Cout.) A. Camus and Rosmarinus
officinalis L. (Table 1). Aerial parts were collected between June
and July 2009, from wild populations growing at Herdade da Ribeira
Abaixo, Grândola region, southwestern Portugal (38º 8’ N - 8º 33’
W). The plant material was identified in the Herbarium of the Lisbon
Botanical Garden (LISU) and in the Lisbon Agronomic Institute
Herbarium (LISI), where vouchers specimens are deposited.
The aerial parts were shadow dried at room temperature and in
some cases different parts were separated as leaves and fruits
(Table 1). The crude extracts were prepared by sequentially
extracting 100 g of dried powdered plant material with 300 ml of nhexane,
dichloromethane, ethyl acetate, methanol and water for 24
h, at room temperature with occasional shaking. After filtration, the
extracts were concentrated under reduced pressure at 40 to 45ºC,
and stored at 4ºC until use. The yield of the dried extracts (as w/w
percentage of the starting dried material) is available in Table 2.
Screening for antimicrobial activity
The extracts were screened for in vitro activity against Grampositive
bacteria: Staphylococcus aureus ATCC 6538, S. aureus
standard ATCC 43866 (MRSA), ATCC 700699 (Vancomycin
resistant S. aureus; VRSA) and ATCC 106760 (MRSA and VRSA),
Staphylococcus epidermidis ATCC 12228 and Enterococcus
faecalis CIP 104676; Gram-negative bacteria: Pseudomonas
aeruginosa CIP 9027, Klebsiella pneumoniae ATCC 9997,
Salmonella thyphimurium CIP 6062; M. smegmatis CIP 607 and the
yeast C. albicans ATCC 10231.
Plant extracts were dissolved in dimethyl sulfoxide (DMSO) to a
final concentration of 1 mg/ml. Concentrations of plant extracts
were used in a range of 500 to 0.9 μg/ml.
The antibacterial activity evaluation was performed by
determination of the minimum inhibitory concentration (MIC) by the
serial broth microdilution method as described by the National
Committee for Clinical Laboratory Standards (CLSI; 2008). The
inhibition of bacteria growth was evaluated by measuring the well´s
turbidimetry on absorvance microplate reader (ELX 808, Biotek) at
630 nm. Endpoint values were established according to Cos et al.
(2006a; 2006b) and samples with a MIC value ≤ 100 μg/ml were
considered to have antibacterial activity.
Phytochemical screening
To characterize the major compound classes present in the
extracts, preliminary phytochemical analysis was carried out
through thin layer chromatography (TLC) on silica gel. After a semiquantitative
application of the extracts, the TLC plates were
developed with appropriated mixtures of solvents. Spots were
revealed with the following spray-reagents: Dragendorff reagent for
alkaloids, anisaldehyde-sulfuric acid reagent for terpenes, Natural
Products-Polyethylene Glycol (NEU) reagent for flavonoids and fast

Table 2. Plant data and MIC values (µg/ml) of plant extracts.
Species extracts (% w/w)
(% w/w)
S. aureus
S.
epidermidis
E.
faecalis
MIC (µg/ml)
M.
smegmatis
K.
pneumoniae
S.
typhimurium
Lai et al. 2383
P.
aeruginosa
6538 MRSA VRSA MRSA/VRSA
A. anisochilus (aerial parts)
n-hexane (0.2) 62.0 125.0 30 15.0 125.0 125.0 125.0 250.0 250.0 250.0 250.0
CH2Cl2 (0.9) 62.0 125.0 125 15.0 125.0 125.0 125.0 250.0 250.0 250.0 125.0
AcOEt (0.3) 62.0 125.0 125 62.0 125.0 125.0 125.0 250.0 250.0 250.0 250.0
MeOH (7.7) 125.0 ⎯ ⎯ ⎯ 250.0 125.0 125.0 250.0 250.0 250.0 125.0
H2O (2.0) 30.0 125.0 125 125.0 125.0 125.0 125.0 250.0 250.0 250.0 250.0
A. anisochilus (fruits)
n-hexane (3.0) 62.0 30.0 30.0 15.0 125.0 125.0 62.0 250.0 250.0 250.0 125.0
CH2Cl2 (2.3) 62.0 62.0 30.0 62.0 250.0 125.0 125.0 250.0 250.0 250.0 125.0
AcOEt (0.5) 62.0 125.0 62.0 62.0 125.0 125.0 30.0 250.0 250.0 250.0 250.0
MeOH (3.3) 250.0 ⎯ ⎯ ⎯ 250.0 125.0 30.0 250.0 250.0 250.0 250.0
H2O (4.1) 30.0 125.0 125.0 62.0 250.0 125.0 30.0 250.0 250.0 250.0 250.0
E. lusitanica (leaves)
n-hexane (0.8) 125.0 125.0 ⎯ ⎯ 250.0 125.0 125.0 250.0 250.0 250.0 250.0
CH2Cl2 (2.3) 125.0 125.0 ⎯ ⎯ 62.0 125.0 125.0 250.0 250.0 250.0 62.0
AcOEt (1.7) 30.0 30.0 62.0 62.0 15.0 30.0 30.0 250.0 250.0 250.0 250.0
MeOH (6.4) 15.0 15.0 7.5 3.5 62.0 125.0 62.0 250.0 250.0 250.0 125.0
H2O (2.3) 15.0 15.0 15.0 7.5 125.0 125.0 62.0 250.0 250.0 250.0 250.0
L. stoechas subsp. luisieri
(aerial parts)
n-hexane (0.7) 62.0 125.0 62.0 125.0 125.0 62.0 62.0 62.0 250.0 250.0 62.0
CH2Cl2 (2.0) 62.0 125.0 62.0 62.0 125.0 125.0 30.0 250.0 250.0 250.0 250.0
AcOEt (1.6) 62.0 62.0 62.0 125.0 125.0 125.0 250.0 250.0 250.0 250.0 250.0
MeOH (2.5) 62.0 62.0 62.0 62.0 62.0 125.0 15.0 250.0 250.0 250.0 250.0
H2O (4.6) 62.0 62.0 62.0 250.0 250.0 62.0 30.0 250.0 250.0 250.0 62.0
P. broteroi (leaves)
n-hexane (1.6) 62.0 62.0 125.0 62.0 250.0 250.0 125.0 250.0 250.0 250.0 250.0
CH2Cl2 (1.6) 250.0 ⎯ ⎯ ⎯ 250.0 125.0 125.0 250.0 250.0 250.0 250.0
AcOEt (1.8) 1.9 1.9 15.0 7.5 30.0 62.0 62.0 250.0 250.0 250.0 250.0
C.
albicans

2384 J. Med. Plants Res.
Table 2. Contd.
MeOH (5.4) 15.0 3.8 3.8 1.9 15.0 62.0 62.0 250.0 250.0 250.0 250.0
H2O (1.8) 15.0 1.9 3.8 3.8 15.0 62.0 30.0 250.0 250.0 250.0 125.0
P. broteroi (fruits)
n-hexane (2.9) 125.0 ⎯ ⎯ ⎯ 250.0 250.0 250.0 250.0 250.0 250.0 250.0
CH2Cl2 (1.5) 62.0 62.0 30.0 30.0 250.0 125.0 62.0 250.0 250.0 250.0 250.0
AcOEt (1.1) 62.0 125.0 30 15.0 30.0 250.0 62.0 250.0 250.0 250.0 250.0
MeOH (10.9) 15.0 3.8 3.8 7.5 15.0 62.0 125.0 250.0 250.0 250.0 250.0
H2O (0.6) 62.0 1.9 7.5 7.5 15.0 125.0 15.0 250.0 250.0 250.0 125.0
Q. faginea subsp. broteroi
(leaves)
n-hexane (1.0) 250.0 ⎯ ⎯ ⎯ 250.0 125.0 62.0 250.0 250.0 250.0 250.0
CH2Cl2 (0.9) 62.0 125.0 62.0 15.0 250.0 125.0 125.0 250.0 250.0 250.0 250.0
AcOEt (0.9) 62.0 125.0 62.0 30.0 62.0 125.0 62.0 250.0 250.0 250.0 125.0
MeOH (0.4) 15.0 62.0 3.8 62.0 30.0 125.0 62.0 250.0 250.0 250.0 125.0
H2O (9.4) 15.0 125.0 3.8 62.0 30.0 125.0 62.0 250.0 250.0 250.0 125.0
R. officinalis (aerial parts)
n-hexane (2.4) 125.0 ⎯ ⎯ ⎯ 125.0 125.0 125.0 250.0 250.0 250.0 250.0
CH2Cl2 (3.1) 62.0 125.0 30.0 62.0 125.0 125.0 30.0 250.0 250.0 250.0 250.0
AcOEt (2.5) 30.0 62.0 15.0 62.0 30.0 30.0 30.0 250.0 250.0 250.0 250.0
MeOH (4.7) 62.0 125.0 30.0 62.0 125.0 125.0 62.0 250.0 250.0 250.0 250.0
H2O (6.9) 62.0 125.0 250.0 250.0 125.0 125.0 30.0 250.0 250.0 250.0 250.0
blue salt reagent for phenolic compounds, prepared
according to Wagner and Blader (1996). Results were
displayed semi-quantitatively in a range between absence
(−) and strongly present (+++).
RESULTS
A total of forty extracts prepared from six different
“montado” species were screened for their
antibacterial activity. The results are showed in
Table 2. The best results were obtained against
standard and multiresistant S. aureus strains.
80% of the crude extracts inhibit the development
of the S. aureus standard and from those, 30%
displayed low MIC values (

exhibiting the polar extracts of P. broteroi leaves and
fruits the lowest MIC (15 µg/ml). Similar results were
obtained when the E. faecalis was tested, being 20% of
the extracts actives with MIC values ranging from 30 to
62 µg/ml. The development M. smegmatis was inhibited
by 62% of the tested extracts (MIC 15 to 62 µg/ml), being
P. broteroi fruits water extracts the most active. With
exception of the L. stoechas subsp. luisieri n-hexane
extract (Table 2), no activity was found when Gramnegative
bacteria were assayed. The E. lusitana ethyl
acetate and the L. stoechas subsp. luisieri n-hexane and
water extracts were the only ones displaying moderate
activity against C. albicans growth.
P. broteroi leaves extracts (ethyl acetate, methanol and
water), P. broteroi fruits water and methanol and E.
lusitanica and R. officinalis ethyl acetate extracts
displayed the broadest antibacterial activity being active
against all the Gram-positive standard and multiresistant
tested bacteria and against M. smegmatis.
The phytochemical data are displayed in Table 3. The
analyzed plants are not rich in alkaloids, displaying A.
anisochilus vestigial amounts. P. broteroi (leaves), L.
stoechas subsp. luisieri, E. lusitanica, Q. faginea subsp.
broteroi and R. officinalis extracts have a high content in
flavonoids/phenolics. Terpenes were also detected in
some of the screened extracts.
DISCUSSION
The present study showed the potential use of “montado”
flora as a source of antimicrobial agents. Different
extracts of chosen “montado” species exhibited
antimicrobial activity against standard and resistant
microbial strains. Our results revealed that all plant
extracts exhibited stronger activities against Grampositive
bacteria and M. smegmatis. The Gram-negative
antibacterial activity was not significant. These bacteria
possess outer membrane that is highly hydrophobic,
providing these microorganisms with a permeability
barrier. This partially explains the greater resistance
observed by Gram-negative bacteria when exposed to
antibacterial drugs (Stavri et al., 2007) and to the plant
extracts tested herein. The antifungal activity against the
yeast C. albicans was also not significant, with the
exception of E. lusitanica ethyl acetate extract and L.
stoechas subsp. luisieri n-hexane and aqueous extracts,
which showed a moderate activity.
The relationship between the antimicrobial activity and
the phenolic and flavonoid compounds is well known and
this might be in part, responsible for the results showed
by the plants extracts (Cazarolli et al., 2008). Only the A.
anisochilus (aerial parts and fruits) ethyl acetate and
methanol extracts displays positive results for alkaloid
screening, which is in agreement with the data of the
Leguminosae family and of other Adenocarpus species
Lai et al. 2385
(Veen et al., 1992).
In some plant extracts, the high content of terpenes
should not be neglected and might also play an important
role in the inhibition of the bacterial development (Cowan,
1999). The two Labiatae species, L. stoechas subsp.
luisieri and R. officinalis are the richest in terpenes,
phenolics and flavonoids and so almost all their extracts
displayed moderated activity against the tested Grampositive
bacteria. In Lavandula genus, the essential oils
have different chemiotypes, being that L. stoechas subsp.
luisieri essential oils are very peculiar, possessing rare
necrodane derivatives (necrodane type) (González-
Coloma et al., 2006) to who are attributed some of its
biological activities. The mechanisms by which essential
oils can inhibit microorganisms may be related with their
hydrophobicity. Some of the components of the essential
oils act as membrane permeabilitzers (Nicolson et al.,
1999), making it more permeable to the uptake of the
antimicrobial agents, including antibiotics (Helander et al.,
1998).
R. officinalis, the other Labiatae included in our study,
is a very popular herb in the Mediterranean region.
Interesting results were obtained with the ethyl acetate
extract of this species, such as the antimicrobial activity
against S. aureus resistant to vancomycine. The
antibacterial activity of the R. officinalis leaves extracts
against highly drug-resistant Gram negative Bacilli was
previously mentioned (Abdel-Massih et al., 2010).
Oluwatuyi et al. (2004) report on the isolation of abietane
diterpenes on its aerial parts chloroform extract which
seemed to be responsible for the antibacterial activity
against strains of S. aureus possessing efflux
mechanisms of resistance.
Q. faginea subsp. broteroi leaves ethyl acetate and
methanolic extracts also displayed interesting results on
the antimicrobial assays. The phytochemical screening
revealed that those extracts have a high content in
phenolics and flavonoids. The presence of those
compounds is well known in Quercus genus and the
antibacterial activity has been reported in some species
(Andrensek et al., 2004), and could explain the MIC value
obtained in this work.
The most important results were obtained with the P.
broteroi (both leaves and fruits) and E. lusitanica more
polar extracts (ethyl acetate, methanol and water).
Almost all of those extracts displayed very low MIC
values against all the Staphylococcus strains. A synergic
effect between the terpenes, phenolics and flavonoids is
probably related with the antibacterial activity of these
extracts.
In conclusion, our work revealed that several extracts
of the chosen “montado” species displayed very low MIC
values against a large panel of Gram-positive bacteria.
The activity on different microorganism seems to depend
on the extract chemical profile but in general the activity
increases with the polarity of the extracts. Further

2386 J. Med. Plants Res.
Table 3. Qualitative evaluation of extracts chemical composition.
Plants extracts Terpenes Phenolics Flavonoids Alkaloids
A. anisochilus (aerial parts)
n-hexane +++ + ++ -
CH2Cl2 + + - -
AcOEt + + + +
MeOH + - - +
H2O - + - -
A. anisochilus (fruits)
n-hexane +++ + ++ -
CH2Cl2 + + - -
AcOEt + + + +
MeOH ++ + - +
H2O - + - -
E. lusitanica (leaves)
n-hexane +++ ++ ++ -
CH2Cl2 ++ +++ - -
AcOEt ++ + +++ -
MeOH + + +++ -
H2O - - ++ -
L. stoechas subsp. luisieri (aerial parts)
n-hexane +++ ++ ++ -
CH2Cl2 +++ ++ ++ -
AcOEt +++ ++ + -
MeOH ++ ++ +++ -
H2O ++ ++ + -
P. broteroi (leaves)
n-hexane ++ ++ +++ -
CH2Cl2 ++ ++ +++ -
AcOEt ++ + ++ -
MeOH ++ +++ ++ -
H2O ++ + ++ -
P. broteroi (fruits)
n-hexane +++ + ++ -
CH2Cl2 ++ - - -
AcOEt +++ +++ + -
MeOH + + + -
H2O - + + -
Q. faginea subsp. broteroi (leaves)
n-hexane ++ + - -
CH2Cl2 ++ + - -
AcOEt ++ +++ +++ -
MeOH ++ +++ ++ -
H2O + + + -
R. officinalis (aerial parts)
n-hexane + + + -
CH2Cl2 ++ ++ +++ -

Table 3. Contd.
AcOEt ++ ++ ++ -
MeOH + + + -
H2O + ++ - -
experiments will be necessary to assess the potential of
some species, including E. lusitanica and P. broteroi,
which seems to be quite interesting species being worthy
of a detailed phytochemical characterization.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2388-2395, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.1007
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Genetic diversity and population structure of
Dactylorhiza hatagirea (Orchidaceae) in cold desert
Ladakh region of India.
Ashish R. Warghat 1 *, Prabodh K. Bajpai 1 , Ashutosh A. Murkute 2 , Hemant Sood 3 , Om P.
Chaurasia 1 and Ravi B. Srivastava 1
1 Defence Institute of High Altitude Research, DRDO, Leh-Ladakh, India, 194101.
2 Directorate of Onion and Garlic Research, ICAR, Pune, India, 410505.
3 Jaypee University of Information Technology, Waknaghat, Solan, India, 173215.
Accepted 24 February, 2012
Random amplified polymorphic DNA (RAPD) analysis was used to characterize the genetic diversity
and population genetic structure within and among nine natural populations of Dactylorhiza hatagirea,
a critically endangered or rare terrestrial medicinal orchid in cold desert of Ladakh region. Out of the
177 bands generated from twenty random primers, 174 were polymorphic. The genetic diversity of D.
hatagirea that was revealed by observed number of alleles (Na), expected number of alleles (Ne), Nei’s
diversity index (H), Shannon’s diversity index (I), amplificated loci, polymorphic loci and the percentage
of polymorphic loci (PPL). Pair-wise population genetic distances ranged from 0.05 to 0.23. Analysis of
molecular variance (AMOVA) revealed that 57% RAPD of variability was partitioned among population.
The Principal coordinate’s analysis (PCoA) and UPGMA supported the grouping of all 96 accessions of
nine populations into four cluster groups. However, a moderate genetic differentiation among
population was detected based on different measures (Nei’s genetic diversity analysis: Gst = 0.2538;
AMOVA analysis: Fst = 0.254)
Key words: Conservation biology,endangered orchid, genetic diversity, genetic structure.
INTRODUCTION
Dactylorhiza hatagirea (D.don) (Family: Orchidaceae) is a
terrestrial ground dwelling perennial herb, the stem is
erect, hollow and obtuse, and bears palmately lobed and
lanceolate leaves with sheathing leaf base. The
cylindrical and terminal spike bears rosy purple flowers
with green bracts. Flowers are 1.7 to 1.9 cm long with
curved spur. The inflorescence consists of a compact
raceme with 25 to 50 flowers developed from axillary
buds. The dark purple spotted lip of the flower is rounded
and lobed (1 to 5). The plants store a large amount of
water in their tuberous roots to survive in arid conditions
(Chaurasia et al., 2007).
Nowadays, more and more species have become
threatened or extinct in the wild or in natural habitat, and
plant conservations concerned with rare and endangered
*Corresponding author. ashishwarghat@hotmail.com.
species are faced with an intimidating task (Holsinger and
Gottlieb, 1991). The maintenance of genetic variation is
one of the major objectives for conserving endangered
and threatened species (Avise and Hamrick, 1996). For
the management strategies, Markers are now routinely
used to characterize genetic variation among and within
populations which contribute towards conservation
programme concerned with the critically endangered
species (Milligan et al., 1994). According to our survey on
this species, it is estimated that not more than 1000
individuals survived in habitat. At present, nine
populations were studied but some of the populations rise
towards declined stage. The threats to the species are
related to certain anthropogenic activities as well as
habitat destruction. In addition, population number and
size are declining at an alarming rate in the last decade
and genetic diversity is likely to be reduced. Considering
the populations number, size and particular distribution of
this critically endangered species, D. hatagirea should be

Figure 1. Map of Ladakh region of India.
assigned in a high priority for their preservation and
protection. In this study, random amplified polymorphic
DNA (RAPD) markers were used to detect variation at
the population level in samples collected from Ladakh
regions of India. The purpose of this study is to
investigate the levels of variation among populations of
Dactylorhiza hatagirea.
MATERIALS AND METHODS
Study sites and sampling
Nine populations from Ladakh regions were surveyed (Figure 1).
Young leaf tissues of 96 individuals of leaves were selected
randomly for molecular analysis. The number of samples taken in
each population was depending on geographic distribution (Table
1). The sampling stratagem was to trace several sites in different
parts of the investigated area in order to cover diverse growing
habitats which represent the richly capable yet relatively stable
primitive environment in the sampled regions.
RAPD analysis
Warghat et al. 2389
Total genomic DNA was extracted from Dactylorhiza leaves using a
cetyltrimethylammonium bromide (CTAB) method (Doyle and
Doyle, 1987) with minor modifications. The quantity and quality of
isolated total genomic DNA was determined using 0.8% agarose
gel electrophoresis in 0.5 × TAE buffer for mobility related to known
concentrations of lambda DNA. Twenty random decamer primers
from integrated device technology (IDT) Tech. USA (Table 2) were
used for RAPD amplification following the protocol of Williams et al.
(1990). Amplification reactions were performed in volumes of 17 µL
containing 10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl2, 50 mM KCl,
200 µM of each deoxynucleotide triphosphates (dNTPs), 0.4 µM
primer, 20 ng template DNA and 0.5 unit of Taq polymerase
(Sigma-Aldrich, USA) with the following program: initial denaturation
at 94°C for 5 min, followed by 1 min denaturation at 94°C, 1 min
denaturation at specific annealing temperature (37°C), 1 min
extension at 72°C for 39 cycles, and 5 min at 72°C for a final
extension.
Amplification product were electrophoresed on 2.0% agarose gel
(Life Science technologies, USA) and run at constant voltage (80V)
in 1× TAE for 3 h, visualized by staining with ethidium bromide (0.5
µg ml -1 ) and a total of 2.5 µl loading buffer (6x) was added to

2390 J. Med. Plants Res.
Table 1. Geographic localities and sample sizes of naturally distributed D. hatagirea.
S/N Population name Population code Longitude Latitude Altitude (ft) Sample sizes
1 Bogdang P N34°48'.198 E77°02'.453 9240 ± 25.8 10
2 Skampuk Q N34°35'.238 E77°34'.481 10490 ± 17.4 15
3 Skurru R N34°40'.229 E77°18'.031 10295 ± 20.8 15
4 Hunder S N34°35'.043 E 77°28'.592 10357 ± 18.0 10
5 Turtuk T N34°50'.849 E76°49'.720 9240 ± 25.8 10
6 Tirith U N34°32'.378 E77°38'.481 10443 ± 26.9 10
7 Sumur V N34°31'.128 E77°34'.481 10120 ± 12.7 10
8 Changlung W N34°55'.884 E77°28'.276 10982 ± 39.7 10
9 Staksha X N34°55'.885 E77°28'.276 11081 ± 49.2 6
Table 2. List of primers used for RAPD amplification.
Primer
Primer sequence
(5’ 3’)
Total number of fragments amplified Percentage of polymorphic loci Resolving power
S21 CAGGCCCTT C 597 100 12.44
S22 TGCCGAGCT G 545 100 11.35
S23 AGTCAGCCA C 341 100 7.10
S24 AATCAGCCA C 566 88.9 11.79
S25 AGGGGTCTT G 508 100 10.58
S26 GGTCCCTGA C 530 100 11.04
S27 GAAACGGGTG 423 100 8.81
S28 GTGACGTAG G 512 80 10.67
S29 GGGTAACGC C 362 100 7.54
S30 GTGATCGCA G 287 100 5.98
S31 CAATCGCCG T 485 100 10.10
S32 TCGGCGATA G 410 100 8.54
S33 CAGCACCCA C 558 100 11.63
S34 TCTGTGCTG G 455 100 9.48
S35 TTCCGAACC C 476 100 9.92
S36 AGCCAGCGA A 310 100 6.46
S37 GACCGCTTG T 459 100 9.56
S38 AGGTGACCG T 511 100 10.65
S39 CAAACGTCG G 380 100 7.91
S40 GTTGCGATC C 430 100 8.96
Total 9145 98.30 -
each reaction before electrophoresis. After electrophoresis, the gels
were documented on a gel documentation system (Alpha Innotech,
Alphaimager, USA). Molecular size of the amplicon was estimated
using 100 and 500 bp DNA ladders (‘Bangalore Genei.India’).
Data analysis
The resulting presence/absence binary data matrix was analyzed
using POPGENE version 1.31 (Yeh et al., 1999). Jaccard’s
similarity coefficient (J) was used to calculate genetic similarity
between pair of individuals. The similarity matrix was subjected to
cluster analysis by unweighted pair group method with arithmetic
mean (UPGMA) using the SAHN clustering module and
dendrogram was generated using the program NTSYS-pc software
ver. 2.02 (Rohlf, 1992). POPGENE software version 1.31 was used
to describe the structure of studied populations with their
geographic location. Observed number of alleles (Na), effective
number of alleles (Ne), Nei’s genetic diversity (H), Shannon’s
information index (I), number of polymorphic loci (NPL) and
percentage polymorphic loci (PPL) across were analyzed (Zhao et
al., 2006).
The non-parametric analysis of molecular variation (AMOVA)
(Excoffier et al., 1992) was performed using squared Euclidean
distances among all samples to partition the variation into two
hierarchical levels; individual and population. GenAlEx software
was used to calculate a principal coordinates analysis (PCA) that
plots the relationship between distance matrix elements based on
their first two principal coordinates (Peakall and Smouse, 2001).
According to Prevost and Wilkinson (1999) the resolving power (Rp)
of a primer is: Rp = Σ IB where IB (band informativeness) takes the
value of: 1–[2* (0.5–P)], P being the proportion of the 96 genotypes

Warghat et al. 2391
Table 3. Summary of genetic variation statistics for all loci of RAPD among the Dactylorhiza populations with respect to their
distribution.
Markers Sampling sites Sample size H (mean + SD) I (mean + SD) PPL
Bogdang 10 0.1184 ± 0.1706 0.1820 ± 0.2528 36.72
Skampuk 15 0.2346 ± 0.1647 0.3673 ± 0.2284 81.36
Skurru 15 0.1633 ± 0.1869 0.2499 ± 0.2704 51.41
Hunder 10 0.1925 ± 0.1791 0.2977 ± 0.2601 61.58
RAPD Turtuk 10 0.2216 ± 0.1909 0.3629 ± 0.2716 68.36
Tirith 10 0.2036 ± 0.1873 0.3109 ± 0.2689 62.15
Sumur 10 0.2068 ± 0.1884 0.3151 ± 0.2700 62.71
Changlung 10 0.1736 ± 0.1854 0.2666 ± 0.2679 54.80
Staksha 6 0.1375 ± 0.1881 0.2055 ± 0.2749 37.29
Mean 0.185767 0.284211 -
H= Nei’s genetic diversity, I=Shannon’s information index, PPL= percentage of polymorphic loci.
Table 4. Inter-population genetic distances calculated by Nei’s method.
Population Staksha Changlung Sumur Tirith Turtuk Hunder Skurru Skampuk Bogdang
Staksha **** 0.103 0.079 0.089 0.160 0.102 0.174 0.150 0.224
Changlung 0.118 **** 0.053 0.036 0.065 0.092 0.126 0.077 0.137
Sumur 0.095 0.068 **** 0.044 0.081 0.058 0.086 0.061 0.108
Tirith 0.105
a
0.051 0.060 **** 0.062 0.079 0.101 0.066 0.112
Turtuk 0.177 0.081 0.098 0.079 **** 0.090 0.118
a
0.051 0.129
Hunder 0.119 0.107 0.076 0.096 0.108 **** 0.084 0.067 0.135
Skurru 0.186 0.137 0.099 0.113 0.131 0.096 **** 0.042 0.072
Skampuk 0.166 0.092 0.077 0.082 0.068 0.084 0.053 **** 0.063
Bogdang
a
0.236 0.148 0.121 0.124 0.142 0.148 0.080 0.075 ****
Above diagonal values are Nei’s unbiased genetic distances, those below the diagonal are Nei’s genetic distances. a values in bold are
maximum or minimum genetic distances.
containing the band. Nei’s analysis of gene diversity among
population (Nei, 1978) was carried out with counting total genetic
diversity (Ht), within species diversity (Hs), genetic diversity
between populations (Gst) and estimation of gene flow (Nm) from
parameters. Fst index (Wright, 1951) was measured via this formula
(Lynch and Milligan, 1994).
RESULTS
A total of 177 reproducible bands were produced using
the 20 RAPD primers (8.8 Bands per primer) of which
174 were polymorphic (PPL= 98.30%). RAPD genetic
diversity analysis revealed the highest values of Nei’s
genetic diversity (0.23), Shannon information index (0.36)
and polymorphic loci (81.36%) among accession from
Skampuk population and lowest values of Nei’s genetic
diversity (0.11), Shannon information index (0.18) and
polymorphic loci (36.72 %) among accession from
Bogdang population (Table 3). Nei’s (1978) classified
levels of genetic distance at < 0.05 as low, between 0.05
and 0.15 as medium and > 0.15 as high. Thus, the
Bogdang accession varied in narrow range while, the
Skampuk accessions were more diverse. Pair-wise Nei’s
distances (Nei’s, 1973) were calculated for all
populations. The greatest inter-population average
distance (0.23) was between Bogdang and Staksha.
While, the corresponding least distance (0.05) was
between Skampuk and Turtuk (Table 4). The dendrogram
was obtained from UPGMA cluster analysis based on
RAPD data was presented in (Figure 2). The dendrogram
showed four main clusters (I, II, III and IV). The cluster I
consisted of samples from populations Bogdang,
Skampuk and Skurru. Cluster II consisted of samples of
population Tirith, Sumur and Changlung. Cluster III
consisted of Hunder and some populations of Tirith,
Sumur, Skampuk, Skurru and Turtuk. cluster IV and
consisted of Staksha and some population of Skampuk,
Skurru, and Turtuk.
Population genetic structure
Genetic analysis of RAPD marker showed that the
highest genetic identity (0.964) existed between

P1 0 M W
2392 J. Med. Plants Res.
0.07 027 0.27 048 0.48
Coefficient
068 0.68 089 0.89
007
Figure 2. Dendrogram of 96 individuals of nine population of D. hatagirea based on UPGMA analysis of RAPD polymorphisms.
populations Changlung and Tirith. While, the lowest
(0.790) occurred between the population of Bogdang and
that of Staksha (Table 5). The Genetic differentiation
among populations (Gst) was, estimated to be 0.2538,
which indicated that 25.38% of the genetic variability was
distributed among populations (Table 6). The level of
gene flow (Nm) was estimated to be 1.4324 individual per
generation between populations. According to results of
AMOVA analysis, there were highly significant (P

Table 5. Inter-Population genetic identity calculated by Nei’s method.
Warghat et al. 2393
Population Staksha Changlung Sumur Tirith Turtuk Hunder Skurru Skampuk Bogdang
Staksha **** 0.902 0.924 0.915 0.852 0.903 0.840 0.861 0.799
Changlung 0.889 **** 0.948
a
0.964 0.937 0.913 0.881 0.925 0.872
Sumur 0.909 0.934 **** 0.957 0.923 0.943 0.917 0.941 0.897
Tirith 0.900 0.950 0.941 **** 0.940 0.924 0.904 0.936 0.894
Turtuk 0.838 0.923 0.906 0.924 **** 0.914 0.889 0.951 0.879
Hunder 0.888 0.899 0.927 0.909 0.898 **** 0.920 0.935 0.874
Skurru 0.830 0.872 0.906 0.893 0.877 0.908 **** 0.959 0.931
Skampuk 0.847 0.912 0.925 0.921 0.935 0.920 0.948 **** 0.939
Bogdang
a
0.790 0.862 0.886 0.883 0.867 0.863 0.923 0.928 ****
Above diagonal values are Nei’s unbiased genetic identities, those below the diagonal are Nei’s genetic identities . a values in bold are maximum or minimum genetic identities.
Table 6. Overall genetic variability across all the 96 accessions of Dactylorhiza based on RAPD analysis.
Marker H I Ht Hs Gst NPL PPL Fst Nm
RAPD 0.2535 ± 0.1649 0.3984 ± 0.2106 0.2535 ± 0.027 0.1892 ± 0.015 0.2538 174 98.30 0.254 1.4324
H = Nei’s genetic diversity, I = Shannon’s information index, PPL= percentage of polymorphic loci, Ht = total genetic diversity, Hs = population diversity; Gst = gene differentiation, Fst
= Wright inbreeding coefficient, Nm = gene flow.
D. hatagirea. Of the total genetic diversity, 57%
was attributed to be among populations while the
rest 43% was attributed to the differences within
Table 7. AMOVA analysis based on RAPD among the populations of Dactylorhiza.
Source of Variation Among population Within population Total
d.f. 8 87 95
Marker RAPD
S.S.D. 46.917 34.000 80.917
Variance component 0.517 0.391 0.908
Percentage 57% 43% 100 %
P-value < 0.001 < 0.001 < 0.001
Where d.f.= degree of freedom, S.S.D.= sum of square deviation, P-value = probability of null distribution.
populations (Table 7). The positioning of 96
samples based on principal coordinates (PCO)
analysis is shown in Figure 3. The scatter plot
showed mixed population of D. hatagirea, which
was consisted with the results of dendrogram
analysis.

2394 J. Med. Plants Res.
DISCUSSION
Genetic diversity
Principal coordinates
Figure 3. Two dimensional plot of Principal component analysis of nine population of Dactylorhiza using
RAPD analysis.
According to Hamrick and Godt (1989), there is strong
relationship between geographic range and genetic
diversity. There is evidence that a low level of genetic
diversity is a common feature of endangered endemic
plant species (Gitzendanner and Soltis, 2000). D.
hatagirea is a critically endangered, self compatible and
terrestrial orchid. Being terrestrial orchid, populations are
small and spatially isolated. According to population
genetics predict that population of such species, affected
by a number of evolutionary factors including mating
system, gene flow, founder effect, random genetic drift
and seed dispersal (Hamrick and Godt, 1989). We
compared D. hatagirea and this hypothesis with related
genus Gymnadenia which is also an endangered and
endemic species (Mi Yoon Chung, 2009) and our result
of RAPD analysis shows that low level of genetic diversity
within populations and remarkable genetic differentiation
among populations in D. hatagirea. In our field survey,
every year large amount of population of D. hatagirea was
destroyed because of increasing mating opportunity and
habitat fragmentation. Thus, habitat fragmentation under
human disturbance is an important factor leading to low
genetic diversity within population of D. hatagirea. So the
low rate of natural recruitment observed today, together
with increased habitat fragmentation and isolation of
population, is seriously contributing to the low level of
genetic diversity.
Genetic structure
The overall degree of genetic differentiation of D.
hatagirea, as estimated by Gst (0.2587) is slightly higher
than out crossing plants (0.23). According to an AMOVA
analysis, a significantly genetically differentiation among
populations was estimated (Fst = 0.259), where most
genetic variation existed among populations. The genetic
structure of plant populations reflects the interaction of
various factors, including long–term evolutionary history
of the species, genetic drift, mating system and gene flow
(Hogbin and Peakall, 1999). If populations are small and
isolated from one another, the genetic drift could be
capable of influencing the genetic structure and
increasing differentiation among populations (Ellstrand
and Elam, 1993). The gene flow (Nm, 1.4703) of D.
hatagirea per generation was also slightly higher than
outcrossed animal pollinated species (Nm = 1.154) could
be due two geographic barrier (Nubra and Shyok river)
and different environmental conditions.
In this study, we investigated variability among
populations. Based on RAPD analysis, we revealed low
genetic variation within the population and moderate
genetic differentiation among the population. Therefore,
every population deserves specific conservation
attention, as habitat destruction and management for
conservation will have greater effects on population
richness.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2396-2401, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.1190
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
In vitro antioxidant capacities of rice residue
hydrolysates from fermented broth of five mold strains
Wei Tian 1 , Qinlu Lin 1,2* and Gao-Qiang Liu 2,3*
1 College of Food Science and Technology, Hunan Agricultural University, Changsha 410128, P. R. China.
2 National Engineering Laboratory for Rice and By-product Further Processing, Central South University of Forestry and
Technology, Changsha 410004, P. R. China.
3 College of Life Science and Technology, Central South University of Forestry and Technology, Changsha 410004,
P. R. China.
Accepted 22 February, 2012
Rice residue was fermented by five different strains of molds, namely, Aspergillus oryzae, Mucor
racemosus, Rhizopus oligosporrus, Aspergillium niger and Penicillium glaucum. Antioxidant activities
of the fermented products, rice residue hydrolysates (RRHs) were evaluated using ferric reducing
antioxidant power (FRAP) assay, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and 2,2′-azinobis (3ethylbenzothiazoline-6-sulfonic
acid) diammonium salt (ABTS) assay, respectively. Among five types of
RRHs, RRHs from fermented broth of A. niger (RRHsIV) exhibited higher antioxidant potential than
those of other RRHs in the same concentration level, regardless of the applied assays. Moreover, there
was no correlation between reducing power and total phenonic contents in the five RRHs. However,
FRAP values were highly correlated with phenol contents.
Key words: Rice residue, hydrolysates, antioxidant capacity, fermentation.
INTRODUCTION
Reactive oxygen species (ROS) including oxygencentered
radicals and some non-radical derivatives of
oxygen cause oxidative stress to cells. Oxidative stress
can be defined as an imbalance between pro-oxidant/free
radical production and opposing antioxidant defenses. It
has been reported that acute and chronic oxidatives
stress implicated in degenerative diseases, such as
athrosclerosis, diabetes mellitus, ischemia/reperfusion
injury, Alzheimer‟s disease, inflammatory diseases,
carcinogenesis, neurodegenerative diseases,
hypertension, ocular diseases, pulmonary diseases and
hematological diseases (Maxwell, 1995; Opara,
2004).ROS can be scavenged by exogenously obtained
antioxidants, which include synthetic antioxidants and
natural antioxidants.
The use of synthetic antioxidants, because of their
potential health hazard and toxicity, is under strict
regulation. Thus, there is recently an upsurge of interest
*Corresponding author. E-mail: gaoliuedu@yahoo.com.cn.
Tel/Fax: +86 731 8562 3498.
in antioxidative compounds in many natural resources.
Hydrolysates derived from dietary source have been
demonstrated to possess potent antioxidant capacity. It is
likely due to an array of antioxidative components (low
molecular peptides, oligosaccharides and Maillard
reaction products) formed during hydrolytic process
(Mendis et al., 2005; Wang et al., 2010; Dittrich et al.,
2003).
Rice residue is the by-product of rice in the processing
of starch sugar and monosodium glutamate, which
contains more than 50% protein as well as 40%
polysaccharide and dextrin, according to our previous
determination. Whereas, it is usually used as an animal
feed without efficient utilization in China. Microbial
sources have been shown to be a potential means of
producing natural antioxidants (Ishikawa, 1992). It has
been reported that many molds produced antioxidants
that can be extracted from broth culture filtrates by ethyl
acetate (Yen and Lee, 1996). Yen et al. (2003) reported
that antioxidant activity of ethyl acetate extracts from rice
koji showed a marked antioxidants activity on radical
scavenging effect and inhibitory effect on peroxidation of
linoleic. Due to its abundant nitrogen and carbon sources,

ice residue is an ideal raw material for producing
antioxidant hydrolysates by fermentation. To the best of
our knowledge, so far, there is no report on antioxidant
substances from rice residue hydrolysates (RRHs)
fermented with molds.
In this study, RRHs were produced during fermentation
by five different strains of molds, namely, Aspergillus
oryzae, Mucor racemosus, Rhizopus oligosporrus,
Aspergillium niger and Penicillium glaucum. Furthermore,
we investigated the total antioxidant capacity of
fermented prepared RRHs via 1,1-diphenyl-2picrylhydrazyl
(DPPH) assay, 2,2′-azinobis (3ethylbenzothiazoline-6-sulfonic
acid) diammonium salt
(ABTS) assay and ferric reducing antioxidant power
(FRAP) assay. In addition, we evaluated relationship
between RRHs concentration and antioxidant potency.
The objective of the present study was the in vitro
antioxidant capacities of different RRHs fermented with
different strains of molds. The obtained information will be
helpful not only in optimization of fermentation process
for further study, but also in providing a new way to
improve the utilization value of rice residue.
MATERIALS AND METHODS
Rice residue is industrially produced by Hunan JinJian Cereals
Industry Co., Ltd (Hunan, China). A. oryzae, M. racemosus, R.
oligosporrus, A. niger and P. glaucum were purchased from
Institute of Microbiology Chinese Academy of Science (Beijing,
China).
The 6-hydroxy-2,5,7, 8-tetramethylchroman-2-carboxylic acid
(Trolox), ABTS, 2,4, 6-tripyridyls-triazine (TPTZ) and DPPH were
purchased from Sigma Chemical Co. (St. Louis, MO, USA). All
other chemicals and reagents were of analytical grade.
Preparation of RRHs
The five different strains of molds were maintained in solid potato
dextrose agar medium at 4°C, respectively. For primary
fermentation, 14 ± 0.5 g rice residues were mixed with 14 ± 0.5 ml
distilled water. After sterilization at 121°C for 30 min, the mixture
was inoculated by A. oryzae, M. racemosus, R. oligosporrus, A.
niger and P. glaucum as seed mediums, respectively. Fermentation
was carried out at 28 ± 0.5°C for 7 days.
For secondary fermentation, 150 ml sterile water was added into
five sorts of seed mediums previously mentioned, respectively.
Then, all the samples were incubated at 42 ± 0.5°C, in an electroheating
standing-temperature cultivator for 7 days. After secondary
fermentation, the fermented broth was centrifuged at 4000 g, 10
min and the supernatant was dried to powder by freeze
dehydration, which was for usage of analysis.
Determination of total phenolic compounds
The content of total phenolic compounds of RRHs fermented by five
different strains of molds was determined according to the method
described by Singleton et al. (1999).
Briefly, 0.5 ml of RRHs solution was transferred to a volumetric
flask and the volume was adjusted to 46 ml by addition of distilled
water. One milliliter (1 ml)
Tian et al. 2397
of Folin-Ciocalteu reagent was then added to this mixture. After 3
min, 3 ml of sodium carbonate solution (20 g/l) was added to the
volumetric flask. Subsequently, the mixture was shaken
mechanically for 2 h at room temperature. The absorbance was
measured at 760 nm using a ultraviolet (UV)-vis spectrophotometer.
The content of total phenolic compounds of RRHs fermented by five
different strains of molds was calculated using a standard curve
prepared with gallic acid.
Determination of degree of hydrolysis (DH)
DH is operationally defined as the percentage of free N-terminal
amino groups cleaved from proteins, which is calculated from the
ratio of α–amino nitrogen to total nitrogen (You et al., 2010).
According to the method of Nilsang et al. (2005), the amino nitrogen
content was determined by a formaldehyde titration method. The
total protein content was determined by Keldahl method (Chandi
and Sogi 2007). The equation of DH is shown as follows:
DH = [Amino nitrogen content (mg/ml) / total protein content
(mg/ml)] × 100
Antioxidant activities of RRHs
FRAP assay
The FRAP assay was carried out according to the procedure of
Benzie and Strain (1996). FRAP assay measures the change in
absorbance at 593 nm owing to the formation of a blue colored
Fe(II) -tripyridyltriazine compound from colorless oxidized Fe(III)
form by the action of electron donating antioxidants. Briefly, the
FRAP reagent was prepared from acetate buffer (0.3 mol/L, pH
3.6), 10 mmol/L TPTZ solution in 40 mmol/L HCl and 20 mmol/L
iron(III) chloride solution in proportions of 10: 1:1 (v/v), respectively.
3 ml of working FRAP reagent prepared daily was mixed with 100 ul
of diluted sample; the reaction mixture was recorded at 593 nm,
after 30 min incubation at 37°C. The standard curve was depicted
using iron(II) sulfate solution. FRAP values were expressed as
mmol of Fe(II) equivalents/kg. All the measurements were carried
out in triplicate and the mean values were calculated.
DPPH assay
The DPPH radical scavenging activity of RRHs was determined
using the method proposed by Von Gadow et al. (1997). Aliquot
(100 ul) of the tested RRHs sample was placed in a cuvette, and 2
ml of 0.1 mmol/L methanolic solution of DPPH radical was added.
Absorbance measurements commenced immediately. The
decrease in absorbance at 517 nm was determined after 15 min for
all samples. Methanol was used to zero spectrophotometer. The
absorbance of the DPPH radical without antioxidant (control) was
measured daily. Methanolic solutions of Trolox were tested. All
determinations were performed in triplicate. The percentage
inhibition of the DPPH radical by the samples was calculated
according to the formula of Yen and Duh (1994):
% inhibition = [(AC (0) )-AA (t) /AC (0) ] × 100
Where AC (0) is the absorbance of the control at t = 0 min, AA (t) is the
absorbance of the antioxidant at t = 15 min
ABTS assay
The method is based on the ability of antioxidant molecules to

2398 J. Med. Plants Res.
Table 1. Total phenonic compounds, degree of hydrolysis and FRAP value.
RRHs Frap Value (umol/g) DH Total Phenonic Content GAE (ug/g)
RRHsI 15.96 ± 0.30 46.69 ± 0.60 63.13 ± 0.84
RRHsII 12.54 ± 0.17 45.31 ± 0.70 60.14 ± 0.23
RRHsIII 20.74 ± 0.80 45.54 ± 0.13 66.01 ± 0.55
RRHsIV 34.01 ± 0.22 49.07 ± 0.15 56.62 ± 0.36
RRHsV 9.87 ± 0.45 38.17 ± 0.02 50.11 ± 0.47
RRHsI, RRHsII, RRHsIII, RRHsIV and RRHsV represent that RRHs are fermented by A. oryzae, R.
oligosporrus, M. racemosus, A. niger and P. glaucum, respectively. Each value is expressed as mean ±
standard deviation (n = 3).
quench the long-lived ABTS radical cation (ABTS· + ), a blue-green
chromophore with characteristic absorption at 734 nm. The addition
of antioxidants to the preformed radical cation reduces it to ABTS,
determining a decolorization. A stable stock solution of ABTS· + was
produced by reacting a 7 mmol/L aqueous solution of ABTS with
2.45 mmol/L potassium persulfate (final concentration) and allowing
the mixture to stand in the dark at room temperature for 12 to 16 h
before use. At the beginning of the analysis day, an ABTS· + working
solution was obtained by the dilution in ethanol of the stock solution
to an AC of 0.70 ± 0.02 AU at 734 nm (Pellegrini et al., 2003). All the
tests were conducted in triplicate. The percentage inhibition of the
ABTS radical by the samples was calculated according to the
formula:
% inhibition= [(A C ) – A A /A C ] × 100
Where A C is the absorbance of the control, A A is the absorbance of
the antioxidant sample.
Statistical analysis
All the data were express as means ± standard deviations (SD)
from three independent replicates. Results were evaluated by
analysis of variance (ANOVA) with MINTAB 16. Difference was
considered significant when P-value was < 0.05.
RESULTS AND DISCUSSION
Total phenolic compounds
It has long been reported that natural phytochemicals in
fruits and vegetables have antioxidant activity. Among
those substances, the phenolic compounds widely
distributed in fruits and vegetables have the ability to
scavenge free radicals, superoxide and hydroxyl radicals
by single-electron transfer (Li et al., 2005). The total
phenonic contents of RRHs are shown in Table 1.
Among the five different RRHs, RRHsIII displayed the
highest total phenonic content (P < 0.05), followed by
RRHs I, RRHsII, RRHIV and RRHsV. Hence RRHsIII had
higher reducing power than RRHs I, RRHsII and RRHsV.
Interestingly, the total phenolic content of RRHsIV was
lower, whereas its reducing power (FRAP value = 34.01 ±
0.22; P < 0.05) was highest for all the RRHs. Therefore,
there was no correlation (R 2 = 2.6%) between reducing
power and total phenonic content in the five RRHs.
Although, some researchers reported that FRAP value
were highly correlated with phenol content (Rodríguez et
al., 2010; Wang and Lin 2000), our result is in agreement
with many other studies. Sun and Ho, 2005) reported that
there was no correlation found between the AAC value
tested by the bcarotene bleaching method and total
phenolics content, nor between the antioxidant activity
tested by the Rancimat method and total phenolics
content. Free phenolic compounds and antioxidant
capacity in some of vegetables exhibited a positive, but
not very strong (Chu et al., 2002). The ambiguous
relationship between phenolic compounds and
antioxidant activity can be explained as follows: (1)
phenolic compounds cannot include all the antioxidants
(Kahkonen et al., 2001), the antioxidant activity observed
could possibly be due to existence of some other
antioxidants in RRHs, such as low molecular peptides,
oligosaccharides, organic acid and Maillard reaction
products, etc. (2) different method to measure antioxidant
activity with various mechanisms may lead to different
observations. Five different RRHs may contain various
complex antioxidant components, which have different
antioxidant potency.
Antioxidant activities of RRHs
Determination by FRAP assay
The FRAP assay is simple, precise, sensitive and
inexpensive, and gives fast and reproducible results. It is
widely used for evaluating both individual antioxidants
and their mixtures. The reducing power of five types of
RRHs determined at 593 nm is shown in Figure 1. In this
assay, the higher optical density (OD) value at 593 nm
indicated the higher reducing power. As shown in Figure
1, the concentration-dependent profile of reducing power
was obvious for all the tested RRHs. Regarding the five
RRHs, RRHsIV showed the highest activity apparently (P
< 0.05), followed by RRHsIII, RRHsI, RRHsII and
RRHsV. As we know, A. niger is one of important strain
of molds in fermentation industry, and is used today in
various industrial processes for the manufacture of citric
acid (Schreferl-Kunar et al., 1989; Kurbanoglu and
Kurbanoglu 2004). It has been reported that citric acid is

DPPH• scavenging ratio(%)
OD value at 593nm
Figure 1. Reducing power of five typs of RRHs. Each value is the mean ± SD
of triplicate measurements.
70
60
50
40
30
20
10
0.7
0.6
0.5
0.4
0.3
0.2
0.1
Figure 2. DPPH redical scavenging of five typs of RRHs. Each value is the mean
± SD of triplicate measurements.
a kind of metal chelator, which deactivate trace metals
that are free or salts of fatty acids by the formation of
complex ions or coordination compounds, thereby
terminate radical chain reactions. Lee and Lee (2002)
reported that citric acid was used as antioxidant to
prevent browning in plant tissue culture. In previous
study, our data showed that total acid content of RRHsIV
was the highest (0.30%; P < 0.05) followed by RRHsII
(0.16%), RRHsIII (0.09%), RRHsI (0.04%) and RRHsV
(0.03%). The organic acids (citric acid, gluconic acid,
gallic acid etc) in RRHsIV probable were the major
contributors to the antioxidant capacity. On the other
hand, DH of RRHsIV was 49.07%, which is the highest (P
< 0.05) for all the RRHs as shown in Table 1. It was
implicated that RRHsIV might contain more low molecular
peptides, which had antioxidant activity. A large number
of literatures have reported that antioxidant peptides were
0
0
0 0.2 0.4 0.6 0.8 1
Concentration(mg/ml)
0 5 10 15 20 25
Concentration(mg/ml)
RRHsI
RRHsII
RRHsIII
RRHsIV
RRHsV
RRHsI
RRHsII
RRHsIII
RRHsIV
RRHsV
Tian et al. 2399
purified from protein hydrolysates, and the sequences of
amino acid were discovered (Chen et al., 1996; Anisimov
et al., 1997; Je et al., 2005). The antioxidant peptides in
RRHsIV need further study.
Determination by DPPH assay
The DPPH method is a valid, easy, accurate, sensitive
and economic method to evaluate scavenging activity of
antioxidants of fruits and vegetables juices or extracts.
When the antioxidants that can provide hydrogen exist,
they donate hydrogen to the free radicals so that the
radicals remove the odd electron to turn to unreactive
ones (Wang et al., 2006). The DPPH radical scavenging
effects of five RRHs are shown in Figure 2. The DPPH·
scavenging ratio was positively and significantly
correlated with concentration. From RRHsI to RRHsV,

2400 J. Med. Plants Res.
Figure 3. ABTS redical scavenging of five typs of RRHs. Each value is the mean ± SD of
triplicate measurements.
correlation coefficients were 0.79, 0.83, 0.81, 0.84 and
0.83, respectively. The experimental data reveal that
RRHsIV have a stronger effect of scavenging free radical
(P < 0.05) than other RRHs. It is likely due to higher
reductive ability of RRHsIV. A number of studies found
that a relation should be located between antioxidant
activity and the reducing power (Duh, 1998; Yu et al.,
2002; Li et al., 2008). Our results are in agreement with
these literatures. Thus, reductive ability may indirectly
reflect capacity of scavenging free radical.
Determination by ABTS assay
ABTS assays are widely used for assessing the
antioxidant capacity of both hydrophilic and lipophilic
compounds, food products, extracts and biological fluids.
The method is rapid and easy to perform, avoids
unwanted reactions and does not require drastic
conditions to generate radicals (Singh and Singh 2008).
Figure 3 showed all of RRHs samples which possessed
definite antioxidant activity. The ABTS· scavenging ratio
of all the RRHs also correlated with concentration
strongly (correlation coefficients > 0.96). As we expected,
RRHsIV exhibited a higher ability (P < 0.05) for
scavenging ABTS radical than those of RRHsI, RRHsII,
RRHsIII and RRHsV. The scavenging ability of the five
samples against ABTS radical decreased in the order of
RRHsIV > RRHsIII > RRHsI > RRHsII > RRHsV.
Conclusions
ABTS•scavenging ratio(%)
70
60
50
40
30
20
10
0
-10
In the present study, three in vitro assays were applied to
evaluate the antioxidant potency of five RRHs fermented
by different strains of molds. Overall results showed that
0 1.5 3 6 10 12
Concentration(mg/ml)
all the RRHs have antioxidant capacity and RRHsIV
exhibited a higher antioxidant activity than those of four
other RRHs. Considering this, rice residue is an abundant
resource and not efficiently utilized in China, RRHs may
be developed as a new food additive. Further studies are
required to study the in vivo antioxidant activity and
isolation of active compounds of RRHs.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the financial support
Program of the “Twelfth Five-year Plan” for Science and
Technology Research of China (Project No.
2012BAD34B02) and Special Fund for Agro-scientific
Research in the Public Interest of China (Grant No.
200903043-2).
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RRHsII
RRHsIII
RRHsIV
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2402-2409, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.1328
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Determination of scavenging effect of Chinese
medicinal herbs on hydroxyl radical using a new
chemiluminescence system
Cai Zhuo*, Qiu Xia-lin, Liang Xin-yuan, Mo Li-shu and Jiang Cai-ying
College of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, P. R. China.
Accepted 17 February, 2012
A novel flow injection chemiluminescence (FI-CL) method was developed for determination of
scavenging effect of Chinese medicinal herbs on hydroxyl radical (·OH). It was shown that a strong
chemiluminescence (CL) signal was observed when methylene bule (MB) was mixed with Fenton
reagent in an acidic medium. The CL intensity was decreased significantly when the extract of Chinese
medicinal herb was added to the reaction system and partially scavenged the hydroxyl radicals in the
solution. The extent of decrease in the CL intensity had a good stoichiometrical relationship with herb
concentration. Based on this, we developed a new method for the determination of scavenging effect of
Chinese medicinal herbs on hydroxyl radical using a flow injection chemiluminescence (FI-CL)
technique. The proposed method was successfully applied to the evaluations of hydroxyl radical
scavenging capacity of 18 kinds of Chinese medicinal herbs. The results showed that Rhus chinensis
Mill has the highest antioxidant activity.
Key words: Flow injection, chemiluminescence, hydroxyl radical, Chinese medicinal herbs.
INTRODUCTION
Hydroxyl radical (•OH) is by far the most damaging free
radical; its action can have devastating effects within the
body. Ageing, cancer, radiation damage and
phagocytosis are associated with it (Cheng et al., 2002;
Knight, 1995). Therefore, the investigation of natural
antioxidants and their capacity of scavenging hydroxyl
radicals have always attracted attentions. Many Chinese
medicinal herbs contain chemical active composition
such as polyphenols, flavonoids, saponins and
polysaccharides (Chen et al., 2006), therefore, they are
the potential resource of natural antioxidants which have
many significant advantages over the synthetic
antioxidants. It had been found that the effectiveness of
Chinese herbal medicines is closely associated with their
antioxidant capacity (Pan et al., 2004; Yang et al., 2006).
In fact, some anti-free radical active substances have
been screened out from the Chinese medicinal herbs
(Cai et al., 2004). Thus, it is important for medicine and
*Corresponding author. E-mail: zcai01@yahoo.com
food industries to develop a simple and sensitive method
for quantitative evaluation of hydroxyl radical scavenging
ability of plants. Several analytical methods such as
electron spin trap (Espinoza et al., 2009), highperformance
liquid chromatography (HPLC) (Pezo et al.,
2008), capillary electrophoresis (CE) (Kati et al., 2009),
ultraviolet spectrophotometry (UV) (Chen et al., 2008),
spectrophotometry (Yang et al., 2006) and fluorescence
(Qu et al., 2004) have been utilized for this purpose.
However, both HPLC, CE and fluorescence methods
involve laborious processes or time-consuming
procedures; while the UV and spectrophotometry
methods suffer from lack of high sensitivity.
The chemiluminescence (CL) method was widely
employed in the analysis of pharmaceutical compounds
due to its simplicity, ease of manipulation, and high
sensitivity (Kricka, 2003); it therefore could be an
effective tool in determination of the scavenging effect of
Chinese medicinal herbs on hydroxyl radical.
Methylene blue, a flourescent dye usually adopted in
spectroscopic analysis (Sheng et al., 2008; Zhang et al.,
2005) was chosen as the chemiluminescent reagent in

our new CL system. Fenton reaction is a typical reaction
of generating hydroxyl radical and often used to evaluate
the hydroxyl radical-scavenging capacity of plant active
constituents (Julio et al., 2009; Ivan et al., 2009).
It was observed that a strong CL emission signal
occurred when methylene blue reacted with the hydroxyl
radicals generated from Fenton reagent in an acidic
medium. The water extract of Chinese medicinal herb
was found to be able to inhibit the CL intensity by partially
scavenging the hydroxyl radicals in the solution. The
extent of CL intensity reduction exhibited a good
stoichiometric relationship with the herbs concentration.
Based on this, we established a new flow injection
chemiluminescence (FI-CL) method for the evaluation of
hydroxyl radical-scavenging capacity of Chinese
medicinal herbs.
EXPERIMENTAL
Reagents and solutions
All the chemicals used were of analytical grade and deionized water
was used throughout the experiments. A stock solution of 2.0 × 10 -2
mol/L methylene blue (Shenyang Reagent Plant) was prepared by
dissolving 0.7478 g methylene blue in 5 ml of deionized water and
diluting to 100 ml. Hydrogen peroxide (Guang Zhou Xinjian Fine
Chemical Plant) was stored in a fridge at 4°C, and diluted to a
suitable concentration just before usage. A stock solution of 1.5 ×
10 -3 mol/L ferrous iron (Fe 2+ ) was prepared daily by dissolving
0.1471 g ferrous ammonium sulfate 6-Hydrate (Guangdong
Shantou Xilong Chemical Co., Ltd.) in 50 ml of sulfuric acid solution
(0.1 mol/L) and diluting to 250 ml with deionized water. The sulfuric
acid stock solution (0.1 mol/L) was prepared by diluting 2.72 ml of
98% concentrated sulfuric acid (Shanghai Shiyi Chemicals Reagent
Co.,Ltd.) to 500 ml with deionized water.
Sample preparation
18 commonly used Chinese medicinal herbs, with major active
components varying from phenolic acids to fatty acids, were
carefully selected and purchased from local traditional Chinese
medicine shop for this study. The scientific names, family, used
parts, and representative components of the herbs are listed in
Table 1.
Considering that Chinese medicinal herbs are traditionally boiled
in water to produce a soup for patients to drink, therefore herb
samples were prepared using boiling water extraction method. 1.0 g
smashed plant was soaked with 80 ml of deionized water in a
conical flask for 20 min and then extracted at 100°C for 40 min, the
extract was filtered and the residue was washed with 5 ml hot water
twice; the washings and the filtrate were mixed together and diluted
to 100 ml with deionized water.
Apparatus
The CL measurements were performed using a FI-CL analyzer
(IFFL-E, Xi’an Remex Analysis Instrument Co., Ltd. China). This
analyzer consisted of two peristaltic pumps and a six-way injection
valve equipped with a 75 μl sample loop (Figure 1). A PTFE tubing
(0.8 mm i.d.) was used to connect all components in the flow
system. The flow-cell was a coil of glass tubing (1 mm i.d., total
length 100 mm), positioned in front of the detection window of a
Zhuo et al. 2403
sensitive photomultiplier tube (PMT) which was operated at a
negative high pressure of 750 V. The CL signal collected by the
PMT was recorded with a computer employing CL analysis
software.
All ultrasonic oscillations of reagents were performed using an
ultrasonic cleaner (KQ-2200E, Kunshan Ultrasonic Instrument Co.,
Ltd.).
Procedure
As shown in the schematic diagram of experimental manifold
(Figure 1), the solutions of Fe 2+ and sample (or deionized water)
were mixed by a 3-way mixing valve and propelled by the peristaltic
pump (P1) into the flow-cell as carrier stream at the flow rate of 4.7
ml/min; H2O2 and methylene blue solutions were merged together
by another 3-way mixing valve and propelled by the peristaltic pump
(P2) at the flow rate of 7.0 ml/min through a 10 cm tubing into a sixway
valve which was then injected 75 μl of the mixed solution into
the carrier stream. The light output from the flow-cell was detected
by a photomultiplier tube (PMT).
The scavenging rate of a Chinese medicinal herb was calculated
based on the decrement of the CL emission intensity (ΔI) according
to the following equation:
Scavenging rate (s) = [(I0-Is)/ I0]×100% (1)
Here, I0 and Is are the CL emission intensities in the absence and
presence of Chinese medicinal herb, respectively.
RESULTS AND DISCUSSION
Kinetic curve of CL reaction
The kinetic characteristic curve of the reactive system is
shown in Figure 2. The CL signal generated from the
mixed solution of methylene blue and Fenton reagent in
an acidic medium reached its maximum intensity at 3.5 s
and then extinguished immediately within 3 s thereafter
(Figure 2a), indicating that the luminescence reaction
was a rapid reaction. In the presence of Rubia cordifolia
L. extract, a relatively weaker CL signal with the same CL
emission time and similar peak shape was obtained
(Figure 2b).
Selection of the experimental manifold and apparatus
parameter
Various types of the flow injection (FI) manifolds were
investigated; the results showed that the maximal CL
emission signal was obtained when using the manifold
depicted in Figure 1; therefore, this manifold was
employed in this study.
The flow rate is important to FI-CL detection. If the flow
rate is too high or too low, a suitable CL emission signal
can not be obtained. The effects of flow rate on the CL
intensity were examined in the range of 3.5 to 7.5 ml/min.
It was shown that the CL intensity was kept at a constant
with the increase in the flow rate of the carrier stream
delivered by P1, while it was significantly enhanced with

2404 J. Med. Plants Res.
Table 1. Chinese medicinal herbs used in this study.
Plant Chinese
name
Scientific name Family Part used
Major representative
components
1 Wu Bei Zi Rhus chinensis Mill. Anacardiaceae Gall Gallotannin, gallic acid
2 He Zi Terminalia chebula Retz. Combretaceae Fruit Ellagitannins, gallic acid
3 Lian Qiao Forsythia suspense (Thunb.) Vahl. Oleaceae Fruit Forsythigenol, forsythin, rutin
4 Jin Yin Hua Lonicera japonica Thunb. Caprifoliaceae Floral bud
Chlorgenic acid, luteolin, luteolin-7glucoside
5 Yue Ji Hua Rosa chinensis Jacq. Rosaceae Flower
Quercetin, catechin, anthocyanins,
gallic acid, tannins
6 Sheng Di Rehmannia glutinosa Libosch. Scrophulariaceae Root Ferulic acid, caffeic acid
7 Gou Qi Zi Lycium barbarum L. Solanaceae Fruit Coumarins (scopoletin)
8 Huang Qín Scutellaria baicalensis Georgi Labiatae Root
Baicalein, baicalin, chrysin,
wogonin
9 Dang Gui Angelica sinensis (Oliv.) Diels Umbelliferae Root Vanillin, p-cresol, ferulic acid
10 Ye Ju Hua Chrysanthemum indicum L. Asteraceae Inflorescence
Acaciin, luteolin, flavone
glycosides, coumarins
11 Gan Cao Glycyrrhiza uralensis Fisch. Leguminosae Root Glycyrrhizin, liquiritin, liquirigenin
12 Yin Xing Ye Ginkgo biloba L. Ginkgoaceae Leaves
Keampferol, luteolin, myricetin,
quercetin, isorhamnetin, syringetin
13 Huang Qi Astragalus mongholicus Bge. Leguminosae Root Astragalin, calycosin, formononetin
14 He Shou Wu Polygonum multiflorum Thunb. Polygalaceae Root
Chrysophanol, emodin, rhein,
physcion, tannins, resveratrol
15 Wu Wei Zi
Schisandra
chinenesis(Turcz.)Baill.
Magnoliaceae Fruit
Schizandrins, schizatherins,
wulignan
16 Qian Cao Rubia cordifolia L. Rubiaceae Root Purpurin, alizarin, munjistin
17 Jin Qian Cao Lysima chiachristinae Hance Primulaceae Whole plant
Isorhamnetin, quercetin,
kaempferol
18 Huo Ma Ren Cannabis sativa L. Moraceae Seeds
Figure 1. Schematic diagram of the flow injection
chemiluminescence analysis system. a, Fe 2+ and H2SO4 solution; b,
sample solution or H2O; c, H2O2 solution; d, methylene blue; P1, P2,
peristaltic pump; V, 6 way injection valve; F, flow cell; W, waste
solution; PMT, photomultiplier tube; HV, negative high voltage; COM,
computer.
Oleic acid, linoleic acid, linolenic
acid

1
2
Time (s)
Figure 2. Kinetic curve of chemiluminescence 1. 1.5 × 10 -3 mol·L -1 Fe 2+ + 0.5%
H2O2 + 3.2 × 10 -5 mol·L -1 methylene blue; 2. 1 + R. cordifolia L. extract.
H2SO4 (mmol L -1 )
Figure 3. The effect of H2SO4 concentration on CL intensity.
the increase in the flow rate of H2O2 and methylene blue
mixed solution propelled by P2. As a result of a
compromise between reagent consumption and CL
intensity, a flow rate of 4.7 ml/min for the carrier stream
and a flow rate of 7.0 ml/min for H2O2 and methylene blue
mixed solution were adopted respectively in our
experiments.
Effect of sulfuric acid concentration
Zhuo et al. 2405
The effect of the concentration of sulfuric acid used for
preparing Fe 2+ solution on the CL intensity was
investigated in the range of 1.0 × 10 -4 to 5.0 × 10 -3 mol/L.
The results (Figure 3) showed that the CL intensity
increased with the increase of sulfuric acid in the solution,

2406 J. Med. Plants Res.
Fe 2+ (mmol L -1 )
Figure 4. The effect of Fe 2+ concentration on CL intensity.
H2O2 (mmol L -1 )
Figure 5. The effect of H2O2 concentration on CL intensity.
and reached a maximal point at 1.0 × 10 -3 mol/L. Thus,
1.0 × 10 -3 mol/L sulfuric acid was used in the CL reaction
system.
Effect of Fe 2+ concentration
Ferrous iron (Fe 2+ ), which functions as the catalyst in the
Fenton reagent, is closely associated with the yield of
hydroxyl radicals. The influence of Fe 2+ concentration on
the CL intensity was studied in the range of 2.0 × 10 -4 to
2.5 × 10 -3 mol/L. The result (Figure 4) showed that the CL
intensity reached its maximal point when the
concentration of Fe 2+ was 1.5 × 10 -3 mol/L. Thus, 1.5 ×
10 -3 mol/L Fe 2+ was adopted in our CL system.
Effect of hydrogen peroxide concentration
Hydrogen peroxide is the main source of hydroxyl radical;
its concentration could exert a comparative strong impact
on the CL intensity. The effect of H2O2 concentration was
investigated in the range of 0.02 to 0.08 mol/L. The
results (Figure 5) demonstrated that the CL intensity
increased sharply with increasing concentration of H2O2
and then decreased when the H2O2 concentration
reached 0.05 mol/L. The probable reason responsible for
the decrease in CL intensity was that the excessive
hydrogen peroxide reacted directly with hydroxyl radicals
resulting in the reduction of hydroxyl radical concentration
in the solution, and indirectly inhibited the reaction of
hydroxyl radical with methylene blue (Shen et al., 2007).
In this study, the H2O2 concentration of 0.05 mol/L was
used for the CL reaction.
Effect of methylene blue concentration
The effect of methylene blue concentration was
investigated in the range of 8.0 × 10 -6 to 8.0 × 10 -5 mol/L.
It was shown that (Figure 6) the CL intensity increased
sharply when the methylene blue concentration ranged
from 8.0 × 10 -6 to 3.2 × 10 -5 mol/L and changed slowly
after 3.2 × 10 -5 mol/L. Thus, 3.2 × 10 -5 mol·L -1 methylene
blue was chosen for the CL reaction in this research.
Validity of new CL system
Thiourea is a specific and powerful hydroxyl radical
scavenger (Long et al., 2006), thus, it was employed as
the reference material for the evaluation of validity of the
new CL system in the determination of radical scavenging
effect. A series of thiourea solutions of different
concentration were prepared for the effective examination
of the proposed method. The results showed that the
scavenging rate increased with increasing thiourea
concentration (Figure 7), indicating that thiourea
concentration and hydroxyl radical scavenging rate had a
significant dose-effect relationship. Therefore, this system
can be used for the determination of the capacity of
hydroxyl radical scavenger.
Measurement of radical scavenging capacity
10 ml of herb extract was diluted to 100 ml (200 ml for R.
chinensis Mill) with deionized water, the scavenging rate
was measured under the optimal experimental conditions,
and the result was shown in Table 2. As can be seen from
the table, the radical scavenging ratios of herb extracts
vary from 97.7 to 25.6%, of the 18 Chinese medicinal
herbs tested, the gall of R. chinensis Mill exhibited the
highest potency in scavenging hydroxyl radical, while the
seeds of Cannabis sativa L showed the lowest radical
scavenging capacity. It is reported in literature that the
radical scavenging capacity of a herb is related to its
phenolic contents (Guo et al., 2008; Liu and Ng, 2000;

Wong et al., 2006). It can be seen from Table 1 that the
major active components of R. chinensis Mill are
polyphenols (gallotannin) and phenolic acids (gallic acid),
the main active components of other herbs, ranking lower
than R. chinensis Mill but still within in the top ten of the
list, also include phenolic compounds such as tannins,
phenolic acids, flavonoids, and simple phenols, yet the
major active components of Cannabis sativa L are
polyunsaturated fatty acids including oleic acid, linoleic
acid and linolenic acid. These results indicated that
phenolic compounds may contribute significantly to the
radical-scavenging activities of the herbs, which is in
agreement with the results of Jiang et al. (2011) and Lee
et al. (2008).
Possible mechanism of the CL reaction
The possible mechanism of CL reaction of the proposed
Figure 6. The effect of methylene blue concentration on
CL intensity.
Figure 7. Effects of thiourea concentration on ·OH
scavenging rate.
Zhuo et al. 2407
CL system could be explained as follow: When methylene
blue (MB) reacted with the hydroxyl radicals produced
from Fenton reagent, a certain amount of energy was
released and absorbed by the unreacted methylene blue
in the solution to form the excited-state methylene blue,
which emitted CL when returned to the ground state; the
CL emission signal was inhibited when Chinese herb
extract was added and partially scavenged the hydroxyl
radicals in the CL system.
The reaction pathway can be summarized as the
following:
Fe 2+ + H2O2 → Fe 3+ + ·OH + OH -
Fe 3+ + H2O2 → Fe 2+ + ·OOH + H +
·OOH + H2O2→ ·OH + H2O+ O2
MB + ·OH →product + energy
MB +energy → (MB) *
(MB) * → MB + hv
[Herb-OH] + ·OH→ [Herb-O] + H2O

2408 J. Med. Plants Res.
Conclusion
Table 2. The scavenging ratio on ·OH of extracts of 18 Chinese medicinal herbs (n = 5).
Chinese medicine herb Scavenging rate (%) RSD (%)
R. chinensis Mill. 97.7 0.21
T. chebula Retz. 96.8 0.10
F. suspensa (Thunb.) Vahl. 92.5 0.22
L. japonica Thunb. 91.5 0.59
R. chinensis Jacq. 89.5 1.6
R. glutinosa Libosch. 87.5 1.0
L. barbarum L. 86.8 1.1
S. baicalensis Georgi 86.7 1.7
A. sinensis (Oliv.) Diels 84.4 0.68
C. indicum L. 83.4 3.0
G. uralensis Fisch. 79.4 0.65
G. biloba L. 78.4 1.4
A. mongholicus Bge. 75.7 0.78
P. multiflorum Thunb. 74.2 1.4
S. chinenesis(Turcz.)Baill. 62.8 4.5
R. cordifolia L. 58.2 4.6
L. chiachristinae Hance 39.1 5.2
C. sativa L. 25.6 3.9
A new flow-injection chemiluminescence method for
determination of scavenging capacity of Chinese
medicinal herbs to hydroxyl radicals based on the
inhibition effect of herb water extract on the CL intensity
in methyleme blue-Fe 2+ -H2O2 system was established.
The scavenging rates of the herbs could be measured
even with a simple setup. It was believed that the
reaction of methylene blue with hydroxyl radical was
responsible for the CL emission in the CL system, while
the degree of the reduction of the CL intensity resulted
from the scavenging action of herb water extracts on
hydroxyl radical had a stoichiometric relationship with
herb concentration in the solution. The proposed method
is not only simple and convenient, but also stable and
user-friendly. It had been applied to the determination of
hydroxyl radical scavenging capacity of 18 Chinese
medicinal herbs with satisfactory results.
ACKNOWLEGEMENTS
We gratefully acknowledge financial support from the
Guangxi Science Foundation of China (No.
2010GXNSFA013001).
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2421-2437, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.1395
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
A survey of medicinal plants in mangrove and beach
forests from sating Phra Peninsula, Songkhla Province,
Thailand
Oratai Neamsuvan*, Patcharin Singdam, Kornkanok Yingcharoen and Narumon Sengnon
Faculty of Traditional Thai Medicine, Prince of Songkla University, Hat yai, 90110, Thailand.
Accepted 18 November, 2011
This study aimed to survey medicinal plants in mangrove and beach forests from Sating Phra
Peninsula, Songkhla Province. Three representative districts including Sing Ha Nakhon, Sating Phra
and Ranode were selected. Semi-structured interview was conducted to six local healers for asking
about local names, parts of use, preparation and properties. Plant specimens also were collected.
Identification was done and the specimens were deposited at The Prince of Songkla University
herbarium (PSU). A total of 110 species belonging to 100 genera and 51 families was found. Among
them, 69 species were only found in the beach forests, 35 species were only found in the mangrove
forests and 6 species could be found in both areas. Fabaceae was the most important family in term of
species used. Herb was the most frequently used habit of plants. Most plant species were used for
curing fever (18.52%), skin diseases (10.65%) and gastrointestinal tract problems (10.19%), respectively.
Interestingly, 34 species relate to pharmacological activities, while 13 species have never been
investigated. Therefore, their biological activity should be investigated to support utilization of herbal
medicine.
Key words: Medicinal plant, mangrove forest, beach forest, Sating Phra Peninsula, Songkhla Province.
INTRODUCTION
A survey of medicinal plants has been carried out
throughout Thailand, especially the studies based on
knowledge of minority ethnic groups due to their
traditional and cultural identity. Since almost minorities
have settled down in various kinds of forests such as
evergreen, deciduous dipterocarp, or mixed deciduous
ones, then medicinal plants which are endemic to those
areas have been studied. However, some interesting
types of forest such as beach and mangrove which are
occupied by few dwellers have been neglected for the
investigation.
Mangrove forest is a vegetation group occupying the
intertidal zone in tropical shorelines or estuaries
(Chanyong, 2009), that is, the west and east coast of
Thailand. It is only in the peninsular Thailand that it is
composed of 932 km on the Gulf of Thailand (East coast)
*Corresponding author. E-mail: oratai.n@psu.ac.th. Tel: 0066-
811420012. Fax: 0066-74282709.
which spread over 173,310 km 2 and 710 km on Andaman
coast (West coast) with an estimated area of 155,591
km 2 (Plathong and Plathong, 2004). Generally, plants in
mangroves survive and even thrive in saline condition of
coastal areas, and most of them are evergreen
vegetation.
Mangroves are not only important for serving as
bleeding and nursing ground for marine species and
reducing the devastation impact of natural disasters, such
as tsunamis and hurricanes (Giri et al., 2008), but they
are also the socioeconomically important ecosystem,
especially for inhabitants of coastal regions
(Bandaranayake, 1998) who depend on them for fuel
(Day et al., 1987), food, medicine, and other basic
necessities (Cornejo et al., 2005).
Beach forest is plant community growing along sandy
shores and up to high tidal zone which is exposed to salt
spray. The vegetation is found on sand dunes,
sometimes on sand gravel or rock. Beach forests may be
open which composed of dense grasses, shrubs and
herbs. On the other hand, it may be grove or forest with

2422 J. Med. Plants Res.
close canopy. Plants can tolerate salt spray (Halophytes),
strong wind and drought (Rueangphanich, 2005). For
Gulf of Thailand, beach forests can be found around
shorelines from Chonburi to Trad province and the
seacoast from Petchburi province to Malaysia border at
Narathiwat province. For Andaman coast, it can be found
from Ranong to Satun province.
Beach forest is a crucial natural resource which is
important for the ecosystem of any economy. Nowadays,
Thai coastal areas have been exploited severally. The
beautiful beaches have not only been used for attracting
tourism, but they also play the important role on other
ecosystems (Defeo et al., 2009) such as supporting
several macrofauna and microfauna populations
(Goncalves, 2009), serving as nursery area for juvenile
fishes, nesting sites for shorebirds, bait and food
organisms, as well as wave dissipation and associated
buffering against extreme events (storms, tsunamis)
(Defeo et al., 2009).
Although mangrove and beach forests are greatly
important for ecosystems and being human, mangrove
areas in Thailand have rapidly decreased from 3,679,000
km 2 in 1961 to 1,686,825.6 km 2 in 1993. The major cause
of this situation is conversion of mangrove forests to
aquaculture especially the shrimp farming (Giri et al.,
2009; Chuenpagdee, 2003). In addition, the beach areas
are also lost by conversion to tourist attractions and
tourist residences, that is, resorts (Chuenpagdee, 2003).
Mangrove and beach forests are composed of many
plant species. However, the survey of medicinal plants in
mangrove and beach forests has been scattered and
documented as a minor part of medicinal plant books. For
example, Upho (2005) studied about ethnobotany of
Buddhist and Muslim Thais in some locations in the lower
part of Southern Thailand, then a few districts of Trang
province located on mangrove and beach forest were
included as a part of that study. The study found only 16
species of medicinal plants. In addition, Thaewchatturat
(2000) studied about ethnobotany of Mogen ethnic group
in Phang nga Province in which 4 species of mangrove
and 27 species of beach plants were used for herbal
medicine. According to previous reports, there is a small
number of used plants from mangrove and beach forest,
while some documents indicated that 48 mangrove and
77 beach species have been found in Southern Thailand
(Working Group of academic standard for Pilot National
Park, 2007) and beach forest only was found to contain
167 species in Peninsular Thailand (Laongpol et al.,
2009).
Recent studies on medicinal plants in mangrove and
beach forests in Thailand were focused on Andaman
coast, but there was no any close study in Gulf of
Thailand in spite of a large number of folk healers
existing (Golomb, 1988). Therefore, it is interesting to
study the medicinal plants from the East coast of
Thailand. In this study, we decided to survey mangrove
and beach forests from Sating Phra peninsula, Songkhla
province. In the past, there were the extensive areas of
mangrove and beach forests in Sating Phra peninsula.
However, nowadays, these forests have been decreased
by destruction through various forms such as building,
tourism and shrimp farming (Trisurat, 2006).
Consequently, mangrove and beach plants are
decreased both in species richness and abundance.
Hence, it is urgent to study the utilization of medicinal
plants before the disappearance of beach and mangrove
forests along with losing of plant species and knowledge
of ethnobotany.
The objective of this study was to collect and survey
the use of medicinal plants in mangrove and beach
forests from the local healers established in Sating Phra
peninsula, Songkhla province.
MATERIALS AND METHODS
Study area and local healers
Sating Phra peninsula (Figure 1), located on peninsular Thailand,
composes of 4 districts: Ranode, Sating Phra, Krasaesin and
Singha Nakhon. It is bordered to the north by Hua Sai district,
Nakhon Si Thammarat province, to the south by Mueang Songkhla
district, Songkhla province, to the east by Gulf of Thailand, and to
the west by Songkhla lagoon. The total area is approximately
1,228.2 km 2 . Most of local people are Buddhist. Their occupations
mostly are agriculture namely, farming and fishing (Songkhla
Statistical Office, 2010). A prominent landscape of this study area is
sand bars lying between Gulf of Thailand and Songkhla lagoon.
In this study, three districts were selected; Singha Nakhon,
Sating Phra and Ranode. Studied mangrove forests were from
Baan Ta Hin village in Sating Phra district, Baan Cha Lae village,
Baan Bo Pab village and Baan Hua Khao village in Singha Nakhon
district. Studied beach forests were from Hat Kaew beach in Singha
Nakhon, Muang Ngam beach, Di Luang beach and Maharatch
beach in Sating Phra district, and Bo Tru beach as well as Pak
Rawa beach in Ranode district. Six local healers also were
selected.
Field study
The field study was conducted in July 2010 to November 2010,
once monthly. The semi-structured interview was used for asking
the local healers about local name of medicinal plants, plant part
used, how it is used and its properties. The folk healers were
interviewed at their houses and also during collection of the
specimens in the fields. To confirm the plant properties, one type of
use was mentioned by at least 2 healers.
Herbarium specimens
All medicinal plants utilized by the local healers were photographed
and then collected for making voucher specimens according to
Chayamarit’s (1997) method. The voucher specimens were
deposited at PSU Herbarium, Department of Biology, Faculty of
Science, and Faculty of Traditional Thai Medicine Herbarium,
Prince of Songkla University.
Medicinal plants identification
The collected specimens were identified with the aids of relevant

Figure 1. Study site: Thailand map represent Songkhla province (a+b) as well as Sathig Phra
peninsular (b) (Left) and 3 selected districts in Sathig Phra peninsular (Right) (N=northern,
C=central, SW=southwestern, NE=northeastern, E=eastern, SE=southeastern, and
PEN=peninsula).
literature e.g. Flora of Thailand, Flora Malesiana, Flora of China
and Flora of British India.
Data analysis
The data were analyzed by descriptive statistics and interpretation.
The results were also compared to the close studies.
RESULTS
Plant use
Totally, 110 medicinal plants species belonging to 100
genera in 51 families were collected (Table 1). Thirty five
species (31.82%) were found only in mangrove forests.
Sixty nine species (62.73%) were found only in beach
forests. In addition, 6 species (5.45%) were found in both
types of forests. The higher number of species in beach
forest than mangrove one is congruent with the survey of
plant diversity in Tarutao National Park (Working Group
of academic standard for Pilot National Park, 2007).
The families most frequently used were Euphorbiaceae
(10 species), Fabaceae (9 species) and Rubiaceae (5
species), respectively. In addition to the mostly used
families, Fabaceae and Rubiaceae are also grouped in
Neamsuvan et al. 2423
the largest family in terms of species (Clayton and
Renvoize, 1986), this reflects that people tend to use
plant resource present in their environment. It is recorded
that these 3 families were always used in other parts of
the world such as Kenya (Bussmann et al., 2006), Nepal
(Kunwar et al., 2010), Uganda (Kamatenesi et al., 2011)
and Peru (Luziatelli et al., 2010).
Ninety five species (86.36%) were dicotyledons, twelve
species (10.91%) were monocotyledons and tree species
(2.73%) were ferns.
According to plant habit, herbs were most frequently
used with 30 species (27.27%), followed by trees with 29
species (26.36%), shrubs with 24 species (21.82%),
climber with 22 species (20%) and epiphytic plants with 5
species (4.55%). The most use of herbs as medicinal
plants is also in agreement with the study of Coe and
Anderson (1996), as well as Luziatelli et al. (2010).
Disease/symptom to treat
The 110 medicinal plants were classified into 26
categories according to disease or symptom to treat
(Table 2). However, most categories based on species
number were fever with 40 species (18.52%), skin
diseases with 23 species (10.65%) and gastrointestinal

2424 J. Med. Plants Res.
Table 1. Medicinal species list found in Sating Phra Peninsula, Songkhla Province.
Botanical name H1 Specimen Local name H2 Part/preparation/administration/disease
Acanthaceae
Acanthus ebracteatus Vahl NS 001 Whole plant/ decoction/ oral/cancer
Amaranthaceae
Achyranthes aspera L. H NS 024 Phanngu khao B Whole plant/ decoction/ oral/ fever
Alternanthera sessilis (L.) R. Br. ex DC. H NS 069 Phak ped khao B
Whole plant/ decoction/ oral/ menstrual disorder
Whole plant/ poultice/ topical/ infant convulsion, parasites, fever
Amaranthus viridis L. H NS 081 Phak khom B Whole plant/ decoction/ oral/ fever
Annonaceae
Melodorum siamensis (Scheff.) Bân C NS 115 Nom maeo M Stem and leaves/ decoction/ oral/ diarrhea and dysentery
Uvaria rufa Blume C NS 033 Nom kwai M
Leaves/ decoction/ oral/ joint and muscle pain
Wood/ decoction/ oral/ fever
Asclepiadaceae
Calotropis gigantea (L.) Dryander ex
W.T. Aiton
S NS 056 Rak Khao B
Latex/ juice/ topical/ decayed tooth, warts and corns;
Leaves/ poultice/ inhalation/ sinusitis and snuffy nose
Tyrophora indica (Burm.f.) Merr. C NS 061 Ton pan rak B Whole plant/ decoction/ oral/ abscess and contusion
Asteraceae
Chromolaena odoratum (L.) R.M.King &
H.Rob.
H NS 101 Sapsuea B
Pluchea indica( L.) Less. S NS 092 Khlue tale
Leaves/ poultice/ topical/ wound (bleeding)
Roots/ decoction/ oral/ diabetes mellitus
B, Leaves/ bath/ oral/ diabetes mellitus
Leaves/ decoction/ oral/ dysuria, kidney stone, hemorrhoid
M
Leaves/ poultice / topical/ parasites
Vernonia cinerea (L.) Less. H NS 076 Ya khrao maeo B Leaves/ poultice / topical/ wound (bleeding)
Wedelia biflora (L.) DC. C NS 046 Benchamart -numkhem
M Leaves/ poultice / topical/ wound
Whole plant/ poultice / topical/ prickly heat
Avicenniaceae
Avicennia alba Blume T NS 002 Samae khao M Heart wood/ decoction/ oral/ blood tonic
Avicennia officinalis L. T NS 001 Samae dum M Heart wood/ decoction/ oral/ fatigue

Table 1. Contd.,
Capparidaceae
Capparis sepiaria L. C NS 042 Nam ngai M Wood/ powder/ topical/ contusion
Casuarinaceae
Casuarina equisetifolia J.R. & G.Forst T NS 103 Son thale B Root/ decoction/ oral/ headache, encephalitis
Celastraceae
Pleurostylia opposita (Wall.) Alston S NS 107 Thing thuad B Root, barks/ decoction/ oral/ malaria
Salacia chinensis L. S NS 064 Kumpang jed chan B
Barks/ decoction/ topical/ toothache
Stem/ decoction/ oral/ fatigue
Combretaceae
Lumnitzera racemosa Willd. T NS 008 Fard dok khao M Wood/ powder/ wound
Combretaceae
Combretum quadrangulare Kurz T NS 044 Sakae M
Whole plant/ decoction/ oral/ parasites
Seed/ raw/ oral/ parasites
Stem/ charcoal/ sauna/ post partum
Neamsuvan et al. 2425
Commelinaceae
Commelina benghalensis L. H NS 074 Ya nam dub fai B
Whole plant/ decoction/ oral/ fever; whole plant/ poultice/ topical/
abscesses, contusion
Murdannia sp. H NS 095 Bae phu B Whole plant/ decoction/ oral/ fever
Convolvulaceae
Cuscuta reflexa Roxb. Ep NS 053 Foithong B Whole plant/ decoction/ oral/ fatigue
Ipomoea pes-caprae (L.) R. Br. H NS 019 Phak bung thale B
Leaves/ poultice / topical/ wound caused by jellyfish
Leaves/ juice/ oral/ constipation
Tetracera sp. C NS 043 Pod
B, Stem/ decoction/ oral/ liver diseases and splenopathy,
M Joint and muscle pain
Euphorbiaceae
Breynia sp. S NS 111 Kangpla dang B Roots/ decoction/ oral/ fever
Bridelia stipularis (L.) Blume S NS 029 Sa ai B Wood/ decoction/ oral/ malaria
Euphorbia heterophylla L. H NS 066 Phak bung dong B Leaves/ raw/ oral/ constipation
Euphorbia hirta L. H NS 017 Namnom- ratchasi B
Leaves/ raw/ oral/ constipation
Whole plant/ decoction/ oral/ Herpes zoster, Lactogogue

2426 J. Med. Plants Res.
Table 1. Contd.
Excoecaria agallocha L. T NS 084 Tatum thale M
Micrococca mercurialis(L.) Benth. H NS 051 Tamyae maeo B
Microstachys chamaelea (L.) Müll. Arg. S NS 079 Phraow- nok khoom B
Latex/ juice/ oral/ constipation
Heart wood/ decoction/ oral/ blood tonic
Whole plant/ poultice / topical/ wound
Whole plant/ decoction/ oral/ vomiting
Fruits/ raw/ oral/ joint and muscle pain
Whole plant/ decoction/ oral/ diabetes mellitus
Mallotus hymenophyllus Airy Shaw S NS 036 Prik M Leaves/ poultice / topical/ fever
Shirakiopsis indicum (Willd.) Esser T NS120 Samore thale M Leaves/ decoction/ steam bath/ diet
Sauropus bacciformis (L.) Airy Shaw S NS 093 Phraow- nokkhoom B Whole plant/ decoction/ oral/ parasites
Fabaceae
Abrus precatorius L. C NS 106 Maklam ta nu B
Senna sophera L. S NS 080 Phak khet B
Roots/ Decoction/ Oral/ Fever (decrease high-bodily temperature)
Seeds/ decoction/ oral/ thirsty relief
Leaves/ poultice / topical/ athlete’s foot
Seeds/ decoction/ oral/ thirsty relief
Leaves/ decoction/ oral/ constipation
Whole plant/ decoction/ oral/ toxin in the body
Senna tora L. S NS 067 Khilek jued B Whole plant/ decoction/ oral/ fever
Crotalaria retusa L. S NS 048 Hinghai B Whole plant/ decoction/ oral/ fever, toxin in the body
Dalbergia candenatensis (Dennst.) Prain C NS 027 Sakkhi M heart wood/ decoction/ oral/ blood tonic
Derris scandens (Aubl.) Pittier C NS 038 Thaowan priang M Roots/ decoction/ oral/ cancer
Derris trifoliata Lour. C NS 009 Thopthaep nam M
Leaves/ decoction/ oral/ constipation
Stems/ decoction/ oral/ joint and muscle pain
Indigofera tinctoria L. S NS 040 Khram M
Pithecellobium dulce (Roxb.) Benth. T NS 005 Makham tate M
Flagellariaceae
Flagellaria indica L. C NS 121 Wai ling B
Leaves/ poultice/ topical/ inflammation of abscess
Whole plant/ decoction/ oral/ fever
Bark/ decoction/ topical / toothache, gingivitis
Roots/ decoction/ oral/ menstrual disorder
Leaves/ decoction/ oral/ cardiotonic, antenatal care
Rhizome/ decoction/ oral/ fever, malaria, jaundice

Table 1. Contd.
Guttiferae
Calophyllum inophyllum L. T NS 026 Krating M
Garcinia hombroniana Pierre T NS 114 Wa B
Labiatae
Clerodendrum inerme (L.) Gaertn. S NS 045 Sam ma li nga M
Hyptis suaveolens (L.) Poit. S NS 097 Maenglak kha B
Leucas zeylanica (L.) R. Br. H NS 073 Brek
Vitex rotundifolia L. f. S NS 057 Kontiso tale B
Fruits/ oil/ topical/ joint and muscle pain
Flowers/ oil/ topical/ hair and scalp damage
Fruits/ raw/ oral/ constipation
Latex/ juice/ oral/ to stimulate vomiting
Bark/ decoction/ topical/ toothache
Leaves/ decoction/ bath/ prickly heat and pruritic rash
Leaves/ poultice/ topical/ inflammation of abscesses
Whole plant/ decoction/ oral/ fever, fatigue
Seeds/ dessert/ oral/ constipation
B, Whole plant/ decoction/ oral/ menstrual disorder
M Leaves/ poultice/ topical/ asthma, wound (bleeding)
Leaves/ decoction/ oral/ carminative, malaria, fever
Roots/ decoction/ oral/ joint and muscle pain
Liliaceae
Asparagus racemosus Willd. C NS 117 Rak samsip B Roots/ decoction/ oral/ fatigue, antenatal care
Gloriosa superba L. C NS 105 Dong dung B Rhizome/ powder/ oral/ hemorrhoid
Malvaceae
Hibiscus tiliaceus L. T NS 011 Po thale M Bark, wood, roots/ decoction/ oral/ dysuria
Sida acuta Burm. f. S NS 083 Ya khat mon B
Whole plant/ decoction/ oral/ joint and muscle pain,
Roots, stem/ powder/ topical/ Herpes zoster
Sida cordifolia L. S NS 059 kledpla kradi B Whole plant/ decoction/ oral/ fever
Melastomataceae
Melastoma malabathricum L. S NS 122 Blae M Roots/ decoction/ oral/ fever, wound, abscess
Neamsuvan et al. 2427
Meliaceae
Xylocarpus granatum J. Koenig T NS 031 Ta boon M Bark/ Decoction/ Oral/ Mucous and bloody dysentery, diarrhoea

2428 J. Med. Plants Res.
Table 1. Contd.
Menispermaceae
Tiliacora triandra Diels C NS 104 Ya nang B Root/ decoction/ oral/ fever, toxin in the body
Myrtaceae
Melaleuca cajuputi Roxb. T NS 014 Samet khao B
Rhodomyrtus tomentosa (Aiton) Hassk. S NS 098 Thoh/ Pha ya rak dam B
Inflorescence/ raw/ oral/ apthus ulcer, follicular pharyngitis, fever
Leaves/ decoction/ bath/ itching
Fruit/ raw/ oral/ diarrhea
Roots/ decoction/ oral/ tonic
Syzygium gratum (Wight) S.N.Mitra T NS 050 Samet daeng B Inflorescence/ raw/ oral/ carminative
Nyctaginaceae
Boerhavia diffusa L. H NS 022 Phak khom hin B Whole plant/ decoction/ oral/ dysuria, menstrual disorder
Olacaceae
Olax scandens Roxb. C NS 035 Joh to
B, Whole plant/ decoction/ oral/ abscesses
M Fruits/ raw/ oral/ abscesses
Oleaceae
Jasminum nervosum Lour. C NS 068 Bleh tuan M Leaves/ decoction/ topical/ apthus ulcer
Pandanaceae
Pandanus odoratissimus L. f. T NS 116 Lam chiak B Root/ decoction/ oral/ dysuria, toxin i the body
Passifloraceae
Passiflora foetida L. C NS 018 Ka tok rok B Whole plant/ decoction/ oral/ cough, expectorant, dysuria, fever
Poaceae
Chrysopogon aciculatus (Retz.) Trin. H NS 086 Ya chaoshu B whole plant/ decoction/ oral/ dysuria, kidney stone renal disease
Dactyloctenium aegyptium (L.) Willd. H NS 094 Ya paak kwai B Whole plant/ decoction/ oral/ fever, dysuria
Perotis indica (L.) Kuntze H NS 110 Ya hang krarok B Whole plant/ decoction/ oral/ dysuria
Spinifex littoreus (Burm. f.) Merr. H NS 087 Ya looklom B Roots/ decoction/ oral/ joint and muscle pain
Polypodiaceae
Drynaria sparsisora (Desv.) T. Moore Ep NS 113 Wao ta le B
Rhizome/ decoction/ oral/ fever
Rhizome/ powder/ topical/ wound (snake bites)

Table 1. Contd.
Pyrrosia piloselloides (L.) M.G. Price Ep NS 013 Bia lan B
Portulacaceae
Portulaca pilosa L. H NS 085 Sao chiang mai B
Whole plant/ decoction/ bath/ psoriasis
Whole plant/ decoction/ oral/ fever, dysuria
Neamsuvan et al. 2429
Whole plant/ poultice/ topical/ wound
Whole plant/ decoction/ oral/ urinary system, menstrual disorder
Rhamnaceae
Colubrina asiatica (L.) Brongn. C NS 006 Pak wan tale M Whole plant/ decoction/ oral/ abscesses
Ziziphus mauritiana Lam. T NS 088 Phut sa B
Roots/ juice/ topical/ conjunctivitis
Leaves/ poultice/ topical/ hair and scalp damage
Ziziphus oenopolia (L.) Mill.
var. oenopolia
C NS 096 Yap yio B
whole plant/ decoction/ oral/ dysuria, kidney stone
Leaves/ decoction/ oral/ blood tonic
Fruits/ raw/ oral/ expectorant
Rhizophoraceae
Bruguiera cylindrica (L.) Blume T NS 004 Thua khao M Flowers/ decoction/ oral/ expectorant
Rhizophora apiculata Blume T NS 003 Kongkang bailek M Fruits/ decoction/ oral/ fever
Rhizophora mucronata Lam. T NS 007 Kongkang Baiyai M
Rubiaceae
Catunaregam spathulifolia Tirveng. T NS 118 Nam khet B
Morinda elliptica (Hook. f.) Ridl. T NS 010 Yo pa M
Fruits/ decoction/ oral/ fever;
Bark/ decoction/ oral/ dysentery;
Roots/ decoction/ oral/ dysuria, kidney stone
Fruits/ poultice/ topical/ athlete’s foot
Wood/ decoction/ oral/ cancer
Wood/ decoction/ oral/ parasites, to release lochia;
Fruits/ decoction/ bath/ fever, seborrheic dermatitis
Hedyotis corymbosa (L.) Lam. H NS 090 lin ngu B Whole plant/ decoction/ oral/ fever, wound (snake bites), cancer
Oldenlandia heynei Oliv. H NS 100 Lin ngu lek B Whole plant/ decoction/ oral/ fever, wound (snake bites), cancer
Spermacoce articularis L. f. H NS 063 pik malang wan B Whole plant/ decoction/ oral/ kidney stone
Rutaceae
Glycosmis pentaphylla (Retz.) DC S NS 089 Khoei tai
B
Roots/ decoction/ oral/ fever;
Bark/ decoction/ oral/ abscesses;
Fruits, flowers/ poultice/ topical/ scabies, herpes simplex, Herpes
zoster

2430 J. Med. Plants Res.
Table 1. Contd.
Sapindaceae
Allophylus cobbe (L.) Raeusch. S NS 039 To sai M
Cardiospermum halicacabum L. C NS 077 Poo om B
Roots, wood/ decoction/ oral/ joint and muscle pain
Leaves/ poultice/ topical/ fever
Whole plant/ decoction/ oral/ fever
Roots/ decoction/ oral/ wound (snake bites), constipation
Fruits/ decoction/ oral/ choleretic
Mischocarpus sundaicus Blume T NS 015 Si fun B Roots/ decoction/ oral/ fever, malaria
Sapotaceae
Pouteria obovata (R. Br.) Baehni T NS 112 Ram tua phu B Fruits/ decoction/ oral/ menstrual disorder
Schizoeaceae
Lygodium microphyllum (Cav.) R. Br. Ep NS 055 Li phao yung B
Roots/ decoction/ oral/ fever
Whole plant/ decoction/ oral/ orchitis, cancer, joint and muscle pain
Scrophulariaceae
Lindernia ciliata (Colsm.) Pennell H NS 109 Ya kra tai jam B Whole plant/ decoction/ oral/ fever, dysuria
Lindernia crustacea (L.) F. Muell. H NS 054 Ya kled hoi B Whole plant/ decoction/ oral/ fever
Solanaceae
Physalis minima L. H NS 102 Thong theng B
Solanum trilobatum L. C NS 123
Sonneratiaceae
Sonneratia caseolaris (L.) Engl. T NS 082 Lam phu M
Whole plant/ poultice/ oral/ tonsillitis
Fruits/ raw/ oral/ sore throat
Ma waeng- B, Leaves/ poultice/ topical/ Herpes zoster
khruea M
Whole plant/ decoction/ oral/ dysuria, diabetes mellitus
Fruits/ raw/ oral/ fever, apthus ulcer, sore throat
Roots/ powder/ topical/ Herpes simplex
Fruits/ raw/ oral/ wound, diarrhea
Sterculiaceae
Heritiera littoralis Aiton T NS 041 Ngon kai thale M Wood/ decoction/ oral/ menstrual disorder
Tiliaceae
Corchorus trilocularis L. H NS 072 Nguag pla mo B Whole plant/ decoction/ oral/ hypotension
Microcos tomentosa Sm. T NS 037 Plab pla M
Fruits/ raw/ oral/ toxin in the body;
Fruits/ decoction/ oral/ mouth sore

Table 1. Contd.
Urticaceae
Pouzolzia pentandra (Roxb.) Benn. H NS 025 Khob cha nang M
Whole plant/ decoction/ oral/ fever;
Whole plant/ decoction/ topical/ toothache
Verbenaceae
Phyla nodiflora (L.) Greene H NS 070 Ya lek khood B Whole plant/ bath/ oral/ joint and muscle pain
Whole plant/ decoction/ oral/ parasites, fever,
Stachytarpheta jamaicensis (L.) Vahl S NS 078 Ya pan ngoo kheaw B Dysuria, wound
Whole plant/ poultice/ topical/ abscesses
Vitex peduncularis Wall. T NS 021 Non B
Vitaceae
Cayratia trifolia (L.) Domin C NS 071 Thao khan khao
Neamsuvan et al. 2431
Whole plant/ decoction/ oral/ joint and muscle pain, fever
Whole plant/ decoction/ topical/ mouth sore
B Stem/ decoction/ oral/ expectorant,
M Menstrual disorder, abscess
Zygophyllaceae
Tribulus terrestris L. H NS 058 Khok kra soon B Whole plant/ decoction/ oral/ dysuria, kidney stone, fever
H1= Habit (C=climber, Ep=epiphyte; H=herb, S=shrub, T=tree); H2= habitat (B=beach forest, M=mangrove forest.
tract problems with 22 species (10.19%),
respectively.
Fever or pyrexia was the common illness with
high-bodily temperature, weakness and
headache. Then, the medicinal plants to get rid off
that symptoms were applied such as roots of
Casuarina equisetifolia L. used to cure headache
and encephalitis, whole plant of Commelina
benghalensis L. used as antipyretic, and roots of
Abrus precatorius L. used to decrease high-bodily
temperature.
In skin disease, the symptoms were oozing
eczema due to lymphatic disorder, herpes simplex
and herpes zoster. Then, the medicinal plants to
get rid off that symptoms were such as latex of
Calotropis gigantea L. used to cure warts and
corns, leaves of Indigofera tinctoria L. used to
cure inflammation of abscess, and fresh fruits of
Catunaregam spathulifolia Tirveng. used to cure
athlete’s foot.
In gastrointestinal tract problems, the symptoms
were grouped, including constipation, diarrhea,
dysentery and hemorrhoid. The medicinal plants
used to cure these were such as leaves of
Euphorbia heterophylla L. and roots of
Cardiospermum halicacabum L. used as laxative.
Plant parts used
There were 9 plant parts used by traditional
healers for treatment of diseases and (Table 3).
However, whole plant was the most frequently
utilized for 48 species (29.63%), followed by
root/rhizome for 27 species (16.67%) and leaves
for 26 species (16.05 %), respectively.
Notably, some species could be used for more
than one plant parts either for healing one or
different diseases. For example, root and bark of
Pleurostylia opposita (Wall.) Alston also could be
used for curing malaria. In contrast, leaf of
Chromolaena odoratum (L.) R.M. King & H.Rob.
was used to stop bleeding, while its root was
used for diabetes mellitus.
Traditionally, whole plant refers to 5 parts of
plant: Root, stem, leaf, flower and fruit. In case of
small or herbaceous plant, it means really whole
plant. However, it is only representative of those

2432 J. Med. Plants Res.
Table 2. Diseases or symptoms to be cured by medicinal plants
in Sating Phra Peninsula, Songkhla Province.
Diseases/symptoms Frequency Percentage
Fever 40 18.52
Skin diseases 23 10.65
Gastrointestinal tract 22 10.19
Problems
Urinary system 17 7.87
Wound 16 7.41
Joint and muscle pain 13 6.02
Menstrual disorder 11 5.09
Dental hygiene 10 4.63
Respiratory 7 3.24
Toxin in the body 4 1.85
Thirsty relief 2 0.93
Jaundice 2 0.93
Hair and scalp 2 0.93
Syndrome
Fatigue 8 3.70
Cancer 7 3.24
Parasites 5 2.32
Malaria 5 2.32
Midwifery 6 2.78
Blood tonic 4 1.85
Diabetes mellitus 4 1.85
Visceral organ 3 1.39
Damage
Infant convulsion 1 0.46
Diet 1 0.46
Cardiotonic 1 0.46
Eye problems 1 0.46
Hypotension 1 0.46
Table 3. Plant part used.
Plant part Frequency Percentage
Whole plant 48 29.63
Root/rhizome 27 16.67
Leaf 26 16.05
Fruit 17 10.49
Wood/heart wood 16 9.88
Bark 8 4.94
Stem 7 4.32
Flower/inflorescence 6 3.70
Seed 4 2.47
Latex 3 1.85
Table 4. Methods for herbal preparation.
Method Frequency Percentage
Decoction 97 64.24
Poultice 23 15.23
Raw 15 9.93
Powder 6 3.97
Juice 5 3.31
Bath 2 1.36
Oil 1 0.66
Charcoal 1 0.66
Dessert 1 0.66
Table 5. Administration for medicinal plants.
Administration Frequency Percentage
Oral 99 71
Topical 34 24
Bath 3 2
Inhalation 1 1
Sauna 1 1
Steam Bath 1 1
5 parts if it is shrub or tree.
Herbal preparations
There were 9 herbal preparations documented from this
study (Table 4). The most frequently used preparation
was decoction for 97 species (64.24%), followed by
poultice for 23 species (15.23%) and raw for 15 species
(9.93%). It should be noted that some plants were
prepared with more than one method for treating different
disease. For example, Alternanthera sessilis (L.) R. Br.
ex DC. with whole plant was prepared as decoction for
curing menstrual disorder, whereas it may also be
prepared as poultice to cure intestinal parasitism in
childhood.
According to preparation method, decoction was
classified into 3 kinds. Firstly, medicinal materials in clean
drinking water would be boiled until liquid decreased to
be a one third, then it was used for drinking. Secondly,
medicinal materials in clean water were boiled until steam
is obtained, then it was used for taking a bath. Finally,
medicinal materials in clean drinking water were boiled
until steam is obtained, then it was drunk as tea.
In addition, poultice was crushed, pinched, chopped, or
pounded medical materials for mostly applying on skin.
Raw was the utilization of any plant parts without
processing. In this study, plant parts were eaten as raw
vegetables or fruits. Powder was prepared by grinding
plant parts. This received more fine granules than
poultice method, and it was mostly used for skin disease.

Juice was gathered from extraction of any plant parts. It
might be from squeezing or damaging materials to get
watery sap or latex. The method of bath entails placing
plant parts in hot water or parboiling them. This method
was performed with plant parts which were eaten as
parboiled vegetables. Oil was made by extraction
method, while dessert was prepared by putting plant
parts in syrup.
Herbal administration
Local drugs were administered through 6 routes (Table
5): Oral, topical, inhalation, sauna, steam bath and bath.
Oral administration was the most frequently used route
with 99 plant species (71.22%), followed by topical
administration with 34 species (24.46%) and bath with 3
species (2.16%), respectively. It showed that some plants
could be administered with more than one routes.
Pouzolzia pentandra (Roxb.) Benn. was an example that
its decoction of whole plant was oral administered as
fever relieving, while that decoction was also topical
administered by keeping it in mouth to cure toothache.
Administration by oral route is in agreement with many
previous studies in various tribes around the world (Coe
and Anderson, 1996; Kamatenesi et al., 2011; Collins et
al., 2007).
DISCUSSION
The most common disease
From the study, it showed that the symptom or disease
that local healers knew many plants for curing were
pyrexia or fever. This is related to the report of Ministry of
Health Thailand (2010) which indicated that pyrexia of
unknown origin was a disease having high morbidity rate
in Thailand since 1983. Moreover, it was reported that
people in Southern Thailand suffered from pyrexia of
unknown origin with 704.04 people per 1,000 people a
year. Therefore, it reflected the truth why local healers
knew many plants for curing this symptom or disease. In
this study, however, the fever mostly mentioned by local
healers was Khai-phid-nam.
Khai-Phid-Nam
Khai is a fever in Thai language. Khai-phid-nam is a kind
of fever, caused by a return of fever after patients took
their bath and were absolutely relieved of the last illness.
The chief complaint of this fever is lower temperature at
both feet, while higher one at other body parts.
According to healers’ knowledge, there were 7 species
of medicinal plants used for treating Khai-Phid-Nam
including Bridelia stipularis (L.) Blume, Breynia sp.,
Flagellaria indica L., Melaleuca cajuputi Roxb., Solanum
Neamsuvan et al. 2433
trilobatum L., Glycosmis pentaphylla (Retz.) DC,
Mischocarpus sundaicus Blume and Sida cordifolia L.
Comparison to related study
The folk knowledge of herbal utilization obtained from this
survey was compared to 2 closely related studies. The
former Thaechatturat’s study (2000) about using
medicinal plants of Morgan tribe in Phang-nga province,
the resembling properties to present study were found in
2 species, namely C. odoratum (L.) R.M. King & H.Rob.
used for wound treating and Morinda elliptica (Hook. f.)
Ridl. used as anthelmintic. The latter Upho’ s study
(2005) about using medicinal plants of Buddhist and
Muslim Thais in Trang province, a total of 12 species
with resembling properties to this study was found,
namely C. odoratum (L.) R.M. King & H.Rob. used for
wound healing, Vernonia cinerea (L.) Less. used to stop
bleeding, Ipomoea pes-caprae (L.) R. Br. used for curing
toxin from jellyfish, Euphorbia hirta L. used as
lactogogue, Senna sophera L. used as antidote,
Chrysopogon aciculatus (Retz.) Trin. used for gravel
treatment, Tiliacora triandra Diels used for relieving fever,
Oldenlandia corymbosa L used to treat cancer,
Glycosmis pentaphylla (Retz.) DC used to treat herpes
simplex as well as herpes zoster, Lygodium microphyllum
(Cav.) R. Br. used to relief fever. Moreover, 2 species
namely Xylocarpus granatum J. Koenig and
Rhodomyrtus tomentosa (Aiton) Hassk. were used as
antidiarrhoeal.
Biological confirmation
From this study, 34 medicinal plants showed that their
folk properties were concordant to pharmacological
activities studied previously (Table 6). For example,
Abrus precatorius L, the traditional healer used to cure
Athlete’s foot while the biological research showed that
its leaf extract (Adelowotan et al., 2008), root extract
(Mistry et al., 2010) and seed extract (Prashith Kekuda et
al., 2010) were potentially against the Gram positive
organism Staphylococcus aureus, causing pus and ulcer
(Franklin, 1998).
Alternanthera sessilis (L.) R. Br.ex DC was used to
cure fever. Simultaneously, Praveen et al.’s (2010) study
showed that its extract has potential to cure antipyretic
activity. In addition, Johnson et al.’s (2010) study showed
that leaves extracts are more effective against Proteus
vulgaris, Streptococcus pyogenes, Bacillus subtilis and
Salmonella typhii.
However, there were 13 species that have never been
studied about biological activities including Drynaria
sparsisora (Desv.) T. Moore, Jasminum nervosum Lour.,
Uvaria ridleyi King, Fimbristylis sericea R. Br., Lindernia
ciliata (Colsm.) Pennell, Lindernia crustacea (L.) F.
Muell., Allophylus cobbe (L.) Raeusch., Microstachys

2434 J. Med. Plants Res.
Table 6. The species with biological confirmation
Scientific name
Traditional healer uses (plant
part/ disease)
Abrus precatorius L. Leaves/ athlete's foot
Acanthus ilicifolius L. Whole plant/ cancer
Pharmacological activities (References)
Antibacterial activity (Adelowotan Bobbarala and Vadlapudi,
2009; Prashith et al., 2008; Mistry et al., 2010; Kekuda et al.,
2010); antifungal activity (Prashith Kekuda et al., 2010)
Antitumour activity (Babu et al., 2002); antioxidant activity
(Babu et al., 2001)
Alternanthera sessilis (L.) R.
Br.ex DC.
Whole plant/ fever Antipyretic activity (Praveen et al., 2010)
Antibacterial activity (Johnson et al., 2010)
Calophyllum inophyllum L. Fruits/ joints and bones pain Antiinflamamtory activity (Shah et al., 2006)
Rhizophora apiculata Blume Fruits/ fever Antiviral activity (Jassim and Naji, 2003)
Rhizophora mucronata Lam.
Rhodomyrtus tomentosa(Aiton)
Hassk.
Fruits/ fever;
stem bark/ diarrhea, mucous;
bloody dysentery,
Antibacterial activity (Jelager et al., 1998)
Fruits/ diarrhoeal Antibacterial activity (Surasak et al., 2009)
Sida cordifolia L. Whole plant/ fever Antibacterial activity (Mahesh and Satish, 2008)
Solanum trilobatum L.
Tiliacora triandra Diels Stem/ fever
Tribulus terrestris L.
Vernonia cinerea (L.) Less.
Xylocarpus granatum J. Koenig
Leaves/ herpes simplex; Analgesic activity (Annamalaia et al., 2009)
Fruits/ sore throat, fever
Antiinflammatory and analgesic activity (Ramakrishna et
al., 2011)
Whole plant/ diabetes mellitus
Antibacterial activity (Swapna Latha & Kannabiran, 2006);
antidiabetic activity ( Doss et al., 2009)
Antimalarial activity (Saiin and Markmee, 2003); antipyretic
activity (Jongchanapong et al., 2010)
Whole plant/ Dysuria, Diuretic activity (Al-Ali et al., 2003)
Kidney stone CaOx crystallization inhibition (Aggarwal et al., 2010)
Whole plant/ wound
Antiinflammatory activity (Mazumder et al., 2003);
Antibacterial activit(Gupta et al., 2003 )
Whole plant/ smoking cessation Smoking cessation (Wongwiwatthananukit et al., 2009)
Stem bark/ diarrhoeal, mucous
bloody dysentery
Antibacterial activity (Alam et al., 2006)
Ziziphus mauritiana Lam. Leaves/ seborrheic dermatitis Antibacterial activity (Abalaka et al., 2010)
Calotropis gigantean(L.) W.T.
Aiton
Latex/ toothache,corns
Senna tora L. Whole plant/ fever
Antibacterial activity (Alam et al., 2008; Subramanian and
Saratha, 2010) ; wound healing (Waya et al., 2009)
Antibacterial activity (Roopashree et al., 2008);
antibacterial activity (Chavan et al., 2011)
Cayratia trifolia (L.) Domin Stem/ used as emmenagogue PGE2 inhibition (Siriwatanametanona, 2010)

Table 6. Contd.
Chromolaena odoratum L. Leaves/ bleeding Would healing (Phan et al., 2001)
Clerodendrum inerme (L.)
Gaertn.
Leaves/ fever, exanthemathous
prickly heat and puritic rash
Neamsuvan et al. 2435
Antiinflammatory and Analgesic activity (Yankanchi and Koli,
2010); antibacterial activity (Chahal et al., 2010)
Combreta quadrangulare Kurz. Seeds/ anthelmintic Anthelmintic activity on Ascaridia galli (Sritong et al., 2005)
Commelina benghalensis L. Whole plant/ fever Antibacterial activity (Bagchi et al., 1999)
Crotalaria retusa L. Whole plant/ fever Antibacterial activity (Gangoue-pieboji et al., 2006)
Derris scandens (Aubl.) Pittier Roots/ cancer Antimigration of cancer cells (Laupattarakasem et al. 2007)
Euphorbia heterophylla L. Whole plant, leaves/ purgative Laxative activity (Falodun and Agbakwuru, 2004)
Euphorbia hirta L.
Whole plant/ lactogogue; Lactogogue activity (Blanc et al., 1963);
Whole plant/ herpes zoster Antiviral activitiy (Gyuris et al., 2009)
Hyptis suaveolens (L.) Poit. Seeds/ antidiarrhoeal Antimicrobial activity (Nantitanon et al, 2007)
Ipomoea pes-caprae (L.) R. Br.
Leaves/ toxin from jellyfish;
Leaves/ insect bites
Lippia nodiflora (L.) Michx. Whole plant/ Joint and muscle pain
Neutralization of toxic effects (Pongprayoon et al., 1991)
Antiinflammatory and analgesic activity (Forestieri et al.,
1996 )
Lumnitzera racemosa Willd. Wood, roots, stems/ wound healing Antibacterial activity (Souza, 2010)
Melaleuca cajuputi Roxb. Inflorescence/ wound in oral cavity Antibacterial activity (Khare, 2007)
Morinda elliptica(Hook. f.) Ridl.
Physalis minima L.
Pithecellobium dulce(Roxb.)
Benth.
Friuts,stem bark/ fever, oozing
eczema due to lymphatic disorder
and seborrhoeic dermatitis;
Roots/ fever causing convulsion
Antibacterial activity (Ali et al., 2000)
Whole plant/ tonsillitis Antiinflammatory and analgesic activity (Khan et al., 2009);
Fruits/ sore throat antibacterial activity (Shariff et al., 2006)
Stem bark/ toothache, gingivitis
Antiinflammatory activity (Sahu and Mahato, 1994);
antiinflammatory and antibacterial activity(Chandran and
Balaji, 2008)
Plucea indica Less. Whole plant/ dysuria Diuretic effect (Nilvises et al., 1989)
chamaelea (L.) Mull. Arg., Perotis indica (L.) Kuntze, L.
microphyllum (Cav.) R. Br., Pouteria obovata (R. Br.)
Baehni, Oldenlandia heynei Oliv. and Bridelia stipularis
(L.).
Therefore, it is interesting to study about
pharmacological activities of the rest.
It should be noted that only the old local healers know
different utilization of medicinal plants whereas many
plants are rapidly destroyed by human activities,
nowadays. Therefore sustainable conservation should be
conducted for preserving both indigenous knowledge and
medicinal plants.
ACKNOWLEDGEMENT
The authors are grateful to Research and Development
office, Prince of Songkla University for supporting the

2436 J. Med. Plants Res.
budget during the period of operation.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2438-2442, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.1435
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
High-performance liquid chromatography (HPLC)
determination of five active ingredients in the calyces
of Physalis Alkekengi L. var. franchetii (mast.) Mskino
Baoli Xu, Chengguo Ju, Huijie Guan, Liang Xu, Nan Xu and Bing Wang*
Liaoning University of Traditional Chinese Medicine, 77 shengming 1 Road, DD Port, Dalian, Liaoning Province,
P. R., 116600, China.
Accepted 11 January, 2012
A simple high-performance liquid chromatographic (HPLC) assay using the internal standard method is
developed for the simultaneous determination of five active ingredients. The analyzed compounds from
the calyces of Physalis alkekengi L. var. franchetii (mast.) Mskino include Luteoloside, Luteolin,
Physalin A, Physalin O and Physalin P. HPLC analysis is performed on an Agilent C18 analytical column
(150×4.6 mm and 5 μm) using solvent (A) acetonitrile and (B) 0.2% aqueous phosphoric acid as the
mobile phase with Ultraviolet (UV) absorption at 350 and 220 nm. The flow rate was 1.0 ml/min. The
calibration curves of the five active ingredients are linear (r 2 > 0.9991) over the concentration range of
10 to 120 μg/ml. The mean recoveries were 97.59 to 101.04%. The results indicate that the HPLC method
developed can easily be applied to the determination of five active ingredients in the calyces of P.
alkekengi L. var. franchetii (mast.) Mskino.
Key words: Calyces of Physalis alkekengi L. var. franchetii (mast.) Mskino, flavonoid, physalin.
INTRODUCTION
The calyces of Physalis alkekengi L. var. franchetii
(mast.) Mskino major recorded in the Chinese
Pharmacopoeia is a well-known traditional Chinese
medicine (TCM) for the treatment of sore throat and
urinate inability. The content of the luteolin not less than
0.1% in the dry calyces in accordance with the provisions
of the Pharmacopoeia of PRC 2010 (The Pharmacopoeia
of PRC, 2010). Flavonoids and physalins (Matsuura et
al., 1970; Kawai et al., 1987, 1988, 1993; Kawal et al.,
1995; Kazushi and Shii, 1992) are the main ingredients in
the calyces of P. alkekengi. Flavonoids and physalins are
the class of anti-inflammatory compounds (Xu et al.,
2009; Melissa et al., 2005; Angelica et al., 2005). The aim
of this study is to develop an assay to fully evaluate the
contents of flavonoids and physalins in the calyces from
different locations, wilds and purchases order to exploit
and utilize them reasonably.
*Corresponding author. E-mail: YZBwang@lnutcm.edu.cn. Tel:
+8641187586003.
MATERIALS AND METHODS
Plant material
Dried calyces of P. alkekengi var. franchetii were collected in six
provinces of China (Heilongjiang, Jilin, Liaoning, Shanxi, Shandong
and Anhui) from October 5 to 15, 2010. Voucher specimens were
maintained at Liaoning University of TCM, China.
Reagents
The stand substances of the Luteoloside bought from Dida
Technology Company Limited (Guizhou, China). The stand
substances of the Luteolin were bought from State Food and Drug
Administration (Beijing, China). The three standard substances of
physalin were isolated from the calyces of P. Alkekengi including
Physalin A, Physalin O and Physalin P by Prof. Nan Xu and Prof.
Liang Xu (Liaoning University of Traditional Chinese Medicine,
China). The purity of all the stand substances exceeds 98%. HPLCgrade
acetonitrile was purchased from Kermel Chemical Reagent
Company Limited (Tianjin, China), and the water used in all
experiments was purified by a Milli-Q Ultrapure Water System
(Millipore, MA). All other chemicals were of analytical reagent grade
purchased from Kermel Chemical Reagent Company Limited
(Tianjin, China).

Figure 1. HPLC chromatogram of five standard substances (1, Luteoloside; 2, Luteolin; 3, Physalin A; 4, Physalin P; 5,
Physalin O) (A) and the calyces of Physalis Alkekengi L. var. Franchetii (B).
Chromatographic system and conditions
HPLC analysis was carried out with an Agilent 1100 series HPLC
(Palo Alto, CA) incorporating a UV detector. The analytes were
determined at 30°C on an analytical column (Agilent C18, 150×4.6
mm, 5-μm particle size) (Agilent Technologies, Palo Alto, CA). The
mobile phase consisted of the solvent (A) acetonitrile and (B) 0.2%
aqueous phosphoric acid (v/v) using a gradient elution of 20 to 23%
(A) at 0 to 13 min and 23 to 31% (A) at 13 to 37 min and then
returned to initial condition for a 5 min re-equilibration, with total run
time 42 min. The mobile phase was passed under vacuum through
a 0.45-μm membrane filter before use. The analysis was carried out
at a flow rate of 1 ml/min with the detection wavelength set at 350
nm at 0 to 24 min and at 220 nm at 24 to 37 min.
Sample preparation
Crushed dried calyces of P. alkekengi are the 50 mesh, to the
conical flask with lid, 0.5 g of the powder of the calyces and 25 ml
of carbinol were add, then extracted in an ultrasonic bath for 1 h.
The filtrate concentrated to 25 ml volumetric flask. The filtrate were
filtered with 0.45 μm membrane filer to obtain the filtered solution
and an aliquot (20 μl) of filtrate was injected into the HPLC system.
RESULTS AND DISCUSSION
HPLC analysis
Our attempts to use the method with isocratic elution for
the determination of five active ingredients were
unsuccessful. The gradient elutionmethod was, therefore
used for the separation of the ingredients. During method
Xu et al. 2439
development, the mobile phase composition varied using
different combinations of methanol, acetonitrile and
phosphoric acid. It demonstrated a longer retention time
with a decrease in the organic composition. Here, the
mobile phase consisting of (A), acetonitrile and (B) 0.2%
phosphoric acid were chosen to achieve good peak
shape, satisfactory resolution, and relatively short
analysis time. Figure 1 show typical chromatograms of
the standard substances (A), and the calyces of P.
Alkekengi (B). The retention times of Luteoloside,
Luteolin, Physalin A, Physalin P and Physalin O, were
approximately 6.7, 21.9, 27.9, 32.2 and 33.1 min,
respectively, and the total chromatographic run time was
37 min.
Through the pre-test we found the maximum absorption
of the five active ingredients at 220 nm, but the
Luteoloside and Luteolin were not well separated from
adjacent interference peaks and the baseline was not
smooth. Luteoloside and Luteolin also had the maximum
absorption at 350 nm, but Physalin A, Physalin P and
Physalin O were not detected. So the detection
wavelength set at 350 nm at 0 to 24 min and at 220 nm at
24 to 37 min was chosen in the assay and suitable for the
simultaneous determination of the five active ingredients.
A mixture of methanol-water (70:30, v/v) was chosen as
the extraction solvent. A simple ultrasonic method was
used for sample preparation, which was timesaving and
may not lead to losses of the five active ingredients. The
sample clean-up steps to a minimum were a primary
concern; thus, the filtrate, after ultrasonication, was

2440 J. Med. Plants Res.
Table 1. Results of recovery experiments (%).
NO. Luteoloside Luteolin Physalin A Physalin P Physalin O
1 100.8 100.0 99.8 99.5 97.4
2 100.0 99.9 99.1 98.9 99.9
3 98.7 95.9 100.8 97.3 100.5
4 104.7 99.2 99.1 100.5 99.4
5 99.2 97.3 98.4 98.7 98.7
X±SD 100.8±0.024 98.5±0.018 99.4±0.009 98.9±0.011 99.2±0.012
RSD (%) 2.37 1.81 0.92 1.07 1.23
Table 2. Results of stability experiments.
Time (h) Luteoloside Luteolin Physalin A Physalin P Physalin O
0 1698.8 92.6 1684.0 74.1 139.2
6 1697.2 90.8 1684.5 68.5 142.0
12 1706.3 90.8 1683.2 68.5 138.7
18 1698.0 91.0 1682.4 68.3 138.2
24 1695.4 92.3 1682.0 69.2 139.0
X±SD 1699.1±4.196 91.5±0.877 1683.2±1.050 69.2±1.287 139.2±1.490
RSD (%) 0.24 0.96 0.06 1.86 1.07
Table 3. Results of the calyces from different locations (mg/g).
No. Location Luteoloside Luteolin Physalin A Physalin P Physalin O Total
s01 Fuhai, Qiqihaer, Heilongjiang 0.86 0.05 3.79 - 0.31 5.00
s02 Mohe, Yichun, Heilongjiang 0.45 0.06 1.46 - 0.58 2.54
s03 Xinchun, Yichun, Heilongjiang - 0.05 0.54 - 3.18 3.77
s04 Nancha, Yichun, Heilongjiang 0.48 0.11 8.07 - 0.31 8.98
s05 Yimianpo, Shangzhi, Heilongjiang 4.47 0.18 4.08 - 0.13 8.87
s06 Nanwaizi, Gongzhuling, Jilin 1.97 - 4.08 - - 6.04
s07 Guojiadian, Lishu, Jilin 1.22 0.03 4.93 - 0.60 6.77
s08 Shilingzi, Lishu, Jilin 1.20 0.02 5.30 - 0.19 6.70
s09 Erhedian, Dunhua, Jilin 1.23 - 2.41 - 0.13 3.77
s10 Dapucaihe, Dunhua, Jilin 2.12 0.02 4.18 - 0.52 6.83
s11 Weijing, Dongliao, Jilin 1.14 0.02 7.29 - 0.14 8.59
s12 Dongfeng, Liaoyuan, Jilin 1.10 0.05 6.03 - 0.43 7.62
s13 Xiaosiping, Liaoyuan, Jilin 1.13 0.08 4.99 - 0.23 6.43
s14 Meihekou, Jilin 1.05 0.02 6.21 - 0.15 7.42
s15 Shanchengzhen, Meihekou, Jilin 1.28 - 5.06 - 0.13 6.47
s16 Wangou, Baishan, Jilin 1.62 0.04 3.73 - 0.66 6.03
s17 Ermenshi, Baishan, Jilin 3.11 0.18 6.01 - 0.22 9.53
s18 Dahua, Baishan, Jilin 0.92 - 4.12 - 0.20 5.24
s19 Tonghua, Jilin 1.00 - 4.02 - 0.46 5.48
s20 Liming, Tonghua, Jinlin 0.50 - 3.05 - 0.55 4.11
s21 Daquanyuan, Tonghua, Jilin 0.97 0.06 3.37 - 0.11 4.51
s22 Xifeng, Tieling, Liaoning 0.65 0.04 1.34 - 0.61 2.63
s23 Tukouzi, Fushun, Liaoning 0.85 0.16 4.65 - 0.53 6.18
s24 Caoshi, Fushun, Liaoning 1.25 - 3.54 - 0.12 4.91
s25 Yingermen, Fushun, Liaoning 2.86 0.02 3.35 - 0.10 6.34
s26 Qingyuan, Fushun, Liaoning 0.43 0.12 2.63 - 1.05 4.23

Table 3. Contd.
Xu et al. 2441
s27 Beisanjia, Fushun, Liaoning 1.25 0.03 6.65 - 0.30 8.22
s28 Hongtoushan, Fushun, Liaoning 1.24 - 4.44 - 0.13 5.80
s29 Dongling, Shenyang, Liaoning 1.90 0.02 1.90 - - 3.82
s30 Hunhe, Shenyang, Liaoning 1.71 0.03 2.12 - 0.08 3.94
s31 Hunhepu, Shenyang, Liaoning 2.67 0.08 4.10 - 0.30 7.16
s32 Baiqingzai, Shenyang, Liaoning 3.02 0.03 2.92 - 0.15 6.12
s33 Cuigangzi, Heishan, Liaoning 0.55 - 2.45 - 0.21 3.22
s34 Qianxing, Heishan, Liaoning 0.79 0.01 2.90 - 0.24 3.95
s35 Zhangjiahuang, Heishan, Liaoning 2.34 - 2.12 - 1.10 5.56
s36 Lijiazhen, Heishan, Liaoning 1.25 0.03 3.00 0.32 0.24 4.84
s37 Yonglingzhen, Xinbin, Liaoning 1.61 0.02 3.50 - 0.10 5.22
s38 Qianjincun, Xinbin, Liaoning 0.77 - 4.14 - 0.16 5.06
s39 Guchengzhen, Huairen, Liaoning 1.03 - 3.17 - 0.29 4.49
s40 Shuanglingzicun, Huairen, liaoning 2.08 - 3.37 - 0.11 5.56
s41 Heigouxiang, Huairen, Liaoning 1.14 0.04 4.93 - 0.74 6.86
s42 Xiaoshizhen, Benxi, Liaoning 1.73 0.03 6.34 - 0.20 8.30
s43 Kazuo, Chaoyang, Liaoning 1.15 0.05 3.61 - 0.13 4.95
s44 Zhongtunxiang, Jinzhou, Liaoning 1.42 0.03 7.31 - 0.40 9.16
s45 Dawaxian, Panjin, Liaoning 2.37 0.09 6.64 - 0.78 9.87
s46 Sanshijiazizhen, Lingyuan, Liaoning 0.74 0.16 2.78 - 1.12 4.78
s47 Qianshan, Anshan, Liaoning 0.57 - 6.44 - 0.37 7.38
s48 Heishankexiang, Huludao, Liaoning 2.66 0.03 4.86 - 0.16 7.72
s49 Yangmadianzixiang, Huludao, Liaoning 0.85 0.04 9.86 - 0.20 10.95
s50 Yingkou, Liaoning 0.94 0.03 2.37 - 0.16 3.50
s51 Gaizhou, Yingkou, Liaoning 0.35 0.02 2.70 - 0.14 3.21
s52 Xiongyue, Yingkou, Liaoning 0.34 - 8.12 - 0.12 8.57
s53 Donggang, Liaoning 1.44 0.09 8.88 - 0.29 10.70
s54 Pulandian, Dalian, Liaoning 1.04 0.05 3.90 - 0.11 5.09
s55 Yuerjiancun, Datong, Shanxi 0.61 0.14 9.42 - 0.75 10.91
s56 Shikuxiang, Lucheng, Shanxi 1.07 0.07 4.71 - 0.69 6.55
s57 Qumenzhen, Yiyuan, Shandong 0.08 - 0.62 - 0.70 1.40
s58 Tiefou, Huaibei, Anhui 0.13 - 0.84 - 0.23 1.20
s59 Fulizhen, Shuzhou, Anhui 0.96 - 3.58 0.31 0.25 5.10
s60 Fulizhen, Shuzhou, Anhui 0.61 - 3.55 - 0.60 4.76
PS: “-” represented the contents had not been detected.
directly analyzed to decrease assay variability, and five
active ingredients showed good resolution and high
recovery.
Method validation
Linearity
The five calibration curves in the concentration ranges of
Luteoloside, Luteolin, Physalin A, Physalin P and
Physalin O were 0.532~2.66, 0.04~0.2, 0.48~2.4,
0.04~0.2 and 0.36~1.8 μg/mL, respectively.
The regression equations and coefficients were: y =
236.44x + 198.43, r = 0.9997; y = 371.34x + 95.48,
r = 0.9995; y = 76.337x + 25.217, r = 0.9996; y = 79.788x
– 8.81, r = 0.9997; y = 104.53x + 38.59 and r = 0.9997,
respectively, where y is the peak area of the five active
ingredients and x is the concentration of the five active
ingredients.
Recovery
For a validation of the extraction recoveries of the five
active ingredients, the analysis for each active ingredient
was carried out in five replicates. The results showed that
the mean extraction recoveries were acceptable,
suggesting that there was negligible loss during the
calyces extraction (Table 1).

2442 J. Med. Plants Res.
Stability
The stock solutions of the five active ingredients were
found to be stable at room temperature based on the
peak area of the five active ingredients over the time
range of 0 to 24 h. (Table 2).
Simultaneously determine five active ingredients
HPLC incorporating UV detector was employed to
simultaneously determine the five active ingredients in
dried calyces of P. alkekengi were collected in six
provinces of China. The powdered calyces were treated
as in the “Sample preparation” section. The results
indicated the variation of the five active ingredients in the
calyces from different locations (Table 3). According to
the Pharmacopoeia of PRC, the samples were qualified
except the sample of s03 which have been collected from
Xinchun, indicating that Luteoloside in relatively stable in
the calyces.
However, according to the overall contents. The sample
s49 which been collected from Yangmadianzixiang
contained the five active ingredients was the highest in all
samples. According to the Table 3 the Luteolin, Physalin
P and Physalin O had low levels and instability. Physalin
A had high content and stability and it was the unique
ingredients in the calyces. So we can establish the quality
standards of the calyces that determined content of
Physalin A.
Conclusion
A simple HPLC method was developed for the
simultaneous determination the five active ingredients in
dried calyces of P. alkekengi. The linearity, recovery and
stability of the developed method were validated,
respectively. From the results of this study, we know that
the contents of the five active ingredients in the calyces
major varied from different locations. In a word, the
results can be taken as the evidence for the exploitation
and utilization of the calyces reasonably.
ACKNOWLEDGMENTS
Thanks for the guidance of Prof. Yin haibo and Prof. Ying
xixiang during the experiment.
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Physalis in China. Chinese Wild Plant Resources, 28(1): 21-23.

Journal of Medicinal Plants Research Vol. 6(12), pp. 2443-2447, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.1509
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Betalains from stem callus cultures of Zaleya decandra
L. N. Burm. f. - A medicinal herb
M. Radfar, M. S. Sudarshana and M. H. Niranjan*
Medicinal Plant Tissue Culture Laboratory, Department of Studies in Botany, University of Mysore, Manasagangotri,
Mysore, 570006 Karnataka, India.
Accepted 1 December, 2011
The plant synthesizes various medicinal important compounds; similarly in vitro derived callus also
synthesizes the compounds. Hence, callus can also be used as an alternative to whole plant for the
production of secondary metabolites. A preliminary experiment was conducted using stem explants of
Zaleya decandra (Aizoaceae) on MS, B5 and Whites media containing different concentrations of auxins
and cytokinins in order to test the best suitable medium for callogenic potentiality. Murashige and
Skoog (MS) medium elicited a better response than other media used. The callus formation and the
nature of callus varies depending upon the nature of the growth regulators used. The various
concentrations and combinations of dichlorophenoxy acetic acid (2,4-D), 6-benzylaminopurine (BAP),
thidiazuron (TDZ), kinetin (Kn), naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA), indole-3butyric
acid (IBA) and coconut milk were tested. Of these, combination of 2,4-D (1.0 mg/L) and TDZ (2.0
mg/L) was found to be the best in inducing pigmented callus. As the concentrations of 2,4-D and TDZ
increased upto 5 mgL -1 , callogenic potentiality of the explants decreased. Phenotype color ranged from
white/green through yellow, mezenda, violet red to red representing different types of pigments. The
pigment when subjected to laboratory test using a spectrophotometer confirmed as betalains.
Key words: Callus, betalain, growth regulators, medicinal plant, Zaleya decandra.
INTRODUCTION
Natural products have been a major source of new drugs
(Vuorela et al., 2004). The use of medicinal plants to treat
human diseases has its roots in pre-historical times.
Medicinal plants are used by 80% of the world population
as the only available medicines especially in developing
countries (Hashim et al., 2010). Unfortunately, we know
very little or still unaware of a number of herbs whose
potential is yet to be fully exploited (Kamboj, 2000). In the
last few decades, the traditional system of medicine has
become the interest of many scientists as a tool to
*Corresponding author. E-mail: niranmhniran@gmail.com.
Abbreviations: BAP, 6-benzylaminopurine; NAA,
naphthaleneacetic acid; MS, Murashige and Skoog; 2, 4-D,
dichlorophenoxy acetic acid; Kn, kinetin; CW, coconut water;
IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; TDZ,
thidiazuron; FW, fresh weight; OD, optical density; DF, dilution
factor; LCMS, liquid chromatography- mass spectrometry;
NMR, nuclear magnetic resonance
improve the global health issues. At the same time, the
needs for the basic scientific investigation of medicinal
plants using indigenous medical systems have become
more significant and in the world of technology, the
importance of it is much more highlighted.
Zaleya decandra L. is a prostrate weed belonging to
the family Aizoaceae. It is distributed in the tropical and
subtropical regions of the world and southern parts of
India. The root is used for the treatment of hepatitis,
asthma and orchitis, and also, the decoction of the root
bark is credited with properties of aperients. The juice of
the leaves is dropped into the nostrils to relieve partial
headache (Nadkarni, 1996; Kirtikar and Basu, 1991). Z.
decandra callus culture was considered as a prospective
producer of betalains with high radical scavenging
activity. Betalains comprise a class of nitrogen containing
plant pigments found in the cell sap of plants belonging to
ten families of Caryophyllales (Benson and Laudermilk,
1957; Johri and Bhattacharyya, 1998).
Biotechnological approaches, specifically plant tissue
culture plays a vital role in search for alternatives to

2444 J. Med. Plants Res.
production of desirable medicinal compounds from plants
(Ramachandra and Ravishankar, 2002). On a global
scale, medicinal plants are mainly used as crude drugs
and extracts. Several of the more potent and active
substances are employed as isolated compounds,
including many alkaloids such as morphine (pain killer),
codeine (antitussive), papaverine (phosphordiesterase
inhibitor) and various types of cardiac glycosides (heart
insufficiency) (Wink et al., 2005). The capacity for plant
cell, tissue, and organ cultures to produce and
accumulate many of the same valuable chemical
compounds as the parent plant in nature has been
recognized almost since the inception of in vitro
technology. The strong and growing demand in today’s
market place for natural, renewable products has
refocused attention on in vitro plant materials as potential
factories for secondary phytochemical products, and has
paved the way for new research exploring secondary
product expression in vitro. Plant-produced secondary
compounds have been incorporated into a wide range of
commercial and industrial applications, and fortuitously,
in many cases, rigorously controlled plant in vitro cultures
can generate the same valuable natural products. Plants
and plant cell cultures have served as resources for
flavors, aromas and fragrances, biobased fuels and
plastics, enzymes, preservatives, cosmetics
(cosmeceuticals), natural pigments, and bioactive
compounds (Karuppusamy, 2009).
In the present investigation, this paper describes the
isolation of the secondary metabolite (betalain pigment)
from the callus mass of Z. decandra by altering various
growth hormones and adjuvants.
MATERIALS AND METHODS
Z. decandra L. were collected from in and around Mysore University
campus, Karnataka and maintained as stock plants in the
Departmental garden. Stem explants were utilized for investigation.
Explants were thoroughly washed in running tap water for 30 min
followed by 5% (v/v) liquid detergent laboline for 5 min. Then
washed in distilled water and followed by surface sterilization in
0.1% (w/v) mercuric chloride solution for 2 to 3 min for stem
explants. After surface disinfection the material was thoroughly
washed in sterile distilled water 4 times. Then stem explants were
aseptically cut into pieces of required sizes and they were
inoculated on MS medium supplemented with different
concentrations and combinations of auxins and cytokinins. All the
media contained 3% (w/v) sucrose. All the cultures were maintained
at the temperature of 25±2°C under 16 h photoperiod and
maintained for the callus formation.
For realizing the pigments the following experiment has been
done. The weight of violet red coloured callus obtained has been
measured. 862 mg of fresh weight (FW) of callus having violet red
colour was aseptically removed from the culture flask, macerated
and extracted the pigment with cold water (temperature

Figure 1. Pigmented callus initiation from stem explants on MS medium
supplemented with 1.0 mgL -1 (2,4-D) and 2.0 mgL -1 (TDZ) after 8 weeks
of culture.
Table 1. Effect of 2,4-D and TDZ on pigmented callus formation from stem explants of Z. decandra on MS medium.
Radfar et al. 2445
Growth regulator (mg/L)
2,4-D TDZ
Nature of callus Intensity of pigmented callus formation
0.5 0.5 Pink, creamy, compact +
1.0 1.0 Pink, creamy, compact +
0.5 1.5 Red, fragile +
1.0 2.0 Dark red, soft +++
Intensity of pigmented callus: + (low), ++ (moderate), +++ (high).
noted that some authors have attributed the high
antioxidant activity of crude betalain-containing extract to
their high concentration of flavonoids (Lee et al., 2002).
Betalains reportedly have diverse, desirable activities
(Lila, 2004), including anti-inflammatory (Lee et al., 2006)
hepatoprotective (Galati et al., 2005) and cancer chemopreventative
activities (Kapadia et al., 1996).
Recently it has been reported (Sreekanth et al., 2007)
that betanin induces apoptosis in human chronic mylloid
leucemia cells. Hence, betalains are likely to be highly
suitable in natural colorants for preparing healthy foods
and their consumption is likely to increase. Plant cell and
tissue cultures are attractive alternative sources of
bioactive plant substances, including betalain pigments
(Ramachandra and Ravishankar, 2002). The
biotechnological production of food colorants using plant
in vitro cultures offers several advantages over the
conventional cultivation of whole plants, notably the
ability to maintain aseptic, controlled conditions (Vanisree
et al., 2004).
Various factors affecting plant cell culture, such as
concentration of essential nutrients, stress factors, light,
incubation period and concentration of growth regulators,
were found to be important determinants of secondary
metabolite production. The results obtained from the
growth of Z. decandra callus culture, biosynthesis of
betalains and the uptake of the main nutrients showed
that it was distinguished by certain physiological
peculiarities, which is related to the biosynthesis of
betalains. Betalains exhibit anti cancer activity and
antioxidant properties (Gentile et al., 2004).
Conclusion
In vitro propagation of medicinal plants with enriched
bioactive principles and cell culture methodologies for
selective metabolite production is found to be highly
useful for commercial production of medicinally important
compounds. The increased use of plant cell culture
systems in recent years is perhaps due to an improved
understanding of the secondary metabolite pathway in

2446 J. Med. Plants Res.
Figure 2. Pigmentation in subcultured callus on MS medium supplemented with BAP
(1.0 mgL -1 ) and CM (20%), after 8 weeks of subculture.
Table 2. Effect of subcultured pigmented callus on MS medium supplemented with different growth regulators and an
adjuvant.
Growth regulators (mg/L)
BAP IBA Kn
Adjuvant
CW (%)
Intensity of pigmentation on subcultured callus
0.5 - - 15 +
1.0 - - 20 +++
1.5 - - 40 ++
2.0 - - 60 +
- 0.5 0.5 15 ++
- 0.5 1.0 30 +++
- 0.5 1.5 45 ++
- 0.5 2.0 60 +
Intensity of pigmentation = + (low), ++ (moderate), +++ (high).
economically important plants. Advances in plant cell
cultures could provide new means for the cost-effective,
commercial production of even rare or exotic plants, their
cells, and the chemicals that they will produce. Therefore
in vitro system can be noticed as a suitable method for
the production of betalains pigment. Further confirmation
with liquid chromatography- mass spectrometry (LCMS)
and nuclear magnetic resonance (NMR) are
recommended for betalains.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2448-2452, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.1550
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Anti-Candida activity of ethanolic extracts of Iranian
endemic medicinal herbs against Candida albicans
H.A. Rohi Boroujeni 1 , A. Ghasemi Pirbalouti 1 *, B. Hamedi 1 , R. Abdizadeh 1 and F. Malekpoor 2
1 Shahrekord Branch, Islamic Azad ‎University, Researches Centre of Medicinal Plants and Ethno-veterinary, Rahmatieh,
POBox: ‎‏166‏‎, Shahrekord, Iran.
2 Department of Biology, Faculty of Basic Science, Tarbiat Moallem University (Kharazmi), Tehran, Iran.
Accepted 14 December, 2011
It has long been known that herbs and their extracts have antimicrobial activities. Heracleum
lasiopetalum Boiss., Satureja bachtiarica Bunge., Thymus daenensis Celak., Echiophora platyloba L.,
Dracocephalum multicaule Benth., Kelussia odoratissima Mozaff. and Achillea kellalensis Boiss. are
Iranian endemic plant species that have been traditionally used as medicinal herbs and spices in
different regions of Iran especially Central Zagross. Seven ethanolic extracts of endemic medicinal
herbs and one extract of native medicinal herb (Stachys lavandulifolia Vahl.) collected from
Chaharmahal va Bakhtiari province of Iran were assayed for the in vitro antifungal activity against
Candida albicans (ATCC1023), using agar dilution methods. Most of the extracts showed relatively high
anti-Candida activity against the tested fungi with the diameter of inhibition zone ranging between 8 and
17 mm. The extracts of S. bachtiarica and T. daenensis exhibited high inhibitory effect against C.
albicans. The extracts of S. bachtiarica and T. daenensis were characterized using HPLC, the major
components of S. bachtiarica and T. daenensis were carvacrol and thymol, respectively. The minimum
inhibitory concentration (MIC) values for active extract range between 25 and 50 µg/ml. In conclusion, it
can be said that the extract of some of the Iranian endemic medicinal plants (S. bachtiarica and T.
daenensis) could be used as natural anti-Candida.
Key words: Candida albicans, endemic medicinal herbs, antifungal activity, Satureja bachtiarica, Thymus
daenensis.
INTRODUCTION
Infectious diseases especially of fungal origin are major
health hazard all over the world and in some cases they
cause premature deaths, that is, almost 50,000 people
per day (Mulligen et al., 1993) and they have increased
markedly during the last decade (Terell, 1999; Meis and
Verweji, 2001). Candidal infection represents one of the
most rapidly increasing healthcare infections ‎with a
significant mortality rate in hospitalized patients (Jarvis,
1995‎;‎ Wey et al., 1989).‎ These fungal infections are
becoming more prevalent worldwide because the size of
the ‎immunocompromised patient population is rising, and
despite appropriate anti-fungal ‎therapy, mortality from
candidemia is over 30% (Morgan et al., 2005)‎. Candida
*Corresponding author. E-mail: ghasemi@iaushk.ac.ir.
species are now recognized as major agents of hospital
acquired infection (Douglas, 2003). Candida species are
the most common fungal pathogens of humans and are
the ‎causative agents of oral and vaginal candidiasis,
giving rise to severe morbidity in ‎millions of individuals
worldwide (Calderone and ‎ Fonzi‎, 2001; Ruhnke, 2002).‎
Candida albicans is the organism most often associated
with serious fungal infection and it is showing increased
resistance to traditional antifungal agents (Hawser and
Douglas, 1995; Jarvis, 1995). C. albicans is a diploid
fungus that grows both as yeast and filamentous cells,
and ‎is‎ a causal agent of opportunistic oral and genital
infections in humans (Ryan and Ray‎, 2004; Enfert and
Hube, 2007). ‎C. albicans is one of the leading causes of
opportunistic fungal infections in immunocompromised
individuals, including AIDS patients, transplant recipients,
and cancer patients ‎(Scherer and Magee‎, 1990‎;‎

Table 1. Iranian medicinal plants used in this study.
Boroujeni et al. 2449
Scientific name Family name Local name Type Parts used Uses/ailments treated
Satureja bachtiarica Bung. Lamiaceae Marzeh-e- Koohi Endemic Aerial parts
Edible as vegetable, flavoring, indigestion,
cough, anti-bacterial
Thymus daenensis Celak. Lamiaceae Avishan-e- Koohi Endemic Aerial parts
Dracocephalum multicaule Montbr and Auch. Lamiaceae Zarrin giah, Zeravihi Endemic Aerial parts
Stachy slavandulifolia Vahl. Lamiaceae Lolopashmak, Chay-e- Koohi Native Aerial parts
Green tea, spice, culinary, cough, anti-
bacterial, carminative
Sedative, analgesia, inflammatory, anti-
bacterial, anti-septic, foot pain
Green tea, anti-bacterial, skin diseases,
menorrhagia
Achillea kellalensis Boiss. and Hausskn. Asteraceae Golberenjaz Endemic Flowers Wound, carminative, indigestion
Kelussia odoratissima Mozaff. Apiaceae Kelus Endemic Leaves
Edible as vegetable, flavoring, indigestion,
rheumatism
Heracleumlasiopetalum Boiss Apiaceae Goolpar, Keresom Endemic Fruit Anti-septic, spice and condiment
Echinophora platyloba DC. Apiaceae Khosharizeh Endemic Aerial plant Anti fungal, spice and culinary
Shepherd et al., 1985)‎. However, this fungus
frequently causes a range of mucosal infections
such as oral ‎thrush and vaginitis (Ruhnke, 2002).‎
The remedial uses of commercially available
antifungal drugs have induced varieties of toxic
side effects (Chotomongakol and
Sukeepaisarncharoen, 1997; Rukayadi et al.,
2008); therefore, there is a distinct need for the
discovery of new, safer, and more effective
antifungal agents (Frontling and Rathway, 1987).
Plant derived medicines have been part of
traditional health care in most parts of the world
for thousands of years, and nowadays there is
increasing interest in plants as sources of agents
to fight microbial diseases (Portillo et al., 2001;
Natarajan et al., 2003).
Numerous Iranian folklore herbs for example:
Heracleum lasiopetalum, Satureja bachtiarica,
Thymus daenensis, ‎Echiophora platyloba,
Dracocephalum multicaule, Kelussia
odoratissima, Achillea kellalensis and Stachys
lavandulifolia have been utilized as traditional
medicines by the indigenous people of
Chaharmahal va Bakhtiari, Southwest Iran
(Ghasemi Pirbalouti, 2009). There is no information
or report on the anti-Candida properties of these
plants. Hence, the present first-time investigation
was carried out to test the antifungal activity of
ethanolic extracts against C. albicans, which can
cause candidiasis in human beings. It is
necessary to establish the scientific basis for the
therapeutic actions of traditional plant medicines
as these may serve as the source for the
development of more effective drugs. This study
aimed to determine the anti-Candida activity of the
extracts of eight plant species, which are endemic
Iranian plants.
MATERIALS AND METHODS
Plant material
The seven Iranian endemic plants and one native herb
were collected from mountain areas of Central Zagross,
Chaharmahal va Bakhtiari district, during May to
September, 2010 (Table 1). Their identity was confirmed
and voucher specimens were deposited at the Research
Centre of Medicinal Plants, Islamic Azad University,
Shahrekord Branch, Iran.
Sample preparation
Harvested flowering aerial parts (leaves and flowers) were
dried at room temperature for one week. The extracts were
obtained by stirring 100 mg of ground samples with 30 ml

2450 J. Med. Plants Res.
Table 2. Anti-Candida activity of Iranian endemic medicinal herbs.
Plants
50
Concentrations (µg)
25 12.5 6.25 3.12
Satureja bachtiarica Bung. 17.6 a 13.1 10.7 - -
Thymus daenensis Celak. 16.3 12.4 10.5 - -
Dracocephalum multicaule Montbr and Auch. 15.8 12 9.8 - -
Stachys lavandulifolia Vahl. 15.6 11.9 9.6 - -
Echinophora platyloba DC. 15.4 12.3 10.6 - -
Achillea kellalensis Boiss. and Hausskn. 13.8 12.1 8 - -
Kelussia odoratissima Mozaff. 15.6 12 8 - -
Heracleum lasiopetalum Boiss. 13 10 - - -
a: The inhibition zone diameter (mm) for all extracts of plants.
of pure ethanol (analytical grade; Merck, Germany) for 30 min.
Samples were filtered by a Whatman no 4. filter paper.
Reagents and chemicals
Methanol (HPLC grade), ethanol (analytical grade), acetonitrile
(analytical grade) and water (HPLC grade) were purchased from
Merck Co. (Darmstadt, Germany). The standard of thymol and
carvacrol acid were purchased from ROTH (Karlsruhe, Germany).
Preparation of standard solution
Stock standard solutions were prepared by accurately weighing
22.3 mg thymol reference standard and 16.4 mg carvacrol into
separate 50 ml volumetric flasks, and dissolving in acetonitrile/water
(50:50, v/v). Working standard solutions (1, 2.5 and 5 ml) were
prepared by dilution from the stock standard solution. The mixture
was stirred carefully and refluxed in a water bath at 90°C for 1 h.
Identification of phenolic compounds using HPLC
The isolation and analysis method for thymol and carvacrol were
conducted according to previously published protocols
(Hajimehdipoor et al., 2010; Shekarchi et al., 2010). The obtained
mixture was injected to HPLC system (Kanauer, Germany). An HP
1000 series liquid chromatography system comprising vacuum
degasser, quaternary pump, autosampler, thermostatted column
compartment and diode array detector was used. Column Machery-
NAGEL, Nucleosin-100-5 C18, Loop 20 µl was maintained at 30°C.
Solvents used for separation were water (eluent A) and acetonitrile
(eluent B).
The gradient program was as follows: 70% A/30% B, 0 to 5 min;
42% A/58% B, 5 to 18 min; 70% A/ 30% B, 18 to 30 min. The
calibration curves (correlation coefficient) for thymol and carvacrol
were Y = 89322x -382440 (r 2 = 0.998) and Y = 74919x - 247838 (r 2
= 0.994), respectively. Samples were filtered through a 0.45 µm
membrane filter before injection.
The flow rate was kept at 1 ml min -1 . The injection volume was 20
µl, and peaks were monitored at 330 nm. The chromatographic
peaks of thymol and carvacrol were confirmed by comparing their
retention times and UV spectra with that of their reference standard.
Working standard solutions were injected into the HPLC and peak
area responses were obtained. Standard graphs were prepared by
plotting concentration versus area. Quantification was carried out
from integrated peak areas of the samples using the corresponding
standard graph.
Fungal strain
The activity of extracts was assayed against isolate of C. albicans
(ATCC1023). The Candida grown overnight at 36°C in RPMI 1640
with 1-gluamin without bicarbonate sodium with MOPS (0.165 µ, pH
= 7.5) plates, and inoculums for the assays was prepared by
diluting scraped cell mass in solution, adjusted to McFarland scale
0.5 and confirmed by spectrophotometer reading at 600 nm. Cell
suspensions were finally diluted to 10 6 colony forming units
(CFU)/ml for use in the assays (Table 1).
Antifungal test
The disc diffusion method of Iennette (1985) was used with some
modification to determine the rate of growth inhibition of fungi by the
examined plant extracts. Sabouraud dextrose agar (Merck,
Germany) was used to prepare the culture medium and autoclaved
at 121°C for 15 min. Plates (8 cm diameter) were prepared with 10
ml agar inoculated with 1 ml of each microbial suspension. The
extracts were dissolved in dimethyl sulfoxide (DMSO, 20 µl) before
testing for antifungal activity. Sterile paper discs (6 mm in diameter)
were impregnated with 60 µl of dilutions of known extracts
‎concentrations (3.12 to 50 µg/disc) and incubated at 37°C for 48 h.
Microbial growth inhibition was determined as the diameter of the
inhibition zones around the discs (mm). The growth inhibition
diameter was the average of three measurements, taken at three
different directions. All tests were performed in triplicate. The
minimum inhibitory concentration (MIC) value was determined using
serial dilution assay. The MIC was defined as the lowest
concentration of the compound to inhibit the growth of 50% of
microorganisms. Each tube was inoculated with 5 ml of microbial
suspension at a density of 10 6 CFU/ml and incubated at 37°C for 48
h. The growth of microorganisms was observed as turbidity
determined by the measure of optical density at 600 nm (Eppendorf
spectrophotometer, AG, Germany). Extract-free solution was used
as a negative control. Control tubes were incubated under the same
condition. All assays were carried out in triplicate.
RESULTS AND DISCUSSION
The growth inhibition value of extracts on fungal strains is

Table 3. Minimum inhibitory concentrations (MIC) and minimum fungicidal
concentrations (MFC) of extracts against C. albicans.
Plants MIC (μg/ml) MFC (μg/ml)
Satureja bachtiarica Bung. 25 50
Thymus daenensis Celak. 25 ‎>50‎
Dracocephalum multicaule Montbr and Auch. 50 ‎>50‎
Stachys lavandulifolia Vahl. >50 ‎>50‎
Echinophora platyloba DC. 50 ‎>50‎
Achillea kellalensis Boiss. and Hausskn. ‎>50‎ ‎>50‎
Kelussia odoratissima Mozaff. ‎>50‎ ‎>50‎
Heracleumlasiopetalum Boiss. ‎>50‎ ‎>50‎
shown in Table 2. The extracts from the different plant
species studied showed antifungal ‎activities, with the
diameters of the inhibition zone ranging from 8 to 17 mm.
There were significant differences (p ≤ 0.05) in the
antifungal ‎ activities of the plant extracts. Among the
plants tested, the extracts of Satureja bachtiarica and
Thymus daenensis showed the best antifungal ‎ activity.
These were followed by the ethanol extracts of
Dracocephalum multicaule‎, Kelussia odoratissima and
Stachys lavandulifolia‎ (Table 2). The results showed that
most of the extracts could effectively inhibit the growth of
C. albicans.
Subsequent experiments were conducted to determine
the minimal inhibitory concentration of all selected plant
extracts. The MIC values for active extracts ranged
between 25 and 50 µg/ml. Among the plants tested, T.
daenensis and S. bachtiarica showed the best antifungal
activities (Table 3). Also, the ethanol extract of
Echinophora platyloba and Dracocephalum multicaule
showed promising antifungal activities against C. albicans
(Table 3). The results obtained appeared to confirm the
antifungal potential of the plants investigated. The
extracts ‎of T. daenensis and S. bachtiarica ‎ showed the
best MIC value and activity against yeast used.
In this study, the extracts ‎from S. bachtiarica and T.
daenensis ‎ ‎exhibited inhibitory effect on fungal growth,
suggesting that the studied plant extracts ‎are potentially a
safe and natural source of antifungal agents. All the
extracts showed varying degrees of antifungal activity on
the yeast tested. Some of these plants were more
effective than traditional antimicrobial to combat the
pathogenic microorganisms studied.
The result of identification of phenolic compounds using
HPLC showed that the major components of S.
bachtiarica and T. daenensis were carvacrol and thymol,
respectively. Some studies claim that the phenolic
compounds present in spices and herbs might also play a
major role in their antimicrobial effects (Hara-Kudo et al.,
2004). Previous studies (Shan ‎et al., 2005) showed that a
highly positive linear relationship exists between
antioxidant and antimicrobial activity and total phenolic
content in some spices and herbs. Many herb and spice
Boroujeni et al. 2451
extracts for example S. bachtiarica and T. daenensis
contained high levels of phenolics and exhibited
antimicrobial activity (Sefidkon and Jamzad, 2000; Sajjadi
and Khatamsaz, 2003). Previous report (Rasooli et al.,
2006) on the antimicrobial activity of the essential oils of
some Thymus spp., most of them possessing large
quantities of phenolic monoterpenes, have shown activity
against viruses, bacteria, food-derived microbial strains
and fungi. The essential oil and extract of some aromatic
plants (for example mint family, Lamiaceae) with a higher
percentage of cavracrol and thymol have a higher
efficacy against microbial (Rasooli et al., 2006). The
results obtained represent a worthwhile expressive
contribution to the characterization of the anti-Candida
activity of plant extracts of traditional medicinal plants
from the Iranian flora. The extracts of T. daenensis and
S. bachtiarica leaves and flowers had antifungal
‎activities. The present study suggests that the extracts ‎of
these plants are a potential ‎source of natural antifungal
agents. ‎Evaluations of the extracts against other
important human pathogens are also being conducted.
After this screening experiment, further work should be
performed to describe the antifungal activities in more
detail as well as their activity in vivo. In addition,
phytochemical studies will be necessary to isolate the
active constituents and evaluate the antifungal activities
against a wide range of fungi population.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2453-2457, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.1641
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Acute toxicity of Clarias gariepinus exposed to Datura
innoxia leaf extract
Ayuba V. O. 1 *, Ofojekwu P. C. 2 and Musa S. O. 2
1 Department of Fisheries and Aquaculture, University of Agriculture, Makurdi, Benue State, Nigeria.
2 Department of Zoology University of Jos, Jos, Plateau State, Nigeria.
Accepted 1 February, 2012
Acute toxicity test was carried out with aqueous extract of Datura innoxia leaf on Clarias gariepinus
fingerlings for 96 h under laboratory conditions using static bioassays with continuous aeration. The
LC50 of the exposed fingerlings was 120.23 mg/L with lower and upper confidence limits of 96.18 and
150.29 mg/L, respectively. The fish exhibited loss of balance, respiratory distress, vertical and erratic
movement, accumulation of mucus on the body surface and gill filament and death. Water quality
parameters were within recommended acceptable limits.
Key words: Datura innoxia, Clarias gariepinus, static bioassays, 96 h LC50.
INTRODUCTION
Different plants assume different importance in different
societies. Solanaceae plants occupy a prominent position
as source of drugs in medicine, pharmacology but many
are toxic when used in excess. Toxin concentrations in a
plant can vary tremendously, often by 100 times or more,
and can be dramatically affected by environmental stress
on the plant (drought, heat/cold, mineral deficiencies, etc)
and disease. Different varieties of the same plant species
can also have different levels of toxins and nutritional
value (D'Mello, 2000). Effect of naturally-occurring plantmade
toxins found at low levels in many foods and drugs
for humans and animals is based on laboratory tests
using toxin concentrations much higher than the
concentrations normally found in food and drug.
Datura innoxia, which belongs to the family
Solanaceae, has been reported by Djibo and Bouzon
(2000) to contain hallucinogenic properties as the flowers
and seeds were used for voluntary intoxication in Niger.
Dereck (2002) listed D. innoxia as one of the poisonous
houseplants as well as a sedative. The toxicity of most
plant extracts varies depending on the type and animal
species involved. This is due in part to the phytochemical
composition of the extract and also the very great
variation in susceptibility between individual animals.
Ayuba et al. (2011) reported the phytochemical
*Corresponding author. E-mail: vayuba2001@yahoo.com.
components of the plant, D. innoxia, to include atropine
scopolamine, saponins, phenols, flavonolds and cardiac
glycocides.
Biologically, the African catfish, Clarias gariepinus, is
undoubtedly an ideal aquaculture species. It is widely
distributed, not only in African countries but also in the
Netherlands; it thrives in diverse environments,
temperate to tropical (Hecht et al., 1996). It is hardy and
adaptable principally as a consequence of its air
breathing ability, feeds on a wide array of natural prey
under diverse conditions, is able to withstand adverse
environmental conditions, is highly resistant to diseases,
and is highly fecund and easily spawned under captive
conditions. It has a wide tolerance of relatively poor water
quality and possibly the most exciting feature of the
species is its potential for highly intensive culture without
prerequisite pond aeration or high water exchange rates
and its excellent meat quality (Hecht et al., 1996), hence
its choice for this study.
MATERIALS AND METHODS
Fresh samples of D. innoxia leaves were collected from the
University of Agriculture, Makurdi, Nigeria and brought to
hydrobiology and Fisheries Research Laboratory, University of Jos
Nigeria, where they were washed and air dried to constant weight.
The dried samples were pounded using a clean laboratory mortar to
a fine powder which was then sieve through 0.25 mm sieve. 500 g
of the resultant powder was dissolved in 2 L of water at room

2454 J. Med. Plants Res.
Table 1. Ingredient and proximate composition of diet fed to
C. gariepinus.
Ingredient (%) Diet
Fish meal 30
Soyabean meal 35
Bone meal 10
Rice bran 15
Corn oil 3
*Vitamin and mineral mix 5
Starch 2
Proximate composition (% DM)
Crude protein 44.5
Crude fat 8.6
Crude fibre 5.1
Ash 16.4
NFE 25.5
Table 2. Mortality rate of C. gariepinus fingerlings exposed to acute concentrations of D. innoxia leaf extract.
Concentration
(mg/L)
Log
Time (h) for 50%
mortality
Mean total
mortality (%)
Mean probit value
200.00 2.30 36 (0.00) 90 (10.00) 6.28 (3.72)
180.00 2.25 36 (0.00) 80 (0.00) 5.84 (0.00)
160.00 2.20 48 (0.00) 55 (5.05) 5.13 (3.35)
140.00 2.15 54 (6.00) 50 (0.00) 5.00 (0.00)
120.00 2.08 90 (0.00) 50 (10.00) 5.00 (3.72)
100.00 2.00 - 25.(0.05) 4.87 (3.35)
0.00 0.00 - - -
temperature (23± 0.5°C) for 24 h. The extract was filtered through
Whatman’s filter paper (No. 1) with the aid of a vacuum pump. The
filtrate was freeze dried and used for the experiment.
Healthy specimens of C. gariepinus fingerlings weighing 10.3(± 0.
35) g were collected from Rock water fish farm Jos. They were
acclimatized for two weeks under laboratory conditions; during that
period, fish were fed 4% of their body weight with laboratory
formulated feed (Table 1); their mortality during acclimation period
was less than 3%.
After acclimation, the freeze-dried extract was dissolved in
distilled water and introduced into 18 glass aquariums each
measuring 60 cm × 30 cm × 30 cm containing dechlorinated well
aerated municipal tap water at a concentration 200.00, 180.00,
160.00, 140.00, 120.00 and 100.00 mg/L while 0.00 mgL -1 served
as control. Each concentration had 3 replicates. Fish were
randomly selected and stocked at 10 fingerlings per glass
aquarium.
Fish were staved for 24 h prior to and throughout the exposure
period which lasted 96 h. Static bioassay techniques as described
by Reish and Oshida (1987) were used. The following physiochemical
parameters (temperature, dissolve oxygen, pH, total
alkalinity, ammonia-nitrogen and Free carbon dioxide) using
methods described by APHA et al. (1985) were measured at the
beginning and daily thereafter. Aeration was provided prior to
exposure and during exposure in all experimental aquaria. The
behaviour of the fish was observed before and during exposure
period. The tanks were examined for mortality every 6 h until the
end of the 96 h exposure period. Fish were considered dead when
the opercula and tail movements stopped and there was no
response to a gentle prodding. Dead fish were removed
immediately from test solutions to avoid fouling the test media.The
96 h LC50 was determined as a probit analysis using the arithmetic
method of percentage mortality data. The lower and uppers
confidence limits of the LC50 were determined as described by
UNEP (1989). Result obtained were subjected to statistical analysis
with Duncan’s multiple range F-test to test for significant difference
(P < 0.05) between the various concentrations of D. innoxia and the
control.
RESULTS
The results of the mean mortality rates of the fish C.
gariepinus exposed to D. innoxia leaf extract are
presented in Table 2. The logarithmic-probability curves
of the mean mortality rates are presented in Figure 1. It
was observed during the exposure that fish placed in
media devoid of the extract survived the 96 h exposure

Mean probit value
6.5
6
5.5
5
4.5
4
3.5
y = 4.463x - 4.3017
r 2 = 0.7501
1.95 2.00 2.05 2.10 2.15 2.20 2.25 2.30 2.35
Log concentration (mg/L)
Figure 1. Linear relationship between mean probit mortality and log concentration of C. gariepinus
exposed to various concentrations of D. innoxia leaf extract for 96 h.
period. No mortality was observed in the group exposed
to 140 mg/L and below within the first 24 h of exposure.
The 96-h LC50 of C. gariepinus exposed to various
concentrations of D. innoxia leaf extract was 120.23 mg/L
with lower and upper confidence limits of 96.18 and
150.29 mg/L, respectively.
The regression equation of the relationship was
calculated to be Probit y = -4.302 + 4.463 log Conc. x and
on R square value (r 2 ) of 0.7501. These expressions, that
is, the regression equation r 2 value indicated that mortality
rate of the test fish and concentrations of leaf extract are
positively correlated. This shows that the mortality rate of
the fish increased with increase in the concentrations of
D. innoxia leaf extract.
During the exposure period, the test fish exhibited
various behavioral patterns before death occurred.
Restlessness, loss of balance (that is, overturning), air
gulping, and convulsion were frequently observed.
However, these observations were minimal in the groups
of fish exposed to 100 mg/L of the leaf extract. These
observations were not noticed in the groups of fish
exposed to the media devoid of D. innoxia leaf extract.
The opercula ventilation rate and tail beat frequencies
per minute are not presented because all the test fish
exhibited irregular opercula ventilation rate and tail beat
Ayuba et al. 2455
frequency. This may not be unconnected with the
presence of assessory organs, which enables it breathe
in atmospheric oxygen other than dissolved oxygen.
During the exposure period of the acute toxicity test, it
was observed that the mean values of the water quality
parameters (Table 3) were not significantly different (P >
0.05) for temperature and pH. Even though dissolved
oxygen, free carbon dioxide and total alkalinity values
differ significantly (P < 0.05) compared to the control
values, they were still within acceptable limits
(Mackereth, 1963).
DISCUSSION
Physical changes observed in C. gariepinus exposed to
acute toxicity of D. innoxia leaf extract included loss of
balance, respiratory disorder gulping of air and erratic
swimming before death; this is in line with the findings of
Omoregie et al. (1998), Ayuba and Ofojekwu (2002),
Tawari-Fufeyin et al. (2008); Ololade and Oginni (2010);
Okomoda et al. (2010) who made similar observations
when they exposed C. gariepinus to different toxicants.
Behavioural responses of fish to most toxicant and
differences in reaction times, according to Bobmanuel et

2456 J. Med. Plants Res.
Table 3. Mean water quality parameters obtained during the exposure of C. gariepinus fingerlings to acute concentrations of D. innoxia leaf extract
for 96 h.
Parameter
200.00 180.00
Concentrations (mg/L)
160.00 140.00 120.00 100.00 0.00
Temperature (°C) 22.97(0.65) 22.98(0.6) 22.96(0.65) 22.94(0.66) 22.92(0.6) 22.90(0.6) 22.90(0.6)
Dissolved oxygen (mg/L) 5.24(0.27) 5.62(0.21) 6.24(0.10) 6.26(0.13) 6.30(0.19) 6.34(0.28) 6.94(0.34)
Free carbondioxide (mg/L) 3.80(0.27) 3.58(0.76) 3.32(0.62) 3.18 (0.64) 2.88(0.73) 2.14(0.49) 1.48(0.58)
Total alkalinity (mg/L) 32.60(1.72) 31.80(2.0) 30.50(2.01) 27.00(0.89) 25.80(0.9) 22.80(1.6) 19.88(0.3)
pH 6.94(0.05) 7.00(0.04) 7.00(0.04) 7.06(0.05) 7.06(0.08) 7.02(0.04) 7.00(0.07)
Ammonia (unionized NH3) 0.23(0.03) 0.22(0.01) 0.22(0.01) 0.22(0.01) 0.22(0.01) 0.22(0.01) 0.22(0.01)
al. (2006), are due to effect of chemicals, their
concentrations, species, size and specific environmental
conditions Figure 1 which shows the results obtained
from this research revealed that the 96 h LC50 for the
African catfish, C. gariepinus exposed to D. innoxia leaf
extract was 120.23 mg/L with lower and upper confidence
limits of 96.18 and 150.29, mg/L, respectively. The 96 h
LC50 had earlier been reported for C. gariepinus by
Ayuba and Ofojekwu (2002) to be 204.17 mg/L for D.
innoxia root extract, while Ayotunde et al. (2010) reported
the 96 h LC50 of C. gariepinus fingerlings exposed to
Carica papaya seed powder to be 12.9 mg/L; Abalaka
and Auta (2010) also recorded 296.14 and 225.48 mg/L
for aqueous and ethanol extracts of Parkia biglobosa pod
respectively for C. gariepinus. Fafioye et al. (2010) who
worked on the toxicity of aqueous and ethanol extracts of
Parkia biglobosa and Raphia vinifera on C. gariepinus
reported the 96 h LC50 for aqueous and ethanol extracts
of Parkia biglobosa to be 2.8 and 2.4 ppm respectively.
While for R. vinifera aqueous and ethanolic extracts, the
values were 3.4 and 3.2 ppm, respectively. The
difference in the result of the present study and those of
these researchers may be due to the difference in
toxicants, their concentrations, environmental conditions,
age and the size of C. gariepinus.
There was no death recorded in the control aquaria,
also none in concentrations, 100 and 120 mg/L before 72
and 60 h, respectively. This could be that the fish showed
some tolerant to the D. innoxia leaf extract at certain
level; this agrees with the findings of Datta and Kaviraj
(2002), and Fafioye et al. (2004) who reported that C.
gariepinus is tolerant to pollutants. It is widely known that
the toxicity test of pollutant varies with the test organism
and chemical quality of the test water. Gaafar et al.
(2010) observed that toxicants in the aquatic environment
may not necessarily result in the outright mortality of
aquatic organisms but can result in several physiological
dysfunctions in the fish.
Mucus accumulation was observed on the body surface
and gill filament of exposed fish during the present study.
This might be as a result of increase in the activity of
mucus cells due to subsequent exposure to pollutants.
This agrees with the reports of Oti (2002) and Adeyemo
(2005) who made similar observations when they
exposed C. gariepinus fingerlings to cassava mill effluent.
The water quality parameters of the test solutions, though
fluctuated during the bioassay and were significantly
different from those of the control tanks, were within
suggested tolerance range (Mackereth, 1963) and so
could not have affected the mortality of the test fish. The
behavioral alterations that occurred before death in this
study may be as a result of nervous impairment due to
blockade of nervous transmission among the nervous
system and various affector sites, failing organs and
retarded physiological processes in fish body functions
(Shah, 2002). This may have resulted from enzyme
dysfunction and paralysis of respiratory muscle or
depression of respiratory centre and disturbances in
energy pathways leading to depletion of energy (Gabriel
et al., 2010). Since aquatic organisms are in continuous
contact with polluted medium, breathing and feeding is
impaired due to the damages to respiratory and other
organs of the body (Omoregie et al., 1998).
Mortality which is a non response of an organism to the
toxic effects of hazardous substances does present a
standard form for ranking toxicity and enable the
prediction of the effect of potential hazards. Fishes
generally produce detoxifying enzymes when exposed to
toxicants, but this may be hardly possible in acute
exposures which do not allow enough time for the fish to
induce detoxifying enzymes as means of increasing
immunity against the toxic effects of the chemical. The
inability of the fish to detoxify the toxicant and excrete the
resultant metabolites, besides direct damage by the
toxicant to the epithelial cells of gills, possible destruction
of liver and internal asphyxiation may account for the
rapid mortality recorded in acute lethal studies of this
nature (Farah et al., 2004).
Conclusion
The present study revealed that D. innoxia leaf extract
causes a regular trend in mortality of C. gariepinus
fingerlings which increased with increased concentration.
The use of D. innoxia leaf in aquatic environment

should be done with caution.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2458-2467, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.1643
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Dietary supplementation of green tea by-products on
growth performance, meat quality, blood parameters
and immunity in finishing pigs
Md. Elias Hossain, Seok Young Ko and Chul Ju Yang*
Department of Animal Science and Technology, Sunchon National University, Suncheon, Jeonnam 540-742, Korea.
Accepted 20 February, 2012
Green tea has a long-standing reputation for its health-promoting properties. It has a useful content of
amino acids, proteins, vitamins, tannins and polyphenols, such as epicatechin, epigallocatechin,
epigallocatechin gallate and gallocatechin. This study evaluated the potential use of green tea byproducts
in finishing pig diet. A total of 100 finishing pigs were assigned to 5 dietary treatments with 4
replications for 6 weeks in a completely randomized design. The treatments were as follows: control
(basal diet), antibiotic (basal diet with 0.003% chlortetracycline), and basal diet with 0.5, 1 or 2% green
tea by-products (GTB). In this experiment, a poor weight gain and feed conversion ratio was observed
for the GTB-2% group when compared with the antibiotic group. Both crude protein and crude fat
contents of the meat were inversely proportional to each other in the GTB groups and 0.5 to 1% level
differed with the antibiotic group. The slaughter weight and shear value were higher in GTB-1 and 0.5%,
group respectively, while a lower heating loss and higher tenderness were observed in the GTB-2%
group. Supplementation of the pig diet with GTB reduced the thiobarbituric acid reactive substances
values of the meat, and increased white blood cells (WBC) and red blood cells (RBC) content compare
to others. Although, spleen weight was decreased in the GTB-1 and 2% groups, spleen cells growth and,
IL-6 and TNF-α production were improved by the addition of GTB to the feed. These combined results
indicated that 0.5 to 1% GTB hold great promise to use as feed additive for finishing pigs.
Key words: Green tea by-products, growth, meat, blood, immunity, pigs.
INTRODUCTION
Recent consumer scares over animal production
practices have renewed interest in using alternative
ingredients to antibiotics, particularly those from plants,
which are perceived as ‘natural’ and ‘safe’ by consumers.
However, plant extracts are in many commercial
preparations currently used in animal production, and
produce antimicrobial (Jamroz et al., 2003), antioxidant
(Cross et al., 2007), antitoxin effects (Platel and
Srinivasan, 2000); and are able to improve digestibility
(Rao et al., 2003), stimulate enzyme activity (Platel et al.,
2002) and immune functions (Ko and Yang, 2008).
Consumption of ready-made tea drinks such as green tea
*Corresponding author. E-mail: yangcj@scnu.kr. Tel: 82-61-750-
3235. Fax: 82-61-750-3239.
(Camellia sinensis) has increased markedly in ecent
years in Southeast and East Asia. World green tea
production was 0.968 million metric ton (MMT) in 2006
and the projected production in 2017 is 1.571 MMT
(Hicks, 2009). Consequently, the amount of tea byproducts,
which are the residues of tea drink
manufacturing at beverage companies, has been
increasing.
In fact, in 2003, more than 200,000 tons of used tea
leaves were produced from all kinds of tea, of which
approximately 90,000 tons were from green tea and
40,000 tons from black tea (Kondo et al., 2005). Used
green tea leaves from factories are disposed of as
compost, dumped into landfills or burned, which causes
both an economical and environmental problem. It is
important to reuse tea wastes in livestock production to
reduce the use of artificial additives and environmental

effect. Green tea has over 200 bioactive compounds and
contains over 300 different substances (Labdar, 2010).
Numerous in vitro and in vivo studies on green tea
preparations have demonstrated the antimutagenic,
anticarcinogenic and antioxidant properties of the
polyphenolic compounds, which are mainly composed of
four types of catechin: epicatechin, epigallocatechin,
epigallocatechin gallate and gallocatechin (Yamamoto et
al., 1997). The predominant green tea component has
been shown to improve body weight gain and feed
efficiency in pig (Ko et al., 2008), cattle (Sarker et al.,
2010) and broiler (Biswas and Wakita, 2001).
In addition, this component maintains microflora
balance and displays antimicrobial effects against
pathogenic bacteria (Hara-Kudo et al., 2005). It has been
shown that green tea polyphenols have strong
antioxidation properties (Nishida et al., 2006). It reduces
thiobarbituric acid reactive substances (TBARS) values
and maintain oxodative stability of pig meat (Ko et al.,
2008), broiler meat (Yang et al., 2003) and egg yolk
(Uuganbayar et al., 2005). Yang et al. (2003) reported
that cholesterol levels were decreased and fatty acids of
plasma and meat were improved, when the animals were
fed different levels of green tea by-products (GTB).
Supplementation of GTB and green tea with probiotics
has no negative effect on the blood components of beef
cattle and calves (Lee, 2005; Sarker et al., 2010). Ko and
Yang (2008) found that adding 0.5% GTB in the finishing
diet produced positive effects on the humoral and cellmediated
immunity of pigs.
In the present study, we evaluated the activity of GTB
to determine the specific effects of this component on
growth performance, carcass characteristics, meat quality,
blood parameters and immune response in finishing pigs
and also to determine the effective level in finishing pig
diet.
MATERIALS AND METHODS
Animals, diets and experimental design
A total of 100 crossbreed (Landrace × Large White) castrated male
finishing pigs with an average body weight of 77 ± 0.4 kg were
assigned to 5 dietary treatments in a completely randomized design.
Each treatment had 4 replications with 5 pigs per replication. The
pigs were reared in the experimental farm at Sunchon National
University following the guidelines for the care and use of animals
in research (Korean Ministry for Food, Agriculture, Forestry and
Fisheries, 2008). All animals were fed experimental diets for 6
weeks.
The five dietary treatments were as follows: control (basal diet),
antibiotic (basal diet with 0.003% chlortetracycline) and basal diet
with 0.5, 1 and 2% GTB. Used green tea, residues from green tea
drink manufacturing were collected from the green tea experimental
station (Boseong, South Korea), dried, grounded and used for
experiment. GTB was analyzed and the main constituents of were
found as moisture 10.15%, crude protein 20.16%, crude fat 3.08%,
crude fiber 19.20%, crude ash 5.22% and catechins 7.17%. All diets
were formulated to meet or exceed nutrient requirements of
finishing pigs (NRC, 1998). The formula and chemical composition
Hossain et al. 2459
of the basal diet used in this experiment are provided in Table 1.
Measurements and analysis
Growth performance, carcass characteristics and oxidative
stability of meat
Body weights were measured on a biweekly basis from the
beginning to the end of the experiment. Feed intake was
determined by measuring feed residue on a biweekly basis from the
start of the experiment. Feed conversion ratio (FCR) was obtained
by dividing the feed intake by body weight gain. At the end of the
experiment, pigs were transported to the slaughter house (Naju,
Korea). Carcass weight and back fat thickness were measured, and
carcass grade were determined according to Animal Products
Grade System (Korea Institute for Animal Products Quality
Evaluation, 2011). All the pork carcasses in South Korea are
graded both in quality and conformation terms. The quality of pork
carcasses is graded 1 + , 1 and 2 based on the marbling, lean color
and conditions of belly streaks. The conformation terms of pork
carcasses is graded A, B and C by assessing carcass weight, back
fat thickness, balance, muscle, fat condition and so on. Carcass
grades in this study were expressed as 3 (extremely good), 2
(good) and 1 (bad) in A, B and C grades of conformation terms. The
thiobarbituric acid reactive substances (TBARS) value of pork was
assayed using the methods described by Vernon et al. (1970) with
slight modifications. Four gram of loin meat mixture was blended at
full speed for 1.5 min with 10 ml of solution containing 20%
trichloroacetic acid in 2 M phosphoric acid. The resulting sediment
was transferred to 100 ml volumetric flask containing 8 ml distilled
water and diluted by shaking and homogenized. The mixture was
filtrated through Whatman No. 1 filter paper. Then 5 ml filtrate was
transferred to a 20 ml test tube and 5 ml of 2-thiobarbituric acid
(0.005 M in distilled water) was added. The solution was shaken in
a water bath at 80°C for 30 min. After cooling, the color
development was measured at 530 nm using a VIS-
Spectrophotometer (Model 20D + , Milton Roy, PA, USA).
Thiobarbituric acid reactive substances (TBARS) values were
expressed as micromole of malondialdehyde per hundred gram of
meat.
Meat composition, quality and sensory evaluation
Moisture, crude protein, crude fat, and ash percentage of the meat
samples were analyzed according to Association of Analytical
Communities (AOAC, 2000) methods. Color values on a freshly cut
surface (3 cm thick slice) of loin meat were measured using a CR-
301 Chroma Meter (Minolta Co., Osaka, Japan) for International
Commission on Illumination (CIE) standard of lightness (L), redness
(a) and yellowness (b). Muscle pH values were determined using a
pH meter (ATI 370, Orion Research Inc, MA, USA). The meat
samples were broiled in a water bath at a temperature of 70°C for
30 min, surface dried, and weighed. Cooking loss was determined
by expressing the cooked sample (B) weight as a percentage of the
precooked sample (A) weight. Cooking loss (%) = [(A−B)/A] × 100.
Water holding capacity was determined according to the procedure
described by Wardlaw et al. (1973). Shear force was measured
using the Instron Universal Testing Machine (Model 4465, Instron
Corp., Norwood, MA, USA), which was equipped with a Warner-
Bratzler shear device. From each cooked meat sample, a 0.5 × 4.0
cm (approximately 2.0 cm 2 ) cross section was cut for shear force
measurements. The meat samples were placed at right angles to
the blade. The crosshead speed was 100 mm/min and the full scale
load was 50 kg. Sensor evaluation of pork meat was
organoleptically evaluated by ten trained judges from the

2460 J. Med. Plants Res.
Department of Animal Science and Technology at Sunchon National
University on the three point hedonic scale for juiciness, tenderness
and flavor.
Biochemical and hematological parameters of blood
Blood samples were collected from the jugular vein of the pigs by
venipuncture on the last experimental day, 3 h after feeding. To
analyze the biochemical composition of plasma, blood samples
were separated by centrifuging for 20 min at 1500 (× g) and were
total protein, cholesterol, glucose, albumin, globulin,
albumin/globulin (A/G) and blood urea nitrogen (BUN)
concentrations were measured using a blood analyzer (COBAS
MIRA: Roche, Germany). Number of white blood cell (WBC,
10 3 /mm 3 ), red blood cell (RBC, 10 6 /mm 3 ) and hemoglobin
concentration (g/dl) were determined using a hematological
analyzer (XE-2100, Automated Hematology Analyzer, Sysmex
America, Inc.) within 3 h after blood sampling.
Spleen cells culture and proliferation
The immune response of pig spleen cells was analyzed at the end
of the experiment. At one third areas of the spleens of pigs, a 1 cm 3
tissue sample was extracted and split into single cell on bovine
serum medium (RPMI-1640). Next, NycoPrep TM 1.077A was used
to remove the dead cells and red blood cells. Spleen cells were
cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal
calf serum, 2 mM l-glutamine, 100 units/ml penicillin and 100 μg/ml
Table 1. Ingredients and chemical composition of the basal diet.
Ingredients (%, as-fed basis) Amount
Corn 44.65
Wheat 25.00
Wheat bran 4.00
Soybean meal 13.50
Lupin seeds 3.00
Limestone 0.77
Tricalcium phosphate 1.10
Salt 0.25
Vitamin-mineral premix 1
0.56
Animal fat 2.50
Molasses 4.50
L-lysine 0.17
Chemical composition (% dry matter)
Crude protein 15.00
Calcium 0.78
Available phosphorus 0.55
Lysine 0.80
Methionine 0.27
Metabolisable energy (kcal/kg) 3160
1 Provided the following nutrients per kg of diet: vitamin A, 6000IU;
vitamin D3, 800IU; vitamin E, 20IU; vitamin K3, 2 mg; thiamin, 2 mg;
riboflavin, 4 mg; vitamin B6, 2 mg; vitamin B12, 1 mg; pantothenic
acid, 11 mg; niacin, 10 mg; biotin, 0.02 mg; Cu, 21 mg; Fe, 100 mg;
Zn, 60 mg; Mn, 90 mg; I, 1.0 mg; Co, 0.3 mg; Se, 0.3 mg.
streptomycin, 1% (v/v) nonessential amino acids, and 0.05 mM 2mercaptoethanol
(Gibco Paisley, UK) at 37°C, 5% CO 2 condition.
Viable cells were counted in a hemocytometer by trypan blue
exclusion.Triplicate cultures were performed in 96-well flatbottomed
tissue culture plates (Costar, Cambridge, MA, USA) in a
final volume of 200 µl per well containing 5 × 10 5 cells in the
presence of different concentrations of LPS (lipopolysaccharide; 1.0,
3.0 and 10 μg/ml–Sigma-Aldrich, Saint Louis, MO, USA) or Con A
(concanavalin A; 0.1, 0.3 and 1.0 μg/ml–Sigma-Aldrich, Saint Louis,
MO, USA). Cultures were then incubated for 3 days at 37°C, 5%
CO2. Cell proliferation was examined by MTS assay using the
Celltiter 96 ® AQueous One solution Cell Proliferation Assay Kit
(Promega, Madison, WI, USA) following the manufacturer’s
instruction. The optical densities were measured in a microplate
reader (Optimax, Molecular Devices, USA) at a wavelength of 490
nm. Absorbance was corrected by a prepared triplicate set of
control wells (without cells) containing the same volumes of culture
medium and CellTiter 96 ® AQueous One Solution Reagent, as in the
experimental wells. We subtracted the average 490 nm absorbance,
from the “no cell” control wells, from all other absorbance values to
yield corrected absorbances.
T cell subsets analysis
Spleen lymphocytes (1 × 10 6 cells/ml) were stained with fluoresein
isothiocyanate (FITC)-labeled anti-mouse CD3 and phycoerythrin
(PE)-labeled anti-mouse CD4 + monoclonal antibodies for helper
(CD3 + , CD4 + ) T cells, or fluoresein isothiocyanate (FITC)-labeled
anti-mouse CD3 and fluoresein isothiocyanate (FITC)-labeled

Table 2. Effects of dietary green tea by-products (GTB) on growth performances of finishing pigs.
Parameter
Control Antibiotic GTB-0.5%
Hossain et al. 2461
GTB-1.0% GTB-2.0%
Average initial weight (kg/pig) 77.20 ± 0.61 77.60 ± 0.69 77.47 ± 0.47 76.87 ± 0.71 77.07 ± 0.24
Average final weight (kg/pig) 113.33 ab ± 0.77 116.50 a ± 0.29 111.87 ab ± 2.07 112.80 ab ± 1.47 111.07 b ± 1.75
Average weight gain (kg/pig) 36.13 ab ± 1.37 38.90 a ± 0.98 34.40 ab ± 1.60 35.93 ab ± 1.29 34.00 b ± 1.51
Average feed intake (kg/pig) 131.67 b ± 8.82 47.50 ab ± 1.44 135.00 b ± 7.64 140.00 ab ± 7.69 158.20 a ± 0.92
Feed conversion ratio (feed/gain) 3.64 b ± 0.13 3.80 b ± 0.06 3.92 b ± 0.05 3.89 b ± 0.08 4.67 a ± 0.20
a,b Means with different superscripts within same row are significantly different (P < 0.05). Data are presented as the mean ± SE.
anti-mouse CD8 + monoclonal antibodies for cytotoxic (CD3 + ,
CD8 + ) T cells (Phar Mingen, San Diego, CA, USA) for 30 min at 4°C.
The lymphocytes were collected by centrifugation at 160 ×g for 5
min and rinsed three times with PBS containing 10% FBS. The
washed lymphocytes were fixed with 2% paraformaldehyde, and
approximately 1 × 10 4 cells were analyzed using a Coulter Epics XL
Flow Cytometer (Beckman Coulter, Inc. CA, USA).
Cytokines (IL-6 and TNF-α) analysis
Splenocytes (5 × 10 5 cells/ml) were cultured in 96-well flat-bottomed
tissue culture plates in a final volume of 200 µl per well containing
RPMI 1640 medium supplemented with 10% (v/v) fetal calf serum,
2 mM L-glutamine, 100 units/ml penicillin and 100 μg/ml
streptomycin, 1% (v/v) nonessential amino acids, and 0.05 mM 2mercaptoethanol,
and stimulated with LPS (10 μg/ml) or Con A (1.0
μg/ml) for 24 h. The cell culture supernatants were collected, stored
at -20°C. The cell culture supernatants were assayed f or IL-6 and
TNF-α, using the Porcine IL-6 Quantikine ELISA kit (Cat. No.
P6000) and Porcine TNF-α Quantikine ELISA Kit (Cat. No. PTA00)
according to the manufacturer’s instructions (R&D Systems,
Minneapolis, MN, USA). Optical density of each well was measured
within 30 min, using a microplate reader (Optimax, Molecular
Devices, USA) set to 450 nm (correction wavelength set at 570 nm).
Each experiment was run in duplicate and the results represent
means of three repeat experiments.
Statistical analysis
Data were analyzed using the general linear models of SAS (2003)
to estimate variance components with a completely randomized
design. Duncan’s multiple comparison tests were used to examine
significant differences among the treatment means. The level of
significance was set at P < 0.05. Data are presented as mean
values ± SE.
RESULTS
Growth performance, carcass characteristics and
meat quality
Body weight, weight gain, feed intake and feed
conversion ratio of different dietary groups are given in
Table 2. The initial body weight did not differ significantly
but the final body weight and body weight gain
were lower (P < 0.05) in the GTB-2% group when
compared to the antibiotic group. GTB-2% group
exhibited a higher feed intake relative to GTB-0.5% and
the control group and the FCR was also low when
comparing to the other groups (P < 0.05). Different levels
of GTB affect the proximate composition of loin meat
(Table 3). When pigs were fed a diet containing GTB-2%,
the crude fat decreased to the same level as the
antibiotic group (P < 0.05). The GTB 0.5% group had a
lower crude protein content, which was followed by the
GTB-1% group (P < 0.05); however, higher value (GTB-
2%) was not different than the antibiotic group.
Table 4 shows that the pigs in control group had a
higher (P < 0.05) slaughter weight than the GTB-0.5 and
2% groups. There were no significant differences (P >
0.05) in back fat thickness, carcass grade, water holding
capacity and pH among the groups; however, the shear
value was higher in the GTB-0.5% group relative to the
other GTB groups and the heating loss was lower in the
GTB-2% group when compare to the others groups (P <
0.05). No differences in meat color, juiciness and flavor
were observed due to supplementation with GTB,
although the tenderness was lower in GTB-0.5% group
(P < 0.05).
Biochemical and hematological parameters of blood
and oxidative stability of meat
The effects of green tea by-products on the biochemical
and hematological parameters of finishing pigs are
presented in Table 5. There were no significant
differences in total cholesterol, glucose, total protein,
albumin, globulin, albumin/globulin (A/G) ratio, BUN and
hemoglobin contents of the blood when the diets
contained 0.5 to 2% of GTB. Although no significant
differences were observed, the cholesterol level tended
to decreased (P > 0.05) with an increase in the GTB
content. WBC and RBC were increased (P < 0.05) at the
highest GTB concentration (2%). TBARS values for the
different treatment groups at different weeks are
presented in Figure 1.
Significant differences (P < 0.05) were observed
among treatments except fresh, 4th week and average

2462 J. Med. Plants Res.
Table 3. Effects of dietary green tea by-products (GTB) on loin meat composition of finishing pigs.
Parameter (%) Control Antibiotic GTB-0.5%
GTB-1.0% GTB-2.0%
Moisture 72.95 ± 0.62 71.45 ± 0.62 72.27 ± 1.00 72.19 ± 0.42 73.53 ± 0.44
Crude ash 1.21 ± 0.03 1.13 ± 0.02 0.93 ± 0.05 1.14 ± 0.10 0.94 ± 0.16
Crude fat 2.10 b ± 0.35 2.66 b ± 0.28 4.21 a ± 0.19 3.87 a ± 0.20 2.19 b ± 0.34
Crude protein 23.04 ab ± 0.34 23.49 a ± 0.61 21.12 c ± 0.23 22.05 bc ± 0.38 23.27 ab ± 0.54
a,b,c Means with different superscripts within same row are significantly different (P < 0.05). Data are presented as the
mean ± SE.
Table 4. Effects of dietary green tea by-products (GTB) on carcass characters and meat quality in finishing pigs.
Parameter 1
Control Antibiotic GTB-0.5%
GTB-1.0% GTB-2.0%
Slaughter wt (kg/pig) 93.60 a ± 2.32 92.00 ab ± 1.73 85.17 c ± 2.85 91.33 ab ± 1.43 87.17 b ± 0.79
Back fat (mm) 19.60 ± 0.68 23.83 ± 1.08 21.83 ± 2.02 22.17 ± 1.82 21.17 ± 1.30
Carcass grade 1.80 ± 0.49 2.33 ± 0.33 2.67 ± 0.21 2.33 ± 0.42 2.6 7 ± 0.21
Shear value (kg) 3.14 ab ± 0.12 3.14 ab ± 0.10 3.38 a ± 0.19 2.97 b ± 0.05 2.86 b ± 0.07
Heating loss (%) 33.50 a ± 0.66 34.25 a ± 0.52 32.58 a ± 0.75 32.55 a ± 0.26 30.74 b ± 0.37
WHC (%) 57.34 ± 0.82 57.29 ± 0.16 56.83 ± 1.03 56.96 ± 0.55 57.91 ± 0.60
pH 5.78 ± 0.09 5.61 ± 0.01 5.66 ± 0.04 5.64 ± 0.05 5.64 ± 0.07
Meat color
CIE L
51.54±1.10 54.24 ± 0.76 52.69 ± 3.18 54.55 ± 1.23 5 0.43 ± 1.81
CIE a
9.62 ± 0.54 9.98 ± 0.51 9.40 ± 1.19 9.32 ± 0.33 9.3 0 ± 0.35
CIE b
5.62 ± 0.68 6.47 ± 0.66 5.58 ± 0.94 6.50 ± 0.30 5.6 5 ± 0.90
Juiciness 3.73 ± 0.23 4.00 ± 0.12 3.80 ± 0.35 4.35 ± 0.22 4.08 ± 0.05
Tenderness 4.38 a ± 0.15 4.28 ab ± 0.17 3.70 b ± 0.34 4.35 a ± 0.05 4.48 a ± 0.13
Flavor 4.20 ± 0.12 4.05 ± 0.10 4.28 ± 0.18 4.25 ± 0 .17 4.35 ± 0.05
a,b,c Means with different superscripts within same row are significantly different (P < 0.05). Data are presented as the mean ± SE.
1 The carcass grades were assessed on three points: 3, extremely good; 2, good; and 1, bad. WHC = water holding capacity; CIE
= international commission on illumination; L = lightness; a = redness; b = yellowness.
values. These results demonstrated the consistency in
the decrease of these values when higher levels of GTB
were added. The antibiotic supplemented group showed
a higher TBARS content in the 2nd week, whereas the
TBARS content was higher in the control group during
the 1st and 3rd week.
Spleen cells growth and cytokines production
Table 6 shows the immunity of spleen cells obtained from
finishing pigs. When higher levels (1 to 2%) of GTB were
added to the diet, the spleen weight was lower than the
control group (P < 0.05). Production of helper and
cytotoxic T cells among the dietary groups were not
significant (P > 0.05), although higher values were seen
in the GTB-0.5% and 1% group. As the dose of Con A
and LPS increased, cytokine secretion from spleen cells
increased. In both media, supplemented groups showed
a higher value compare to the control group (P < 0.05).
IL-6 and TNF-α production from spleen cells stimulated
by LPS and Con A were positively influenced by dietary
levels of GTB (Figure 2). When stimulated with 1.0 μg/ml
Con A, IL-6 production was significantly increased in the
GTB-1% group relative to the antibiotic and control group
(P < 0.05), while IL-6 production by spleen cells with LPS
(10.0 μg/ml) was higher in the GTB-2% group when
compared to the GTB-0.5% and control group (P < 0.05).
Data obtained from this experiment showed that TNF-α
production from spleen cells treated with 1.0 μg/ml Con A
and 10.0 μg/ml LPS were significantly higher for the GTB-
1% and GTB-0.5% group, respectively, when compare to
the antibiotic and control group (P < 0.05).
DISCUSSION
Body weights are commonly used for monitoring the
nutritional status and growth of animals (Ndlovu et al.,
2007). The different levels of green tea-by products in the
experimental diets affected weight gain of the pigs. This
result was supported by Yang et al. (2003) and Sayama

Hossain et al. 2463
Table 5. Effects of dietary green tea by-products (GTB) on blood biochemical and hematological parameters in
finishing pigs.
Parameter 1
Control Antibiotic GTB-0.5%
GTB-1.0% GTB-2.0%
Glucose (mg/dl) 52.10 ± 5.60 55.90 ± 3.63 60.70 ± 5.81 55.60 ± 7.52 65.20 ± 5.93
Cholesterol (mg/dl) 130.80 ± 5.37 123.00 ± 3.09 128.70 ± 5.45 123.30 ± 6.47 121.30 ± 5.96
Total protein (g/dl) 7.18 ± 0.20 7.49 ± 0.15 7.39 ± 0.22 7.34 ± 0.16 7.30 ± 0.25
Albumin (g/dl) 3.03 ± 0.17 3.25 ± 0.06 3.18 ± 0.13 3.10 ± 0.08 3.02 ± 0.13
Globulin (g/dl) 4.15 ± 0.11 4.24 ± 0.11 4.21 ± 0.12 4.24 ± 0.10 4.28 ± 0.16
A/G
0.74 ± 0.05 0.77 ± 0.02 0.76 ± 0.03 0.73 ± 0.02 0.7 1 ± 0.03
BUN (mg/dl) 17.70 ± 1.16 20.30 ± 0.82 18.30 ± 0.68 18.80 ± 1.11 18.50 ± 0.92
WBC (10 3 /mm 3 ) 18.85 bc ± 0.46 18.57 c ± 0.55 20.45 ab ± 0.35 20.49 ab ± 0.73 20.87 a ± 0.67
RBC (10 6 / mm 3 ) 6.75 b ± 0.20 6.70 b ± 0.17 6.94 ab ± 0.047 7.21 ab ± 0.37 7.52 a ± 0.15
Hemoglobin (g/dl) 13.03 ± 0.19 13.08 ± 0.32 13.43 ± 0.15 13.68 ± 0.94 13.58 ±0.25
a,b,c Means with different superscripts within same row are significantly different (P < 0.05). Data are presented as the mean ±
SE. 1 A/G = albumin/globulin; BUN = blood urea nitrogen; WBC = white blood cell; RBC = red blood cell.
MDA (µmol/100g meat)
10
8
6
4
2
0
Control Antibiotic GTB-0.5% GTB-1% GTB-2%
a
a
bc ab
c
ab
d
abc
bc
c
a
b
bc
cd
d
Fresh 1st week 2nd week 3rd week 4th week Average
Preservation time
Figure 1. Effects of dietary green tea by-products (GTB) on thiobarbituric acid reactive
substances (TBARS) values in loin meat. TBARS values were expressed as micromole of
malondialdehyde (MDA) per 100 g of meat. Data are presented as the mean ± SE. Bars within
a time class not sharing a common letter are significantly different (P < 0.05).
et al. (2000), who found that higher levels of green tea
and body weight gain were negatively related in broilers
and rats. In contrast, Ko et al. (2008) found no significant
differences in the weight gain of finishing pigs when their
diet contained GTB. In addition, Sarker et al. (2010)
found a positive relationship in green tea probiotics when
fed to weaning calves. In our experiment, feed intake and
FCR were not affected up to 1% GTB. Ko and Yang
(2008) and Shomali et al. (2011) found no significant
differences in feed intake and FCR in pigs and broilers
but Biswas and Wakita (2001) reported a lower feed
intake and better FCR when 1% green tea powder was
added to the feed for broilers. In general, the body weight
gain and FCR tended to decrease with the addition of
GTB. This was probably due to the high tannin content,
which interferes with protein and starch digestion, and
high fiber content of GTB. Some studies have reported
that the catechin contents of green tea, mainly
epicgallocatechin gallete, could inhibit digestive lipase
activity and affect the lipid metabolism of animals
(Sayama et al., 2000; Weisburger, 2001). Both crude
protein and crude fat contents of meat were inversely
proportional to each other, indicating that if the crude fat
content was high then the crude protein content was low

2464 J. Med. Plants Res.
Table 6. Effects of dietary green tea by-products (GTB) on spleen cell related immunity in finishing pigs.
Parameter
Control Antibiotic GTB-0.5%
GTB-1.0% GTB-2.0%
Spleen weight (g) 181.50 a ± 24.02 145.75 ab ± 20.0 140.17 ab ± 20.01 132.42 b ± 5.42 132.50 b ± 9.38
Helper cell (%) 13.23 ± 0.87 12.88 ± 2.13 12.95 ± 0.74 13.12 ± 1.34 10.23 ± 1.38
Cytotoxic cell (%) 25.73 ± 2.18 23.63 ± 3.21 26.93 ± 1.56 26.33 ± 2.66 21.75 ± 2.05
Growth of spleen cells stimulated with concanavalin A (Con A)
Con A-0.1 (µg/ml) 0.59 b ± 0.01 0.92 a ± 0.02 0.87 a ± 0.04 0.90 a ± 0.09 1.01 a ± 0.10
Con A -0.3 µg/ml) 0.66 d ± 0.01 0.94 bc ± 0.02 0.88 c ± 0.05 1.09 a ± 0.07 1.03 ab ± 0.05
Con A -1.0 µg/ml) 0.81 b ± 0.01 1.00 a ± 0.04 0.95 ab ± 0.05 1.10 a ± 0.09 1.00 a ± 0.04
Growth of spleen cells stimulated with lipopolysaccharide (LPS)
LPS-1.0 µg/ml) 0.56 b ± 0.02 0.74 a ± 0.04 0.79 a ± 0.06 0.85 a ± 0.09 0.84 a ± 0.07
LPS-3.0 ( µg/ml) 0.68 b ± 0.02 0.86 ab ± 0.02 0.85 ab ± 0.06 1.01 a ± 0.09 1.02 a ± 0.09
LPS-10.0 (µg/ml) 0.79 b ± 0.01 0.95 b ± 0.01 1.01 ab ± 0.08 1.02 a ± 0.09 1.26 a ± 0.15
a,b,c,d Means with different superscripts within same row are significantly different (P < 0.05). Data are presented as the mean ± SE.
Concentration (pg/mL) (pg/ml)
250
200
150
100
50
0
b
a
ab
b
ab
c
ab
b
ab
a
c
a
ab
bc
abc
Control
Antibiotic
GTB-0.5%
GTB-1%
GTB-2%
a
bc ab ab
c
IL-6 in Con A IL-6 in LPS TNF-α in Con A TNF-α in LPS
(1.0 µg/mL) µg/ml) (10.0 µg/mL) µg/ml) (1.0 µg/mL) µg/ml) (10.0 µg/mL) µg/ml)
Figure 2. Effects of dietary green tea by-products (GTB) on IL-6 and TNF-α production by
spleen cells in concanavalin A (Con A) and lipopolysaccharide (LPS) medium. Data are
presented as the mean ± SE. Bars within a con A or LPS concentration not sharing a
common letter are significantly different (P < 0.05).
(Davis et al., 1975). Ko and Yang (2008) observed the
same phenomena when using green tea probiotics in the
diet of finishing pigs. In this study, a similar result was
observed for the GTB groups. Some researchers
reported that the pigments included in the diets affect the
color of meat and fat (Kim et al., 2006; Lee et al., 2009).
It was believed that the yellowness of the meat and back
fat increased when GTB was added to the diet since the
pigments of GTB percolated through the meat. Lee
(2005) reported that the addition of 0.02% green tea to
the diet had no effects on back fat, carcass grade and
meat color of the beef cattle. However, the addition of 2%
green tea to the diet increased the yellowness and
redness of the egg yolk (Uuganbayar et al., 2005). Jin et
al. (2003) and Kim (2000) reported that the aroma, flavor,
juiciness and overall acceptability of meat were affected
by the ingredients of diets. Lee et al. (2009) also reported
a positive effect of adding Eucommia ulmoides leaf to the
diet in the sensory evaluation of meat. Although slaughter
weight was some different, a positive effect on sensory
evaluation and overall meat quality will be achieved by
incorporation of GTB into the diet.
Biswas and Wakita (2001) and Kondo et al. (2004)
observed a significant decrease in the serum cholesterol
contents in broiler and lactating cow when broiler diets
contained 0.5 to 1.5% green tea powder and 5% of GTB

in cow diet, which is similar to the results of this study.
Green tea polyphenolics may favor the slow digestion of
carbohydrates, which prevents sharp spikes of insulin in
the blood and favors fat-burning over fat-storage (Zink,
2011). We observed no differences in the plasma
biochemical composition with the addition of GTB, but
some improvements in hematological parameters were
observed. Lee (2005) found that supplementation of
0.02% GTB had no effect on the blood components in
beef cattle. In contrast, Lee et al. (2009) found improve
hematological and plasma biochemical parameters in
pigs fed E. ulmoides leaves. However, Sarker et al.
(2010) also found no significant differences among green
tea probiotic and others group in regards to WBC, RBC
albumin, globulin and A/G in pre and post weaning calves
except albumin in the post-weaning period.
There is increasing interest in using the antioxidant
compounds found in herbs and spices because they
improve the flavor of food, retard the oxidative
degradation of lipids and play an important role in the
prevention of diseases (Achinewhu et al., 1995; Nakatani,
2000). Green tea studies showed that green tea extracts
displayed a dose-dependent inhibitory activity against
end stage of lipid peroxide decomposition product
formation, and early lipid oxidation (Pearson et al., 1998;
Yamane et al., 1999). Epigallocatechin gallate has been
found to be over 100 times more effective in neutralizing
free radicals than vitamin C and 25 times more powerful
than vitamin E (Harold and Graham, 1992). It also has
other antioxidants, such as butylated hydroxyanisole,
butylated hydroxytoluene and resveratol. Ko et al. (2008)
and Ko and Yang (2008) reported that meat obtained
from pigs fed diets containing GTB and green tea
probiotics had lower TBARS values, which was similar to
the results observed in this study. Decreases in TBARS
values were also found in broiler when the GTB content
was increased from 0.5 to 1% level (Yang et al., 2003).
The spleen contains lymphocytes (mainly T cells and B
cells) and macrophages, which engulf and destroy
bacteria, dead tissue and foreign matter, and remove
them from the blood passing through the spleen
(Ezekowitz and Hoffman, 1998). Sayama et al. (2000)
reported that 2% green tea supplementation to the rat
diet depressed the spleen weight of the rats. The data
obtained from current study showed that 0.5 to 2% GTB
supplementation had an effect on spleen weight of
finishing pigs. Pigs like other species contain CD4 + and
CD8 + T lymphocytes in their peripheral blood and
secondary lymphoid organs. These cells have been
shown to express CD3 (Yang and Parkhouse, 1998) and
have helper and cytolytic functions, respectively (Martins
et al., 1993). However, unlike humans and mice, swine
also have a prominent CD4 + and CD8 + lymphocyte
population, comprising between 8 and 64% of the circulating
pool of small resting T-lymphocytes (Zuckermann
and Husmann, 1996). Helper and cytotoxic cells were
numerically increased by the addition of 0.5 to 1% GTB in
Hossain et al. 2465
this study. The role of green tea components in improving
cell-mediated and humoral immunity was previously
investigated (Chae et al., 2004; Shin et al., 2004). Con A
and LPS were shown to induce mitosis in T cells and B
cells of many different specificities or colonial organ
(Tanaka et al., 2005; Arens et al., 2004). Therefore, the
growth reaction of spleen cells was checked by
stimulating T cells with Con A and B cells with LPS, which
specifically proliferates only T cells and B cells among the
spleen cells. This experiment demonstrated that addition
of GTB had a positive effect on humoral and cellmediated
immunity. Spleen cells secrete several types of
cytokines in response to stimulation with Con A or LPS,
which can be used to classify the immune response. One
of the most important functions of IL-6 and TNF-α is the
initiation of a response known as the acute-phase
response. This involves a shift in the proteins secreted by
the liver into the blood plasma and results from the action
of IL-6 and TNF-α on hepatocytes (Kawai et al., 2004).
The result of this experiment is an agreement with
previous studies (Ko et al., 2008; Ko and Yang, 2008),
who reported that the amount of IL-6 and TNF-α
production by spleen cells increased when finishing pigs
were fed diets containing GTB and green tea probiotics. It
can be assumed that the bioactive components of green
tea are responsible for these outcomes.
Conclusions
Based on the observations of this study, the addition of
different GTB levels had an effect on some tested
parameters. Growth performances were negatively and
carcass composition was positively, affected at higher
GTB concentrations (2%). Carcass characters, meat
quality and hematological parameters did not consistently
change with GTB concentration. Strong antioxidative
activities were observed in the GTB groups. Our results
also indicate that the addition of GTB to the diets of
finishing pigs increases immune related cells, and
enhances Il-6 and TNF-α production. Future studies are
needed to elucidate the mechanisms for potentially
enhanced immunity and to investigate its antimicrobial
effect in challenged pigs.
ACKNOWLEDGEMENT
The authors would like to thank the Agricultural R and D
Promotion Center (ARPC) of the Korea Rural Economic
Institute (KREI) for their financial assistance (project no:
1380003137).
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2468-2473, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.1710
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Influence of salt stress on growth, pigments, soluble
sugars and ion accumulation in three pistachio
cultivars
H. Abbaspour 1 *, H. Afshari 1 and M. A. Abdel-Wahhab 2
1 Department of Biology, Damghan Branch, Islamic Azad University, Damghan, Iran.
2 Food Toxicology and Contaminants Department, National Research Center, Dokki, Cairo, Egypt.
Accepted 10 February, 2012
In this study, three pistachio cultivars were treated with different concentration of salt solution to
provide a reference for the cultivation of salt tolerant rootstocks. The results revealed that increasing
the salinity stress resulted in decreasing growth performances of the plants in the three pistachio
cultivars. This decrease was not significant in salinity of 100 mM but was significant in other salinities
compared to control. In all salinity levels and control conditions, Akbari (Ak) cultivar exhibited larger
growth performances of the plants in comparison with the other two cultivars; this state is more
sensible in salinities of 200 and 300 mM. Ak cultivar assumed significantly higher pigment amount than
the other two cultivars in high salinities. Impact of salinity on reduction of chlorophyll amount was
significant in all cultivars starting from salinity of 200 mM. Moderate and high salinity led to an increase
in amount of total soluble sugars in Ak cultivar while no significant contrast was achieved between the
other two cultivars compared to control conditions. The impacts of different salinities on Na and Cl
were nearly similar and the amounts of these elements increased with exceeding salinity stress in
different cultivars compared to control condition. The high stress level resulted in remarkable
enhancement of Cl and Na amounts in Ouhadi (Ou) and Kalehghouchi (Ka) cultivars. However, the low
and moderate salinities increased the concentration of potassium in AK cultivar compared to the other
two cultivars. Therefore, higher plant growth and photosynthetic pigments, lower Na and Cl content,
greater accumulation of soluble sugars and K could explain the greater salt-tolerance of AK compared
to that of Ka and Ou.
Key words: Pistachio cultivars, salinity, growth, ion accumulation.
INTRODUCTION
Agricultural productivity is severely affected by soil
salinity and the damaging effect of salt accumulation in
agricultural soils has become an important environmental
concern (Jaleel et al., 2007b). Over 800 million hectares
of land worldwide is affected by salinity (Munns, 2005),
comprising nearly 7% of the worlds total land area.
Irrigation systems are particularly prone to salinization,
*Corresponding author. E-mail: Abbaspour75@yahoo.com,
Abbaspour@damghaniau.ac.ir. Tel: 09131923217, 0232-
5235214. Fax: 98-232-5235214.
Abbreviations: Ak, Akbari; Ou, Ouhadi; Ka, Kalehghouchi;
mM, milimolar.
with nearly one third of irrigated land being severely
affected. Every year, more and more land becomes nonproductive
due to salt accumulation. Iran, the second
largest country in the soil salinity which is considered a
serious agricultural problem, accounting for at least 20%
of the Middle East and has 165 million hectares irrigated
lands (Munns and Tester, 2008). Approximately, 90% of
the country is classified as arid and semi-arid region,
most of which suffers from low rainfall, high evaporationtranspiration,
salinization, shortage of fresh water, soil
erosion, excessive heat and desertification. In Iran, the
pistachio nut tree (Pistacia vera L.) is an important and
valuable crop which is usually grown under saline
conditions (Sheibani, 1994). Land salinization is a major
limiting factor for conventional crop productivity in the

country (Cheraghi, 2004). Salinity in soil or water is one
of the most damaging abiotic stress factors limiting crop
(Debez et al., 2006). High concentration of salts in the
soils immediately imposes on plants the osmotic stress
effect due to lower soil water potential leading to
retardation of water uptake. When exposed for longer
period, salinity entails ionic stress when plants absorb
and accumulate toxic level of Na + and Cl -
in the
cytoplasm. Salinity also induced secondary stresses such
as nutritional imbalance and oxidative stress (Zhu, 2002).
For worldwide crop production, the detrimental effects of
salinity on plant growth are associated with low osmotic
potential of soil solution (water stress), nutritional
imbalance, specific ion effect (salt stress), or a
combination of these factors (Levit, 1980; Ashraf and
Harris, 2004). All of these agents cause adverse
pleiotropic effects on plant growth and development at
physiological and biochemical levels (Munns, 2002) and
the molecular level (Tester and Davenport, 2003).
To survive in hyper-saline soil, plants have evolved
complex mechanisms that contribute to the adaptation to
osmotic and ionic stresses caused by high salinity. These
mechanisms include osmotic adjustment that is usually
established by intake of inorganic ions as well as
accumulation of compatible solute (Osmoprotectants).
Inorganic ions are sequestered in the vacuole, while
organic solutes are compartmentalized in the cytoplasm
to balance the low osmotic potential in the vacuole
(Rontein et al., 2002). Furthermore, plants have to
employ a wide range of biochemical and molecular
mechanisms including alteration of photosynthetic
pathway, change in membrane structure, stimulation of
phytohormones and induction of antioxidative enzymes
(Parida and Das, 2005; Mudgal et al., 2010). Difference
in salt tolerance exists not only among variant genera
and species, but also within the different organs of the
same species (Bankar and Ranjbar, 2010). Comparing
the response of cultivars of one species to salinity
provides a convenient and useful tool for unveiling the
fundamental mechanisms involved in salt tolerance.
The objective of this investigation was to evaluate the
effects of salt stress on growth, soluble sugars and ion
accumulation of three pistachio cultivars, to provide a
reference for the cultivation of salt-tolerant trees.
MATERIALS AND METHODS
Three types of pistachio seeds (Pistacia vera L. Cultivars Akbari,
Ouhadi and Kalehghouchi) were used in this study. Seeds were
collected from the Anar area, Kerman province, which is one of the
main pistachio growing regions in Iran with saline condition. The
trial was conducted at Biology Department, Islamic Azad University,
Damghan Branch, Damghan, Iran during 2011. The seeds were
surface-sterilized for 10 min in 10% H2O2, washed three times with
tap water to remove any trace of chemicals that could impair seed
germination and were placed on sterile vermiculite at 18 to 25°C to
germinate. After 21 days of germination, seedlings were sown in
plastic post containing sandy soil (pH 7.6, 3.8% silt, 14.6% clay,
81.6% sand, and electrical conductivity (ECe) = 0.98 ds/m).
Abbaspour et al. 2469
Seedlings were transplanted in 20 ×15 cm plastic pots containing a
mixture of salinity clay: sand (1:5 v/v) (four seedlings/pot).
Each pot contained 3 seedlings which were sufficiently irrigated
with urban water every 4 days. All pots were placed in a
greenhouse condition with mean relative humidity 45 to 60%, day /
night temperature 18 to 30°C and photoperiod from 14 to 16 h
(photo-synthetically active radiation of approximately of 430 to 460
mol/ms).
Seedlings were grown under these conditions for 4 weeks before
initiation of NaCl treatments and salinity treatments were 0, 100,
200, and 300 mM of NaCl. The soil was salinized in step-wise
manner to avoid subjecting plants to an osmotic shock. After 55
days of salt treatment, the plants were harvested and separated
into shoots and roots. After complete washing with distilled
deionized water, fresh masses of shoots and roots and the other
morphological parameters such as shoot height, number of
branches and leaves were measured in fresh samples. Total leaf
area was calculated with an AM-200 leaf-area meter. Total
chlorophyll and cartenoid were extracted and estimated from fresh
leaves according to the standard method of Arnon (1949).
Chlorophyll was extracted in 80% (v/v) aceton from 1 g of fresh leaf
sample in the dark at room temperature. Absorbance was
measured at 663 and 645 ηm in a UV/VIS spectrophotometer.
Chlorophyll concentration was calculated using the equation:
Chl 0.
0202 × A645 + 0.00802 × A663
Cartenoid concentration was calculated using the equation:
Car = (A436× V) / 196 × b)
Where V is volume of dilution, b is the length of cell (1 cm). The
value 196 is coefficients of specific absorption. The remaining
samples were then over-dried at 80°C for 48 h so as to record dry
masses. A flame photometer was used for Na + and K +
determination. Phosphorus was analyzed colorimetrically (Boltz and
Lueck, 1958) and Cl content was determined by atomic absorption.
Fresh roots and leaves were collected for the determination of
total soluble sugars content. Soluble sugar content was determined
by 0.1 ml of the alcoholic extractor reacting with 3 ml freshly
prepared anthrone (2000 mg anthrone + 100 ml 72% H2So4) then
was placed in a boiling water bath for 10 min as described by
Irigoyen et al. (1992). After cooling, the absorbance was read to be
equal to 620 nm.
Statistical analysis
The experiment was arranged in a completely randomized design
(CRD) in a two-factor factorial arrangement with 3 replications (n =
3). All parameters were investigated by analysis of variance
(ANOVA) using SPSS software. The means were compared by the
Tukey’s test at a 0.05 confidence level.
RESULTS
Growth performances of the plants were estimated by
measuring plant height, leaf area, number of leaves and
branches, and total fresh and dry weights. With increase
in salinity stress, dry weight of shoots decreases in the
three pistachio cultivars. However, this decline was not
statistically significantly different in salinity of 100 mM but
was significant different in the other salinities compared
to the control plant. In all salinity levels and control
conditions, Akbari cultivar exhibited larger dry weight

2470 J. Med. Plants Res.
Table 1. Fresh and dry weight (g), number of leaves and branches, leaf area (cm 2 ) and plant height (cm) of pistachio plants
under NaCl stress.
Plant
height
Leaf
area
Number of
branches
Number
of leaves
Root weight
Fresh Dry
Shoot weight
Fresh Dry
Cultivars
27 a 91.04 a 7 a 43 a 3.19 a 4.86 a 8.2 a 5.38 a Ak
28 a 95.4 a 7 a 46 a 1.9 ce 3.08 c 7.9 a 4.9 a Ka
27 a 93.2 a 8 a 48 a 2.01 ce 3.19 c 7.4 a 5.05 a Ou
22 ab 85.44 a 6 ac 40 a 2.4 ae 3.4 ac 5.6 b 4.7 a Ak
21 ab 87.35 a 8 a 41 a 1.2 bc 2.78 c 5.4 b 3.9 a Ka
24 ab 86.2 a 7 a 44 a 1.15 bc 2.67 c 5.2 b 3.8 a Ou
19 bd 77.37 ae 4 cd 29 c 1.7 bce 2.7 c 3.9 bd 2.7b e Ak
10 c 61.30 de 3 d 21 c 0.87 bd 2.2 dc 2.6 de 1.5 c Ka
12 c 64.35 de 3 d 24 c 0.98 bd 2.1 dc 2.3 de 1.6 c Ou
17 d 65.43 e 3 d 23 c 1.2 bd 2.2 dc 3.94 bd 1.9 ce Ak
9 c 37.3 c 2 d 10 d 1.1 d 0.49 d 1.58 e 0.23 d Ka
10 c 35.4 c 3 d 13 d 1.05 d 0.45 d 1.32 e 0.19 d Ou
Within each column, means superscript with different letters are significantly different (P≤ 0.05).
compared to the other two cultivars. Moreover, this state
was more sensible in salinities of 200 and 300 mM and
was statistically significant at P≤ 0.05. No significant
difference was observed in the dry weight of shoots
between the two cultivars, that is, Ka and Ou in all salinity
levels and control conditions. Fresh weight was
significantly declined in all salinity levels compared to
control plant. However, a significant difference in fresh
weight was only observed between Ak cultivar and the
other two cultivars in the salinity of 300 mM (Table 1).
Root weight was higher in Ak cultivar than the other two
cultivars under the control and salinity stress conditions.
However, this increase was statistically significant only
under the control conditions. The results presented in
Table 1 also indicated that root weight was decreased
with exceeding stress in all pistachio cultivars. This
decrease was statistically significant in salinities over 200
mM. No significant contrast was observed between the
two cultivars, Ou and Ka regarding the fresh and dry
weights of root under the control and different salinity
conditions.
No significant difference in number of branches was
observed between the control and salinity level of 100
mM among different pistachio cultivars. However, under
stress condition at 200 and 300 mM, number of branches
showed a significant decrease in all varieties. This
decrease was more pronounced in the higher level of
salinity and in Ou and Ka cultivars compared to Ak
cultivar in different salinities. On the other hand, no
significant difference was observed between Ak cultivar
and the other two varieties regarding the number of
leaves for the control and low or medium salinity
conditions. However, number of leaves in Ak cultivar was
Salinity
levels (mM)
0
100
200
300
significantly higher in the high salinity level compared to
the other two cultivars (Table 1). It is of interest to
mention that the number of leaves was decreased in all
the different pistachio cultivars as salinity stress was
increased. This decrease was statistically significant only
at the medium and higher salinity levels compared to the
control plants. Moreover, the reduction leaves number in
Ou and Ka cultivars was more intense from moderate
salinities onwards (Table 1).
No tangible difference was observed for plant height
between the control and the low salinity conditions
among different cultivars. However, Ak cultivar had a
significant increase in plant height under the moderate
and high salinities in comparison with the two other ones.
However, the plant height of different pistachio cultivars
was shortened with exceeding salinity stress; this
reduction was more intense in Ou and Ka cultivars under
the moderate and high salinities (Table 1).
The impact of chloride-sodium on leaf area revealed no
considerable difference among different cultivars under
the control and low salinity conditions. However, the
difference between the three cultivars was intensified with
the increase in salinity stress since this difference
between Ak cultivar and two other cultivars was
significant in high salinity. Ak cultivar showed the
maximal leaf area among all varieties although this
parameter was reduced in all varieties as stress was
increased and it was more noticeable for Ou and Ka
cultivars under high salinity (Table 1). The effect of
chloride-sodium on the chlorophyll and cartenoid content
in leaves is presented in Table (2). These results
indicated that no significant difference in cartenoid
pigment was observed among different pistachio cultivars

Abbaspour et al. 2471
Table 2. Chlorophyll, cartenoides content (mg/g f.w.) and soluble sugars (mg/g d.w.) of three pistachio cultivars as
affected by salinity treatment.
Salinity levels (mM) Cultivar Total chlorophyll Cartenoides Soluble sugars
Ak 1.2
0
a 0.89 a 22 a
Ka 1.25 a 0.77 a 26 a
Ou 1.30 a 0.93 a 24 a
100
200
300
Ak 0.89 ab 0.78 a 25 a
Ka 0.85 ab 0.79 a 24 a
Ou 1.03 ab 0.81 a 27 a
Ak 0.73 bc 0.82 a 33 b
Ka 0.54 cd 0.71 a 22 a
Ou 0.59 cd 0.76 a 24 a
Ak 0.75 bc 0.22 b 35 b
Ka 0.38 d 0.27 b 23 a
Ou 0.41 d 0.31 b 20 a
Within each column, means superscript with different letters are significantly different (P≤ 0.05).
under the control and different chloride-sodium salinity
conditions. However, at salinity of 300 mM, a significant
reduction in cartenoid amount was observed in different
cultivars. The results indicated chloride-sodium did not
induce any remarkable difference in total chlorophyll
pigments among different cultivars under the control and
low-moderate salinity conditions. However, Ak cultivar
showed a significantly higher pigment amount than the
other two varieties under high salinities. Moreover, the
impact of salinity on the reduction of chlorophyll amount
was statistically significant in all varieties starting from
salinity of 200 mM (Table 2).
The impact of chloride-sodium salinity on soluble sugar
value indicated that low salinity had no considerable
impact on dissolved sugars in different cultivars.
However, the moderate and high salinity led to an
increase in the amount of total soluble sugars in Ak
cultivar while no significant difference was achieved
between the other two cultivars compared to the control
conditions. The difference in soluble sugars between Ak
and the other two cultivars was significant in salinities of
200 and 300 mM while no significant difference was
observed among the three cultivars under the control and
low salinity conditions with regard to soluble sugars
(Table 2).
The effect of different concentrations of chloridesodium
on the amounts of Na, Cl, K and N in different
cultivars of pistachio shrubs is presented in Table 3. The
results indicated that the impacts of different salinities on
Na and Cl were nearly similar as the amounts of these
two elements were increased with exceeding salinity
stress in different cultivars compared to control condition.
This increase was statistically significant under all salinity
levels in comparison with the control condition. Moreover,
no significant difference was observed between the
amounts of the respective elements in different cultivars
under the control and low-moderate salinity conditions.
However, increasing salinity from 200 to 300 resulted in
remarkable enhancement of Cl and Na amounts in Ou
and Ka varieties. Interestingly, no significant difference
was found in the concentration of Na and Cl elements in
Ak cultivar due to increasing salinity compared to salinity
of 200 mM (Table 3).
Salinity of 100 mM had no significant impact on
reduction of potassium level in Ak cultivar but this
reduction was significant in the other varieties as
compared with the control. The concentration of
potassium was significantly higher (P ≤ 0.05) in Ak
cultivar than the other two cultivars under the low and the
moderate salinities. However, no significant difference
was reported in potassium concentration among different
cultivars in control and high salinity conditions (Table 3).
The results of nitrogen concentration in different cultivars
under the control and salinity conditions revealed
insignificant difference under the control and salinity
conditions. The concentration of this element decreased
in all cultivars as salinity stress increased and the
reduction was statistically significant under the moderate
and high salinities compared to the control conditions
although at low salinity this difference was insignificant
(Table 3).
DISCUSSION
In this study, the responses of three different pistachio
cultivars to salt stress were compared with regard to plant
growth, soluble carbohydrate and ion accumulation. It is
well documented that high salinity can inhibit the growth
and development of plants and even result in their death
(Shi and Wang, 2005; Moghaieb et al., 2004). The
current results (Table 1) demonstrated a significant
reduction in plant growth with increasing soil salinity.
These results are parallel to those reported for different

2472 J. Med. Plants Res.
Table 3. Shoot content of Na and Cl (mol/kg d.m.), K and N (mg/g d.m.) in three pistachio cultivars at
different salinity levels.
Salinity level (mM) Cultivar Na Cl K
Ak 0.054
0
a 0.301 a 236 a
Ka 0.061 a 0.298 a 218 a
Ou 0.056 a 0.291 a 240 a
100
200
300
Ak 0.258 b 0.898 b 195 a
Ka 0.241 b 0.842 b 141 b
Ou 0.269 b 0.901 b 150 b
Ak 0.284 b 0.810 b 140 b
Ka 0.345 b 0.911 b 85 c
Ou 0.301 b 0.943 b 79 c
Ak 0.315 b 0.894 b 120 bc
Ka 0.637 c 1.987 c 71 c
Ou 0.748 c 1.785 c 84 c
Within each column, means superscript with different letters are significantly different (P≤ 0.05).
plants such as carthamus (Abbaspour, 2010), pistachio
(Bankar and Ranjbar, 2010) and wheat (Khan et al.,
2006). In order to improve salt tolerance in pistachio
plants, inter-cultivar variation in pistachio varieties should
be examined to select the promising lines-genotypes.
Differences in growth of pistachio cultivars in response to
salt stress observed in the present study might have
occurred due to variation in a number of biochemical or
physiological traits that are associated with the processes
related to the mechanism of salt tolerance such as
photosynthetic pigments, nutrient homeostasis and
accumulation at compatible solute. The comparison of
different cultivars under investigation showed that the
growth of Ou and Ka varieties were significantly inhibited
and these cultivars cannot survive in 300 mM NaCl.
However, Ak cultivar can grow well at the same degree of
salinity.
In the present study, the photosynthetic pigment
content of pistachio plants was decreased under salt
stress. Photosynthetic pigments play a key role in
maintaining photosynthetic capacity of most plants
(Dubey, 2005). The reduction in leaf chlorophyll content
under NaCl stress has been attributed to the destruction
of chlorophyll pigments and the instability of the pigment
protein complex (levit, 1980). It is also attributed to the
interference of salt ion with the de novo synthesis of
proteins and the structural component of chlorophyll
rather than the breakdown of chlorophyll (Jaleel et al.,
2007a). Moreover, the changes of pigments content
under salt stress are used as parameter for selection of
tolerant and sensitive cultivars in plants (Eryilmaz, 2007).
The three tested cultivars showed differences in the
accumulation of soluble sugar with increasing salinity.
The content of soluble sugars in Ou and Ka did not
changed; while, the respective value was increased in Ak
at medium and high salinities. Thus, it is proposed that
the accumulation of soluble sugars might be important to
cytoplasmic osmotic of Ak and the destructive effects of
salinity are commonly thought to be a result of low water
potential and ion toxicity (Parida and Das, 2005).
Na + is the main poisonous ion in salinized soil and
plants growing in saline conditions generally
compartmentalize Na + into vacuoles resulting in Na +
toxicity in the cytosol. It is essential for maintaining a
number of enzymatic processes (James et al., 2006; Zhu,
2003; Munus, 2002), and the Na + /K + ratio is an important
index representing the salt-tolerance ability of a plant
(Parida and Das, 2005). The current results indicated that
the increasing levels of NaCl induced a progressive
absorption of Na and Cl. Such pattern of accumulation of
the toxic ion has earlier been reported in a number of
plant species referred as “salt accumulators” (Turan et
al., 2010). Accumulation of Cl in the tissue is disruptive to
membrane uptake mechanisms (Yousif et al., 1972). The
reduction in potassium in shoots as result of salt stress
(Table 3) has been observed previously, interpreted as
resulting from competition between this ion and Na
(Karmoker et al., 2008; Turan et al., 2010). According to
Cordovilla et al. (1995), NaCl decreased N concentration
in the shoot tissues and the salinity has a negative impact
on the nitrogen acquisition and utilization (Lewis, 1986).
Similar negative effect of chloride-sodium on NO -
3 was
reported by other authors (Wehrman and Handel, 1984;
Turan et al., 2010).
By comparing the accumulation traits of the three
pistachio cultivars, it is obvious that under the same
degree of salinity, especially in high salinity, Na + , Cl - and
Na + /K + were much lower in Ak than those of Ou and Ka

cultivars which was suggestive of greater capacity of Ak
in controlling Na + . Tolerance of salt stress is greater in Ak
cultivar than that in Ou and Ka cultivars and the
difference might be due to different salt-tolerance
mechanisms. Higher plant growth and photosynthetic
pigments, lower Na and Cl content, greater accumulation
of soluble sugars and K could explain the greater salttolerance
of Ak compared to that of Ou and Ka.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2474-2477, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.1735
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Study on salicin content correlation between taxilli
herba and their willow host plants
Dong Lu 1 , Benwei Su 1 , Yonghua Li 2 *, Kaixin Zhu 1 , Hehuan Pei 1 , Minghui Zhao 1 and Jing Li 1
1 Qinzhou Hospital of Traditional Chinese Medicine, Qinzhou 535000, China.
2 Guangxi Traditional Chinese Medicine University, Nanning, 530001, China.
Accepted 20 February, 2012
This paper aims to study the correlation of salicin content between taxilli herba and its host plants,
willows. The salicin content of the taxilli herba parasitizing in willows and mulberries was determined
by the method of reversed-phase high performance liquid chromatography (RP-HPLC). The salicin
sample was ultrasonically extracted in methanol solution in the chromatographic conditions of an
Ultimate ® XB-C18 column (250 mm × 4.6 mm, 5 μm) at room temperature, with a mobile phase of methanol
0.1% potassium dihydrogen phosphate (32:68, V/V), a flow rate of 1.0 ml·min -1 , and a detection
wavelength of 269 nm. The linear range of salicin was from 2.02 to 404.80 μg·L -1 (r = 1.0000), and the
average recovery rate was 97.76%. The salicin content of willow host trees and of the taxilli herba
parasitizing in them was in the range of 0.84 to 3.29 mg·g -1 and of 1.80 to 10.56 mg·g -1 , respectively, the
latter reaching 355.17% (3.55 times) as high as the former, at the very most. However, no salicin could
be determined in mulberry host trees and the taxilli herba parasitizing in them. Salicin characteristic
components in willow host trees could be multiplied by their taxilli herba, and host trees could affect
the medicinal quality of the taxilli herba parasitizing in them possibly by delivering their characteristic
components.
Key words: Taxilli herba, willow, salicin, reversed-phase high performance liquid chromatography, ultraviolet
detection.
INTRODUCTION
Having antipyretic and analgesic effects, salicin can be
used for the treatment of fever and diseases, like arthritis
(Teruaki et al., 2002). As a natural product, it is widely
found in the family salicaceae, especially in the willow
branches and barks (Zhao et al., 2005). Taxilli herba is
commonly used as a traditional Chinese herbal medicine.
Being the most widely used taxilli herba parasitizes in
mulberries from ancient times, taxilli herba parasitizing in
other host plants like daimyo oaks, beeches, willows,
bigcatkin willows, and maples are still being used (Li et
al., 2009; Li et al., 2006). Even in the current Chinese
Pharmacopoeia, the host plants of taxilli herba are not
clearly defined (National Pharmacopoeia Committee,
2010). In this study, the method of RP-HPLC is adopted
to determine the salicin content of the taxilli herba
*Corresponding author. E-mail:liyonghua185@126.com. Tel:
+867713137585. Fax: +867713140360.
parasitizing in willows and its host plants, with the taxilli
herba parasitizing in mulberries and its host plants as
reference substances. By observing the salicin content of
taxilli herba and its host plants, this study can provide
some experimental evidence for the assessment of the
impact of host plants on their taxilli herba’s medicinal
quality.
MATERIALS AND METHODS
Materials and reagents
The reference substance of salicin was purchased from Chengdu
Mansite Pharmaceutical Co., Ltd., with the batch number of A0130.
Furthermore, ultrapure water was used, and all the other chemical
reagents were analytically pure. The taxilli herba parasitizing in
willows and in mulberries was collected from Qinzhou, Guangxi in
the wild environment; and the standardized planting base of
Qinzhou Institute of Traditional Chinese Medicine respectively in
March 2011. The taxilli herba was identified by researcher Huaxing
Qiu from South China Institute of Botany, the Chinese Academy of

Science as Taxillus chinensis (DC.) Danser and the host plants as
Salix babylonica Linn. and Morus atropurpurea Roxb., respectively.
The samples collected were washed and dried, and their branches
and leaves were separated and ground to a 45-mesh powder for
use.
Instruments and equipment
The instruments and equipments used included: high performance
liquid chromatograph (Japanese Shimadzu Corporation, LC-20AT),
UV detector (Japanese Shimadzu Corporation, SPD-20A),
Ultimate®XB-C18 column (250 mm × 4.6 mm, 5 μm), ultrasonic
cleaner (Kun Shan Ultrasonic Instruments Co., Ltd, KQ-300DB),
analytical balance (AND GH-252), adjustable micro pipette
(Thermo), and 0.45 μm microporous membrane.
Experimental methods
Preparation of the standard solution
Accurately weighed 10.12 mg of salicin reference substance was
added to the methanol solution and diluted to a constant volume of
25 ml, thus forming the mother liquor of standard solution with the
concentration of 404.80 µg·L -1 , which was then kept at a 4°C
refrigerator for use.
Preparation of the sample solution
Accurately weighed 0.2 g of the sample was added to 25 ml of
methanol solution. Then the sample solution was ultrasonically
extracted for 30 min and finally cooled. After filtration by the 0.45
μm microporous membrane, it was kept at a 4°C refrigerator for
use.
RESULTS
Chromatographic conditions and system adaptability
The detection wavelength of the salicin was 269 nm (Hui
and Wang., 2004); the mobile phase was methanol 0.1%
potassium dihydrogen phosphate (32:68, V/V); the
column temperature was room temperature; and the flow
rate was 1.0 ml·min -1 . The chromatograms of both the
reference substance and the sample are shown in Figure
1.
Linear relationship
The mother liquor of the reference solution was diluted by
methanol to form a series of salicin reference solutions
with concentrations of 2.02, 10.12, 50.60, 101.20, 202.40
and 404.80 µg·L -1 in turn. Then, 10 μl of each salicin
reference solution with the aforementioned concentrations
was injected into the HPLC system to determine the
peak areas, which were used for the regression analysis
of the salicin concentrations. Finally, standard curves
were drawn to calculate the regression equation, which
Lu et al. 2475
was Y = 2065.7x + 5046.2, with a correlation coefficient
(r) of 1.0000. As a result, it could be concluded that there
was a good linear relationship between the
concentrations and their corresponding peak areas when
the salicin ranged in concentration from 2.02 to 404.80
µg·L -1 . The limit of detection (LOD) was 1.09 µg·L -1 and
the limit of quantification was 3.32 µg·L -1 (three times of
the noise).
Precision testing
Accurately weighed 10 μl of the same sample solution
was repeatedly injected into the HPLC system for six
times to determine the salicin content, respectively. The
RSD was 0.26% (n = 6), proving the good precision of the
equipments.
Stability testing
The same sample solution was accurately weighed and
injected seven times into the HPLC system at 0, 2, 4, 6,
8, 12 and 24 h, respectively to determine the salicin
content. The RSD was 1.36% (n = 7), showing that the
salicin sample solution was stable within 24 h.
Reproducibility testing
First, six portions of sample were accurately weighed.
Then after being extracted and injected, they were used
to determine the salicin content. The RSD was 0.9% (n =
6), indicating that the current method had a good
reproducibility.
Average recovery rate testing
Five portions of the accurately weighed sample whose
salicin content had been determined were added to a
certain amount of salicin reference substance. After they
had been extracted and injected, the Salicin content was
determined again. The average recovery rate was
97.76%, and the RSD was 1.56% (Table 1), proving that
the current method had a high recovery rate and met the
experimental requirements.
Determination of salicin content in samples
The samples of taxilli herba and of its host plants were
accurately weighed (0.2 g) and used for the preparation
of the sample solution by the methods mentioned earlier.
Then 10 μl of each sample was injected into the HPLC
system to determine the salicin content, whose results
are shown in Table 2.

2476 J. Med. Plants Res.
Figure 1. Chromatograms of salicin reference substance, taxilli herba, and the host plants. 1: Salicin; A: Chromatograms of
salicin reference substance; B: Chromatogram of branches of willow host plants; C: Chromatogram of leaves of willow host
plants; D: Chromatogram of branches of taxilli herba (in willow host plants); E: Chromatogram of leaves of taxilli herba (in
willow host plants); F: Chromatogram of branches of mulberry host plants; G: Chromatogram of leaves of mulberry host
plants; H: Chromatogram of branches of taxilli herba (in mulberry host plants); I: Chromatogram of leaves of taxilli herba (i n
mulberry host plants).
Table 1. Salicin average recovery rate.
Quality of
samples (g)
DISCUSSION
Content of
salicin (μg)
Amount of added
standards (μg)
Measured
value (μg)
Recovery
rate (%)
0.1013 158.84 202.40 356.66 97.74
0.1025 160.72 202.40 356.16 96.56
0.1019 159.78 202.40 354.44 96.18
0.1006 157.74 202.40 353.08 100.00
0.1012 158.68 202.40 357.70 98.33
Relative salicin content between taxilli herba and
their host plants
The taxilli herba medicinal materials recorded in Chinese
Pharmacopoeia are the dry leaves and branches of
T. chinensis (DC) Danser. The relative salicin content
between taxilli herba and their host plants is listed in
Table 2. The current experiment results of the salicin
content determination show that salicin components
inherent in willow host trees can be multiplied by their
taxilli herba and the accumulating amount of salicin is
different in different parts of the medicinal materials. The
ratios of salicin content in the leaves and branches of
taxilli herba medicinal materials to their corresponding
parts in willow host plants are in the range of 214.29 to
355.17% (2.14 to 3.55 times).
Average
recovery rate (%)
RSD (%)
97.76 1.56
Impact of host plants on the quality of taxilli herba
It can be seen from the determined results of the salicin
content of the taxilli herba parasitizing in willows and
mulberries and its host-plants, that salicin is the inherent
component in willow host plants, and that taxilli herba
contains salicin because of its parasitizing in willows.
However, no salicin component can be found in the taxilli
herba parasitizing in mulberries owing to the fact that its
mulberry host plants contain no salicin. The experiment
results are consistent with one of the authors’ reports that
the 1-deoxynojirimycin inherent in mulberry host plants
could be accumulated by the taxilli herba parasitizing in
them (Hu et al., 2011; Li et al., 2011). The experiment
results again indicate that host plants are likely to be the
key factors affecting taxilli herba medicinal materials’
quality, and that host trees can affect the medicinal
quality of the taxilli herba parasitizing in them by

Table 2. Determination of salicin content in taxilli herba medicinal materials and their host plants.
Samples
no.
1
2
3
Samples
Samples
weight (g)
Retention
time (min)
Peak
areas
Contents of
salicin (mg·g -1 )
Lu et al. 2477
Relative
contents (%) a
Branches of Willow 0.2036 6.044 16759 0.84
Leaves of Willow 0.2048 5.968 17596 0.87
Branches of Taxillus chinensis 0.2016 6.003 35595 1.80 214.29
Leaves of Taxillus chinensis 0.2020 6.007 61376 3.09 355.17
Branches of Willow 0.2017 6.016 20990 1.06
Leaves of Willow 0.2012 5.936 65024 3.29
Branches of Taxillus chinensis 0.2039 6.023 60481 3.02 284.91
Leaves of Taxillus chinensis 0.2011 5.933 208725 10.56 320.97
Branches of mulberry 0.2031 / / /
Leaves of mulberry 0.2023 / / /
Branches of Taxillus chinensis 0.2038 / / /
Leaves of Taxillus chinensis 0.2048 / / /
a Relative content (%) = [(content of salicin in parasitic loranthus) / (content of salicin in the same part of it host tree)] × 100%.
delivering their inherent components.
Quality control of taxilli herba medicinal materials
Given the fact that salicin is an inherent component in
willow host plants, and that taxilli herba contains salicin
owing to its parasitizing in willows, the method
established in the current study could be used to control
the medicinal materials quality of the taxilli herba
parasitizing in its willow host plants, as well as to
distinguish the medicinal materials of the taxilli herba
parasitizing in its willow host plants from those of nonwillow
trees.
Conclusion
Salicin characteristic components in willow host trees
could be multiplied by their taxilli herba, and host trees
could affect the medicinal quality of the taxilli herba
parasitizing in them possibly by delivering their
characteristic components.
ACKNOWLEDGEMENTS
This research was supported by both the National Natural
Science Foundation Project (81173537) and the Natural
Science Foundation Project of Guangxi
(2010GXNSFA013264).
REFERENCES
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deoxynojirimycin component correlation between medicinal parasitic
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Li YH, Ruan JL, Chen SL, Lu D, Zhu KX, Zhao MH, Pei HH (2009).
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People’s Republic of China. Chin. Med. Sci. Technol. Press, Beijing,
pp. 280-281.
Li YH, Bu BW, Zhang XJ, Zhu KX, Pei HH, Zhao MH, Lu D(2011).
Correlation of DNJ between Taxilli Herba and its host-plants. China J.
Chin. Mater. Med., 36(15): 2102-2106.
Teruaki A, Tetsuro Y, Kyoich K, Masao H (2002). Evaluation of salicin
as an Antipyretic Prodrug that does not cause Gastric Injury. Planta
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58.

Journal of Medicinal Plants Research Vol. 6(12), pp. 2478-2487, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.1753
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Antimicrobial activity and chemical composition of
essential oils of four Hypericum from Khorasan, Iran
Motavalizadehkakhky Alireza
Department of Chemistry, Neyshabur Branch, Islamic Azad University, Neyshabur Branch, Neyshabur, Iran.
E-mail: Amotavalizadeh@yahoo.com. Tel: +989153510342 or +985516611720.
Accepted 24 January, 2012
The essential oils obtained by hydrodistillation from the flowers, leaves, stems and roots or from four
species Hypericum include Hypericum perforatum L., Hypericum hyssopifolium Vill., Hypericum
helianthemoides (Spach) Boiss. and Hypericum scabrum L. from plants growing wild in Khorasan,
Northeast of Iran, were analyzed by gas chromatography (GC) and gas chromatography/mass spectral
(GC/MS). In the oil of flowers, leaves, stems and roots from H. perforatum, 47, 43, 30 and 34 (0.06 to
0.1%w/w); from H. hyssopifolium 42, 48, 45 and 44 (0.05 to 0.09%w/w); from H. helianthemoides 50, 49,
39 and 46 (0.05 to 0.09%w/w); and from H. Scabrum 58, 43, 41 and 40 (0.04 to 0.1%w/w) components
were identified, respectively. In the light conclusion and despite the morphological characters, H.
perforatum L., H. hyssopifolium and H. scabrum could be placed in the pinene group, and H.
helianthemoides in the ß-caryophyllene group. The in vitro antimicrobial activity of essential oils were
also examined against seven microbial strains (gram positive and gram negative) by disc agar diffusion
method and in general, the oils showed moderate activity against all tested microorganisms.
Key words: Antimicrobial activity, Essential oils composition, Iran, Khorasan, Hypericum SP, βcaryophyllene,
α-pinene.
INTRODUCTION
The genus Hypericum L. (Family Hypericaceae) consists
of over 460 species, which occur in all temperate parts of
the world (Robson NKB 2003; Hosni K et al. 2008).
Seventeen species of them are found in Iran, three of
which are endemic: Hypericum fursei N. Robson,
Hypericum dogonbadanicum Assad, and Hypericum
asperulum Jaub.f Spach (Rechinger, 1980; Mozaffarian,
1996). Among them most commercially important
member of this genus, Hypericum perforatum L., St.
John’s wort, demonstrated antidepressant, antimicrobial,
antiseptic, antihelmintic antiviral, and anticancer activities
(Mills et al., 2000; Barnes, 2001; Fnimh, 2001). H.
perforatum also demonstrated anti dermatophyte activity
(Larypooret al., 2009). This genus contains different
natural product classes, including naphthodianthrones,
prenylated phloroglucinols, xanthones, flavonoids,
biflavonoids, tannins, proanthocyanidins, and phenolic
acids (Javidnia et al., 2008). H. perforatum L., Hypericum
hyssopifolium Vill., Hypericum helianthemoides (Spach)
Boiss. and Hypericum scabrum L. are four Hypericum
species from Khorasan provinces, Iran, whose essential
oils from flowers, leaves, stems and roots have been
subjected to analysis in this study. Many studies of the
essential oil content of Hypericum have been performed.
Essential oils and volatile constituents that have been
most frequently reported from Hypericum include the
aliphatic hydrocarbons n-nonane and n-undecane; the
monoterpenes α- and β-pinene; and the sesquiterpenes
β-caryophyllene and caryophyllene oxide (Crockett,
2010).
An examination of H. perforatum plants growing in 10
defined habitat types in Lithuania allowed the
identification of three distinct chemotypes, dominated
respectively by β-caryophyllene, caryophyllene oxide and
germacrene D (Mockute et al., 2003, 2007). A similar
study performed in southeastern Poland identified
significant differences among both the content and
composition of essential oils from plants growing in 16
habitats, although two major constituents (2methyloctane
and α-terpineol) were produced by
representatives of most populations (Crockett, 2010). An
examination of 11 accessions of H. perforatum leaves

and flowers growing in a single population in Lithuania
indicated that β-caryophyllene and caryophyllene
oxide dominated in leaves, while spathulenol,
tetradecanol and viridiflorol were dominant constituents of
the flowers (Radusiene et al., 2005). In June 2008 we
studied H. perforatum plants growing in northeastern of
Iran (northwestern of Neyshabur) revealed α- and βpinene
and α- and β-selinene as the primary volatile
constituents of the leaves and flowers, while germacrene
D was predominant in the oil extracted from the stems
and roots (Motavalizadehkakhky et al., 2008). The aerial
parts of wild H. perforatum were collected during the
flowering period, especially in different regions of
Western Europe (France, Italy, Portugal, Spain, Greek,
Serbia), but also in Turkey, Uzbekistan, Lithuania as well
as in China and India (Bertoli et al., 2011).
H. perforatum collected from Serbia (Saraglou et al.,
2007) contains an important quantity of α-pinene (8.6%),
while the same species from the Rujan mountains did not
contain α-pinene (Gudzic et al., 2001). α- and β-pinene
are major components in the oil of H. perforatum from
Greece (Petrakis et al., 2005).
The amount of mono- and sesquiterpenes, seem
reduced in H. perforatum from Turkey (Demirci et al. ,
2005; Cirak et al., 2010). The main components in the oil
of H. prforatum from Italy were 2-methyl octane (21.1%),
germacrene-D (17.6%) and α -pinene (15.8%) (Pintore et
al., 2005). Samples of French H. scabrum plants were
rich in sesquiterpenes (Mathis et al., 1964), while the oil
of the same species collected in Turkey consisted of 13
monoterpene hydrocarbon (85%) and α- pinene was the
major component (72%).
The predominance (45.3%) of α-pinene was also
confirmed in dried flowering aerial parts of H. scabrum
collected from Iran (Morteza-Semnani et al., 2005).
Hyperican content in flower and leaves of eight
Hypericum (helianthemoides, hyssopifolium, scabrum,
perforatum,...) species from Iran determined by HPLC
(Jaymand et al., 2008). Chemical composition of leaves
and flowers and fruits of H. perforatum from Kashan in
Iran were determined by gas chromatography-mass
spectrometry (Akhbari et al., 2009). Analysis of oil
resulted in identification of 55 compounds (91.4%), for
leaves, which α-pinene (29.33%) was the main
components.
The analysis for flower and fruit part resulted in the
identification of 26 compounds (95.96 %), which α-
Amorphene (15.86%), α-pinene (11.34%), Thymol
(7.27%) and α-Campholene aldehyde (6.63%) were the
main components. Chemical composition of aerial parts
of H. perforatum and H. scabrum from Tajikistan were
analyzed by GC-MS. Sixty-six compound were identified
in the oil of H. prforatum with Germacrene D (13.7%), αpinene
(5.1%), (E)-Caryophyllene (4.7%), n-dodecanol
(4.5%), Caryophyllene oxide (4.2%), Bicyclogermacrene
(3.8%), Spathulenol (3.4%) as the main constituents.
Twenty-six compounds were identified in the oil of H.
Alireza 2479
scabrum L. with α-pinene (44.8%), Spathulenol (7.1%),
Verbenol (6.0%), trans-Verbenol (3.9%), and γ-
Muurolene (3.5%) as the abundant compounds
(Sharopov et al., 2010).
Many recent example of antibacterial or antifungal
activity of essential oils can be found in the Hypericum
genus, not only for H. prforatum. In fact, several
Hypericum species native to different region have been
investigated on several types of bacteria and fungi
(Warnke et al., 2009; Buchbauer et al., 2010, 2004; Pauli
et al., 2010).
Essential oil from H. maculatum Crantz. in Serbia
showed a large spectrum and a strong activity as
antimicrobial agent especially against Staphylococcus
aureus, Escherichia coli, Pseudomonas aeruginosa,
Salmonella enteritidis, Klebsiella pneumoniae, Bacillus
subtilis, Sarcina lutea (Gudzic et al., 2002).
The antimicrobial activities of α- and β-pinene, as well
as β-caryophyllene, have been well-documented and, as
these compounds represent dominant components in the
essential oils of many Hypericum species, such effects
are not unexpected. Further investigations with essential
oils, volatile fractions and infused oils from Hypericum
species would be of interest due to the ex vivo antiinflammatory
activity and in vivo gastroprotective effects
that have been demonstrated with H. perforatum infused
oils (Zdunic et al., 2009; Lavagna et al., 2001).
The essential oil of fresh aerial parts of Hypericum
richeri Vill. subsq. grisebachii obtained by hydrodistilation
was analyzed by GC and GC-MS. One hundred and five
constituents identified and tested against a panel of
microbial strains by broth microdilution assay and it was
found to was also moderate effect against all tested
microorganisms (Dordevic et al., 2011).
The essential oils of H. scabrum, H. scabroides and H.
triquetrifolium were studied for the first time for their
antimicrobial activity against nine organisms. All the
essential oils exhibited some broad spectrum
antibacterial activity, at a concentration of 80 µg/ml. The
essential oils of Hypericum species showed antibacterial
activity against the tested organisms and a yeast (Kizil et
al., 2004). The composition of the hydrodistilled oils
obtained from aerial parts of H. hyssopifolium subsp. and
H. heterophyllum Vent. were analyzed by means of GC
and GC-MS, and 66 compounds were determined in
total. The oils of H. hyssopifolium, is rich in
monoterpenes consists α-pinene (57.3%), β-pinene
(9.0%), limonene (6.2%) and α-phellandrene (4.4%). The
oils were tested for antifungal activity using microbial
growth inhibition assays in vitro against 10 agricultural
pathogenic fungi. In general, the oils showed moderate
activity against several fungal species (Cakir et al., 2004).
The chemical composition of the essential oils of nine
taxa from seven sections of Hypericum L. (Guttiferae, H.
perforatum subsp. perforatum, H. perforatum subsp.
veronense, H. calycinum, H. montanum, H. richeri subsp.
richeri, H. hyssopifolium, Hypericum hirsutum, Hypericum

2480 J. Med. Plants Res.
Table 1. Weight of plants, time and yield percentage of hydrodistillation.
Parts of
plant
Weight
(g)
H. perforatum H. hyssopifolium H. helianthemoides H. scabrum
Time
(h)
Yield
(%)
Weight
(g)
Time
(h)
Yield
(%)
Weight
(g)
Time
(h)
Yield
(%)
Weight
(g)
Flower 128 3 0.1 120 2.8 0.09 132 3.2 0.09 120 3.5 0.1
Leaves 150 3 0.09 165 3 0.08 180 3 0.08 220 3 0.09
Stems 110 2.5 0.08 100 3 0.06 120 3.5 0.06 110 3 0.06
Roots 100 3.5 0.06 95 3.5 0.05 90 3.5 0.05 110 3.5 0.04
hircinum subsp. majus, and Hypericum tetrapterum)
occurring in central Italy was analyzed by gas
chromatography (GC) / flame ionization detector (FID)
and GC/MS. A total of 186 compounds were identified,
accounting for 86.9 to 92.8% of the total oils. The major
fraction of the oil was always represented by
sesquiterpene hydrocarbons (30.3 to 77.2%), while
quantitative differences occurred between the other
classes of volatiles depending on the taxa considered.
Chemical composition of the nine Hypericum entities with
respect to the taxonomical classification was discussed.
Essential oils obtained from six taxa, were also tested for
their antimicrobial properties against five different
microbial strains by the broth-microdilution method, and
they were found to have significant activity (expressed as
MIC) (Maggi et al., 2010).
The volatile constituents, obtained from air-dried aerial
parts of fruiting Hypericum elongatum were analyzed by
GC/MS method. Thirty four components of about 96.50%
of total oil were identified. α-Pinene (80.43%), γ-
Terpinene (4.23%) and β-Pinene (2.59%) were the
principal components (87.16%). The essential oil and
hydroalcoholic extract were evaluated for antibacterial,
antifungal and anti-yeast activities by using disc diffusion
method (Ghasemi et al., 2007).
MATERIALS AND METHODS
Plant material
Four Hypericum were collected from Khorasan-Razavi and
Khorasan-Shomali Provinces, Iran, in June 2010. H. perforatum L.,
H. hyssopifolium Vill., H. helianthemoides (Spach) Boiss. and H.
scabrum L. were collected from Kharve in Este of Neyshabur [in last
study we investigated H. perforatum L. from northeastern of
Neyshabur (Motavalizadehkakhky et al., 2008)]; Bojnord; Mashhad
(Akhlamad) and Chenaran, respectively. The plants were air dried
and dried samples were crushed, then essential oils were obtained
by hydrodistillation of their flowers, leaves, stems and roots,
separately. Voucher specimens of the plant have been deposited in
the herbarium.
Isolation of the Essential oils
90 to 220 g out of any parts (flowers, leaves, stems and roots) of
plants were subjected to hydrodistilation for 2.5 to 3.5 h using an
Time
(h)
Yield
(%)
original Clevenger-type apparatus and yielded from 0.04 to 0.1%
(w/w) of essential oils. After decanting, the obtained essential oils
were dried over anhydrous magnesium sulfate and, after filtration,
stored in refrigerator at - 4°C until tested and analy zed (Table 1).
Analysis of the essential oils
Gas chromatography
Samples of the oils were diluted in acetone (1:9) and 1 µl was used
for analysis. GC-MS analyses of the essential oil was analyzed on
an Agilent Technologies 7890A GC system coupled to a 5975C
VLMSD mass spectrometer with an injector 7683B series device.
An Agilent (9091)-413:325°C HP-5 column (30 m x 320 x 0.25 µm)
was used with helium as carrier gas at a flow rate of 3.35 ml/min.
The GC oven temperature was initially programmed at 50°C (hold
for 1 min) and finally at 300°C (hold for 5 min) at a ra te of 80°C/min
while the trial temperature was 37.25°C.
The column heater was set at 250°C in a split less mode while
the pressure was 10.2 psi with an average velocity of 66.5 cm/s and
a hold-up time of 0.75 min. Mass spectrometry was run in the
electron impact mode (EI) at 70eV. The percentage compositions
were obtained from electronic integration measurements using
flame ionization detector (FID), set at 250°C.
Gas chromatography-mass spectrometry
The essential oils were analyzed by GC-MS on an Agilent
Technologies 7890A GC system coupled to a 5975C VLMSD mass
spectrometer with an injector 7683B series device. An Agilent
(9091) 413:325°C HP-5 column (30 m x 320 x 0.25 µm) was used
with helium as carrier gas at a flow rate of 3.35 ml/min. GC oven
temperature and conditions were as described previously. The
injector temperature was at 250°C. Mass spectra were rec orded at
70 eV. Mass range was from m/z 30 to 500.
Identification of components
Identification of the oil components was based on their retention
indices determined by reference to a homologous series of nalkenes,
and by comparison of their mass spectral fragmentation
patterns with those reported in the literature (Adams, 2007), and
stored on the MS library (NIST 08.L database/ chemstation data
system) with data previously reported in literature (McLafferty and
Stauffer, 1989; Joulain and König, 1998).
The percentages of each component are reported as raw
percentages base on total ion current without standardization. The
chemical compositions of any parts of four Hypericum are
summarized in Tables 2 and 3.

Table 2. Percentage of chemical composition of Hypericum perforatum L. and Hypericum hyssopifolium Vill. oils.
Compound RI
Alireza 2481
H. perforatum L. H. hyssopifolium Vill.
Flower Leaves Stems Roots Flower Leaves Stems Roots
α-Pinene 939 27.5 16.5 2.0 3.5 17.3 13.6 14.0 12.5
Sabinene 975 0.1 5.5 - - 0.4 0.2 0.5 0.9
β-Pinene 979 12.7 2.1 Tr 5.0 4.6 5.0 10.5 7.0
Myrcene 990 0.8 0.2 - - 1.0 0.4 0.9 0.5
α-Phellandrene 1002 0.1 - 0.4 2.4 1.0 1.5 -
p-Cymene 1024 0.2 0.4 - 0.5 0.6 2.1 0.4 -
Limonene 1027 1.0 0.2 - - 2.2 0.6 0.9 2.9
1,8-Cineole 1029 0.3 - 0.5 - 0.4 0.3 0.7 0.2
β-Phellandrene 1031 - 0.2 - 2.7 - - 3.5 0.4
(E)-β-Ocimene 1044 0.7 0.3 - 1.0 0.3 0.9 - 0.3
Terpinolene 1088 - Tr 7.2 - 0.1 - 1.2 0.3
Undecane 1098 - - 1.8 Tr - 0.8 0.8 -
Linalool 1102 0.4 0.3 - - 0.2 1.2 - 0.5
α-Thujone 1107 0.7 0.5 - 0.2 0.1 - 0.6 -
β-Thujone 1112 1.6 - 0.3 - - - 0.4 0.5
Camphor 1141 - - 1.6 - - 0.3 0.3 0.2
Menthone 1152 0.2 0.3 - 0.1 - 0.5 - 0.1
Borneol 1165 0.6 0.4 - - 0.4 1.1 2.0 1.2
Terpinen-4-ol 1174 0.4 0.5 - Tr 0.3 0.5 - 0.2
Carvone 1243 0.5 0.3 0.1 - 0.1 0.3 - -
Linalool acetae 1257 0.2 - 0.1 0.1 0.1 - 0.1 -
Thymol 1290 5.0 3.0 Tr - 0.4 3.1 2.5 5.0
Carvacrol 1299 1.4 0.3 - - 0.5 0.4 0.6 -
α-Cubebene 1348 0.7 0.4 - 0.5 - 0.6 - 0.3
α-Copaene 1376 0.4 0.3 2.0 1.9 - - - -
β-Elemene 1390 0.7 0.3 1.0 2.8 0.4 0.4 1.7 3.1
α-Gurjunene 1409 0.9 0.7 - - - - 0.3 1.6
β-Caryophyllene 1419 4.1 4.5 8.5 4.0 5.5 3.1 6.0 5.5
β-Copaene 1432 0.3 - 0.1 - 0.2 0.6 - 0.7
β-Humulene 1438 - 0.5 - 1.7 - 0.1 0.2 -
(Z)-β-Farnesene 1443 Tr - 0.2 3.6 - 0.6 0.7 1.2
α-Humulene 1454 - 0.4 - 2.2 Tr 2.1 Tr -
(E)-β-Farnesene 1456 5.1 2.7 3.5 - 5.5 6.3 4.2 3.7
β-Acoradiene 1466 0.4 - - 0.3 0.3 - - 2.2
Dodecanol 1470 - - - - 5.3 0.2 4.0 3.2
γ-Gurjunene 1477 0.3 0.8 - Tr - - - -
γ-Muurolene 1479 1.5 - 1.2 4.0 5.2 2.2 3.1 3.7
Germacrene D 1485 0.2 3.5 35.0 19.5 10.2 8.1 6.7 4.2
β-Selinene 1490 7.1 20.2 - 8.5 3.3 4.3 2.8 1.9
α-Selinene 1498 8.0 11.4 - 6.0 2.4 7.1 4.5 2.1
Bicyclogermacrene 1500 2.5 0.4 0.3 0.1 0.2 0.2 - 1.1
(Z)-α-Bisabolene 1507 - - - 1.9 0.1 0.1 0.2 0.1
Germacrene A 1509 0.6 0.3 0.4 0.1 0.7 0.5 0.2 -
γ-Cadinene 1513 0.3 0.2 - 3.0 4.5 3.5 1.7 1.9
Myristicin 1518 0.2 - 7.2 - - - - -
δ-Cadinene 1523 2.4 1.1 7.3 4.6 4.5 3.0 2.5 1.8
α-Calacorene 1545 0.3 0.4 1.4 - 0.1 1.5 0.3 1.2
Elemicin 1557 - 0.5 1.2 - - 0.5 - -
(3Z)-Hexenyl benzoate 1569 0.3 0.4 - - - 0.4 0.4 0.3
Spathulenol 1578 0.4 0.5 2.0 1.7 11.5 7.3 2.5 4.6

2482 J. Med. Plants Res.
Table 2. Contd.
Caryophyllene oxide 1583 1.7 0.4 1.9 2.2 2.0 2.2 1.9 1.9
Junenol 1619 0.2 - Tr 0.1 0.1 0.2 0.6 0.4
5-Cedranone 1630 - - - 3.2 - - - -
β-Eudesmol 1650 0.2 0.2 0.6 - 0.3 4.0 2.5 4.5
α-Cadinol 1654 2.5 0.3 - Tr 1.2 1.0 2.1 0.9
Germacra-4(15),5, 10 (14)triene-1-α-ol
1686 0.4 1.2 - - - 0.5 0.8 0.7
Junicedranol 1692 - - - - 3.2 1.4 2.5 2.2
6,10,14-trimetyl-2pentadecanone
1849 - 3.5 - - - - - -
Unidentified 1956 0.5 - - - - - - -
(E)-Phytol 1943 0.6 - 0.3 - - 0.2 0.5 0.1
Heneicosane 2100 - 6.0 - 1.0 0.6 1.0 1.0 0.9
Number of identified compounds 47 43 30 34 42 48 45 44
Yield of the oil (%) 0.1 0.09 0.08 0.06 0.09 0.08 0.06 0.05
Monoterpenes 43.1 25.4 9.6 12.7 28.9 23.8 33.4 24.8
Oxygenated monoterpenes 11.1 5.6 2.5 0.3 2.4 7.7 7.1 7.9
Sesquiterpenes 35.8 48.1 60.9 64.7 43.1 44.3 35.1 36.3
Oxygenated sesquiterpenes 5.4 2.6 4.5 4.0 17.3 16.6 12.9 15.2
Diterpenes - - - - - - - -
Oxigenated diterpenes 0.6 - 0.3 - - 0.2 0.5 0.1
Others 1.2 10.4 10.3 4.3 6.0 2.9 6.3 4.4
Total 97.2 92.1 88.1 86.0 97.7 95.5 95.3 88.7
Tr : trace(< 0.05%).
Antimicrobial assay of the Oils
In vitro antibacterial assay of the oils carried out according to disc
agar diffusion method (Jirovets, 1999; Kumar, 2004). Antibacterial
activity of the oils were tested against gram positive bacterial strains
such as Bacillus cereus (MTCC430), B. subtilis (MTCC441), S.
aureus subsp. Aureus (MTCC2940); and Gram-negative bacterial
strains such as K. pneumonia (MTCC109), E. coli (MTCC443),
Proteus vulgaris (MTCC426) and Salmonella typhi (MTCC733),
were grown in nutrient broth for 24 h (pH 7.2 to 7.4) and were used
as inoculums. The Mueller-Hinton agar medium were poured into
the plates to uniform depth of mm and allowed to solidify. Then the
microbial suspensions were streaked over the surface of media
using a sterile cotton swab to ensure the confluent growth of the
organism. Aliquots of 10 µl of the oil at 1:2 dilutions in dimethyl
sulfoxide (DMSO) were impregnated on Whatman No. 1 filter paper
discs of 6 mm diameter. These discs were aseptically applied to the
surface of the agar plates at well-spaced intervals. The plates were
incubated at 37°C for 24 h and observed inhibition z ones including
the diameter of the discs were measured. Control discs
impregnated with 10 µl of the solvent DMSO and streptomycin (10
µl /disc), reference for bacteria were used alongside the test discs
in each experiment. The results are presented in Table 3.
RESULTS AND DISCUSSION
Chemical composition of the essential oils
1. H. perforatum L.
The compounds identified in flowers, leaves, stems and
roots of H. perforatum L. essential oils are listed in Table
2.
(a) Flowers: Forty seven constituents accounted for
97.2% of the total flowers oil. α-Pinene (27.5%), β-Pinene
(12.7%), β-Caryophyllene (4.1%), (E)-β-Farnesene
(5.1%), β-Selinene (7.1%), and α-Selinene (8.0%) were
major components. Monoterpenes, oxygenated
monoterpenes, sesquiterpenes and oxygenated
sesquiterpenes were 43.1, 11.1, 35.8 and 5.4%,
respectively.
(b) Leaves: Forty three constituents accounted for 92.1%
of the total leaves oil. α-Pinene (16.5%), Sabinene
(5.5%), β-Pinene (2.1%), β-Caryophyllene (4.5%), β-
Selinene (20.2%), Germacrene D (3.5%), Heneicosane
(6.0%) and α-Selinene (11.4%) were major components.
Monoterpenes, oxygenated monoterpenes,
sesquiterpenes and oxygenated sesquiterpenes were
25.4, 5.6, 48.1 and 2.6%, respectively.
(c) Stems: Thirty compounds identified in stems oil
(88.1%). Terpinolene (7.2%), β-Caryophyllene (8.5%),
Germacrene D (35.0%), Myristicin (7.2%) (E)-β-
Farnesene (3.5%), and δ-Cadinene (7.3%) were main
components. Monoterpenes (9.6%) decreased, but
sesquiterpenes (60.9%) increased while oxygenated
mono- and sesquiterpenes decreased (2.5 and 4.5%,
respectively).
(d) Roots: Thirty four constituents representing 86.0% of

Table 3. Percentage of chemical composition of Hepericum helianthemoides(Spach) Boiss. and Hypericum scabrum L. oils.
Compound RI
Alireza 2483
H. helianthemoides H. scabrum L.
Flower Leaves Stems Roots Flower Leaves Stems Roots
α-Pinene 939 13.5 12.0 10.0 7.0 31.5 33.0 32.5 25.7
Camphene 950 0.5 1.0 4.3 5.1 0.5 0.9 0.8 0.4
trans-Pinane 975 - 2.0 3.5 4.2 0.4 - 0.5 -
β-Pinene 979 1.5 0.6 1.7 3.1 2.9 3.5 4.0 3.7
Myrcene 990 0.4 0.5 0.7 2.0 3.1 4.0 3.2 4.0
α-Phellandrene 1002 0.2 0.5 0.1 - 0.1 - - 0.8
p-Cymene 1024 0.4 0.5 - 0.2 3.6 2.5 - 1.8
Limonene 1029 2.3 1.9 0.4 - 2.8 0.9 0.8 1.0
1,8-Cineole 1030 - 1.0 - - 0.2 0.6 0.6 -
Sylvestrene 1030 - 1.7 0.4 1.9 - - - -
(Z)-β-Ocimene 1037 6.7 3.7 2.8 0.9 1.0 0.9 0.2 0.1
(E)-β-Ocimene 1044 0.6 0.8 - 0.6 0.5 - 0.7 -
p-Mentha-2,4(8)-diene 1088 Tr - 0.2 0.1 0.2 0.2 - 0.1
trans-Sabinene hydrate 1098 - - - - 0.3 - - -
Undecane 1100 0.2 - - - 0.5 0.8 0.5 -
Linalool 1100 0.2 0.6 0.5 0.9 0.2 1.0 2.2 2.1
1,3,8-p-Menthatriene 1110 - - - - 0.9 0.8 1.2 0.6
Myrcenol 1122 - - - - 0.6 - 0.7 -
Camphor 1141 1.2 2.3 4.0 0.5 - - - -
Trans-Verbenol 1144 1.7 1.7 0.9 0.6 2.1 - 0.1 0.4
Menthone 1152 - - - - 2.5 2.2 1.7 1.9
Pinene oxid 1159 0.1 - 0.7 0.3 1.4 0.5 1.5 0.5
Borneol 1165 0.2 0.6 - 0.8 0.9 - - -
Lavadulol 1169 - - - - 0.5 0.8 0.6 -
p-Cymen-8-ol 1184 0.1 1.2 2.2 0.5 0.7 0.9 - 0.5
α-Terpineol 1189 0.4 0.3 0.6 1.0 1.9 2.1 1.9 0.8
Verbenone 1207 0.3 0.2 - - 2.5 0.2 - -
Citronellol 1225 - - - - 0.9 0.8 1.0 1.2
cis-Pulegol 1229 - - - - 0.8 0.9 0.2 0.1
Thymol 1290 0.4 2.5 2.9 3.1 2.9 - 0.9 0.8
Carvacrol 1299 0.9 1.9 1.6 0.5 2.5 2.1 3.1 1.2
Pulegone 1237 1.7 2.6 3.1 1.7 - - - -
2-Ethyl menthone 1286 0.5 0.3 0.4 - - - - -
δ-Terpinyl acetate 1317 - - - - 0.3 - - -
Eugenol 1359 - - - - 0.6 0.5 0.3 -
iso-Longifolene 1390 0.3 0.8 0.7 1.5 - - - -
β-Isocomene 1407 - - - - - 0.2 0.9 1.0
β-Caryophyllene 1419 19.0 10.2 15.9 9.0 2.1 2.5 1.0 2.1
β-Copaene 1432 1.5 7.2 2.5 9.1 0.5 - 0.7 1.1
γ-Elemene 1436 - - - - 0.2 0.8 0.6 -
α-Guaiene 1439 0.1 0.2 - 0.3 1.5 - 1.0 -
(Z)-β-Farnesene 1443 0.2 1.7 0.6 1.5 - - - -
α-Humulene 1454 1.2 2.5 0.9 0.8 1.0 1.6 3.1 0.9
dehydro-Aromadendrane 1462 0.7 0.6 0.3 0.1 0.2 - - -
Dauca-5,8-diene 1472 0.1 - - - - - - -
Dodecanol 1470 0.3 - - - 0.6 0.8 - 1.9
β-Chamigrene 1477 - 0.2 - 0.9 - - - -
α-Amorphene 1481 0.6 - 0.9 1.2 1.3 0.8 0.2 0.1
Germacrene D 1485 2.5 4.2 3.5 3.9 2.5 2.4 3.1 3.0

2484 J. Med. Plants Res.
Table 3. Contd.
β-Selinene 1490 0.8 2.5 3.1 1.2 1.8 3.1 2.6 0.9
α-Selinene 1496 - 1.7 - 1.8 - - - -
Bicyclogermacrene 1500 0.8 - 0.3 - 0.4 0.3 - 0.5
(Z)-α-Bisabolene 1505 - 0.4 - 0.6 0.3 0.3 0.1 0.2
Germacrene A 1509 0.5 0.1 0.5 - 0.4 - - 1.1
γ-Cadinene 1513 2.1 3.2 2.6 2.9 2.9 3.4 1.7 3.9
Myristicin 1518 - - - - 0.4 - - -
δ-Cadinene 1523 1.2 1.7 1.0 1.9 0.8 1.6 - 1.1
Selina-3,7(11)-diene 1546 0.4 0.2 - 0.1 0.5 - - 0.1
Elemicin 1557 - - - - 0.4 0.3 0.2 0.5
(3Z)-Hexenyl benzoate 1569 0.2 0.1 - 0.3 - - - -
Caryolane-8-ol 1572 3.7 2.7 3.2 2.6 0.4 3.1 3.0 2.0
Spathulenol 1578 8.0 6.5 7.0 4.0 4.5 7.0 6.2 5.9
Caryophyllene oxide 1583 2.1 1.1 2.2 1.4 - - - -
Cubeban-11-ol 1595 0.3 0.2 - - - - - -
Epi-Cedrol 1619 - - - - 0.7 0.5 0.2 -
β-Eudesmol 1650 2.7 1.1 1.7 2.1 0.7 3.5 4.2 4.7
α-Cadinol 1654 0.5 0.6 - 0.7 1.8 0.7 - -
14-Hydroxy-9-epi-(E)-
Caryophyllene
1669 4.9 2.2 1.9 2.1 1.2 0.2 0.1 0.6
Tetradecanol 1672 1.4 - - 0.9 - - - -
α-Chenopodiol 1856 - - - - 0.9 0.7 0.1 -
(E)-Phytol 1943 0.1 0.2 - 0.5 0.3 0.2 - 0.1
Heneicosane 2100 Tr 0.5 0.4 0.9 Tr - - -
Number of identified compounds 50 49 39 46 58 43 41 40
Yield of the oil (%) 0.09 0.08 0.06 0.05 0.1 0.09 0.06 0.04
Monoterpenes 26.1 25.2 24.1 25.1 47.5 46.7 43.9 38.2
Oxygenated monoterpenes 7.2 15.2 16.5 9.9 21.5 12.6 14.8 9.5
Sesquiterpene 32.0 37.4 32.8 36.8 16.4 17.0 15.0 16.0
Oxygenated sesquiterpenes 22.2 14.4 16.0 12.9 10.2 15.7 13.8 13.2
Diterpenes - - - - - - - -
Oxigenated diterpenes 0.1 0.2 - 0.5 0.3 0.2 - 0.1
Others 2.6 0.9 0.4 2.1 2.2 1.9 0.7 2.4
Total 90.2 93.3 89.8 87.3 98.1 94.1 88.2 79.4
Tr : trace(< 0.05%).
essential oils were identified in the essential oils of H.
perforatum L. roots .12.7, 0.3, 64.7 and 4.0 were
percentages of mono- , oxygenated mono- , sesqui and
oxygenated sesquiterpenes, respectively.
2. H. hyssopifolium
The compounds identified in flowers, leaves, stems and
roots of H. hyssopifolium essential oils are listed in Table
2.
(a) Flowers: Forty two constituents accounted for 97.7%
of the total flowers oil. α-Pinene (17.3%), β-Pinene
(4.6%), β-Caryophyllene (5.5%), (E)-β-Farnesene (5.5%),
γ-Muurolene (5.2%), Germacrene D (10.2%), Spathulenol
(11.5%), δ-Cadinene (4.5%) and γ-Cadinene (4.5%) were
major components. Monoterpenes, oxygenated
monoterpenes, sesquiterpenes and oxygenated
sesquiterpenes were 28.9, 2.4, 43.1 and 17.3%,
respectively.
(b) Leaves: Forty eight constituents accounted for 95.5%
of the total leaves oil. α-Pinene (13.6%), β-Pinene (5.0%),
β-Caryophyllene (3.1%), β-Selinene (4.3%), (E)-β-
Farnesene (6.3%), Germacrene D (8.1%), Spathulenol
(7.3%) , β-Eudesmol (4.0%) and α-Selinene (7.1%) were
major components. Monoterpenes, oxygenated
monoterpenes, sesquiterpenes and oxygenated
sesquiterpenes were 23.8, 7.7, 44.3 and 16.6%,

Alireza 2485
Table 4. Antibacterial activity (zoon inhibition) of the oil of flowers ,leaves, stems and roots of four Hypericum against four gram positive and gram negative bacteria (%) as compare
Streptomycin (N.T=Not Tested).
Zone of
inhibition
(mm)
Oil in DMSO (1:2)
H. perforatum
H. hyssopifolium
H. helianthemoides
H. scabrum
MTCC
No.
respectively.
(c) Stems: Forty five compounds identified in
stems oil (95.3%). β-Caryophyllene (6.0%),
Germacrene D (6.7%), β-Pinene (10.5%), (E)-β-
Farnesene (4.2%), and α-Selinene (4.5%) were
main components. Monoterpenes (33.4%),
sesquiterpenes (35.1%), oxygenated mono- and
sesquiterpenes (7.1 and 12.9%, respectively)
were identified.
(d) Roots: forty four constituents
representing88.7% of essential oils were identified
in the essential oils of H. hyssopifolium roots,
24.8, 7.9, 36.3 and 15.2 were percentages of
Streptomycin
Gram-positive bacteria Gram-negative bacteria
1 mg/ml B. cereus B. subtillis Staphylococcus Escherichia Klebsiella Proteus Salmunella
- 430 441 2940 443 109 426 733
Flower 11.5 12.5(108) 13(113) 12.5(108) 12.5(109) 11.5(100) 13(113) 10(87)
Leaves 10.5 13(124) 11.5(109) 10(95) 12(114) 11(105) 10(95) 9(86)
Stems 8 10(125) 8(100) 7(86) 7(87) 6.5(81) 7.5(94) 8(100)
Roots 6.5 7(108) 6(86) 5(77) 6(92) 5(77) 5.5(100) 6.5(100)
Flower 12 13(108) 12(100) 13.5(120) 13(108) 12.5(104) 11.5(96) 10(83)
Leaves 11 11(100) 12(109) 11.5(104) 12(109) 12.5(114) 13(118) 10.5(95)
Stems 10.5 9(86) 8.5(81) 9.5(90) 13(124) 12.5(119) 13(124) 11.5(109)
Roots N.T N.T N.T N.T N.T N.T N.T N.T
Flower 13 12.5(96) 12(92) 11.5(88) 13(100) 12.5(96) 11(85) 10.5(81)
Leaves 11.5 10.5(91) 10(87) 9(78) 13(113) 12(104) 10(87) 12(104)
Stems 9 7(78) 6(67) 6(67) 10(111) 8(89) 7.5(83) 7(78)
Roots N.T N.T N.T N.T N.T N.T N.T N.T
Flower 12 14(116) 13(108) 13.5(112) 11(92) 12(100) 12.5(104) 10(83)
Leaves 10 11(110) 11.5(115) 12(120) 11(110) 12(120) 12(120) 11.5(115)
Stems 8 10.5(131) 9(112) 9.5(119) N.T N.T N.T N.T
Roots 6.5 6(92) 5(77) 5.5(85) 6(92) 7(108) 8(123) 5.5(85)
mono-, oxygenated mono-, sesqui and
oxygenated sesquiterpenes respectively. α-
Pinene (12.5%), β-Pinene (7.0%), Thymol (5.0%),
β-Caryophyllene (5.5%), Germacrene D (4.2%) ,
β-Eudesmol (4.5%) and Spathulenol (4.6%) were
main components.
3. H. helianthemoides
The compounds identified in flowers, leaves,
stems and roots of H. helianthemoides essential
oils are listed in Table 3.
(a) Flowers: Fifty constituents accounted for
90.2% of the total flowers oil. α-Pinene (13.5%),
β-Caryophyllene (19.0%), (Z)-β-Ocimene (6.7%),
Germacrene D (2.5%) and Spathulenol (8.0%)
were major components. Monoterpenes,
oxygenated monoterpenes, sesquiterpenes and
oxygenated sesquiterpenes were 26.1, 7.2, 32.0
and 22.2%, respectively.
(b) Leaves: Forty nine constituents accounted for
93.3% of the total leaves oil. α-Pinene
(12.0%),(Z)-β-Ocimene (3.7%), β-Copaene
(7.2%), β- Caryophyllene (10.2%), Spathulenol
(6.5%) and Germacrene D (4.2%) were major

2486 J. Med. Plants Res.
components. Monoterpenes, oxygenated monoterpenes,
sesquiterpenes and oxygenated sesquiterpenes were
25.2, 15.2, 37.4 and 14.4%, respectively.
(c) Stems: Thirty nine compounds identified in stems oil
(89.8%). α-Pinene (10.0%), Camphene (4.3%), Camphor
(4.0%), β-Caryophyllene (15.9%), Germacrene D (3.5%)
and Spathulenol (7.0%) were main components.
Monoterpenes (24.1%), sesquiterpenes (32.8%),
oxygenated mono- and sesquiterpenes (16.5% and
16.0% respectively) were identified.
(d) Roots: Forty six constituents representing 87.3% of
essential oils were identified in the essential oils of H.
helianthemoides roots . 25.1, 9.9, 36.8 and 12.9 were
percentages of mono-, oxygenated mono-, sesqui and
oxygenated sesquiterpenes respectively. α-Pinene
(7.0%), trans-Pinene (4.2%), Camphene (5.1%), β-
Caryophyllene (9.0%), Germacrene D (3.9%), β-Copaene
(9.1%) and Spathulenol (4.0%) were main components.
4. H. scabrum
The compounds identified in flowers, leaves, stems and
roots of H. scabrum essential oils are listed in Table 3.
(a) Flowers: Fifty eight constituents accounted for
98.1% of the total flowers oil. α-Pinene (31.5%), Myrcene
(3.1%), p-Cymene (3.6%), Germacrene D (2.5%) and
Spathulenol (4.5%) were major components.
Monoterpenes, oxygenated monoterpenes,
sesquiterpenes and oxygenated sesquiterpenes were
47.5, 21.5, 16.4 and 10.2%, respectively.
(b) Leaves: Forty three constituents accounted for
94.1% of the total leaves oil. α-Pinene (33.0%), Myrcene
(4.0%), β-Caryophyllene (2.5%), Spathulenol (7.0%), β-
Eudesmol (3.5%) and Germacrene D (2.4%) were major
components. Monoterpenes, oxygenated monoterpenes,
sesquiterpenes and oxygenated sesquiterpenes were
46.7, 12.6, 17.0 and 15.7%, respectively.
(c) Stems: forty one compounds identified in stems oil
(88.2%). α-Pinene (32.5%), Myrcene (3.2%), β-
Pinene(4.0%), α-Humulene (3.1%), β-Eudesmol (4.2%),
Germacrene D (3.1%) and Spathulenol (6.2%) were main
components. Monoterpenes (43.9%), sesquiterpenes
(15.0%), oxygenated mono- and sesquiterpenes (14.8%
and 13.8%) were identified, respectively.
(d) Roots: forty constituents representing 79.4% of
essential oils were identified in the essential oils of H.
scabrum roots. 38.2, 9.5, 16.0 and 13.2 were
percentages of mono-, oxygenated mono-, sesqui and
oxygenated sesquiterpenes respectively. α-Pinene
(25.7%), β-Pinene (3.7%), Myrcene (4.0%), Germacrene
D (3.0%), γ-Cadinene (3.9%), β-Eudesmol (4.7%) and
Spathulenol (5.9%) were main components.
As a light conclusion according to Cakir et al. (2005),
Hypericum species can be divided in two groups, based
on the monoterpene and sesquiterpene content with
emphasis on ß-caryophyllene and α-pinene as the major
and characteristic compounds. Hence, H.
perforatum L., H. hyssopifolium and H. scabrum could be
placed in the pinene group, because α-pinene is major
components in the essential oils of them, and H.
helianthemoides in the ß-caryophyllene group because
it's essential oils is rich in ß-caryophyllene.
Antimicrobial activity
The results of antimicrobial activity of the four Hypericum
genus essential oils against seven bacterial (gram
positive and gram negative) are listed in Table 4. Zone of
inhibition diameters (mm) determinations were obtained
by disc agar diffusion method . In general, the oils
showed moderate activity against all tested
microorganisms.
The H. perforatum L., H. hyssopifolium , H.
helianthemoides and H. Scabrum oils obtained from
flowers, leaves, stems and roots in DMSO (1:2 dilutions)
showed 77 to 125, 81 to 120, 67 to 96 and 77 to 131%
inhibition against gram positive bacteria : B. cereus, B.
subtilis and S. aureus subsp. aureus, respectively, as
compared to the standard, streptomycin at 10 µl/disc
(Table 4).
The H. perforatum L. H. hyssopifolium ,H.
helianthemoides and H. Scabrum oils obtained from
flowers, leaves, stems and roots in DMSO (1:2 dilutions)
showed 77 to 114, 83 to 124, 78 to 113 and 83 to 123%
inhibition against gram negative bacteria : K. pneumonia,
E. coli, P. vulgaris and S. typhi respectively, as compared
to the standard, streptomycin at 10 µl/disc (Table 4).
The antimicrobial potential of four Hypericum genus
essential oils could be explained by its high content of
terpenoids. This activity is suspected to be associated
with the high percentage of sesquiterpenoid fraction,
since it was previously reported that ß-caryophyllene, ßpinene
and caryophyllene oxide possessed moderate to
strong activities against a number of microorganisms
(Magiatis et al., 2002; Bougatsos et al., 2004).
ACKNOWLEDGEMENTS
The author is thankful to Prof. Rustaiyan, Dr. Masoudi,
Dr. Mehrzad, Dr. Taheri, and (MSc. Student) Zohreh
Ebrahimi.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2488-2492, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.1765
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
The antitumor activities of Lentinula edodes C91-3
mycelia fermentation protein on S180
(Mouse sarcoma cell) in vivo and in vitro
Mintao Zhong, Ben Liu, Yingli Liu, Xiaoli Wang, Xingyun Li, Lei Liu, Anhong Ning, Jing Cao
and Min Huang*
Department of Medical Microbiology, Dalian Medical University, 9 Western Section, Lvshun South Road, Lvshunkou
District, Dalian 116044, China.
Accepted 16 February, 2012
Lentinula edodes is one of the most widely used medicinal mushrooms which exhibit significant
antitumor activity. It is mostly studied and several studies have showed that its polysaccharides play
critical roles in the antitumor effects. In this study, we determine the antitumor activity of Lentinula
edodes C91-3 mycelia fermentation protein (LFP91-3) on S180 tumor cells in vivo as well as in vitro. The
results showed that LFP91-3 inhibited the growth of S180 tumor cells in vitro, and the mechanism may be
related to apoptosis induced by LFP91-3. In vivo, LFP91-3 treatment significantly prolonged life span in
S180 tumor-bearing mice. LFP91-3 can be used as an important antitumor element in Lentinula edodes C 91-3
mycelia fermentative liquid.
Key words: Lentinula edodes C91-3 fermentation protein, Lentinula edodes, antitumor activity.
INTRODUCTION
Lentinula edodes, commonly known as the Shiitake
mushroom, is the second most popular and widely
cultivated edible mushroom in the world. As the main
medicinal fungi, its extract has anti-viral, anti-bacteria,
anti-tumor activity and regulation of immune function, etc
(Hearst et al., 2009; Kuppusamy et al., 2009; Sarangi et
al., 2006; Unursaikhan et al., 2006). The strain of
Lentinula edodes was studied in March, 1991. So we
named Lentinula edodes C91-3, and “C” means China.
Numerous studies showed Lentinan could inhibit tumor
cells growth, improve patient’s symptoms, and reduce
adverse reactions. It is now officially used for clinical
medicine as adjuvant chemotherapy drugs (Ooi and Liu,
2000; Kidd, 2005; Zheng et al., 2005; Fang et al., 2006;
Miyaji et al., 2006). However, antitumor activity of
Lentinula edodes on protein level was rarely reported. In
this study submerged fermentation for Lentinula edodes
C91-3 mycelia was done and its antitumor effects on
protein level were determined.
*Corresponding author. E-mail: huangminchao@163.com.
Tel:+86-411-86110007. Fax: +86-411-86110007
MATERIALS AND METHODS
Experimental animals
50 healthy inbred BALB/C mice were selected. Three months old
SPF-grade mice were used having weight 18 to 20 g. We used 20
male and 20 female mice, obtained from the Experimental Animal
Center of Dalian Medical University. This study was approved by
Dalian Medical University Animal Ethical Review Committee, similar
to the guidelines for the use and care of animals that are published
by the National Institute of Health.
Carcinoma cell lines
S180 (mouse sarcoma tumor) cell lines were obtained from the
Department of Microbiology of Dalian Medical University. Cell lines
were grown in RPMI-1640 medium (Gibco) containing 10% fetal calf
serum (China National Medicines Corporation, Ltd). The cells were
incubated at 37°C in a humidified atmosphere containing 5% CO2.
Lentinula edodes C91-3 strain and culture conditions
Lentinula edodes C91-3 strain were obtained from the Department of
Microbiology of Dalian Medical University. It was cultured in the
potato culture medium containing 1% Vitamin B1, 2.0% Agar,
0.15% MgSO4, 0.3% K2HPO4, and 2.0% glucose having pH 6.0.

Table 1. Comparison of survival times of S180 tumor-bearing mice among the 5 treatment groups.
Group Death/total Mediated life span(d) Survival rate (%)
NS 10/10 15.80±2.94 0
CTX 10/10 21.30±2.50* 0
LFP91-3I 8/10 23.00±2.62* 20
LFP91-3II 6/10 28.50±4.60* 40
LFP91-3III 5/10 33.60±3.64* 50
p95%, was determined by trypan blue staining. The
cell concentration was adjusted to a final concentration of 1×10 7
cells per ml. 100 μl cell suspension was added into 96-well plate
per well. After pre-incubating S180 tumor cells for 8 h at 37°C in 5%
CO2 incubator, the control group contains normal saline 100 μl. 100
μl LFP91-3 with a final concentration (5 μg/ml) was added as the
experimental group and incubated with the cells for 24, 48 and 72
h. The cells were collected and the cell concentration was adjusted
to 5 × 10 5 /ml. The cells were washed twice with PBS, centrifuged at
1000 rpm for 5 min and the supernatants were aspirated. The cells
were suspended with 500 μl 1× Binding Buffer, followed by adding 5
μl Annexin V-FITC, 5 μl PI (BioVision Annexin V-FITC Apoptosis
Detection Kit), keep in dark for 5min at room temperature, and then
detected through flow cytometry.
Statistical analyses
The data were analyzed by ANOVA using SPSS software. Results
are reported as mean ± standard deviation and p

2490 J. Med. Plants Res.
Inhibition ratio (%)
Figure 1. Inhibition ratio of S180 cell growth affected by different concentration LFP91-3.
Table 2. Inhibition ratio of S180 cell growth affected by different concentration LFP91-3.
Time (h)
Inhibition ratio (%)
5 μg/ml 10 μg/ml 15 μg/ml
24 8.16 21.42 29.89
48 14.45 29.17 64.14
72 29.91 39.90 74.53
Table 3. Apoptosis ratio of S180 in the initial stage between LFP91-3 experimental group and NS control group.
Time
0 24 48 72
Time (h)
Apoptosis ratio in the initial stage (%)
24 h 48 h 72 h
NS control group 0.82 0.93 1.22
LFP91-3 experimental group 2.56 7.51 8.47
the LFP91-3II-treatment group, in contrast, mortality was
only 60%, with an average survival time of 28.5 days
(12.7 days longer than the control group; p

Figure 2. Apoptosis scatterplot of S180 in the initial stage between LFP91-3 experimental group and NS control group. (UL: upper left quadrant FITC-/PI+:
impaired cell; UR: upper right quadrant FITC+/PI+, dead cell or non-viable apoptotic cell; LL: left lower quadrant FITC-/PI-, living cell: LR: lower right
quadrant FITC+/PI-, apoptotic cell).
Zhong et al. 2491

2492 J. Med. Plants Res.
Figure 3. Apoptosis ratio of S180 in the initial stage between LFP91-3 experimental group and NS control group.
due to malignant tumors (Ebina, 2005). The studies on
anti-tumor ingredients extracted from the fungus or its
metabolites have increased researchers attention. Its
antitumor activity has been recognized internationally.
But there were many research have been done about
polysaccharide extracts from Lentinula edodes (Borchers
et al., 2004; Ooi and Liu, 2000; Kidd, 2005; Zheng et al.,
2005; Fang et al., 2006; Miyaji et al., 2006; Ng and Yap,
2002). To explore its antitumor activity on protein level,
we firstly found that the LFP91-3 gained through
submerged fermentation could kill S180 tumor cells directly
in vitro, and its mechanism related to apoptosis pathway
induced by LFP91-3. In vivo, LFP91-3 extended the life span
of S180 tumor-bearing mice, but the mechanism needs to
be further study. However, LFP91-3 was crude protein
complexes. So further research will focus on the structure
and biochemical character of protein monome and study
the anti-tumor mechanism on apoptosis pathway in detail.
In conclusion, our study provides a new kind of antitumor
material and a new theoretical basis for further study of
the mechanism in antitumor proteins from Lentinula
edodes C91-3 Mycelia Fermentation Protein.
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Apoptosis ratio(%)
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9
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Apoptosis ratio of S180 cells in the initial stage
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0 24h 24 48h 48 72 72h
time Time (h)
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2493-2503, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR12.004
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Effect of plant density on phenology and oil yield of
safflower herb under irrigated and rainfed planting
systems
Roghayeh Shakeri Amoghein 1 , Ahmad Tobeh 1 and Shahzad Jamaati-e-Somarin 2 *
1 Department of Agronomy and plant breeding, Faculty of Agriculture, University of Mohaghegh Ardabili. Ardabil. Iran.
2 Young Researchers Club, Ardabil Branch, Islamic Azad University, Ardabil, Iran.
Accepted 24 January, 2012
In order to study the effect of plant density on phenology and oil yield of safflower herb under irrigated
and rainfed planting systems, an experiment was conducted in Agriculture Research Station of Islamic
Azad University, Ardabil Branch, Ardabil, Iran in 2010 as a factorial experiment based on a randomized
complete block design with four replications. The factors included planting system (irrigation and
rainfed) and plant density (60, 50, 40 and 30 plants.m -2 ). The results showed that the density of 30
plants.m -2 under both planting systems and the density of 40 plants.m -2 under rainfed system had the
highest number of days to 50% emergence. The densities of 60 and 50 plants.m -2 under rainfed system
had the highest number of days to 50% branch-bearing. The density of 60 plants.m -2 under irrigated
system had the lowest number of days to 50% bud-bearing. The plants at the densities of 60 and 50
plants.m -2 under rainfed system initiated flowering earlier than other treatments. The density of 50
platns.m -2 was ranked in the same group with the densities of 40 and 60 plants.m -2 and irrigated system.
under irrigated system, the highest oil percentage was obtained from the lowest density and the lowest
one from the density of 40 plants.m -2 and the densities of 50 and 60 plants.m -2 were ranked in the same
group, while under rainfed system, the highest oil yield was obtained from the highest density under
irrigated system and the lowest one from the densities of 40 and 60 plants.m -2 under rainfed system. As
shown, oil percentage was increased with the decrease in density. The significantly highest seed yield
was produced at the density of 60 plants.m -2 under rainfed system which was ranked in the same group
with the density of 50 plants.m -2 under irrigated system.
Key words: Flowering time, emergence, oil percentage, safflower, yield, yield components.
INTRODUCTION
Out of the prevalent oilseeds, safflower is the only one
native to Iran. Indeed, Iran has a high diversity of
safflower. Safflower (Carthamus tinctorius L.) belongs to
the family of Asteraceae. It has been established in
various climates and its wild species are widespread
throughout Iran (Poordad, 2006). Safflower (Honghua in
China) is a member of the family Compositae or
Asteraceae and an annual herb. It is soft with mild odor
and slightly bitter in taste (Zheng, 1999).
*Corresponding author. E-Mail: jamaati_1361@yahoo.com. Tel:
+989141594490. Fax: +984517714126.
Safflower is both edible and medicinal and is issued by
the Ministry of Health in China and consumed safely
(Guan et al., 1999). The main ingredients of safflower are
safflower glucoside, safflower yellow and safflower
quinone. In addition, it also contains a small amount of
oleic acid, linoleic acid, linolenic acid, flavonoids, amino
acids and polysaccharides (Zheng, 1999). Safflower
grows widely in many areas of China and it is one of the
traditional Chinese medicinal herb in common use with its
flowers to treat coronary heart disease and thrombosis,
remove blood stasis, cure pain and swelling (Zheng,
1999; Zhang et al., 2005).
Moreover, it was reported that safflower had the
functions of anti-thrombosis and hypoxia tolerance, and

2494 J. Med. Plants Res.
can increase coronary flow and improve microcirculation
(Ling, 2002). In recent years, the use of safflower as a
coloring and flavoring agent has been increased as a
food additive in some Asian countries (Nobakht et al.,
2000). Modern pharmacological studies have shown that
the safflower polysaccharide has the activity of anti-tumor
and antioxidative effect (Jin et al., 2004; Shi et al., 2010).
Safflower yellow A has the functions to relieve myocardial
ischemia, protect neuron against hypoxia injury and
attenuate acute lung injury induced by lipopolysaccharide
administration in mice (Jin et al., 2005; Ye and Guo,
2008). Exercise-induced fatigue, due to over-exercise,
refers to the body that cannot maintain its specific level
physiologically or cannot maintain the predetermined
exercise intensity, manifested as mental and physical
fatigue (Wu et al., 2003; Chen et al., 2004; Gao and
Chen, 2003). Exercise-induced fatigue can be recovered
by supplemented energetic substance, releasing
metabolic production and administrated tonics, but these
bring harms to the body even though retarding the fatigue
(Li and Wei, 2005). In addition, some of the drugs are
forbidden by the International Olympic Committee. During
the process of seeking for safe and effective anti-athletic
fatigue methods, the specialty of Chinese herbal
medicine has drawn the attentions of scholars in the
world (Shenhua et al., 2009) and some research results
have been reported in recent years.
Nowadays, given its tolerance to heat and survival at
minimum moisture, its cultivation is agronomically and
economically feasible for local consumption as oilseed
and edible dynes (Aliari et al., 2000). Since it is a native
crop, it has marked characteristics such as adaptability to
arid and semi-arid climates, high-quality oil, and
resistance to abiotic stresses particularly drought stress
(Weiss, 2000). Mundel et al. (1994) classified different
developmental stages of safflower as emergence,
rosette, stem elongation, formation of auxiliary branches,
flowering and maturing. Bagheri (1995) categorized them
as emergence, branching, flowering and maturing,
whereas Mohammadi (1995) and Nejad (1996) named
them as emergence, branching, emergence of
reproductive buds, bud-bearing, head termination and
maturing and Zand (1995) named them as emergence,
stem-bearing, branch-bearing, flowering and maturing.
Seed development proceeds through a set of important
stages such as nutrient storage, drying and dormancy.
Each stage brings about some changes in seed which
affect their yield. The stage at which the seeds reach to
their maximum dry weight on their parental plants is
known as the physiological maturity (Shaw and Loomis,
1950). Yazdi (1996) stated that harvest at complete
maturity, when seed moisture content was 100 g.kg -1 ,
was preferable because of ease of threshing and high
storage capability owing to the optimum moisture content
in both pods and seeds. Also, the results of other studies
(Tavakoli, 2002) have shown that irrigation withdrawal
before and during flowering of safflower resulted in fewer
numbers of seeds per head and that the closer the time
of stress application was to flowering, the more effective
it was on the number of seeds.
Rostami (2004) reported that the application of drought
stress after flowering and pollination slightly decreased
the number of seeds and mainly reduced 1000-seed
weight. In a study on three high, moderate and pooryielding
genotypic groups of safflower in Mashad, Iran,
Zand (1995) showed that they exhibited significantly
different developmental stages. Yasari et al. (2005)
revealed that branch-bearing period was the most
effective period of completion of yield and yield
components. The results of Hashemi and Marashi (1995)
showed that the interaction of genotype and moisture
regime was significant on all traits unless days to
flowering initiation and 50% flowering, number of seeds
per head and seed yield per plant.
Istanbulluoglu (2009) evaluated the effect of irrigation
and water deficit at different developmental stages on
seed yield per ha and 1000-seed weight and showed that
safflower was significantly impacted by water stress at
late-vegetative stage. The highest yield was obtained at
early and late-vegetative growth and seed yield
formation. There were significant differences among
varieties at 1% probability level with respect to flowering
initiation and termination, days to maturity, 1000-seed
weight, oil percentage and yield. Under rainfed farming,
cultivars with short maturity period have the highest seed
yield. Means comparison of seed and oil yield of different
cultivars showed significant differences. Also, as means
comparison revealed, high-yielding cultivars had lower
number of days to maturity than other traits. Means of
varieties with shorter maturing period can tie higher seed
yield with higher seed production (Hatamzadeh et al.,
2003).
In the study of Omidi (2009), irrigation withdrawal at the
end of flowering stage or at the initiation of grain-filling
period not only did not greatly decrease seed yield, but
also saved water. Zareian and Ehsanzadeh (2001)
showed that plant density only significantly affected the
bud-bearing initiation of safflower. In total, days after
sowing and the heat demand for each developmental
stage started to decrease with the increase in the density.
The effect of cultivar was significant on such stages as
emergence, bud-bearing, head production and 50%
flowering, but it did not significantly affect other
developmental stages. The results of the studies on the
effect of plant density per unit area on crops indicate that
the yield of the crops per unit area is change with the
change in plant density (Koocheki, 1997). The effect of
plant density on seed yield was significant too, so that the
number of heads per m 2 was increased as the density
was increased (Fazeli et al., 2007). Planting row spacing
was shown to significantly affect the number of heads per
plant, the number of seeds per head, seed yield and oil
yield (Ghasemi et al., 2006).
The effect of on-row plant spacing was not statistically

Table 1. Results of the analysis of the soil of study field.
Depth Saturation
percentage
EC
(ds.m -1 )
pH Neutralizable
material (%)
Organic C
(%)
Total N
(%)
Absorbable P
(ppm)
Absorbable K
(ppm)
Amoghein et al. 2495
Soil texture
Clay Silt Sand
Soil type
0-30 48 2.66 7.8 4.8 0.97 0.103 4.8 4601 28 41 31 Loam-clay
30-60 45 2.4 8.2 7 0.48 0.056 2 290 24 36 40 Clay
significant on the number of heads per plant, the number
of seeds per head, 1000-seed weight, harvest index, and
seed and oil yield. In the study of Ranjbar et al. (2004),
optimum seeding rate (row spacing and on-row plant
spacing) influenced yield and yield components through
changing density and environmental resources. Majd et
al. (2003) stated that seed yield per unit area and per
plant, yield components, the number of seeds per plant
and kernel percentage were influenced by plant density.
Means comparison for different studied traits at different
plant densities showed that as plant density was
decreased, the number of heads per plant, 1000-seed
weight, the number of seeds per plant, kernel percentage
and seed yield per plant were increased, but the number
of seeds per head and yield per unit area were
significantly decreased.
In this study, safflower is considered as one of the
resistant drought plants and it has excessive drug use
and spice. Also, the Ardabil region in Iran has the highest
talent to cultivate various medicinal plants. So, the
objective of the current study was to examine the effect of
plant density on phenology and oil yield of safflower herb
under irrigated and rainfed systems in Ardabil, Iran.
MATERIALS AND METHODS
The current study was carried out in Agriculture Research Station of
Islamic Azad University, Ardabil Branch, Ardabil, Iran (Alt. 1350 m.,
Long. 48°20´ E, Lat. 38°05´ N) in 2010. All the executive and
laboratory operations were conducted in Department of Agronomy,
University of Mohagheghe Ardabili, Ardabil, Iran. In order to
determine physical and chemical characteristics of the soil of the
study field, it was sampled before field preparation from the depths
of 0 to 30 and 30 to 60 cm. Then, the samples were analyzed in
Water and Soil Laboratory of Islamic Azad University, Ardabil
Branch, whose results are given in Table 1.
Sina was the cultivar of autumn safflower which was used in this
study. It was procured from Agriculture Research Center of Zanjan,
Iran. Imported from ICARDA in 1997, Cv. Sina (line PI-537598) was
first examined in Rainfed Agriculture Research Institute of Iran. It
was one of the superior genotypes and was studied in terms of its
local adaptability. Its evaluation in the fields of Kermanshah,
Lorestan, Northern Khorasan and Ilam, all in Iran, showed that it
performed considerably better than control cultivar (Namely,
Zaraghan 279). Safflower cv. Sina is early-maturing with
intermediate growth type, resistant to drought stress, thorny with
yellow/orange flowers with mean height of 103.5 cm and 1000-seed
weight of 34.7 g.
The study was a factorial experiment based on a randomized
complete block design with four replications. The factors included
plant density and planting system (irrigated and rainfed). Each
block consisted of 8 treatments or plots with the dimensions of
4×2.1 m 2 with 7 rows with row spacing of 30 cm and inter-plant
spacing of 8, 6, 5 and 4 cm used for making the densities of 60, 50,
40 and 30 plants.m -2 . The planting system was created with and
without irrigation.
The study field had been under the cultivation of potato in the
previous year. For the field preparation, the field was first plowed in
autumn and was added with 100 kg ammonium phosphate per
hectare. Then, it was leveled. After testing the germination
percentage of the seeds, they were planted with the on-row spacing
of 8, 6, 5 and 4 cm at the depth of 4 cm on April 9, 2010. At the
same day, the first irrigation was carried out.
The measured traits included days to 50% emergence, 50%
rosette, 50% branch-bearing, 50% boll-bearing, flowering initiation,
50% flowering and flowering termination in order to make
phenological studies possible. To measure seed and oil yield, the
upper and lower 0.5 m of the harvested plants were removed and
the rest was used for this measurement. After harvesting, the plants
were dried in open air. Then, their seeds were separated and
weighed. Next, seed weight per unit area and per hectare was
measured. Afterwards, the oil percentage of the seed samples (32
samples) was determined by Soxhlet method, and finally, oil yield
was measured by the following formula:
Oil yield = oil percentage × weight of dried seed sample
Statistical analysis
To analyze the variance of the data, software MS-TATC and SAS
were used and the means were compared by Duncan Test.
Regression analysis was used to study the relations between
different harvest stages and studied traits as well as to interpret the
observed variation during seeds maturing, and the graphs were
drawn by software MS-Excel. At the end, the coefficient of the
correlations between the studied traits was measured.
RESULTS AND DISCUSSION
Days to 50% emergence
The results of analysis of variance of phenological traits
are shown in Table 2. As shown, the simple effect of
plant density was significant at 5% probability level with
respect to 50% germination. The means comparison for
this trait (Table 3) at the lowest density (30 plants.m -2 )
showed the highest number of days to 50% emergence,
while the simple effect of the density of 60 plants.m -2 had
the lowest number of days after emergence. In the study
of Rahman et al. (2005), the plant density did not affect
germination. In addition, drought stress had no effect on

2496 J. Med. Plants Res.
Table 2. Summary of analysis of variance for some studied traits at final harvest.
Sources of variation df
50% emergence
Mean of squares
50% rosette 50% branch-bearing 50% boll-bearing
Replication 3 6.00 ns 8.28 ns 6.36* 0.75 ns
Planting system (S) 1 2.00 ns 5.28 ns 30.03** 18.00*
Plant density (D) 3 8.66* 3.28 ns 26.94** 4.50 ns
Interaction S × D 3 3.33 ns 4.28 ns 6.86* 4.50 ns
Error 21 2.57 11.85 2.38 2.89
Coefficient of variations (%) 8.11 9.20 2.45 2.31
ns, * and ** show non-significance and significance at 5 and 1% probability level, respectively.
Table 3. Means comparison for the main studied effects on some traits at final harvest based on Duncan Test at 5% probability level.
Treatment
Planting system
Irrigated
Rainfed
Plant density
30 plants.m -2
40 plants.m -2
50 plants.m -2
60 plants.m -2
50% emergence
(days after planting)
19.51 a
20.00 a
21.00 a
20.00 ab
19.50 ab
18.50 a
Means with the same letter(s) showed no significant differences.
seed germination or vigor of corn and sorghum
(Ghassemi-Golezani et al., 1997). Francaneto (1993)
showed that drought stress can reduce the germination of
soybean seeds and that irrigation can be used to
counteract this effect.
Days to 50% rosette
The results of analysis of variance (Table 2) showed no
statistically significant differences in the number of days
to 50% rosette between planting system and plant
density per m 2 as well as the interactions between them.
Means comparison for this trait (Table 3) confirmed this
result. Among the interactions between planting system
and plant density (Figure 1), no statistically significant
differences were observed in this trait. Nonetheless,
rainfed system had lower number of days to 50% rosette
than irrigated system. So, it can be concluded that as the
density was increased from 30 to 60 plants.m -2 , the
number of days to 50% emergence and 50% rosette
decreased.
Days to 50% branch-bearing
According to the results of analysis of variance (Table 2),
50% rosette
(days after planting)
37.00 a
37.81 a
37.62 a
38.00 a
36.50 a
37.50 a
50% branch-bearing
(days after planting)
61.93 b
63.87 a
60.50 c
62.50 b
64.62 a
64.00 ab
50% boll-bearing
(days after planting)
74.25 a
72.75 b
73.12 a
73.12 a
73.12 a
74.62 a
the simple effect of planting system and plant density
resulted in statistically significant differences in the
number of days to 50% branch-bearing at 1% probability
level. Moreover, the interaction between planting system
and plant density was significant at 5% probability level.
Means comparison (Table 3) indicated that 50% branchbearing
was realized earlier under irrigated system than
under rainfed system and that the densities of 50 and 60
plants.m -2 had the highest number of days to 50%
branch-bearing and the density of 30 plants.m -2 had the
lowest one.
The density of 40 plants.m -2 had the highest number of
days to 50% branch-bearing and was ranked in the same
group with the density of 60 plants.m -2 . Among the
interactions (Figure 2), the densities of 60 and 50
plants.m -2 under rainfed system had the highest number
of days to 50% branch-bearing and were ranked in the
most superior group. The density 30 plants.m -2 under
both planting systems and the density of 40 plants.m -2
under irrigated system had the lowest number of days to
50% branch-bearing; and the density of 50 plants.m -2
under irrigated system had the highest number of days to
50% branch-bearing and was ranked in the same group
with the density of 40 plants.m -2 under rainfed system.
Yasari et al. (2005) revealed that the duration of

(
-
)
(
(days after planting)
50% Rosette
brarning
(days after planting)
40
39.5
39
38.5
38
37.5
37
36.5
36
35.5
35
a
a
Irrigated Rainfed
a
a
30 40 50 60
Plant density ( plants . m - 2 )
Figure 1. Diagram of 50% rosette (days after planting) as affected by plant density and planting system.
50% Branch
67
66
65
64
63
62
61
60
59
58
57
c
c
Irrigate Rainfed
c
ab
30 40 50 60
a
ab
Plant density (plant.m -2 )
Figure 2. Diagram of 50% branch-bearing (days after planting) as affected . by plant density and planting system.
branch-bearing period was the most effective period in
completing yield and yield components. Therefore,
supplying the needs of a plant during this period played
an essential role in improving the yield of the plant and
the duration of this period was recommended as a good
criterion for selecting genotypes with higher potential
yields.
Days to 50% boll-bearing
a
a
Amoghein et al. 2497
The results of the analysis of variance (Table 2) revealed
that the planting system (irrigated and rainfed) showed
statistically significant differences in the number of days
to 50% boll-bearing or bud-bearing at 5% probability
level, while means comparison (Table 3) indicated that
a
bc
a
a

2498 J. Med. Plants Res.
Table 4. Summary of analysis of variance for some studied traits at final harvest.
Sources of
variation
df
Flowering
initiation
50%
flowering
Means of squares
Flowering
termination
Oil
percentage
Replication 3 1.20 ns 0.83 ns 1.50 ns 2.51 ns 0.01 ns 112807.65 ns
Planting system (S) 1 32.00 ** 32.00 ** 18.00 * 4.67 ns 0.35 ** 8258550.52 **
Plant density (D) 3 4.70 ns 0.83 ns 3.16 ns 12.03 * 0.01 ns 603124.29 ns
C × D 3 1.08 ns 0.33 ns 7.33 * 2.97 ns 0.01 ns 700845.92 ns
Error 21 2.08 1.21 2.92 4.24 0.02 369363.87
Coefficient of variations (%) 1.59 1.14 1.70 10.56 5.51 25.86
ns, * and ** show non-significance and significance at 5 and 1% probability level, respectively.
Table 5. Means comparison for the main studied effects on some traits at final harvest based on Duncan Test at 5% probability level.
Treatment
Planting system
Irrigated rainfed
Flowering initiation
(days after planting)
50% flowering
(days after planting)
Flowering
termination
(days after planting)
Oil
percentage
(%)
Oil
yield
Oil yield
(kg.ha -1 )
Yield
Seed yield
(kg.ha -1 )
91.68 a 97.12 a 101.37 a 19.87 a 564.38 a 2857.5 a
89.68 b 95.12 b 99.87 b 19.11 a 359.99 b 1841.5 b
Plant density
30 plants.m -2
90.12 a 96.25 a 101.50 a 21.16 a 442.67 a 2058.9 a
40 plants.m -2 91.75 a 96.50 a 100.50 a 18.21 b 413.81 a 2221.0 a
50 plants.m -2 90.12 a 96.00 a 100.50 a 19.24 ab 463.38 a 2422.3 a
60 plants.m -2 90.75 a 95.75 a 100.00 a 19.34 ab 528.88 a 2695.7 a
Means with the same letter(s) showed no significant differences.
irrigated system had the highest number of days to 50%
boll-bearing and was ranked in the most superior group,
but rainfed system had the lowest number of days to 50%
boll-bearing.
Zareian and Ehsanzadeh (2001) showed that among
different phenological stages, plant density significantly
affected only the boll-bearing initiation in safflower. In
total, the number of days after planting and the required
heat demand to reach each developmental stage showed
descending trend with the increase in density.
In the table of correlations (Table 6), 50% boll-bearing
had positive, significant correlation with the number of
initial branches per unit area, the number of seeds per
unit area, biological yield and
harvest index.
Days to flowering initiation
The results of analysis of variance (Table 4) showed that
there was significant difference in the number of days to
flowering initiation between different planting systems at
1% probability level. Means comparison (Table 5) for this
trait revealed that the plant started flowering earlier under
irrigated system, while plant density had no impact on the
initiation of flowering. Istanbulluoglu (2009) showed that
safflower plants were significantly influenced by the latevegetative
stage moisture deficiency. The highest yield
was obtained at early and late-vegetative growth and
seed yield formation. In a study on the effect of drought
stress on safflower cv. Arak 2811, Hashemi Dezfuli
(1994) showed that stress had no effect on flowering
initiation.
Days to 50% flowering
The results of analysis of variance (Table 4) showed
significant differences between the simple effects of
planting system and plant density at 1% probability level.
As shown, there was no significant difference in the
number of days to 50% rosette between planting systems
and plant densities, whereas this difference was
observed after the termination of rosette stage at 50%

Table 6. Simple correlation between final studied traits at final harvest.
Amoghein et al. 2499
Trait 50% branch-bearing 50% flowering Yield Oil percentage Oil yield
50% flowering 0.27 ns 1.00
Yield 0.16 ns -0.03 ns 1.00
Oil percentage 0.29 * -0.20 ns 0.46 ** 1.00
Oil yield -0.23 ns -0.26 ns 0.09 ns 0.24 ns 1.00
ns, * and ** show non-significance and significance at 5 and 1% probability level, respectively.
branch-bearing, flowering initiation and 50% flowering
stages. Means comparison for the number of days to
50% flowering (Table 5) showed that plants reached to
50% flowering earlier under rainfed system than under
irrigated system and plant density did not impact this trait.
As the table of correlations (Table 6) showed, 50%
flowering had positive, significant correlation with the
distance of the first head-bearing branch from ground,
seed weight per head and harvest index.
Days to flowering termination
The results of analysis of variance (Table 4) indicated
statistically significant difference between the simple
effect of planting system and the interactions of the
density and planting system at 5% probability level.
Similar results can be seen in the table of means
comparison (Table 5), so that the plants cultivated under
rainfed system reached to the end of flowering stage
earlier than those planted under irrigated system.
Also, the interaction of planting system and plant
density on this trait showed that the plants cultivated at
the density of 30 plants.m -2 under irrigated system had
the lower density of this treatment was the main reason
for higher number of days to flowering termination of the
plants cultivated at the density of 30 plants.m -2 under
irrigated system; that is, lower density intensified interplant
competition and so, the plants utilized nutrients,
light and water as good as possible and increased their
vegetative growth which led to the increase in the number
of days to flowering termination.
Oil percentage
The results of analysis of variance for oil percentage
(Table 4) showed significant differences between the
simple effects of different plant densities at 5% probability
level. Nonetheless, according to means comparison
(Table 5), the simple effect of planting system was not
significant on oil percentage but among the densities, the
lowest one (30 plants.m -2 ) had the highest oil percentage
and the density of 40 plants.m -2 gave rise to the lowest oil
percentage in their seeds. The oil percentage of the
densities of 50 and 60 plants.m -2 was between these two
extremes.
Denser population of the plants increases oil
percentage but decreases iodine content (Abddollahi and
Zarrinjoob, 2001). Patel and Patel (1996) concluded that
oil percentage was influenced by irrigation regimes and
that with the increase in irrigation, oil percentage was
increased too. Singh et al. (1990) studied the effect of
different irrigation regimes on yield and oil of safflower
and reported that the highest oil percentage was
produced by irrigating the plant at branch-bearing and
seed formation stages. The results of other studies
(Heidari and Asad, 1998; Patel, 1993) also show that
irrigation withdrawal and drought stress reduces oil
percentage of the seeds with the variations being slight
but significant. By increasing the density, Sharikian and
Babayian (2000) observed the decrease in oil percentage
and increase in protein percentage of the seeds which
are in agreement with the results of the current study
(Figure 3).
As the table of correlations (Table 6) shows, oil
percentage had positive, significant relation with the traits
of 50% branch-bearing, the distance of the first headbearing
branch from ground, seed weight per head and
seed weight per m 2 and had positive, very significant
relation with the traits of the number of seeds per m 2 ,
harvest index and yield.
Oil yield
Table 4 shows the analysis of variance for oil yield. It can
be drawn from this table that there was statistically
significant difference between the simple effects of
planting systems (rainfed and irrigated) at 1% probability
level, while according to the means comparison for this
trait (Table 5), the highest oil yield was produced under
irrigated system. But, the simple effect of density did not
affect oil yield. Figure 4 shows the interactions between
plant density and planting systems on this trait, according
which the highest oil yield was obtained from the highest
density under the irrigated system and the lowest one
was obtained from the densities of 40 and 60 plants.m -2
under rainfed system.
As mentioned earlier, oil percentage was increased

2500 J. Med. Plants Res.
Flowering termination (days after planting)
104
103
102
101
100
99
98
97
planting)
a
b
Irrigated Rainfed
b
b
30 40 50 60
Plant density ( plants . m - 2 )
Figure 3. Diagram of flowering termination (days after planting) as affected by plant density and planting system.
Oil yield (days after planting)
800
700
600
500
400
300
200
100
0
bc
bc
bc
c
ab
Irrigated Rainfed
30 40 50 60
ab
Plant density (plant.m -2 )
. -
Figure 4. Diagram of oil yield (days after planting) as affected by plant density and planting system.
with the decrease in the density while oil yield was
increased with the decrease in the density which was
contrary to the results obtained for the oil. Since the
highest seed yield was obtained from the density of 60
plants.m - , it seems that seed yield per hectare was
the reason for the increase in oil yield per hectare at
b
bc
this density. In a study on the stepwise regression for
seed and oil yield including four traits of biological yield,
the number of bolls, the number of auxiliary branches and
the number of seeds per boll, the results of path analysis
for seed and oil yield showed that in order to increase the
yield, firstly seed yield must be increased which is in turn
b
a
b
c

Seed yield(days after planting)
4000
3500
3000
2500
2000
1500
1000
500
0
planting)
bc
Irrigated Rainfed
bc
ab
Amoghein et al. 2501
c c c c
30 40 50 60
Plant density ( plant . m - 2 )
Figure 5. Diagram of seed yield (days after planting) as affected by plant density and planting system.
a function of plant biomass and the number of bolls per
plant (Omidi, 2009). Drought stress significantly
decreased oil yield and percentage (Kafi and Rostami,
2007).
The table of correlation for oil yield (Table 6) showed
that this trait had positive, significant correlation with the
number of seeds per m 2 and harvest index.
Seed yield (kg.ha -1 )
As the results of analysis of variance showed (Table 4),
there was significant difference in seed yield between
planting systems at 1% probability level. Means
comparison for this trait (Table 5) showed that seed yield
was higher under irrigated system than under rainfed
system and the density had no effect on increasing or
decreasing seed yield under both planting systems.
Figure 5 shows the interactions of planting systems and
plant density on this trait which indicates that the highest
seed yield was obtained from the density of 60 plants.m -2
under rainfed system which was ranked in the same
group with the density of 50 plants.m -2 under irrigated
system. The lowest seed yield was obtained from
different planting densities (30, 40, 50 and 60 plants.m -2 )
under rainfed system which was ranked in the same
group with the densities of 40 and 30 plants.m -2 under
irrigated system. Ranjbar et al. (2004) found that the
decrease in seed yield per plant did not make significant
differences in seed yield vs. different seeding rates,
mainly because of the increase in the number of plants
per m 2 . Therefore, 30 seeds.m -2 with the row spacing of
30 cm produced the highest yield and 50 seeds.m -2 with
the row spacing of 40 cm produced the lowest one which
is not in agreement with the results of the current study,
while Azari and Khajehpour (2003) reported that as the
plant density was increased, the plant size and yield
components was decreased, but the increase in the
number of plants per unit area usually compensated the
decrease in total yield.
Istanbulluoglu (2009) reported that the highest
safflower seed yield was obtained from control treatment
(full irrigation) which was 40.5 and 3.74 t.ha -1 for summer
and winter treatments, respectively. Hayashi and Hanada
(1985) concluded that the decrease in seed yield was
brought about by the decrease in the number of bolls per
plant which was in turn caused by the decrease in the
number of branches per plant. Tavakoli (2002) showed
that irrigation withdrawal at flowering stage reduced the
yield which was the result of the decrease in the number
of heads per plant and the number of seeds per head.
Also, Bouchereau et al. (1996) revealed that plants
exposed to moderate moisture stress during their
vegetative growth produced higher seed yield. This yield
was significantly decreased under severe stress applied
during both vegetative and reproductive stages. Stern
and Beech (1965) showed that the highest safflower seed
yield was produced at the density of 610000 plants.ha -1
and it was decreased at densities of less than 288000
plants.ha -1 .
According to the table of final traits correlation (Table
6), yield had very significant, negative relation with
a

2502 J. Med. Plants Res.
harvest index and very significant, positive relation with
oil percentage and yield.
Conclusion
According to the results of the current study, it can be
concluded about oil percentage and yield that the former
was increased with the decrease in density, but the latter
was a function of seed yield.ha -1 , so that the highest oil
yield was produced at the highest density (60 plants.m -2 ).
Also, it can be said that drought stress (rainfed system)
impacted oil yield and percentage, so that both were
decreased under rainfed system.
In the case of oil percentage, it can be drawn that
despite being very slight, but the differences of planting
systems were statistically significant. Therefore, it can be
recommended to cultivate safflower in Ardabil, Iran under
rainfed system with the density of 50 plants.m -2 . Also,
safflower is harvested earlier under rainfed system than
under irrigated system owing to well-ripening and the
opening of the heads and the resulting fall of the seeds
as well as late-season precipitations.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2504-2513, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR12.019
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Astragalus polysaccharides induced gene expression
profiling of intraepithelial lymphocytes in immunesuppressed
mice
Lu Cheng 1 , He Xiaojuan 1 , Xiao Cheng 2 , Guo Yuming 1 , Zha Qinglin 1 , Liu Yuanyan 3 , Liu Zhenli 4 ,
Chen Shilin 5 and Lu Aiping 1 *
1 Institute of Basic Research in Clinical Medicine, China Academy of Chinese Medical Sciences, Beijing, China.
2 Sino-Japan Friendship Hospital, Beijing, China.
3 School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China.
4 Institute of Basic theory, China Academy of Chinese Medical Sciences, Dongzhimen, Beijing, China.
5 Institute of Medicinal Plant Development, China Academy of Medical Sciences, Beijing, China.
Accepted 22 February, 2012
Astragalus polysaccharides (APS) possess a variety of immunomodulatory activities, but the regulation
on mucosal immunity is not fully understood. In this study, immune suppression in mice was induced
with cyclophosphamide treatment and APS was used as an intervention and was administrated at
dosages of 3 g/kg (APS HD) and 1.5 g/kg (APS LD). Intraepithelial lymphocytes (IELs) were isolated from
the intestines. The mRNA expressions from IELs in different groups of mice were detected using gene
expression microarray to explore the gene expression profile of IELs, and what are the unique
pathways related to low and high dosage of APS.
Key words: Astragalus polysaccharides, mucosal immunity, intraepithelial lymphocytes, gene expression.
INTRODUCTION
The dried root of Astragalus mongholicus (Huangqi) has
a long history of medicinal use in traditional Chinese
medicine. Animal experiments and modern clinical trials
have shown that A. mongholicus has excellent
immunomodulating effects (Li, 1991). Even with the
widespread use, a complete understanding of the
biological effects and mechanisms regarding A.
mongholicus has remained largely unknown. The active
pharmacological constituents of A. mongholicus include
various polysaccharides, saponins, and flavonoids.
Among these, Astragalus polysaccharides (APS) have
been most widely studied. APS might induce the
differentiation of splenic DCs with enhancement of T
lymphocyte immune function in vitro (Liu et al., 2011).
Studies have also shown that APS enhances the
immunological function of chicken erythrocytes (Jiang et
al., 2010a). Moreover, APS can modulate the innate
immune response of the urinary tract through inducing
*Corresponding author. E-mail: lap64067611@126.com.
increased TLR4 expression in vitro (Yin et al., 2010).
Apart from these actions, our previous research
suggested that regulation of the enteric mucosal immune
response could be one of the important pathways for
immune modulation by polysaccharides (Zhao et al.,
2010). Likewise, we have demonstrated that herbal
medicines have a modulating effect on the enteric
mucosal immune system (Luo et al., 2010; Xiao et al.,
2009).
The mucosal surface of the intestinal tract is the largest
body surface in contact with the external environment. It
is a complex ecosystem generated by the alliance of
gastrointestinal epithelium, immune cells and resident
microbiota (McCracken and Lorenz, 2001). Intraepithelial
lymphocytes (IELs), as the effector cells of the enteric
mucosal immune system, play a multifaceted role in
maintaining mucosal homeostasis (Ismail et al., 2009)
and may be involved in protective cell-mediated immunity
(Mowat et al., 1986). In general, administration of
Chinese medicine to individuals is typically done orally,
leading to intestinal uptake. Thus, there are extensive
interactions between Chinese medicine and intestinal

tract cells. As IELs are located at this critical interface,
they must balance protective immunity with an ability to
safeguard the integrity of the epithelial barrier (Cheroutre
et al., 2011). So, it is of great interest to detect the genes
expression of IELs by APS.
The use of microarrays to evaluate the transcriptome
has transformed our view of biology. As with genomic
analysis, microarrays are still the best choice for a
standardized genome-wide assay that is amenable to
high-throughput applications (Git et al., 2010). Effective
use of gene expression profiling for biological research
has been done to better understand the nature of
intestinal development in response to a treatment (Fleet,
2007). Immunosuppressant cyclophosphamide is used to
induce the immune-suppressed model (Fang et al.,
2006). It is considered an ideal model for investigating
many aspects of immunological response. Therefore, by
employing immune-suppressed mice, the purpose of the
present study was to examine global alterations in gene
expression of the IELs in response to APS.
MATERIALS AND METHODS
Experimental animals
Twelve healthy male BALB/C mice, 6-8 weeks old, provided by the
China Academy of Chinese Medical Sciences were housed under
constant environmental conditions at 22°C and with a 12 h darklight
cycle. The mice were fed a commercially obtained diet and
allowed ad libitum access to water. Mice were randomly allocated to
four groups: control, cyclophosphamide-induced immunesuppressed
group (Model), APS-treated at a high dose (APS HD),
and APS-treated at a low dose (APS LD). The approval of the
Institutional Animal Ethics Committee was obtained before animal
experiments were carried out.
Induction of immune suppression
Immune suppression in the mice was induced with
cyclophosphamide (CTX) treatment followed a previously described
protocol (Abruzzo et al., 2000; Sun et al., 2002). The mice in the
Model, APS HD, and APS LD groups were injected intraperitoneally
with 80 mg/kg cyclophosphamide (Heng Rui Medicine
Co., Ltd, China) once each day for three days. The mice in the
control group were injected with the same volume of saline.
Administration of APS
APS, a marked drug and proved by the State Food and Drug
Administration, China (SFDA No. Z20040086), purchased from
Tianjin Cinorch Pharmaceutical Co., Ltd. China, were dissolved with
distilled water at a concentration of 250 mg/ml. On the fourth day
after induction, all treated mice were given different dosages of
orally administered APS once a day in the morning and lasting for 3
days according to their experimental groups: 3 g/kg APS in the APS
HD group and 1.5 g/kg APS in the APS LD group. The mice in the
control and model groups were administered an equivalent volume
of saline.
Isolation of IELs
One day after the last dose of APS, blood was taken through the
Cheng et al. 2505
retro-orbital artery from the mice, and then the mice were sacrificed.
The IELs isolation was performed as previously published (Zhao et
al., 2010). In brief, the intestines from the duodenum to the
ileocecal junction were removed and flushed with Ca 2+ and Mg 2+
free HBSS (CMF). Peyer’s patches and the mesentery were
removed, and the intestines were opened longitudinally and cut into
pieces about 10 cm long. The pieces were digested twice for 30
min in CMF containing 10 mM HEPES, 25 mM NaHCO3, 2% FBS,
1 mM EDTA, and 1 mM DTT at 37°C. The eluted cells were
collected and passed through a 74 μm nylon mesh to remove
undigested tissue pieces. The IELs were subsequently separated
from epithelial cells by two centrifugations through a 40/70% Percoll
(Pharmacia) gradient at 600 × g for 20 min. The IELs were
harvested from the interface between the 40 and 70% Percoll
layers.
cRNA labeling
Total RNA was isolated from the IELs using the Trizol extraction
method (Invitrogen, Carlsbad, Canada) as described by the
manufacturer. mRNAs were amplified linearly using the
MessageAmp aRNA Kit (Ambion, Inc., Austin, USA) in
accordance with the instructions of the manufacturer. cRNA was
purified with the RNeasy® Mini Kit (QIAGEN, Hilden, Germany)
based on a standard procedure.
Microarray assay
One color format, whole genome mouse Microarray Kit, 4 x 44K
(Agilent Technologies) was used in this study. Microarray
hybridizations were carried out on labeled cRNAs. Arrays were
incubated at 65°C for 17 h in Agilent's microarray hybridization
chambers and subsequently washed according to the Agilent
protocol. Arrays were scanned at a 5-μm resolution using GenePix
Personal 4100A (Molecular Devices Corporation, Sunnyvale, CA).
Statistics and function analysis
All data were analyzed using the SAS9.1.3 statistical package
(order no. 195557). Differential gene expression was assessed by
ANOVA with the p value adjusted using step-up multiple test
correction to control the false discovery rate (FDR). Adjusted p
values

2506 J. Med. Plants Res.
Figure 1. Body weight changes in the mice. From the second day after CTX
injection, the mice in model group, the mice in the APS HD and APS LD groups
showed pathological weight loss. However, it was noted there was a tendency of
weight increase in the APS groups after day 4. The mice in the APS HD and APS
LD groups grew faster after administration of APS compared with the model
group, although no significant difference was observed in body weight.
Table 1. The significantly expressed genes in each comparison.
Experimental group Number of gene with significant p values
Control versus model 204
APS HD versus model 22
APS LD versus model 17
obvious symptoms. However, it was noted that there was
a tendency of weight gain in APS-treated groups
beginning on the day 4. Administration of APS could slow
down the tendency of body weights decrease. The mice
in the APS HD group and APS LD group grew faster after
the administration of APS compared with the model
group, although no significant differences in body weight
were observed (Figure 1).
Differences in gene expression between groups
The number of genes that were significantly overexpressed
or under-expressed is shown in Table 1.
Comparisons of gene expression between the control
group and model group showed 204 genes to be
significantly regulated by CTX. A comparison of APS HD
group and model group showed that 22 genes were
differentially expressed to a significant. Similarly, 17
genes were observed in APS LD group compared with
model group (Table 1). 204 differentially expressed
genes between model group and control group clearly
separated the model group from the control group.
Among these genes, 95 genes were expressed at a
significantly higher level in the model group. Using
DAVID, 95 up-regulated genes in the model group were
found to be involved in functions such as cellular process,
binding, and cell projection, whereas 109 down-regulated
genes in the model group participated in cellular
metabolic processes, catalytic activity, and cell part. The
GO terms with up-regulated among 95 genes are
indicated in Table 2. The largest group with respect to
number of genes (n=55) was the binding in molecular
function. The other group with a large number of genes
(n=47) was the cellular process in biological process.
Among the 109 down-regulated genes, the GO terms
are indicated in Table 3. There were a number of specific
GO terms that revolved around the cellular constituent
theme of cell and cell part (88 genes). A second
prominent group was the cellular process in biological

Table 2. Gene ontology on up-regulated genes in the model group.
GO classification Specific GO term Number of genes p Value
Response to stimulus 19 8.1E-3
Response to stress 12 8.7E-3
Biological process Cellular process 47 2.3E-2
Cellular developmental process 13 3.4E-2
Negative regulation of biological process 11 4.5E-2
Molecular function
Protein binding 35 3.6E-3
Binding 55 5.2E-3
Ribonucleotide binding 14 2.2E-2
Purine ribonucleotide binding 14 2.2E-2
ATP binding 12 2.5E-2
Adenyl ribonucleotide binding 12 2.7E-2
Purine nucleotide binding 14 2.9E-2
Adenyl nucleotide binding 12 3.7E-2
Purine nucleoside binding 12 3.9E-2
Nucleoside binding 12 4.1E-2
Cellular constituent Cell projection 7 2.6E-2
P values reflect statistical significance of each GO term being over-represented.
Table 3. Gene ontology on down-regulated gene in the model group.
GO classification Specific GO term
Number
of genes
p Value
Cellular process 69 2.97E-07
Cellular macromolecule metabolic process 42 3.93E-05
Biological process Cellular component organization 24 7.06E-05
Macromolecule metabolic process 44 1.24E-04
Cellular metabolic process 48 1.38E-04
Molecular function
Cellular constituent
Nucleoside-triphosphatase activity 12 1.57E-04
Hydrolase activity 24 1.96E-04
Pyrophosphatase activity 12 2.25E-04
Hydrolase activity, acting on acid anhydrides, in
Phosphorus-containing anhydrides
12 2.38E-04
Intracellular part 83 2.75E-12
Intracellular 83 6.96E-11
Intracellular organelle 73 3.29E-09
Organelle 73 3.38E-09
Nucleus 48 1.73E-07
P values reflect statistical significance of each GO term being over-represented.
process containing 69 genes, which included more
specific GO terms such as metabolic process, cellular
metabolic process and macromolecule metabolic
process. The third group was related to various molecular
functions dealing with binding (64 genes).
Cheng et al. 2507
Also, we identified 9 commonly modulated genes in
either APS HD group or APS LD group (Table 4). The
genes were regulated in the same direction (towards the
normal scale). Those genes must be important genes for
elucidating the network how APS affects IELs. To further

2508 J. Med. Plants Res.
Table 4. Shared altered genes between control and model, APS HD and model, APS LD and model.
Gene symbol Accession #
Control vs.
Model (Ratio)
APS HD vs.
Model (Ratio)
APS LD vs.
Model (Ratio)
Related function or disease
Rnf139 NM_175226 ↑(1.57) ↑(1.57) ↑(1.48) Kidney cancer
2310079N02Rik AK086714 ↑(1.73) ↑(1.54) ↑(1.54)
Anapc1 AK090134 ↑(1.52) ↑(1.47) ↑(1.43) Gastric cancer
C030015A19Rik AK028820 ↑(1.44) ↑(1.43) ↑(1.45)
Fbxo3 NM_212433 ↑(1.46) ↑(1.40) ↑(1.55) Oral squamous cell carcinoma
Cpd AK134736 ↑(1.25) ↑(1.33) ↑(1.24) Stimulate NO production
Derl2 NM_033562 ↑(1.26) ↑(1.17) ↑(1.17) Hepatocellular carcinoma
Ddx17 NM_199079 ↓(0.67) ↓(0.73) ↓(0.72) Dysregulated in cancers
Arl6ip2 NM_019717 ↓(0.51) ↓(0.53) ↓(0.54)
Figure 2. Significant molecular networks of 9 common modulated genes generated by the
Ingenuity software. Genes or gene products are represented as nodes, and the biological
relationship between two nodes is represented as a line. Note that the gray symbols
represent gene entries that occur in our data, while the transparent entries are molecules
from the Ingenuity knowledge database, inserted to connect all relevant molecules in a
single network. Solid lines between molecules indicate direct physical relationship between
molecules; dotted lines indicate indirect functional relationships.
understand the correlations among the candidate genes,
bioinformatics analyses were performed using the IPA
software, and these analyses led to the identification of
biological association networks. As shown in Figure 2,

Table 5. Genes uniquely regulated by APS HD or APS LD.
Gene symbol Accession #
Control versus
model (Ratio)
APS HD versus
model(Ratio)
Cdc27 NM_145436 ↑(1.65) ↑(1.49)
1110007A13Rik NM_145955 ↑(1.18) ↑(1.17)
Hmgcr NM_008255 ↑(1.51) ↑(1.42)
Man1a2 NM_010763 ↑(1.44) ↑(1.43)
D530033C11Rik NM_030132 ↑(1.42) ↑(1.57)
Ufm1 NM_026435 ↑(1.30) ↑(1.30)
Zcd2 NM_025902 ↑(1.25) ↑(1.23)
Txndc9 NM_172054 ↑(1.18) ↑(1.21)
Zc3h10 NM_134003 ↓(0.79) ↓(0.76)
Sfi1 NM_030207 ↓(0.65) ↓(0.72)
Sidt2 AK081177 ↓(0.59) ↓(0.69)
Nr3c2 NM_001083906 ↓(0.4) ↓(0.49)
Alpi NM_001081082 ↓(0.31) ↓(0.47)
Cheng et al. 2509
APS LD versus
model(Ratio)
Tubd1 NM_019756 ↑(1.56) ↑(1.43)
AK050250 AK050250 ↑(1.20) ↑(1.34)
Hps4 NM_138646 ↓(0.78) ↓(0.84)
Ulk1 NM_009469 ↓(0.76) ↓(0.73)
Usf2 U01663 ↓(0.72) ↓(0.67)
Utrn NM_011682 ↓(0.47) ↓(0.52)
Fosl2 NM_008037 ↓(0.42) ↓(0.53)
Hbp1 NM_177993 ↓(0.42) ↓(0.41)
the main functionalities for the networks are antigen
presentation, cardiovascular disease, cellular
development, cancer, cell cycle, cell death, RNA damage
and repair, protein synthesis, nutritional disease,
developmental disorder, genetic disorder, neurological
disease, cellular growth and proliferation.
As we here observe dose-dependent difference of
APS, we analyzed special gene expression in IELs. We
found that treatment with APS HD led to additional
changes in gene expression compared to APS LD, as
indicated by the identification of 13 genes regulated
specifically by APS HD. In contrast, 9 unique genes were
observed to be induced solely by APS LD (Table 5). IPA
shows the network about APS HD related genes (Figure
3), and the main functionalities are lipid metabolism,
molecular transport, small molecule biochemistry, cancer,
cell cycle, cell death, RNA damage and repair, protein
synthesis, nutritional disease, developmental disorder,
genetic disorder, neurological disease, cellular assembly
and organization. The network about APS LD related
genes was shown in Figure 4, and the main
functionalities given by Ingenuity for the networks are cell
morphology, cellular function and maintenance, cellmediated
immune response, antigen presentation,
cardiovascular disease, cellular development, cancer, cell
cycle, cell death, RNA damage and repair, protein
synthesis, nutritional disease, developmental disorder,
genetic disorder and neurological disease.
DISCUSSION
The major finding in this study is that APS might play
critical in transcriptional regulation of cancer, since the
gene expressions of Rnf139, 2310079N02Rik, Anapc1,
C030015A19Rik, Fbxo3, Cpd, Derl2, Ddx17, and Arl6ip2,
were significantly regulated to normal levels in the APS
HD and APS LD treated mice, in which 7 were upregulated
and 2 down-regulated.
Cpd is a type 1 transmembrane protein the cycle
between the trans-Golgi network and the plasma
membrane. It performs a wide variety of functions,
ranging from the digestion of food to the selective
biosynthesis of hormones and neuropeptides(Kalinina
and Fricker, 2003, Kalinina et al., 2002). Cpd expression
is enhanced during inflammatory processes and may
stimulate NO production by cleaving Arg from peptide
substrates (Hadkar and Skidgel, 2001). Another hallmark
of response in the APS-treated mice is the up-regulation
of Anapc1, RNF139, Fbxo3, and Derl2 gene expression.
A mitotic gene (Fong et al., 2007), The Anapc1 gene was
found to contain a long open reading frame of 1944
amino acids, encoding a polypeptide with a calculated
molecular mass of 216,087 Da (Starborg et al., 1994). It

2510 J. Med. Plants Res.
Figure 3. Significant molecular network of APS HD related genes. Genes are represented as nodes, and the biological
relationship between two nodes is represented as a line. Note that the colored symbols represent gene entries that occur in our
data, while the transparent entries are molecules from the Ingenuity knowledge database. Red symbols represent up-regulate
genes, green symbols represent down-regulate genes. Solid lines between molecules indicate direct physical relationship
between molecules; dotted lines indicate indirect functional relationships.
is a centromere-associated protein that appears to have
a transient function during mitosis. Anapc1 was identified
and shown to be related to an Aspergillus nidulans mitotic
checkpoint regulator (Jorgensen et al., 1998). Moreover,
Anapc1 may be a possible candidate for causing the
chromosomal instability seen in gastric cancer (Lima et
al., 2008). RNF139 encodes an endoplasmic reticulumresident
E3 ubiquitin ligase that inhibits growth in a
RING- and ubiquitylation-dependent manner (Lee et al.,
2010). RNF139 is similar to the Patched family of
proteins, with a putative sterol-sensing domain and an
extracellular loop capable of interaction with the
hedgehog protein (Cho et al., 2005). Chromosomal
translocation of this gene may be important in the
development of kidney cancer (Gemmill et al., 2002). The
Fbxo3 gene may be associated with oral squamous cell
carcinoma (OSCC) tumorigenesis and/or progression
(Cha et al., 2011), and Fbxo3 can synergistically increase
p53 transcriptional activity (Shima et al., 2008). Derl2, a
member of the Derlin family, is a putative proto-oncogene
and has a direct role in oncogenic transformation (Hu et
al., 2007). Increased expression of Derl2 is confirmed in
hepatocellular carcinomas (Ying et al., 2001). Taken
together, these reports support that the actions of APS
might play critical roles in transcriptional regulation in
cancer.

Cheng et al. 2511
Figure 4. Significant molecular network of APS LD related genes. Genes or gene products are represented as nodes,
and the biological relationship between two nodes is represented as a line. Note that the colored symbols represent
gene entries that occur in our data, while the transparent entries are molecules from the Ingenuity knowledge database.
Red symbols represent up-regulate genes, green symbols represent down-regulate genes. Solid lines between
molecules indicate direct physical relationship between molecules; dotted lines indicate indirect functional relationships.
The DEAD box RNA helicase Ddx17 plays important
roles in multiple cellular processes, including
transcription, pre-mRNA processing/alternative splicing,
and miRNA processing, which are commonly
dysregulated in cancers (Fuller-Pace and Moore, 2011).
Blocking Ddx17 acetylation caused cell cycle arrest and
apoptosis, revealing an essential role for Ddx17
acetylation (Mooney et al., 2010). The ability of Ddx17
suggests that transcriptional regulation in cancer were at
work not only in the up-regulated genes but also in the
down-regulated genes. In addition, the Arl6ip2 gene
found to be down-regulated in APS-treated groups has
been reported in U937 cells exposed to various NO
fluxes (Turpaev et al., 2010). However, with few studies
of the gene, its relationship with the activity of APS
remains unclear. Future studies are needed to test the
exact efficacy of interventions that specifically address
the processes suggested by the present gene expression
studies.
Many of the genes uniquely induced by APS HD or
APS LD represented functional families of genes. In the
APS HD group, the expressions of Cdc27 and Zc3h10
were induced. In the APS LD group, the expressions of
Tubd1 and Fosl2 were modulated. The literature implies
that phosphorylation of Cdc27 is involved in TGF-βinduced
activation of APC (Zhang et al., 2011), and it is
suggested that Cdc27 itself may be a tumor suppressor
(Pawar et al., 2010). Zc3h10 inhibits anchorageindependent
growth in soft agar, suggesting a tumor
suppressor function for this gene (Guardiola-Serrano et
al., 2008). The candidate oncogene Tubd1 is associated
with breast cancer risk (Kelemen et al., 2009). Fosl2 is a
member of the Fos family of AP-1 transcription factors
that is often up-regulated in mammary carcinomas. Fosl2

2512 J. Med. Plants Res.
over-expression is associated with a more aggressive
tumor phenotype and is probably involved in breast
cancer progression in vivo (Milde-Langosch et al., 2008).
Interestingly, these 4 unique genes again show the ability
to regulate cancer development and progression, a
function similar to that of the genes previously mentioned.
In addition, Hmgcr, a unique gene affected by APS HD
which converts HMG-CoA to mevalonate and catalyzes
the rate-limiting step in cholesterol biosynthesis (Ohashi
et al., 2003), and upstream stimulatory factor Usf2, a
unique gene affected by APS LD which regulates the
transcription of genes related to immune response, the
cell cycle, and cell proliferation (Bussiere et al., 2010) are
needed for further exploration on the association between
APS and the activities.
The IPA results showed that the main functionalities for
the networks of APS are antigen presentation, cellular
development, cell cycle, RNA damage and repair, protein
synthesis, cellular growth and proliferation. While the
unique pathways in the network of APS LD include lipid
metabolism, molecular transport, small molecule
biochemistry, and the pathways for APS HD are cell
morphology, cellular function and maintenance, cellmediated
immune response. Antigen presentation is the
key process for immune response, and it is closely
related to RA development (Lebre and Tak, 2009;
Wenink et al., 2009; Yanaba et al., 2008). Cell cycle,
cellular growth and proliferation, and apoptosis are
considered the vital components of various processes
including cell turnover, proper development and
functioning of the immune system, hormone-dependent
atrophy, embryonic development and chemical-induced
cell death related to RA pathogenesis (Elmore, 2007;
Jiang et al., 2010b; Ryu et al., 2010). APS might
demonstrate its pharmacological activity via acting on
these pathways. However, the differences between the
low dosage and high dosage of APS are still waiting for
further validation, though there are evidence
demonstrating the correlation between lipid metabolism,
cell mediated immune response and RA development
(Hansel and Bruckert, 2010; Toms et al., 2010). The
results further support the findings in DAVID analysis.
Though the data presented provides a more
comprehensive picture on how APS mediates biological
effects on IELs, there is a limitation in this study. RT-PCR
did not be conducted for verification. However the
clusters of genes and pathway networks are the main
purpose to demonstrate the complicated biological
networks induced by APS in the mice, and singe gene
verification is hard to meet the requirement. Future
pharmacological studies are needed to test the efficacy of
APS interventions which specifically address the
processes suggested by our microarray analysis.
Conclusions
2310079N02Rik, C030015A19Rik, Rnf139, Anapc1,
Fbxo3, Cpd, Derl2, Ddx17, Arl6ip2 and the related
pathways may be modulated by APS on IELs, and APS
might play critical in transcriptional regulations of cancer.
ACKNOWLEDGMENTS
This research is supported in part by the projects from
Ministry of Sciences and Technology of China (No.
2009ZX09502-019), National Science Foundation of
China, No. 30825047, 30902000, 81001676 and
81001623, E-institutes of Shanghai Municipal Education
Commission No E03008, and Key Laboratory of
Separation Sciences for Analytical Chemistry, Dalian
Institute of Chemical Physics, Chinese Academy of
Sciences No. KL1004.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2514-2519, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR12.042
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Palatability perception of herbal teas: Impact of
extraction time and saccharose
Ain Raal* and Vallo Matto
Department of Pharmacy, University of Tartu, Nooruse 1, Tartu 50411, Estonia.
Accepted 10 February, 2012
The herbal teas have been used as remedies for centuries, but they are believed to be a priori
unpalatable. The palatability of 43 different herbal teas and the effect of the extraction time and a
sweetener (saccharose) on the taste perception of 31 or 25 herbal teas respectively, were evaluated
using the 5 point palatability scale. The palatability scores of the herbal teas varied to a great extent
(from 1.03 to 4.64) but as a general trend the unpalatable teas were considered as distasteful by the
majority of subjects and the scores given to the palatable teas were divergent. The prolongation of the
extraction time from 10 to 45 min had only a very limited effect toward the unpalatableness that effect
was even not found for all herbal teas. While the effect of the saccharose univocally improved the taste
but magnitude of this phenomenon was also moderate. In conclusion, the present study demonstrates
that the favorable palatability but not distastefulness of the herbal teas strongly depends on the taste
perception of the subject, and the time of extraction or addition of saccharose have only a minor effect
on the palatability score of herbal teas.
Key words: Medicinal plants, herbal teas, palatability, extraction time, saccharose, organoleptic analyze.
INTRODUCTION
Perception of taste is an evolutionary preserved
physiological mechanism to evaluate the food and drink
quality (Yarmolinsky, 2009). There are numerous
research works focused on the food taste and palatability,
particularly in the context of food processing/preservation
(van Boekel et al., 2010) or obesity issues (Yeomans,
2004). Contrary, far not so much attention has been paid
on the gustatory aspects or sensations associated with
the intake of nutritional supplies or remedies produced
from natural sources. Orally administered medications
derived from medicinal plants have been used for
centuries to treat various diseases and ailments. Despite
the fact that the modern pharmaceutical industry relies on
chemical synthesis, the utilization of natural medicines is
an increasing trend both in developed and source limited
countries. For convenient daily administration, herbs of
medicinal plants are frequently used in the form of teas,
tisanes, decocts, or infusions However, the herbal teas
are believed to be a priori unpalatable, while the
*Corresponding author. E-mail: ain.raal@ut.ee. Tel.: +372 737
5281. Fax : +372 737 5289.
sensations of astringency or bitterness may limit their use
(Boon and Smith, 2004). The neural pathways of
gustatory sensations are sufficiently determined (Jones et
al., 2006), nevertheless, the perception of palatability of
certain foods or drinks remains partially emotional.
The present open study aimed to characterize the
subjective sensory perception of palatability of various
herbal teas and to evaluate the effect of extraction time or
the use of the saccharose as a sweetener in the
preference of herbal teas.
MATERIALS AND METHODS
Plant material and preparation of herbal teas
All medicinal plants were collected by pharmacy students either
from natural habitats or from special medicinal plant fields of
Estonia. The plants were carefully cleaned on-spot and marked. In
laboratory conditions, the plants were identified again using the
taxonomic guide (Kukk and Kull, 2005), divided into organs and
dried in a dark room at room temperature (22 ± 2ºC) for ten days.
The dried herbs were labeled, packaged in a paper-bag, and stored
at ambient temperature (22 ± 2°C) in a dark and dry stora ge room
until tested. The voucher specimens of the tested plants are stored
at the Department of Pharmacy, University of Tartu, Estonia.

For the comparative purpose, all herbal teas were prepared in
the same manner as follows: one full tablespoon of a dried herbal
material was poured with 200 ml of boiling water, extracted for 10 or
45 min, and filtered before the taste evaluation. This method of
preparation of the herbal teas is recommended by numerous
popular books and Internet-resources and thus widely disseminated
in households. The palatability scoring scale was adopted and
rephrased for the herbal teas from the drinks analog scale
(Macqueen et al., 2003) where originally each of the drinks tested
was scored according to its taste and texture. The palatability of the
herbal teas was evaluated using the 1 to 5 points subjective score
scale according to the following criteria:
5 points: the tested herbal tea has an excellent taste;
4 points: the tested herbal tea “is not bad”;
3 points: the tested herbal tea is not palatable but has an
acceptable taste for a herbal tea as such:
2 points: the tested herbal tea needs taste correction;
1 point: distasteful, the tested herbal tea is unsuitable for oral
consumption.
The method recommended in the European Pharmacopoeia
(European Pharmacopoeia, 2010) for evaluation of bitterness value
was used: each herbal tea was taken into mouth and passed it from
side to side over the tongue for 30 s, then it spitted out and the
mouth was rinsed with water.
Estimation of palatability of herbal teas (experiment 1)
70 subjects (pharmacy students and graduates of both gender, 19
to 45 years) were involved in the open study, the experiment lasted
for several days. Each of the subjects tasted all of the 43 teas (full
list of herbs is given in the Table 1).
Effect of extraction time on palatability of herbal teas
(experiments 2 and 3)
In the experiment 2, 31 pharmacy students of both genders were
involved in the open study. The subjects evaluated the palatability
of herbal teas of 2 different extraction times (10 and 45 min),
respectively. The evaluation criteria were as in Experiment 1. This
experiment was repeated a year later with another group of
students (N=24; Experiment 3) while the list of herbs tested varied
slightly depending on the actual availability of plant material.
Effect of sweetening on palatability of herbal teas (experiment
4)
The herbal teas were prepared as given above; the extraction time
was 10 min. 1 teaspoon of sugar (saccharose, ca 5 g) per 200 ml
tea was added for taste correction. 32 pharmacy students tasted
the herbal teas before and after the sweetening and evaluated the
taste using the 5 point scale as given above.
RESULTS AND DISCUSSION
Our study present unveiled that the palatability of the
commonly used herbal teas varies quite much (Table 1).
As a general trend, the “distasteful” herbal teas were
considered unpalatable by all subjects while the favorable
palatability of the teas was subjective and tended to differ
among the experimenters to a great extent. Though not
Raal and Matto 2515
specifically studied, indeed the bitterness and astringency
are the major limiting factors for the consumption of the
herbal teas. The most unpalatable herbal tea of the
Experiment 1, the Bogbean leaves tea has a bitterness
index 1:500 and it contains also about 7% tannins
thereby being strongly astringent (Weiss and Fintelmann,
2001). Also, several other herbal teas tested are at least
to some extent astringent. While the bitterness is a
univocal criterion, the recent study by Childs and Drake
(2010) reported that the degree of astringency
considered by the subjects as desirable or undesirable
attribute of a beverage is a person-specific feature.
Notably, the Sage leaves and Wormwood herb, both with
a low palatability score in our study, contain high levels of
total polyphenols responsible for the astringency
(Büyükbalci and El, 2008). Further, the same researchers
concluded that the intensity of the astringency is not
necessarily directly related to dislike. It is also well-known
that the emotional and physical status of the subject
contributes to the liking of any taste of the foods and
beverages. This issue is widely studied in sport science,
there are reports showing the role physical and mental
exhaustion on perception and acceptance of fluid and
sport drinks and vice versa (Passe et al., 2009; Passe et
al., 2004). In our study, the experimenters were in
sedentary laboratory conditions that may have impacted
the decision of the palatability of herbal teas, too. In
addition, the environmental and contextual conditions are
known to bias the food or drink palatability (Roefs et al.,
2006; Sakai et al., 2001) but it is not described for the
herbal teas. Intuitively one might expect that the taste
acceptance level of the medicinal herbal teas gains in the
ailing condition of the subject. In future it is necessary to
provide GC-MS or/and HPLC-MS analysis of the herbal
teas investigated organoleptically determining the content
of their biologically active substances which should be
correlate with palatability points between 1 and 5. Also
spectrophotometrical determination of total content of
polyphenols, tannins or flavonoids in infusions could
show interesting results if compared with points given by
students.
The mechanisms of perception of astringency as a
general component that determines the palatability of
teas is well described (Nayak and Carpenter, 2008). The
prolongation of the extraction time of teas may augment
the astringency due to increased solution of polyphenols
but again, the psycho-emotional component plays also an
important role in the perception of palatability (Sakai et
al., 2001). This explains also the moderate discrepancy
of results of the Experiments 2 and 3 (Tables 2 and 3,
respectively). Thus, the amount of extraction of most of
the active compounds groups such as the polyphenols,
tannins, flavonols, coumarins, anthraquinones, saponins,
etc. depends on the duration of the extraction time. The
extraction of those constituents during the extraction
process of 10 minutes is incomplete (Raal and
Kuznetsova, 2007) and generally the extraction rate is

2516 J. Med. Plants Res.
Table 1. Palatability score of herbal teas.
Herb /plant material Average score* Score range
Wild rose fruits (Rosae pseudo-fructus) 4.64 2-5
Peppermint leaves (Menthae piperitae folium) 4.63 3-5
Lime flowers (Tiliae flos) 4.63 3-5
Hawthorn fruits (Crataegi fructus) 4.50 2-5
Primula flowers (Primulae flos) 4.18 2-5
Mullein flowers (Verbasci flos) 4.10 2-5
Catmint leaves (Nepetae catariae folium) 4.07 2-5
Spearmint leaves (Menthae spicatae folium) 3.99 2-5
Raspberry leaves (Rubi idaei folium) 3.99 2-5
Juniper fruits (Juniperi fructus) 3.97 2-5
Creeping thyme herb (Serpylli herba) 3.93 3-5
Lemon balm leaves (Melissae folium) 3.85 2-5
Caraway fruits (Carvi fructus) 3.69 1-5
Liquorice roots (Glycyrrhizae radix) 3.66 1-5
St. John’s wort herb (Hyperici herba) 3.64 1-5
Common plantain leaves (Plantaginis majoris folium) 3.56 2-5
Pineapple weed flowers (Matricariae suaveolentis flos) 3.53 1-5
Cornflower flowers (Cyani flos) 3.51 2-5
Nettle leaves (Urticae folium) 3.50 2-5
German chamomile flowers (Chamomillae flos) 3.50 1-5
Hyssop leaves (Hyssopi folium) 3.47 1-5
Cottonweed herb (Gnaphalii uliginosi herba) 3.47 2-5
Lady’s mantle leaves (Alchemillae folium) 3.44 2-5
Common knotweed herb (Polygoni avicularis herba) 3.36 1-4
Birch leaves (Betulae folium) 3.33 1-5
Primula leaves (Primulae folium) 3.31 1-5
Flax seeds (Lini semen) 3.30 1-5
Pot marigold flowers (Calendulae flos) 3.30 1-5
Coltsfoot leaves (Farfarae folium) 3.18 1-5
Oak bark (Quercus cortex) 2.71 1-4
Hops (Lupuli strobilus) 2.61 2-5
Valerian root (Valerianae radix) 2.60 1-4
Rosemary herb (Ledi herba) 2.60 1-5
Yarrow herb (Millefolii herba) 2.54 1-5
Sage leaves (Salviae officinalis folium) 2.52 1-5
Cowberry herb (Vitis-idaeae herb) 2.36 1-4
Common motherwort herb (Leonuri herba) 2.34 1-4
Tormentil rhizome (Tormentillae rhizoma) 1.97 1-4
Bearberry leaves (Uvae ursi folium) 1.78 1-4
Frangula bark (Frangulae cortex) 1.67 1-3
Iceland moss (Lichen islandicus) 1.48 1-4
Wormwood herb (Absinthii herba) 1.19 1-2
Bogbean leaves (Menyanthidis folium) 1.03 1-2
70 subjects tasted all herbal teas. *5-point taste scale (1=distasteful, 5=excellent taste).
increased if the extraction time is extended.
Nevertheless, this phenomenon is medicinal plant
specific and for an example, in our another study (Raal et
al., 2012) it was found that prolongation of the extraction
time of chamomile tea from 5 min to 12 h in a thermos
bottle did not remarkably increased the content of the
total polyphenols and flavonoids. Usually the German
chamomile inflorescences cultivated in Estonia are rich of

Table 2. Effect of extraction time on palatability score of herbal teas (experiment 2).
Herb /plant material
Average score*
Raal and Matto 2517
10 min extraction 45 min extraction Score reduction/improvement
Blackcurrant fruits (Ribis nigri fructus) 4.83 4.94 +0.11
Oregano herb (Origani herba) 4.43 4.00 -0.43
Lemon balm leaves (Melissae folium) 4.41 4.06 -0.35
Creeping thyme herb (Serpylli herba) 4.36 4.21 -0.15
Cowberry leaves (Vitis-idaeae folium) 4.14 4.21 +0.07
Sage leaves (Salviae officinalis folium) 4.14 3.79 -0.33
Peppermint leaves (Menthae piperitae folium) 4.06 3.78 -0.28
German chamomile flowers (Chamomillae flos) 3.94 3.22 -0.72
Yarrow herb (Millefolii herba) 3.93 3.64 -0.29
Hawthorn fruits (Crataegi fructus) 3.83 3.56 -0.27
Birch leaves (Betulae folium) 3.79 3.29 -0.50
Juniper fruits (Juniperi fructus) 3.71 3.57 -0.14
Hops (Lupuli strobilus) 3.64 3.57 -0.07
Primula flowers (Primulae flos) 3.64 3.23 -0.41
Field horsetail herb (Equiseti arvensis herba) 3.44 3.22 -0.22
Caraway fruits (Carvi fructus) 3.39 3.33 -0.06
Coriander fruits (Coriandri fructus) 3.36 3.07 -0.29
Coltsfoot leaves (Farfarae folium) 3.22 3.11 -0.11
Aniseed fruits (Anisi fructus) 3.21 3.00 -0.21
St. John’s wort herb (Hyperici herba) 3.17 2.28 -0.89
Bearberry leaves (Uvae ursi folium) 3.06 2.78 -0.28
Valerian root (Valerianae radix) 3.06 3.00 -0.06
Flax seeds (Lini semen) 2.94 2.78 -0.16
Pot marigold flowers (Calendulae flos) 2.94 2.65 -0.29
Common thyme herb (Thymi herba) 2.79 2.71 -0.08
Rosemary herb (Ledi herba) 2.36 1.86 -0.50
Frangula bark (Frangulae cortex) 2.29 2.47 +0.18
Common motherwort herb (Leonuri herba) 1.67 1.28 -0.39
Tormentil rhizome (Tormentillae rhizome) 1.61 1.17 -0.43
Bistort rhizome (Bistortae rhizome) 1.39 1.11 -0.28
Iceland moss (Lichen islandicus) 1.38 1.07 -0.31
31 subjects tasted all herbal teas. *5-point taste scale (1=distasteful, 5=excellent taste).
α-bisabolol or bisabolol oxides A and B (Orav et al.,
2010).
The present study demonstrates that the extraction
time is not the crucial determinant for the taste rating of
the herbal teas (Tables 2 and 3), at least as far as the
experimenters were aware that these teas are intended
to be used as remedies. The general trend was that the
prolongation of the extraction time decreased the score of
palatability, but the effect was not great and the
prolongations of the extraction time of the Blackcurrant
and Coriander fruits, Cowberry leaves, Flax seeds, or
Frangula bark teas even somewhat improved the taste.
One has to emphasize that on the 5-point palatability
scale the usual change of the palatability score was only
5 to 20%.
The taste of bitterness is another sensational property
widely associated with teas of medicinal herbs. Though
again at least somewhat contextual and society specific
(6), particularly the bitterness is associated with the
putative efficacy of the medicinal herb derived remedies.
In this context it is surprising that the considerable
sweetening of the herbal teas had also only a minor taste
improving effect (Table 4). Notably only the Blackcurrant
fruits tea achieved the maximum 5 points score, however
the unsweetened entry score of the Blackcurrant fruits
tea in this experiment was also high (4.63). Thus, the
sweetening of the medicinal herb teas is a tool to improve
the tea taste, however, the effect of the addition of
saccharose is only moderate and the unpalatable herbal
teas remain still unpalatable. It is interesting to mention
that all samples known as essential oil drugs (Table 1)
had exclusively the maximum 5 point score (Table 1), but
the minimum score of their teas was sometimes even
only 1 (Caraway fruits, German chamomile flowers,

2518 J. Med. Plants Res.
Table 3. Effect of extraction time on palatability score of herbal teas (experiment 3).
Herb /plant material
Average score*
10 min extraction 45 min extraction Score reduction/ improvement
Flax seeds (Lini semen) 4.56 4.79 +0.23
Bearberry leaves (Uvae ursi folium) 4.16 3.82 -0.34
Primula flowers (Primulae flos) 4.15 3.95 -0.20
German chamomile flowers (Chamomillae flos) 3.98 3.35 -0.63
Blackcurrant fruits (Ribis nigri fructus) 3.89 3.89 0
Juniper fruits (Juniperi fructus) 3.86 3.75 -0.11
Oregano herb (Origani herba) 3.86 3.28 -0.58
St. John’s wort herb (Hyperici herba) 3.82 3.66 -0.16
Creeping thyme herb (Serpylli herba) 3.58 3.28 -0.30
Oak bark (Quercus cortex) 3.42 3.16 -0.26
Caraway fruits (Carvi fructus) 3.26 3.08 -0.18
Sage leaves (Salviae officinalis folium) 3.22 2.86 -0.36
Yarrow herb (Millefolii herba) 3.20 2.83 -0.37
Common thyme herb (Thymi herba) 3.11 3.06 -0.05
Lemon balm leaves (Melissae folium) 3.05 2.51 -0.54
Pot marigold flowers (Calendulae flos) 3.05 2.56 -0.49
Hops (Lupuli strobilus) 3.03 2.89 -0.14
Cowberry leaves (Vitis-idaeae folium) 2.97 2.72 -0.25
Coriander fruits (Coriandri fructus) 2.82 2.85 +0.03
Birch leaves (Betulae folium) 2.78 2.53 -0.25
Rosemary herb (Ledi herba) 2.08 1.83 -0.25
Common motherwort herb (Leonuri herba) 1.55 1.24 -0.31
Iceland moss (Lichen islandicus) 1.33 1.19 -0.14
Wormwood herb (Absinthii herba) 1.11 1.03 -0.08
24 subjects tasted all herbal teas. *5-point taste scale (1=distasteful, 5=excellent taste).
Table 4. Effect of sweetening on palatability score of herbal teas.
Herb /plant material
Average score*
Before sweetening After sweetening Score improvement
Blackcurrant fruits (Ribis nigri fructus) 4.63 5.00 +0.37
Peppermint leaves (Menthae piperitae folium) 4.54 4.80 +0.26
Primula flowers (Primulae flos) 4.07 4.53 +0.46
Lemon balm leaves (Melissae folium) 3.94 4.38 +0.44
Liquorice roots (Glycyrrhizae radix) 3.88 3.88 ±0
Pot marigold flowers (Calendulae flos) 3.88 4.06 +0.18
German chamomile flowers (Chamomillae flos) 3.80 4.07 +0.27
Coltsfoot leaves (Farfarae folium) 3.69 4.06 +0.37
Flax seeds (Lini semen) 3.62 4.00 +0.38
St. John’s wort herb (Hyperici herba) 3.60 3.93 +0.33
Aniseed fruits (Anisi fructus) 3.44 3.88 +0.44
Hyssop leaves (Hyssopi folium) 3.44 3.69 +0.25
Field horsetail herb (Equiseti arvensis herba) 3.44 3.69 +0.25
Hops (Lupuli strobilus) 3.38 4.00 +0.62
Caraway fruits (Carvi fructus) 3.38 3.69 +0.31
Juniper fruits (Juniperi fructus) 3.25 3.69 +0.44
Creeping thyme herb (Serpylli herba) 3.20 3.56 +0.36
Sage leaves (Salviae officinalis folium) 3.20 3.60 +0.40

Table 4. Contd.
Raal and Matto 2519
Hawthorn fruits (Crataegi fructus) 3.20 3.53 +0.33
Rosemary herb (Ledi herba) 3.06 3.50 +0.44
Cowberry leaves (Vitis-idaeae folium) 2.88 3.56 +0.68
Oregano herb (Origani herba) 2.88 3.40 +0.52
Coriander fruits (Coriandri fructus) 2.73 3.07 +0.34
Common thyme herb (Thymi herba) 2.44 2.88 +0.44
Valerian root (Valerianae radix) 2.31 2.88 +0.57
Birch leaves (Betulae folium) 2.13 2.44 +0.31
Oak bark (Quercus cortex) 2.13 2.75 +0.62
Yarrow herb (Millefolii herba) 1.94 2.31 +0.37
Tormentil rhizome (Tormentillae rhizoma) 1.44 2.25 +0.81
Bistort rhizome (Bistortae rhizoma) 1.43 1.69 +0.26
Sweet Flag rhizome (Calami rhizoma) 1.31 1.69 +0.38
Common motherwort herb (Leonuri herba) 1.19 1.50 +0.31
32 subjects tasted all herbal teas. *5-point taste scale (1=distasteful, 5=excellent taste).
Rosemary herb, etc.).
Prolongation of the extraction time of essential oil
bearing or not containing plant material gives principally
similar score reduction (Tables 2 and 3). Also sweetening
of essential oil containing herbal teas did not improve
their palatability remarkably more than in other herbal
teas (Table 4). Analyzing effect of extraction time on
palatability of herbal teas rich of tannins (such as Oak
bark, Tormentil rhizome, Bistort rhizome, etc.) we did not
saw so clear negative trend than, for example, in herbal
teas made from St. John’s wort herb or German
chamomile flowers, and several other drugs containing
different constituents groups. So, the negative effect of
tannins to palatability of herbal infusions seems to be
overestimated.
In conclusion, the present study demonstrates that the
distaste of the herbal teas is univocal. Contrary, the
favorable palatability of herbal teas strongly depends on
the taste perception of the particular subject while the
time of extraction or addition of saccharose as a
sweetener have only a minor effect on the palatability
score.
ACKNOWLEDGEMENT
The authors would like to thank all pharmacy students for
collection of plant materials and for their support
rendered in the laboratory.
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Journal of Medicinal Plants Research Vol. 6(12), pp. 2520-2525, 30 March, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.513
ISSN 1996-0875 ©2012 Academic Journals
Full Length Research Paper
Isolation and purification of terpenoids from Celastrus
aculeatus Merr. by high-speed counter-current
chromatography
Yang Xie 1 , Zongbao Ding 2 , Wenjun Duan 2 and Qingsheng Ye 1 *
1 School of Life Sciences, South China Normal University, Guangzhou 510631, People’s Republic of China.
2 School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, People’s Republic of China.
Accepted 9 May, 2011
Following an initial clean-up step on a silica gel column, preparative high-speed counter-current
chromatography (HSCCC) method was successfully established by using n-hexane–ethyl acetate–
methanol–water (2.3:2:2:1.3, v/v) as the two phases solvent system to isolate and purify terpenoids from
the stem and root bark of Celastrus aculeatus Merr. The isolation was done in less than 260 min and 2.5
mg of nimbidiol (I) and 3 mg of pristimerin (I I) were yielded from 250 mg of the crude extract with the
purity of 95.0 and 97.1%, respectively, as determined by high-performance liquid chromatography
(HPLC). Their structures were identified by using spectroscopic methods including ultraviolet (UV),
electron ionization mass spectrometry (EI-MS), hydrogen nuclear magnetic resonance 1 (HNMR) and
13 CNMR. Nimbidiol and pristimerin were isolated from C. aculeatus Merr for the first time.
Key words: High-speed counter-current chromatography, terpeniods, nimbidiol, pristimerin, Celastrus
aculeatus Merr.
INTRODUCTION
Celastrus aculeatus Merr.(Celastrus) (Guo et al., 2004;
Kim et al., 1998; Jin et al., 2002; Westerheide et al.,
2004) is a Chinese medicine that belongs to the family
Celastraceae and the genus Celastrus. For centuries,
roots, stems and leaves of C. aculeatus have been used
to treat rheumatoid arthritis, osteoarthritis, lower back
pain and so on. However, up to now, few phytochemical
studies on this plant have been described in the
literature. In order to find out the bioactive components,
we have studied the chemical components of C.
aculeatus Merr. Some of the bioactive components are
difficult to separate because of their low contents.
Although preparative HPLC or silica gel column makes
the isolation and purification much easier than before, the
considerable loss of herbal material requires more
efficient methods to provide bioactive components.
*Corresponding author. E-mail: Ye-lab@scnu.edu.cn. Tel:
862085212021. Fax: 862085212021.
Compared to conventional liquid-solid methods, HSCCC
without a solid phase has more advantages. A supportfree
liquid-liquid partition chromatographic technique
eliminates such adsorption problems (Ito, 1981) has been
widely used in preparative separation of natural products
(Yang et al., 2009). In the present study, the HSCCC
method was developed for the separation and purification
of nimbidiol and pristimerin from C. aculeatus Merr. Their
structures were elucidated with EI-MS, 1 HNMR and
13 CNMR. As far as we know, the two compounds were
obtained from C. aculeatus Merr for the first time.
METHODOLOGY
Materials
The roots and stems of C. aculeatus Merr.(were collected from
Guangdong province of China). TBE-300 A high speed countercurrent
chromatograph (Shanghai Tauto Biotech Company). HX-
1050 constant-temperature circulation implements (Beijing
Byoikang Lab. Instrument Company). Waters symmetry C18 column

Table 1. The partition coefficients (K) of the target compounds in different solvent systems with different ratios.
Hexane–ethylacetate–methanol–water
( v/v ) K (Ⅰ) K (Ⅱ) K (Ⅲ)
0.4:2:0.4:2 0.63 0.12 0.82
1:2:1:2 6.97 4.65 2.33
2:2:2:2 7.58 12.64 2.89
2.3:2:2:1.3 1.32 0.45 1.56
(Waters Corp). Nuclear magnetic resonance spectrometer (600 M,
Bruker Company). Fourier transforms infrared spectrometer (Nicolet
Company). HPLC (Agilent Company). Milli-Q Academic ultra-pure
water system (Millipore Company).
EXPERIMENTAL METHODS
Preparation of crude sample
The roots and stems of C. aculeatus Merr. were collected in the
Guangdong province of China and identified by Dr. Ye Hua-gu, a
plant taxonomist at South China Institute of Botany. A voucher
specimen number 10943 was deposited there. Six kilograms airdried
roots and stems were minced with a grinder and then
extracted three times with 95% ethanol at the temperature of 70
Celsius degrees every 3 h. After evaporating of the solvent under
reduced pressure, the ethanol extract was diluted in H2O and then
extracted with petroleum ether. The petroleum ether layer (65 g)
was chromatographed on a silica gel column using petroleum–ethyl
acetate (10:1; 5:1; 1:1; 1:5) as eluent to give four fractions. Fraction
4 (12 g) was used for the subsequent HSCCC isolation and
purification.
Measurement of partition coefficient (K)
The composition of the two phase’s solvent system was selected
according to the partition coefficient (K) and peak resolution of the
target compounds. The measurement of K values was performed
as following procedures according to the literature (Ito, 2005): 3 mg
crude sample was weighed into a test tube to which 1mL of each
phase of the pre-equilibrated two phases solvent system was
added. The test tube was then shaken vigorously for 3 min to
thoroughly equilibrate the sample between the two-phase. An
aliquot of each phase (500 μL) was delivered into a test tube
separately and evaporated to dryness. The residues were diluted
with methanol to1mL and analyzed by HPLC. The K value was
defined as the peak area of target compound in the upper phase
(stationary phase) divided by the peak area of compound in the
lower phase (mobile phase).
Preparation of two phase’s solvent system
In the present study, the two phases solvent system composed of
hexane/ethyl acetate/methanol/water (2.3:2:2:1.3, v/v) was used for
HSCCC separation. The solvent mixture was shaked thoroughly at
room temperature in a separatory funnel and placing overnight
quietly. Before using, the separated two phases were degassed for
30 min, respectively.
HSCCC separation procedure
In the separation process the HSCCC column was first entirely filled
Xie et al. 2521
with the upper organic phase. Then the aqueous mobile phase was
pumped through the column at a flow rate of 1.5 mL/min in the head
to tail direction (reversed mode). After the system reached
hydrodynamic equilibrium, the crude sample dissolved in upper
phase was injected through the sample port. The column was
rotated at 900 rpm. The effluent from the outlet of the column was
monitored continuously with the UV detector at 254 nm and
collected in test tube per 5 min. Each peak fraction was collected
and evaporated under reduced pressure. The residues were
dissolved in methanol for subsequent HPLC analysis. After
separation, retention of the stationary phase was measured by
collecting the column contents into a graduated cylinder by forcing
them out of the column with pressurized nitrogen gas.
HPLC analysis and identification of HSCCC peak fractions
The crude extract and fractions separated by HSCCC were
analyzed by HPLC. HPLC was performed using a waters system
with a waters symmetry C18 column (150×4.6 mm and 5.0 µm). The
wavelength for detection was set at 254 nm according to the UV
spectrums of all the fractions. Flow rate was 0.8 mL/min.
Compound(I) was run in 30 min with methanol from 70 to 100%
(v/v) and water 30% to 0 (v/v) in linear change. Compound (I I) was
run in 30 min with methanol: 0.1%phosphoric acid (88:12) as
mobile phase, and the column temperature was maintained at
30°C. The purities of the collected fractions were determined by
HPLC. Identification of the HSCCC peak fractions was performed
by 1 H NMR and 13 C NMR.
RESULTS AND DISCUSSION
Selection of two phases solvent system and other
conditions of HSCCC
The search for the suitable solvent system which gives a
proper range of K values (partition coefficient) for the
target compound is the crucial first step for successful
HSCCC separation (Ito, 2003). The ideal K values of
target compounds should be from 0.5 to 2.0. Generally,
small K values result in poor peak resolution, while large
K values tend to cause excessive band broadening. For
this crude sample, the most commonly used Hexane–
ethylacetate–methanol–water (HEMW) system at various
volume ratios (0.4:2:0.4:2; 1:2:1:2; 2:2:2:2; 2.3:2:2:1.3,
v/v) were further tested and their K values were
measured. The K values of compounds in different ratios
of HEMW determined by HPLC were listed in Table 1. As
shown in Table 1, when HEMW (0.4:2:0.4:2, v/v) was

2522 J. Med. Plants Res.
Figure 1. The HPLC chromatogram of compound I. Column: Waters symmetry C18 column (150×4.6 mm); column temperature: 25°C: mobile phase: methanol:
water from 70:30 to100:0, flow rate: 0.8 mL /min; detection wave length: 254 nm.
selected as a solvent system, too small K values
would result in poor peak resolution. It can be also
seen that the K values of the HEMW (1:2:1:2, v/v)
and HEMW (2:2:2:2, v/v) were too large, which
lead to a long separation time and broad peaks.
HEMW (2.3:2:2:1.3, v/v) was finally chosen for
HSCCC separation as it gave a reasonable range
of K values and a better resolution of target
compounds. And it was used to isolate and purify
two compounds from C. aculeatus Merr shown in
Figure 3.
In addition, other factors such as the revolution
speed of the separation column and the flow rate
of the mobile phase were also investigated. The
result indicated that a low flow rate could produce
a good separation, but long elution time was
required and peaks became broader. Experiments
result showed that when the flow rate was set at
2.0 ml min -1 , revolution speed was 900 rpm,
retention percentage of the stationary phase could
reach 67% and good separation results could be
achieved. Two compounds (I and II) were
obtained in one-step elution less than 260 min
(HSCCC chromatogram is shown in Figure 3) with
the solvent system composed of n-hexane–
ethylacetate–methanol-water (2.3:2:2:1.3, v/v). An
amount of 2.5 mg of compound I and 13.0 mg of
compound II were separated from 250 mg of the
crude extract.
HPLC analysis and structure identification of
HSCCC peak fractions
As shown in Figure 1 and 2, the HPLC
chromatogram of each HSCCC peak fraction
revealed that two compounds were purified from
the crude extract. The purities of compound I and
compound II were 95.0 and 97.1%, respectively.
The structure identification of the HSCCC peak
fractions was based on EI-MS, 1 H NMR and 13 C
NMR. Data of compound I:
Molecular formula C17H22O3. mp 171 to 175°C.
HR-MS (EI) m/z: 274.1524 m/z (rel. int.%): 274
(M + , 99), 259 (100), 217 (32), 203 (24), 191 (78),
189 (94), 177(57), 163(23), 69 (51), 57(30),
55(24); 1 H-NMR (CDCl3, 600 MHz) δ:7.62 (1H,
br.H-14), 6.83 (1H, br.H-11), 2.54-2.66 (2H,dd,H-
6), 2.15(1H, d,J = 12.6Hz,H-5), 2.15(1H, m),
1.71(H-a,d,J = 13.8Hz, H-1) ,1.64(H-b,m,H-1),
1.48(H-a,d,J = 13.2Hz,H-2), 1.78(H-b,dd,J =
13.2Hz, 4.2 Hz, H-2), 1.22 (H-a,m,H-3), 1.43 (Hb,m,H-3
),1.14(3H, s, H-17), 0.94(3H, s, H-16),
0.88(3H, s, H-15)。 13 C-NMR (CDCl3, 150 MHz) δ:
200.6 (C-7), 152.4 (C-13), 151.4 (C-12), 142.2(C-
9), 123.7 (C-8), 113.6 (C-14), 110.1 (C-11), 49.8

(C-5), 41.3(C-3), 37.9 (C-1), 37.8 (C-10), 35.9 (C-
6),33.2 (C-4), 32.5 (C-2), 23.2 (C-18), 21.3 (C-19),
18.9 (C-20)。IR (KBr, cm -1 ): 3428, 2990,2915,
2820, 1645, 1590, 1505, 1430, 1355, 1323, 1278,
1188, 875. This spectral data of 1 H NMR and 13 C
NMR are in agreement with that of nimbidiol in the
literature (Majumder et al., 1987; Masahiro et al.,
2010).
Data of compound I molecular formula
C30H40O4. mp 217 to 219°C. EI- MS m/z
487[M+Na] + , 465[M+H] + 。IR (KBrυmax / cm -1 ):
3350, 2940, 1723, 1650, 1590, 1520, 1435,
1375,1300,1245,1220, 1205, 1185, 155, 1145,
1095, 1085, 990, 870, 860, 850。 1 H-NMR
(CDCl3): δ 7.03 (1H, d, J=9.2Hz, H-6), 6.98 (1H, s,
Figure 2. The HPLC chromatogram of compound II. Column: Waters symmetry C18 column
(150×4.6 mm); column temperature: 25°C; mobile phase: methanol: 0.1%phosphoric acid (88:12),
flow rate: 0.8 ml/ min; detection wavelength: 254 nm.
H-1), 6.36 (1H, d, J=7.2Hz, H-7), 6.53 (1H, brs,
disappearing after the exchange of D2O, -OH),
3.55 (3H, s, H-31), 2.22 (3H, s, H-23), 1.45 (3H, s,
H-25),1.26 (3H, s, H-26), 1.18 (3H, s, H-30), 1.08
(3H, s, H-28), 0.52 (3H, s, H-27)。 13 C-NMR:δ
119.8 (C-1), 178.6 (C-2), 146.2 (C-3), 117.3 (C-4),
127.6 (C-5), 133. 2(C-6),118.3 (C-7) 160.3 (C-8),
43.2 (C-9) 164.9 (C-10) 33.8 C-11) 29.9 (C-12)
39.6 (C-13), 45.3 (C-14), 28.9 (C-15), 36.6 (C-16),
30.8 (C-17), 44.5(C-18), 31.1 (C-19),40.6 (C-
20), 30.1 (C-21), 35.0 (C-22), 10.5 (C-23), 38.5
(C-25), 21. 8(C-26), 18.5 (C-27), 31.8(C-28),
178.9 (C-29), 32.9 (C-30), 51.8 (C-31). This
spectral data of 1 H NMR and 13 C NMR are in
agreement with that of pristimerin in the literature
(Jiang et al., 1996).
CONCLUSION
Xie et al. 2523
HSCCC was successfully used for the preparative
isolation of terpenoids. In combination with a
suitable extraction and cleanup procedure prior to
HSCCC separation, 2.5 mg nimbidiol and 3.0 mg
pristimerin were obtained from 250 mg of the
crude sample on a preparative scale. Meanwhile,
the isolation of nimbidiol and pristimerin for the
first time from C. aculeatus Merr proved HSCCC
to be a useful tool for the exploring of the
chemical component of traditional medicinal
herbs.

2524 J. Med. Plants Res.
ACKNOWLEDGEMENTS
Figure 3. HSCCC chromatogram of crude extract from C. aculeatus Merr. Two-phase solvent system, n-hexane-ethyl acetate-methanol-water
(2.3:2:2:1.3 v/v); mobile phase, the lower phase; flow rate, 2.0 mL/min; revolution speed, 900rpm; detection wavelength, 254 nm; separation temperature,
20°C; sample size, 250 mg crude sample dissolved in 10 mL of the upper phase and 10 ml of the lower phase. Retention of the stationary phase: 67%
This project was supported by the National
Centerfor Complementary and Alternative
Medicine of NIH (No. FO5AT002013-03) and was
partly supported by Fund for Social Development
Plan Projects of Technology Department of Guang
dong province, No. 2006B35604001, Guangzhou
Technology Projects, No.2007JC0081. The
authors would like to thank South China
Agricultural University for the measurement of
infrared (IR), NMR spectra and EI-MS.
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