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African Journal of
Pharmacy and
Pharmacology
Volume 5 Number 8 August 2011
ISSN 1996-081

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Himanshu Gupta
University of Colorado- Anschutz Medical Campus,
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Molecular Cardiovascular Research Program
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Arizona Health Sciences Center
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Universidade Federal do Pampa
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Prof. Fen Jicai
School of life science, Xinjiang University,
China.
Dr. Ana Laura Nicoletti Carvalho
Av. Dr. Arnaldo, 455, São Paulo, SP.
Brazil.
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Professor of Medicine
Director of Renal Division, Department of Medicine
Peking University First Hospital
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Guangdong Cardiovascular Institute, Guangdong General
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China.
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Faculty of Engineering and Applied Science,
Memorial University of Newfoundland,
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Department of Gynaecology and Obstetrics, the 1st
hospital, NanFang University,
China.
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School of Medicine, Lanzhou University,
China.
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School of Medicine, Taizhou university,
China.
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Research Officer
Laboratory of Molecular Biomedicine,
Institute of Bioscience, Universiti Putra,
Malaysia.
Dr. Fen Cun
Professor, Department of Pharmacology, Xinjiang
University,
China.
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Department of Chemical Engineering, Annamalai
University,
Annamalai Nagar, Tamilnadu,
India.
Prof. Ehab S. EL Desoky
Professor of pharmacology, Faculty of Medicine
Assiut University, Assiut,
Egypt.
Dr. Yakisich, J. Sebastian
Assistant Professor, Department of Clinical Neuroscience
R54
Karolinska University Hospital, Huddinge
141 86 Stockholm ,
Sweden.
Prof. Dr. Andrei N. Tchernitchin
Head, Laboratory of Experimental Endocrinology and
Environmental Pathology LEEPA
University of Chile Medical School,
Chile.
Dr. Sirajunnisa Razack
Department of Chemical Engineering,
Annamalai University, Annamalai Nagar, Tamilnadu,
India.
Dr. Yasar Tatar
Marmara Unıversıty,
Turkey.
Dr Nafisa Hassan Ali
Assistant Professor, Dow institude of medical technology
Dow University of Health Sciences,Chand bbi Road, Karachi,
Pakistan.
Dr. Krishnan Namboori P. K.
Computational Chemistry Group, Computational
Engineering and Networking,
Amrita Vishwa Vidyapeetham, Amritanagar, Coimbatore-
641 112
India.
Prof. Osman Ghani
University of Sargodha,
Pakistan.
Dr. Liu Xiaoji
School of Medicine, Shihezi University,
China.

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Ansell JE, Buttaro ML, Thomas VO (1997). Consensus
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African Journal of Pharmacy and Pharmacology
International Journal of Medicine and Medical Sciences
Table of Contents: Volume 5 Number 8 August, 2011
ences
ARTICLES
Research Articles
Evaluation of the effects of ciprofloxcin or gatifloxacin on
neurotransmitters levels in rat cortex and hippocampus 993
Sayed M. Rawi, Nadia M. S. Arafa and Mansour M. El-Hazmi
Evaluation of estrogen-like activity of Nigella sativa in ovariectomized
rats 1006
Saadat Parhizkar, Latiffah Abdul Latiff, Sabariah Abdul Rahman and
Mohammad Aziz Dollah
A meta-analysis of the efficacy and safety of candesartan in Chinese
patients with mild to moderate essential hypertension 1012
Cui Wen-peng, Du Bing and Qin Ling
Reducing queues in a Nigerian hospital pharmacy 1020
Ndukwe H. C., Omale S. and Opanuga O. O.
Effect of roasting, boiling and microwaving cooking method on
Doxycline residues in edible tissues of poultry by microbial method 1034
Javadi A.
Anti atherosclerotic effects of verjuice on hypocholesterolemic rabbits 1038
Mahbubeh Setorki, Bahar Nazari, Sedighe Asgary, Leila Azadbakht and
Mahmoud Rafieian-Kopaei
Purified alkaloid extract of Scutellaria barbata inhibits proliferation of
hepatoma HepG-2 cells by inducing apoptosis and cell cycle arrest at
G2/M phase 1046
Tie-shan Wang, Li-jing Chen, Zhao-yu Wang, Sheng-tan Zhang and
Jing-ming Lin

Table of Contents: Volume 5 Number 8 August, 2011
ences
ARTICLES
Development of novel herbal cosmetic cream with Curcuma longa
extract loaded transfersomes for antiwrinkle effect 1054
Swarnlata Saraf, Gunjan Jeswani, Chanchal Deep Kaur and
Shailendra Saraf
Preparation of ambroxol hydrochloride carboxymethyl chitosan
microspheres without burst release 1063
Hongfei Liu, Yan He, Zhaoying and Peng Ke
Effect of absorption enhancers on nasal delivery of basic fibroblast
growth factor 1070
Feng Chengcheng, Shao Xiayan, Zhang Chi, Liu Qingfeng, Chen Jie,
Shen Yehong, Zhang Qizhi and Jiang Xinguo
Modulation of monoamines and amino-acids neurotransmitters in
cerebral cortex and hippocampus of female senile rats by ginger and
lipoic acid 1080
Hoda G. Hegazy and Elham H. A. Ali
Induction of apoptosis by Rhizoma Paridis saponins in MCF-7 human
breast cancer cells 1086
Chuan Lu, ChunJiang Li, Dongmei Wu, JingMei Lu, Fan Tu and Lijuan Wang
In-vivo study of stratum corneum water content and transepideramal
water loss using a newly formulated topical cream of Hippophae
rhamnoides fruit extract 1092
Barkat Ali Khan, Naveed Akhtar, Tariq Mahmood, Haji M.Shoaib Khan,
Shahiq-Uz- Zaman, Akhtar Rasul, Muhammad Iqbal, Atif Ali,
Salimullah Khan and Mughal Qayum

Table of Contents: Volume 5 Number 8 August, 2011
ences
ARTICLES
Ameliorative effects of ginger and α-lipoic acid on oxidative stress and
inflammation in senile female rats 1096
Hoda G. Hegazy
Modeling a murine model of immunoglobulin-E (IgE)-mediated
qingkailing injection anaphylaxis 1106
Guoping Liao, Wenshi Li, Shuai He and Zhongyi Zhang
Comparison of the effect of acupressure, fish oil capsules and
ibuprofen on treatment of primary dysmenorrheal 1115
Zafari, M., Tofighi, M., Aghamohammady, A., Behmanesh, F. and
Rakhshaee
The involvement of opioidergic mechanisms in the activity of Bacopa
monnieriextract and its toxicological studies 1120
Abbas M, Subhan F, Mohani N, Rauf K, Ali G and Khan M
Synergy of daptomycin with fusidin against invasive systemic infection
and septic arthritis induced by type VI group B streptococci in mice 1125
Adel Galal El-Shemi and Hani S. Faidah
Anti-nociceptive, anxiolytic and anticonvulsant effects of an aqueous
leaf extract of Leea guineensis G. Don (Family: Leeaceae) 1132
Eric Woode, David Abasiwani Alagpulinsa and Wonder Kofi Mensah Abotsi
HI-6 and obidoxime implication in oxidative stress, antioxidants level
and apoptosis 1145
Miroslav Pohanka, Ladislav Novotny, Josef Fusek and Jiri Pikula

Table of Contents: Volume 5 Number 8 August, 2011
ences
ARTICLES
Triterpenoids from Vernonia auriculifera Hiern exhibit antimicrobial
activity 1150
Joyce Jepkorir Kiplimo, Neil Anthony Koorbanally and Hafizah Chenia
Nutritional and elemental analyses of some selected fodder species
used in traditional medicine 1157
Ali Bahadur, Zubeda Chaudhry, Gul Jan, Mohammad Danish, Atta ur
Rehman, Rafiq Ahmad, Aman khan, Shah Khalid, Irfan ullah, Zahir Shah,
Farman Ali, Tahira Mushtaq and Farzana Gul Jan
Acute toxicity and anti inflammatory effects of supercritical extracts of
Ilex paraguariensis 1162
Tiago R. Pasquali, Sandra M. D. Macedo, Silvane S. Roman, Valéria Dal Prá,
Rogério L. Cansian, Altemir J. Mossi, Vladimir J. Oliveira and
Marcio A. Mazutti
Evaluation of the stem bark of Pistacia integerrima Stew ex Brandis for
its antimicrobial and phytotoxic activities 1170
Shafiq ur Rahman, Muhammad Ismail, Naveed Muhammad, Farhat Ali,
kamran Ahmad Chishti and Muhammad Imran
Antioxidant and antibacterial activity of crude methanolic extract of
Euphorbia prostrata collected from District Bannu (Pakistan) 1175
Mushtaq Ahmad, Abdus Saboor Shah, Rahmat Ali Khan, Farid Ullaha Khan,
Noor Aslam Khan, Mir Sadiq Shah and Muhammad Rashid Khan
Development of new spectrophotometric determination of titanium in
homeopathic pharmacy using Ponceau S as a reagent 1175
G. M. Mastoi, M. Y. Khuhawar, Kulsoom Abbasi, Moina Akhtar, Saba Naz,
Humaira Khan, Arfana Mallah and Zuhra Memon

African Journal of Pharmacy and Pharmacology Vol. 5(8). pp. 993-1005, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP10.223
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Evaluation of the effects of ciprofloxcin or gatifloxacin
on neurotransmitters levels in rat cortex and
hippocampus
Sayed M. Rawi 1 *, Nadia M. S. Arafa 2 and Mansour M. El-Hazmi 1
1 Biology Department, Faculty of Sciences and Arts, Khulais King AbdulAziz University, Saudi Arabia.
2 Associate prof. physiology, Faculty of Education S.D.(Girls) Jazan University Saudi Arabia.
Accepted 07 July 2011
The objective of the present work is to study the possible role of neurotransmitters in the central
nervous system (CNS) side effects due to the administration of ciprofloxacin (80 mg/kg body weight)
and gatifloxacin (32 mg/kg body weight) in male albino rats for 3, 7 and 14 days.The frontal cortex of
ciprofloxacin and gatifloxacin treated groups revealed decrease of glutamate, γ-aminobutyric acid
(GABA), dopamine and serotonin levels and elevation of aspartate, asparagine, glycine, serine and
norepinepherine levels and acetylecholinestrase (AChE) activities in a time related effect. In the
hippocampus area, the results varied in each antibiotic where in ciprofloxacin treated groups, there is
an elevation of asparagine, GABA, glycine, serine, taurine, norepinepherine and dopamine levels and a
reduction of glutamate, aspartate and serotonin levels and AChE activities in a time related effect. In
gatifloxacin treated groups, there is an elevation of glutamate, aspartate, asparagine, GABA, glycine,
taurine and norepinepherine and reduction in the levels of dopamine and serotonin and the AChE
activities. The histopathological examinations showed sever congestion with perivascular oedema in
the blood vessels and capillaries of cerebral cortex as well as of the hippocampus. Overall, the data
suggest that there is a shift in the balance between neurotransmitters towards increased production of
excitatory potency in groups subjected to ciprofloxacin or gatifloxacin administration.
Key words: Ciprofloxacin, Gatifloxacin, Amino Acids, Monoamines, Acetylcholinesterase.
INTRODUCTION
Fluoroquinolones are among the most widely prescribed
antibiotics especially for respiratory and urinary tract
infections. They are generally regarded as safe drugs
associated with mild gastrointestinal and central nervous
*Corresponding author. E-mail: rawisayed@yahoo.com or
s_rawi@yahoo.com Tel: 0020185419545.
Abbreviations: CNS, Central nervous system; AChE,
acetylecholinestrase; NODCAR, National Organization for Drug
Control and Research; HPLC, high performance liquid
chromatography; GABA, γ-aminobutyric acid; MES, maximal
electro shock.
system (CNS) symptoms (Jose et al., 2007; Becnel et al.,
2009). Recent events have brought new attention to
quinolone safety. Four quinolones have been withdrawn
from the US market: temafloxacin, as a result of
hemolysis, renal failure and hypoglycemia; trovafloxacin,
as a result of hepatotoxicity; grepafloxacin, as a result of
torsades de pointes; and sparfloxacin as a result of
phototoxicity and torsades de pointes (Frothingham,
2005).
Ciprofloxacin remains amongst the safest of all
antibiotics with remarkably few reports of serious
reactions over a period of 15 years of use and more than
340 million prescriptions (Ball et al., 1999; Segev et al.,
1999). Ciprofloxacin-associated seizures occur most
commonly in patients with special risk factors that may

994 Afr. J. Pharm. Pharmacol.
cause accumulation of drug or that may decrease the
threshold of epileptogenic activity (Kushner et al., 2001;
Kisa et al., 2005). On the other hand, ciprofloxacin
induced seizures in healthy patients (Darwish, 2008).
Gatifloxacin is one of the newer broad spectrum
fluoroquinolones available and was approved by the US
food and drug administration in December 1999. Ever
since its release in the market, there have been
numerous reports implicating gatifloxacin as a cause of
dysglycemia. This prompted Bristol-Meyer Squibb Co. to
list diabetes mellitus as a contraindication to gatifloxacin
use in the US product labeling and Health Canada to
issue an advisory against the use of gatifloxacin in
patients with diabetes (Jose et al., 2007). Gatifloxacin
showed to be equivalent to ciprofloxacin for the treatment
of acute uncomplicated lower urinary tract infections
(Naber et al., 2004).
In recent years, extensive in vivo and in vitro
experiments have been performed in an attempt to
explain the CNS side effects of quinolones sometimes
observed under therapeutic conditions. These effects
include like dizziness, restlessness, tremor, insomnia,
hallucinations, convulsions, anxiety and depression.
However, the molecular target or receptor for such effects
is still not exactly known. Extensive toxicological and
biochemical experiments have been performed to explain
the CNS effects observed under therapeutic conditions
(Akahane et al., 1993; De Sarro et al., 1999; De Sarro
and De Sarro 2001; Arafa et al., 2004). According to
Freitas et al. (2004) and Cavalheiro et al. (2006), seizure
activity of quinis associated with a wide range of local
biochemical changes, affecting various neurotransmitters
(monoamines, amino acids).
Since status epilepticus is a life-threatening neurologic
emergency which leads to neuronal degeneration of
vulnerable brain regions, we decided to estimate the
effect of antibiotics under investigation ciprofloxacin and
gatifloxacin in concern with their previously investigated
epileptogenic potential in the hippocampal and frontal
cortical areas because the development of spontaneous
seizures is characterized by paroxysmal activity initially
localized in the hippocampal formation and spreading of
paroxysmal activity from hippocampal to cortical
recordings was demonstrated (Cavalheiro et al., 1991).
The frontal cortex and hippocampus areas appeared to
be important in the expression of early convulsive
seizures (Kelly et al., 1999; Ang et al., 2006) in addition
to the important functional association between cortical
regions and the hippocampus in seizure propagation
(Kelly et al., 2002). Also, the frontal cortex and
hippocampus suggested playing a role in inducing
convulsions by quinolones (Motomura et al., 1991).
The present study aims to throw light on the effect of
either ciprofloxacin or gatifloxacin administration under
therapeutic level in male albino rat on the concentrations
of amino acid and monoamine neurotransmitters and
acetylecholinesterase activities in the frontal cortex and
hippocampus brain areas. The study aims to ascertain
the effect of the two antibiotics administration for one,
three, seven and fourteen days. Also the study extended
to include the histopathological effect of the two
antibiotics under investigation during the tested durations
on cortical and hippocampal brain areas.
MATERIALS AND METHODS
Experimental animals
This study was carried out on one hundred eight adult male albino
rats (Rattus norvegicus) with average body weight ranged 100g ±
20 g obtained from the Egyptian Institution of Serum and Vaccine
(Helwan). The experiment conducted in the Department of
Physiology in National Organization for Drug Control and Research
(NODCAR). The male albino rats were housed in iron mesh cages
with seven rats each. Clean sawdust was used to keep the animals
dry and clean throughout the experimental periods. The
experimental animals were allowed to acclimate under the
laboratory conditions two weeks before the beginning of the
experiments. The animals were kept under controlled temperature
of 21°C and 12 h light/ 12 h dark cycle throughout the course of
experiment. A commercial pelleted diet was used during the
experiment and allowed with water ad libitum.
Drug
Ciprofloxacin (Cipro) (C17H18FN3O3•HCl•H2O), manufactured by
Bayer healthcare pharmaceuticals, ciprofloxacin hydrochloride
tablets and Gatifloxacin (TEQUIN) (C19H22FN3O4•1.5 H2O),
manufactured by Bristol-Myers Squibb Company, The antibiotics
were administered by gastric intubation technique daily for fourteen
days. The administered doses calculated equivalent to the human
therapeutic dose according to the Guidance for Industry and
Reviewers (2002).
Experimental design
Animals were divided into three groups using random selection.
The first group (n= 12 rats) were administered 2 ml of distilled water
daily. The second group (n=48 rats) were administered 80 mg/100
g body weight ciprofloxacin dissolved in 2 ml water (cipro-treated
rats). The gatifloxacin-treated rat groups (n=48 rats) were
administered 32 mg/ 100 g body weight gatifloxacin dissolved in 2
ml water. At the end of the experimental periods 3, 7 and 14 days,
twelve animals sacrificed after 12 h from the last dose
administration by rapid decapitation. The brains were dissected out
quickly weighed and cleaned. Four brains from each treated group
served for the histopathological examination according to Banchroft
et al. (1996) and the rest eight brains for the biochemical analysis.
There is a single control group representing the control of the study.
The frontal cortex and hippocampus brain areas were separated
and divided into two halves the first half served for
acetylcholinesterase activity assay according to the modified of
Ellman et al. (1961) method as described by Gorun et al. (1978)
and the second half was homogenized in 75% high performance
liquid chromatography (HPLC) methanol (1/10 weight/volume) using

a homogenizer surrounded with an ice jacket and the homogenates
were used for the determination of the brain contents of amino
acids using the precolumn PTC derivatization technique according
to method of Heinrikson and Meredith (1984) and monoamines
neurotransmitters according to method described by Pagel et al.
(2000).
RESULTS
Data presented in Table 1 recorded the effect of
ciprofloxacin or gatifloxacin on amino acid
neurotransmitters in the frontal cortex and hippocampus
of male albino rats. The data showed a significant
decrease of glutamic acid concentration in the frontal
cortex as a result of treatment with ciprofloxacin and
gatifloxacin throughout the experimental periods as
compared to control. In hippocampus, the ciprofloxacin
treated groups showed a significant decrease after the 7 th
and 14 th days of the experimental periods as compared
with the control. On the contrary, gatifloxacin treated
groups exhibited a significant increase at 0.05 levels in
the 3 rd and 14 th day’s groups as compared to control one.
As regard to frontal cortex aspartic acid concentration,
ciprofloxacin treatment produced a significant increase
(P

996 Afr. J. Pharm. Pharmacol.
Table 1. Effect of ciprofloxacin or gatifloxacin administration on amino acid neurotransmitters contents in rat frontal cortex and
hippocampus.
Frontal cortex Hippocampus
Item Group
Experimental duration (days)
3 7 14 3 7 14
C 9.02±0.25 9.02±0.62 9.02±0.62 9.84±0.75 9.83±0.75 9.83±0.75
Glutamic acid Cip 6.44±0.19* 6.64±0.31* 8.35±0.66* ab 9.86±0.620 8.65±0.15* a 8.22±0.63* ab
Gati 6.33±0.15* 5.25±0.10 *a 6.09±0.25* b 10.80±0.59 * 10.26±0.48 12.30±0.22* ab
Aspartic acid
Asparagine
Serine
GABA
Glycine
Taurine
C 2.18±0.10 2.18±0.10 2.18±0.10 1.93±0.13 1.93±0.13 1.93±0.13
Cip 3.15±0.23* 2.89±0.15 *a 3.55±0.31* ab 1.76±0.09* 1.23±0.07* a 1.97±0.04 ab
Gati 1.86±0.08* 2.43±0.11 *a 2.89±0.16* ab 1.81±0.13* 2.10± 0.11* a 2.13±0.05 *a
C 0.43±0.03 0.43±0.03 0.43±0.03 0.18±0.01 0.18±0.01 0.18±0.01
Cip 0.52±0.04* 0.51±0.04 * 0.76±0.06* ab 0.25±0.01* 0.29±0.02 *a 0.21±0.01* ab
Gati 0.42± 0.02 0.53±0.03 *a 0.55±0.04* a 0.29±0.02* 0.21±0.01* a 0.33±0.01* ab
C 0.47±0.04 0.47±0.04 0.47±0.04 0.23±0.02 0.23±0.02 0.23±0.02
Cip 0.49±0.04 0.45± 0.04 a 0.55±0.04* ab 0.21±0.02 0.20±0.01 * 0.32± 0.01* ab
Gati 0.38±0.02* 0.56±0.03 *a 0.74±0.04* ab 0.20±0.01* 0.21±0.02 * 0.21± 0.01 *
Cip 2.47±0.11 2.47±0.11 2.47±0.11 1.92±0.17 1.92±0.17 1.92±0.17
Gati 1.58±0.10* 1.51±0.04* 1.70±0.15* ab 2.79±0.24* 2.87±0.16 * 2.52±0.08* ab
1.26±0.01* 1.38±0.06* a 1.45±0.04* a 2.94±0.15* 3.02±0.25 * 3.02±0.09*
C 1.71±0.15 1.71±0.15 1.71±0.15 0.99±0.06 0.99±0.06 0.99±0.06
Cip 2.31±0.15* 2.13±0.08* a 2.34±0.24* b 1.32±0.05* 1.12±0.01 *a 1.70±0.14* ab
Gati 1.64±0.02 1.76±0.08 1.88±0.11* a 1.33±0.01* 1.11±0.06 *a 1.05±0.04 a
C 2.00±0.12 2.00±0.12 2.00±0.12 3.36±0.27 3.36±0.27 3.36±0.27
Cip 1.98±0.14 1.78±0.07* a 2.12±0.19 ab 3.59±0.22* 3.27±0.15 a 4.01±0.20* ab
Gati 1.95±0.02 1.99±0.05 1.58±0.06 *ab 4.04±0.07 * 4.17±0.15 * 3.84±0.20 *b
The results are presented as means ± standard deviation, n = 8 rats. *, significant change from the corresponding control value, a , significant
change from the 3 days of treatment group, b , significant change from the 7days of treatment group at 0.05 level.C, control.
Table 2. Effect of ciprofloxacin or gatifloxacine administration on monoamine neurotransmitters contents and acetylcholinesterase
enzyme activity in rat frontal cortex and hippocampus.
Item
Norepinepherine
Dopamine
Serotonin
Group
(n=8)
Frontal cortex
Experimental durations (days)
Hippocampus
3 7 14 3 7 14
C 1.06±0.01 1.06±0.01 1.06±0.01 0.56±0.13 0.56±0.13 0.56±0.13
Cip 0.84±0.07* 0.92±0.08 *a 0.85±0.01* ab 0.73±0.06* 0.982±0.09* a 0.99±0.09* a
Gati 0.72±0.04* 0.72±0.06* 0.86±0.05* ab 0.90±0.07* 0.77±0.05* a 0.70±0.06* a
C 3.36±0.23 3.36±0.23 3.36±0.23 0.66±0.11 0.66±0.11 0.66±0.11
Cip 3.25±0.14 2.43±0.04* a 1.71±0.09* ab 1.60±0.14* 1.65±0.13* 0.92±0.04* ab
Gati 2.75±0.12* 2.74±0.23* 3.00±0.15 *ab 0.65±0.03 0.53±0.02 *a 0.61±0.04
C 85.40±7.71 85.40±7.71 85.40±7.71 244.80±18.36 244.80±18.36 244.80±18.36
Cip 31.80±3.58* 54.90±5.62* a 93.50±13.38* ab 234.00±10.00 161.60±16.71* a 223.70 ± 21.02* b
Gati 58.00±2.62* 49.60±4.21* a 78.30±5.47 *ab 222.60±17.04* 208.50±5.40* 200.10±15.64* a

Table 2. Contd.
AChE
Rawi et al. 997
C 13.27±0.81 13.27±0.81 13.27±0.81 17.52±0.62 17.52±0.62 17.52±0.62
Cip 17.75±1.46* 23.39±1.07* a 32.30±2.30* a b 14.43±0.79* 13.48±0.85* 12.57±1.07* a
Gati 16.02±2.23* 19.99±2.29* a 21.11±1.90* a 15.45±0.92* 14.70±0.71* 12.90±0.94* ab
The results are presented as means ± standard deviation, n = 8 rats. *, significant change from the corresponding control value, a , , significant
change from the 3 days of treatment group, b , significant change from the 7days of treatment group at 0.05 level. C, control.
Figure 1. Normal histology of cerebral cortex (CC) and
covering meninges (m) (H and E x-40).
Figure 2. Normal rat histology of hippocampus (hc). (H and
E x-40).
to the control enzyme activity. On the contrary, in the
hippocampus, the data showed significant decrease in
AchE activity from the 3 rd till the 14 th days of ciprofloxacin
or gatifloxacin administration.
The hispopathological examination the response of
cortex and hippocampus cells to ciprofloxacin and
Figure 3. Three days cip showing vacuolation in the brain
cerebral matix (arrow) associated with congestion of the
blood vessels (v) and perivascular oedema (O). (H and E x-
160).
gatifloxacin administration at different durations is clearly
investigated. The consequence damage to the cortical
area of ciprofloxacin administered animals showed from
the 3 rd dose are vacuolation associated with congestion
and perivascular oedema in the blood vessels and the
meninges showed vascular congestion. Congestion with
perivascular oedema in the blood vessels and capillaries
of cerebral cortex achieved its severe stage at the final
14 th day of ciprofloxacin administration. Also the cerebral
matrix showed diffuse gliosis. Hippocampus of
ciprofloxacin-administered animals showed from the 3 rd
dose congestion in the blood vessels attained the severe
congestion with perivascular oedema in the blood vessels
and capillaries of the hippocampus at the last examined
duration of drug administration (Figures 1 to 8).
Gatifloxacin from the 3 rd dose revealed moderate
changes in the cerebral cortex in the form of neuronal
degeneration, diffuse gliosis, and congestions with
perivascular oedema in the blood vessels and capillaries
with focal gliois at the end of the experiment.
Hippocampus of gatifloxacin administered animals
showed congestion in the blood vessels and capillaries,
degeneration in the some hippocampal neurons after
the3 rd dose. The hippocampus showed vacuolation in the

998 Afr. J. Pharm. Pharmacol.
Figure 4. Three days cip showing congestion of blood vessels
and capillaries (arrow) with perivascular oedema in the
hippocampus. (H and E x-40).
Figure 5. Seven days cip showing vacuolation of cerebral
matrix (v), diffuse gliosis (arrow). (H and E x-40).
Figure 6. Fourteen days cip showing sever congestion of
cerebral blood vessels with perivascular oedema. (H and E
x-40).
Figure 7. Fourteen days cip showing congestion of blood
vessels with perivascular oedema in the hippocampus. (H and
E x-40).
Figure 8. Fourteen days cip showing the magnification of
figure (12) to identify the congestion and perivascular oedema
of blood vessels in the hippocampus (arrow). (H and E x-160).
tissue matrix with degeneration in the neuronal cells and
congestion in the blood vessels and capillaries at the last
dose group of gatifloxacin-administered rat (Figures 9 to
15).
DISCUSSION
In the present study the response of cortex and
hippocampus areas to ciprofloxacin and gatifloxacin at
different durations are clearly investigated through the
determined amino acids and monoamines neurotransmitters
and the recorded activities of the
acetylcholinesterase enzyme. The excitatory potencies of
ciprofloxacin and gatifloxacin recorded throw the elevation

Figure 9. Three days gati showing diffuse gliosis in the cerebral
matrix (C). (H and E x-64).
Figure 10. Three days gati showing congestion with perivascular
oedema in the hippocampus blood vessels and capillaries (arrow).
(H and E x-64).
Figure 11. Three days gati showing degeneration of some
neuronal cells in the hippocampus (arrow). (H and E x- 160).
Rawi et al. 999
Figure 12. Seven days gati showing vacuolation in the
hippocampus tissue matrix (hc) with degeneration in the
neuronal cells (arrow). (H and E x- 160).
Figure 13. Fourteen days gati showing congestion of blood
vessels and capillaries of the cerebral cortex with perivascular
oedema (V) and diffuse gliosis (arrow). (H and E x- 64).
Figure 14. Fourteen days gati showing focal gliois (n) in the
cerebral cortex. (H and E x- 64).

1000 Afr. J. Pharm. Pharmacol.
Figure 15. Fourteen days gati showing congestion in the blood
vessels (V) in hippocampus. (H and E x- 160).
of aspartate and asparagine levels and the increase of
the activities of AChE in spite of decrease of glutamate
and monoamines levels in the cortical area as a result of
either ciprofloxacin or gatifloxacin administrations in a
time related effect. In addition, there was decrease of
GABA and alanine levels in a manner as the
aforementioned effect.
The excitatory potencies of the antibiotics under
investigation varied as regard to the hippocampus area
where ciprofloxacin in the hippocampus area recorded
elevation of asparagines, GABA, glycine, taurine,
norepinepherine and dopamine levels but decrease of
glutamate and serotonin levels in a time related effect. In
addition there are decreases of AChE activities and
aspartate, glutamate levels in a dose related effect. But in
case of gatifloxacin treated groups in the hippocampus
area shows elevation of aspartate, glutamate,
asparagines, GABA, glycine, taurine and
Norepinepherine. Also there are decreases in the
activities of AChE and the levels of dopamine and
serotonin.
The previous findings, which recorded herein in brain
cortical areas from groups subjected to the antibiotics,
are in line with alterations in extrahippocampal regions in
epileptic rat models as previous experimental findings
have also demonstrated that the cortex damaged with
diffuse gliosis in different animal models of acute limbic
seizures. This alterations support the hyperexcitability
with GABAergic inhibition, which could play a crucial role
in seizure generation and expression in epileptic rat
models (Silva et al., 2002).
The alterations in the hippocampus area evidence
provided links early seizures with the later development
of epilepsy and selective hippocampal neuronal loss (Koh
et al., 1999). Also kindled seizures are associated with a
selective degeneration of cortical and hippocampal areas
(Szyndler et al., 2006) which supported by the elevation
of GABA levels in hippocampus tissues of both antibiotics
treated groups which in line with that recorded in kianic
acid induced seizure rat animal model (Bruhn et al.,
1992) and the assumption of Gale (1992) about the
increase of GABAergic transmission can induce
excitatory effects.
The anexiogenic effects due to ciprofloxacin and
gatifloxacin may be supported through the recorded
changes in the concentrations of frontal cortex amino
acids. There were decreases in GABA and glutamate
levels with a concomitant increase in aspartate,
asparagine, glycine, serine and alanine contents which
similar to those recorded in the frontal cortex of epileptic
rat models (Craig and Hartman, 1973; Li et al., 2000;
Szyndler et al., 2006).
The regional differences in GABA level and
acetylcholinesterase activity between decrease of GABA
level and increase of AChE activity in the cortical area
and increase of GABA level and decrease of AChE
activity in the hippocampal area in both antibiotics treated
groups in a dose related effect which mimics that
predicted in rat epileptic models (Appleyard et al., 1986)
and support the proconvulsant effect of the quinolones
previously discussed (Smolders et al., 2002)
Experimental manipulations suggest that in vivo
administration of cholinergic agonists or inhibitors of
AChE increases the concentration of acetylcholine.
Biochemical studies have proposed a role for AChE in
brain mechanisms responsible by development to status
epilepticus through decrease in the AChE activity in the
hippocampus (Freitas et al., 2006)
It is evident from the present study that ciprofloxacin
and gatifloxacin administered groups recorded significant
decrease in the cortical main inhibitory amino acid GABA
levels throughout the experimental periods in a dose
related effect. GABA the principal inhibitory
neurotransmitter in the cerebral cortex maintains the
inhibitory tone that counterbalances neuronal excitation.
When this balance is perturbed, seizures may ensue.
GABA is formed within GABAergic axon terminals and
released into the synapse, where it acts at one of two
types of receptor: GABAA, which controls chloride entry
into the cell, and GABAB, which increases potassium
conductance, decreases calcium entry, and inhibits the
presynaptic release of other transmitters. GABAAreceptor
binding influences the early portion of the
GABA-mediated inhibitory postsynaptic potential,
whereas GABAB binding influences the late portion.
GABA is rapidly removed by uptake into both glia and
presynaptic nerve terminals and then catabolized by
GABA transaminase. Experimental and clinical study
evidence indicates that GABA has an important role in
the mechanism of epilepsy as abnormalities of
GABAergic function have been observed in genetic and

acquired animal models of epilepsy, reductions of GABAmediated
inhibition, activity of glutamate decarboxylase,
binding to GABAA and benzodiazepine sites. GABA
antagonists produce seizures and drugs that inhibit
GABA synthesis cause seizures (Treiman, 2001).
Reduction in inhibitory control due to loss of GABAergic
interneurons, and a decrease in GABA levels and
GABAA receptor sensitivity leads to cortex
hyperexcitability as GABAergic neurotransmission and
epilepsy has long been recognized. Inhibition of GABAA
receptors triggers acute seizures (Prince, 1978; Olsen
and Avoli, 1997; Armijo et al., 2002).
The role of γ-aminobutyric acid (GABA) in anxiety is
well documented (Davis et al., 1994) Some studies
indicate that fluoroquinolones function as GABA receptor
antagonists, (Unseld et al., 1990) and the epileptogenic
action of quinolones has been proposed to be related to
the GABA-like structure of ring substitutes. Quinolones
have an inhibitory effect on the receptor binding of
GABAA, and may thus exert an inhibitory CNS stimulant
action (Akahane et al., 1994; Imanishi et al., 1995).
Benzodiazepine agonists have been reported to
attenuate the central stimulating effects of ciprofloxacin
and pefloxacin (Unseld et al., 1990) Likewise, they
potentiate chemically-induced convulsions, which could
be antagonised by benzodiazepines. (Enginar and
Eroğlu, 1991) The adenosine or GABAA receptor has
therefore been proposed as a possible target for
ciprofloxacin (Dodd et al., 1988).The structural similarities
of the fluoroquinolones to kynurenic acid and other
similar compounds, which are endogenous ligands of the
glutamate receptor, might suggest an interaction of
quinolones with ligand-gated glutamate receptors as well
(Schmuck et al., 1998) which may explain the elevated
hippocampual glutamate level in the gatifloxacin
subjected groups.
The decreased glutamate level in the cortical area of
either ciprofloxacin or gatifloxacin treated groups and the
ciprofloxacin treated group hippocampus area, may be
explained as fluoroquinolones did not bind to the
glutamate or glycine-binding site of the N-methyl-Daspartate
(NMDA) receptor. It has been shown that
fluoroquinolones decrease blocking effects of Mg2+ and
MK-801 binding to the NMDA receptor. Magnesium
chelating properties of fluoroquinolones have been
postulated as mechanisms of fluoroquinolone-induced
atrophy, and the excitatory potency of fluoroquinolones
might also be based on activation of the NMDA receptor
by abolishing the Mg2+ block in the ion channel. This
would prolong the opening time of the channel, thus
increasing intracellular Ca2+ concentration in the neurons
(Sen et al., 2007).
The decrease of glutamate and increase of aspartate
may be explained through the glucose homeostasis
abnormalities (dysglycemia) associated with the use of
Rawi et al. 1001
gatifloxacin (Onyenwenyi et al., 2008) as aspartate
seems to selectively activate the NMDA type of
Glutamate receptors (Curras and Dingledine, 1992).
Electrophysiological experiments using hippocampal
slices have demonstrated that when glucose
concentration was reduced, stimulation of the Schaffer
collaterals gave an Aspartic-mediated NMDA response
(Fleck et al., 1993), indicating a functional role of Asp
released from excitatory nerve endings.
Immunocytochemistry has detected NMDA receptors in
target neuronal cell bodies and dendritic spines contacted
by the type of nerve endings shown here to be enriched
with Asp during hypoglycemia (Takumi et al., 1999). In
hippocampal neurons, a larger number of quanta
transmitters are signaled through NMDA receptors than
through alpha amino 3-hydroxy 5-methyl 4isoxazoleproprionic
acid receptors, and during repetitive
neuronal firing some of the released transmitter can spill
over from the synaptic cleft to activate extrasynaptic
NMDA receptors (Kullmann et al., 1996). Thus, leakage
of aspartate not only from excitatory but also from
inhibitory synapses during conditions of high neuronal
activity, in which the release of aspartate is, enhanced
(Szerb, 1988), may reach NMDA receptors at nearby
sites and cause an enhanced NMDA receptor response
during these conditions. Thus, aspartate could play an
important role in physiologic and pathologic types of
NMDA receptor-mediated transmission. Could such an
aspartate-induced NMDA receptor response be involved
in hypoglycemic neuronal death? Neurons susceptible to
hypoglycemia comprise dentate granule cells, pyramidal
cells in CA1 of hippocampus, and striatal neurons (Auer
et al., 1984).
During hypoglycemia, the excitatory and inhibitory
nerve terminals contacting these neurons are enriched
with Aspartate, which may be released to activate NMDA
receptors on synaptic and extrasynaptic sites. This would
cause an excitotoxic insult, leading to neuronal death
(Wieloch, 1985). In line with this is the recent
demonstration in hippocampal neurons that excitotoxicity
was not only caused by activation of NMDA receptors at
the postsynaptic density but also by activation of NMDA
receptors at extrasynaptic sites (Sattler et al., 2000).
The elevated level of glycine in cortical and
hippocampus areas of rats subjected to ciprofloxacin or
gatifloxacin may be declared the effect of the inhibitory
neurotransmitter glycine on slow destructive processes in
brain cortex slices under anoxic conditions as glycine, the
simplest of the amino acids, is an essential component of
important biological molecules, a key substance in many
metabolic reactions, the major inhibitory neurotransmitter
in the spinal cord and brain stem, and an antiinflammatory,
cytoprotective (Gundersen et al., 2005;
Tonshin et al., 2007). In hippocampus area the experimental
model of epilepsy during kianic acid induced

1002 Afr. J. Pharm. Pharmacol.
epilepsy the results indicated that the levels of glutamate,
aspartate, glycine and GABA were statistically increased
in rat's hippocampus (Liu and Cheng, 1995) which is in
line with our hippocampal results in ciprofloxacin or
gatifloxacin treated groups supporting body of studies
about proconvulsant and anxiogenic effects of
ciprofloxacin and gatifloxacin (Quigley and Lederman,
2004; Bharai et al., 2008) and epileptogenic potential
(Koussa et al., 2007) which extended to that long term
administration of gatifloxacin for 14 days was found to
indicate movement impairing effect in mice (Bharai et al.,
2008).
Metabolic actions of taurine include: bile acid
conjugation, detoxification, membrane stabilization,
osmoregulation, and modulation of cellular calcium
levels. Clinically, taurine has been used with varying
degrees of success in the treatment of a wide variety of
conditions, including: cardiovascular diseases,
hypercholesterolemia, epilepsy and other seizure
disorders, macular degeneration, Alzheimer's disease
(Birdsall, 1998).
About the taurine detected levels which increased and
these may be related to the extracellular increased levels
of excitatory amino acids which led to neuronal, glial and
endothelial impairment through the NMDA receptors
activation and the increase of the intracellular Ca 2+
concentration which is a signal for taurine release, which
in turn blocks NMDA-evoked Ca 2+ entry. This increase in
the extracellular taurine level serves a neuromodulator
that protects against cell damage from the Ca 2+ influx
which followed by Cl - influx resulting in cell swelling and
eventually cell death (Yang et al., 1997). Hence taurine, a
volume regulating amino acid, increased inducing cell
swelling as predicted through the level of taurine
measured or the histopathological examination and this is
in agreement with Stover et al. (1997). The increased
taurine levels in the hippocampus may involve processes
for membrane stabilization, thus favoring recovery after
neuronal hyperactivity. In the hippocampus of epileptic
patients found increases in taurine, glutamate, aspartate
during seizures (Wilson et al., 1996) also it was reported
that the intraperitoneal injection of taurine blocked the
convulsive seizure in rat cortex (Batuev et al., 1997).
Thus the herein recorded increase in taurine levels
could be explained as involvement in the modulation of
spontaneous recurrent seizure activity (Baran, 2006).
It has been suggested that the biogenic amines such
as Norepinepherine, dopamine and serotonin play
important role in the manifestation and inhibition of
convulsions since several animal models of convulsion
may be significantly affected by modifying the levels or
availabilities of these amines in the brain.
The effect of brain amines on convulsion has been
demonstrated to differ depending on the method of
seizure induction. However, there are virtually no detailed
studies investigating the relationship between the type of
stimulation used for convulsion induction and the effects
on brain amines. The main reason is due to the
differences of neurons participated in the response to the
given stimuli to elicit the convulsions. For example the
electrical stimulation none selectively stimulates almost
all kinds of neurons with different neurotransmitters while
the chemical stimulation can distinguish the specialized
receptor to make an excitation in the brain so
monoamines in the hippocampal seizure discharge are
very much dependant on the type of stimulation
employed (Nishi et al., 1981; Meldrum, 1991). It was
found in the kindling model of epilepsy, for instance,
Norepinepherine and serotonin depletion have been
shown to facilitate development of seizures in rats
(Corcoran and Mason, 1980; Cavalheiro et al., 1981;
Bortolotto and Cavalheiro, 1986), and increased
Norepinepherine concentration in hippocampus of rats
subjected to kindling has been demonstrated by
microdialysis (Kokaia et al., 1989). The levels of biogenic
amines such as dopamine, serotonin and nor-adrenaline
in the forebrain region seizures induced by Maximal
Electro shock (MES) method in rats (Balamurugan et al.,
2009). Bidziński et al. (1998) concluded that there is a
functional interaction between brain serotonin and GABA
systems, both at behavioral and biochemical levels, that
is involved in the motor activity habituation process due
to the effect of serotonin depletion in GABAA receptor
down-regulation.
Monoamines levels recorded in the tested antibiotics
shows reduction in norepinepherine, dopamine and
serotonin in the frontal cortex in the ciprofloxacin and
gatifloxacin treated groups. But in hippocampus there
are elevations in Norepinepherine and dopamine and
reduction of serotonin levels in ciprofloxacin subjected
groups. On the other hand there is elevation of
norepinepherine and reduction of dopamine levels in the
gatifloxacin treated groups. These data may be validated
the seizure induction through the assumption about the
pharmacological treatments that lowering monoamine
levels in the brain generally increase the susceptibility to
seizures, while treatments that increase monoamines
decrease the susceptibility (Kiyofumi and Akitane, 1977).
The data recorded about monoamines in the tested
antibiotics may be a supplement data to the previously
mentioned seizure inducing activity of quinolones (Ooie
et al., 1997; Moorthy et al., 2008; Agbaht et al., 2009).
Overall, these data suggest that there is a shift in the
balance between neurotransmitters towards increased
production of excitatory amino acids, and this may be
triggering seizure in rat model (Szyndler et al., 2008). The
excitatory potency reported effect of quinolones
evaluated through the elevation of aspartic acid,
asparagine and norepinepherine in addition to the
reduction of the GABA level and in the frontal cortex and

eduction of the GABA level and in the frontal cortex and
elevation of Norepinepherine contents in hippocampus
tissue in groups subjected to ciprofloxacin. Also the
recorded AChE enzyme activity reduced in hippocampus
gatifloxacin administration excitatory potency recorded
through elevation of frontal cortex Norepinepherine and
glutamic acid and Norepinepherine levels in
hippocampus tissues. Also the recorded AChE enzyme
activity reduced in hippocampus.
Therefore,
the design and development of new
quinolone derivatives with broader antibacterial activity
and better pharmaco-kinetics avoiding CNS side effects
are attractive therapeutic goals. In addition, although
further studies are needed to investigate the mechanism
that causes the CNS side effects of quinolones,
physicians should consider the possible epileptogenic
activity of these compounds when treating patients with
predisposing epileptic factors or when the penetration of
quinolones into the brain via a damaged blood-brain
barrier is enhanced. Thus dosing modifications and
awareness of possible central nervous system adverse
effects are warranted.
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African Journal of Pharmacy and Pharmacology Vol. 5(8). pp. 1006-1011, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP10.257
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Evaluation of estrogen-like activity of Nigella sativa in
ovariectomized rats
Saadat Parhizkar¹ , ²*, Latiffah Abdul Latiff 2,3 , Sabariah Abdul Rahman 2,3 and Mohammad Aziz
Dollah 3
1 Faculty of Health, Yasouj University of Medical Sciences, Iran.
2 Institute of Biosciences, Universiti Putra Malaysia, Serdang 43400, Malaysia.
3 Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang 43400, Malaysia
Accepted 12 April, 2011
The purpose of the current study was to evaluate estrogenic activity of Nigella sativa using
ovariectomized (OVX) rats. Forty OVX rats divided into five groups. N. sativa was administered orally as
a supplement to chaw palate at different doses of 300, 600 and 1200 mg/kg for 21 days to OVX rats and
were compared to either positive (0.2 mg/kg conjugated equine estrogen- CEE) or negative control (1 ml
distilled water). Histopathological changes of the rats’ uterus such as epithelial proliferation and
endometrial glandular hyperplasia were assessed as well as uterus weight and serum estradiol level.
Supplementation with N. sativa resulted in a significant increase in uterine weight as compared to the
OVX controls accompanied by altering the serum estrogen levels. Histopathological evaluation of the
uterine section revealed changes characterized by atrophy of the uterus in the OVX controls, while the
OVX rats supplemented with N. sativa showed increased endometrial response as indicated by
proliferation of the endometrial glands and epithelial hyperplasia as well as epithelial proliferation and
endometrial glandular hyperplasia. The effects of N. sativa were comparable to that in the CEE group. N.
sativa showed the desired effects on the physical, histological and biochemical parameters of the
uterine tissue in OVX rats, thereby indicating its probable beneficial role in the treatment of
postmenopausal symptoms.
Key words: Nigella sativa, estrogenic effect, uterotrophic assay, ovariectomized rat.
INTRODUCTION
Nigella, a genus of the Ranunculaceae family, contains
about 20 species many of which have been used as
traditional medicinal plants. One of these, Nigella sativa,
grows to a height of 60 cm, and is distributed in the South
Europe, North Africa and south West Asia. Its species are
reported to be medicinal plants in the scientific literature
as well as in folklore and horticulture, and their medicinal
values are well documented (Dey, 1980; Boulos, 1983).
The seed of N. sativa has been used as a folk medicine
for the treatment of a number of illness and conditions
that include bronchial asthma, cough, rheumatism,
hypertension, diabetes, inflammation, eczema, fever,
tumor and influenza (Blunden, 2003; Al-Ghamdi, 2001;
Al-Naggar et al., 2003; Nair et al., 1991). Carminative,
*Corresponding author. E-mail: parhizkarsa@gmail.com Tel:
+603-8947 2514 Fax: +603-8947 2534
diuretic, lactogouge and vermifuge properties have been
attributed to a variety of active phytoconstituents in seeds
and its oil (Mahmoud et al., 2002; Al-Hader et al., 1993;
Swamy and Tan, 2001; Badary, 1999; Daba and Abdel-
Rahman, 1998). Several studies on N. sativa seeds (Nagi
and Mansour, 2000) and its different fractions (Bourgou
et al., 2008) have been reported recently but there are no
relevant studies on estrogenic activity of N. sativa.
Because estrogens have typically been used for the
treatment of menopausal symptoms and because N.
sativa have been shown to improve lactation in nursing
mothers and to regulate menstruation among females in
folk medicine, we investigated the potential estrogenic
effects of N. sativa. Therefore, the purpose of the
present study was to investigate the possible estrogenic
effects of N. sativa. The uterotrophic assay in
ovariectomized rats is one of the classical methods to
demonstrate estrogenic activity of chemicals or natural
compounds (Clode, 2006). In this study, uterotrophic

assay, morphological analyses of the uterus and serum
estrogen level were used to study the estrogenic activity
of N. sativa.
MATERIALS AND METHODS
Plant
N. sativa seeds (imported from India) were purchased from a local
shop in Serdang, Malaysia. The seed was identified and
authenticated by Professor Dr. Nordin Hj Lajis, Head of the
Laboratory of Natural products, institute of bioscience, Universiti
Putra Malaysia. Voucher specimens of seeds were kept at the
cancer research laboratory, institute of bioscience, Universiti Putra
Malaysia.
Seeds were cleaned under running tap water for 10 min, rinsed
twice with distilled water and air-dried in an oven at 40°C overnight.
The seeds and rat chow pellet were ground to a powder using an
electric grinder (National, Model MX-915, Kadoma, Osaka, Japan)
for 10 min. Grounded seeds and chow pellet mixed with water into
three doses of 300, 600 and 1200 mg/kg and backed in an oven at
40°C until receiving instant weight.
Chemicals and reagents
Estradiol radioimmunoassay (RIA) kit was purchased from
Diagnostic Systems Laboratories (DSL), USA. Conjugated equine
estrogen (CEE 0.625 mg) was purchased from Wyeth, Montreal,
Canada. CEE (Wyeth Montreal, Canada), prepared in a dosage of
0.2 mg/kg (Genazzani et al., 2004; Oropeza et al., 2005; Araujo et
al., 2006) by dissolving it in distilled water (Genazzani et al., 2004;
Hajdu et al., 1965) and was used as a positive control for the
purpose of comparison with the treated groups. All other reagents
and chemicals were of analytical grade.
Animals
The protocol of the study was approved by animal care and use
committee (ACUC) with reference number of
UPM/FPSK/PADS/BR/UUH/F01-00220 in accordance to “Guide for
care and use of laboratory animals” set by the ACUC of faculty of
medicine and health sciences, Universiti Putra Malaysia. The
experiment was carried out using 16 week-old female albino
Sprague-Dawley rats, weighing 250 to 350 g. They were housed in
cages under standard laboratory conditions within a period of 12 h
light/dark at 29 to 32°C and 50 to 60% relative humidity in the
animal house, faculty of medicine and health sciences, Universiti
Putra Malaysia. The animals were allowed to acclimatize for at
least 10 days before the start of the experiments. The rats had
access to a standard rat chow pellet and drinking water ad libitum.
Hygienic condition was maintained by changing the bedding
weekly. All animal handling were conducted between 08.00 and
10.00 am to minimize the effects of environmental changes. Serum
estradiol and body weight were measured at baseline (day 0), 11th,
and at the end of experiment (21st days). Histological studies were
performed to assess uterine changes at the end of experiment.
Experimental design
Forty rats were ovariectomized in order to induce menopause and
to investigate reproductive changes following N. sativa
supplementation. Their ovariectomy was performed during a
distrous cycle to keep the consistent lowest levels of sex hormones
Parhizkar et al. 1007
in rats. Surgery of the animals was conducted under a combination
of xylazine and ketamine (10 mg/kg + 75 mg/kg, i.p. respectively)
anesthesia. Bilateral ovariectomy was performed via a dorso-lateral
approach with a small lateral vertical skin incision (Parhizkar et al.,
2008). The ovariectomized animals were acclimatized at the
Animal House of Faculty of Medicine and Health Sciences for one
month prior to supplementation.
The ovariectomized rats were divided equally into five groups (8
animals in each group). The grouping of rats includes negative
control (1 ml distilled water by intra-gastric gavage), positive control
(0.2 mg/kg/day conjugated equine estrogen-CEE diluted in distilled
water by intra-gastric gavage) and the rest groups receiving
different doses of N. sativa. The test group consist of low dose N.
sativa-LNS (300 mg/kg NS), moderate dose N. sativa-MNS (600
mg/kg NS) and high dose N. sativa-HNS (1200 mg/kg NS).
Supplementations with N. sativa, CEE and distilled water were
continued for 3 weeks.
Post-mortem and uterine horn histological study
At the end of the experiment, the rats were weighed and sacrificed
under chloroform anesthesia. The uterus was removed and freed
from all connective tissue prior to wet weight recordings. To account
for individual differences in body weight, an adjusted uterine weight
was used for statistical significance calculation. According to Henry
and Witt (2002) the actual organ weight, in grams (g), was divided
by the actual body weight of the female rats before being sacrificed
and multiplied by a standard body weight of 100 g. The adjusted
uterine wet weights were reported in g/100 g of body weight.
Routine histological processes were employed for paraffin
inclusion, sectioning and hematoxylin-eosin staining. Since
previous study by Inuwa and Williams (1996), showed that there
was no significant difference in structure between the two uterine
horns of the same rat and between the various regions of the same
horn. One horn from each rat was randomly selected and cut
transversely into three equal portion known as proximal, middle and
distal. Each portion was prepared as a block and from each block
three ribbons were chosen randomly and examined under a light
microscope (Olympus CK2) and measurements carried out. For
morphological analyses of the uterus, the known E2-induced
features recorded includes; shape and height of the luminal and
glandular epithelial cells, mitotic figures, determination of
hypertrophy and hyperplasia of glands and endometrial epithelium
as well as number and types of glands, presence of leucocytes and
hypervascularity. The thickness of the endometrium and
myometrium were also measured. Appropriate image capture was
made using a light microscope (Olympus CK2) coupled to a camera
(Olympus BX 41). Measurement was carried out with image
analysis software (AxionVision 4.2 RELCarl Zeiss, Jena, Germany).
Statistical analysis
Data were expressed as means ± standard deviation. The data
were analyzed using SPSS windows program version 15 (SPSS
Institute, Inc., Chicago, IL, USA). The one-way analysis of variance
(ANOVA) and general linear model (GLM) followed by Duncan
multiple range test (DMRT) were used to determine which N. sativa
concentration shows the most significant effect. A p-value less than
0.05 (P

1008 Afr. J. Pharm. Pharmacol.
Relative uterus w et weight
(mg/100 g body weight)
200
150
100
50
0
C CEE LNS MNS HNS
Figure 1. Means of uterus relative weight of OVX rats (mg/100 gm body weight)
supplemented with various doses of N. sativa or conjugated equine estrogen.
Treatment: C = control (1 ml distilled water); CEE = conjugated equine estrogen
(0.2 mg/kg); LNS = low dose of N. sativa (300 mg/kg); MNS = medium dose of N.
sativa (600 mg/kg); HNS = high dose of N. sativa (1200 mg/kg) groups. Data is
expressed as mean. *: Significantly different as compared to control at P

A B
C D
E
Figure 3. Photomicrographs showing representative
endometrium of the uterus of ovariectomized rats: A = Control (1
ml distilled water/day), B= conjugated equine estrogen-CEE (0.2
mg/kg/day CEE), C= LNS, low dose N. Sativa (300 mg/kg/day),
D= MNS, medium dose N. Sativa (600 mg/kg/day) and E= HNS,
high dose N. Sativa (1200 mg/kg/day). Arrowheads indicated
different endometrial glands. The squares indicate the area of
uterine lumen and its epithelial cells. H.E. staining (100x).
presence of leukocytes, increased endometrial and
myometrial diameters, increased number of glands,
present of leucocytes (Table 1), as compared to control,
but these changes is more apparent in LNS. In NS
groups, endometrial cells were stimulated but no
pathologic signs were detected. The uterine histology of
rats administered both CEE and NS differed markedly
from those of control animals. The number of normal
glands was increased significantly (P

1010 Afr. J. Pharm. Pharmacol.
Table 1. Summary of physiologic and morphologic finding in uteri of ovariectomized rats supplemented with various doses of N. sativa or
conjugated equine estrogen.
Parameter
C (0)
N. sativa supplementations (mg/kg/day)
LNS (300) MNS (600) HNS (1200)
CEE 0.2
(mg/kg/day)
Endometrial thickness (µm) 251 ± 107 a 359 ± 67 cd 313 ± 49 bc 281 ± 39 ab 365 ±123 d
Myometrial thickness (µm) 369 ± 125 b 483 ± 83 c 343 ± 39 ab 295 ± 57 a 473 ± 132 c
Luminal epithelial cell height (µm) 4.5 ± 1.2 a 15 ± 1.9 d 8.6 ± 1.7 c 5.9 ± 1.8 b 18 ± 3.8 e
Glandular epithelial cell height (µm) 6. 05 ± 2.4 a 13.74 ± 1.24 d 9.24 ± 1.89 c 7.78 ± 2 b 14.42 ± 3.38 d
No. of glands 15.41 ± 4.8 a 30.58 ± 4.43 c 25.20 ± 5.4 b 18 ± 4.7 a 29.08 ± 6.94 c
No. of blood vessels 10.19 ± 0.9 a 14.06 ± 1.4 c 12.12 ± 1.17 b 12.13 ± 1.75 b 15.48 ± 1.87 d
Data are expressed as Mean ± SD for three slides per rats. The data represent the average of three determinations for each slide. Treatment:
C=control group (treated with 1 ml distilled water); CEE= conjugated equine estrogen (treated with 0.2 mg/kg); LNS= low dose of N. sativa (treated
with 300 mg/kg); MNS= medium dose of N. sativa (treated with 600 mg/kg); HNS= high dose of N. sativa (treated with 1200 mg/kg) groups.
abcde: Comparison of the means between rows significant at P

for its financial support of this research project.
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African Journal of Pharmacy and Pharmacology Vol. 5(8), pp. 1012-1019, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP10.358
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
A meta-analysis of the efficacy and safety of
candesartan in Chinese patients with mild to moderate
essential hypertension
Cui Wen-peng 1 , Du Bing 2 and Qin Ling 2 *
1 Department of Nephrology, Second Hospital, Jilin University, Changchun, China.
2 Department of Cardiology, the second part of First Hospital, Jilin University, No. 71 of the XinMin Road, Changchun
City, Jilin Province, China.
Accepted 20 May, 2011
This study was performed to evaluate the efficacy and safety of Candesartan in treating Chinese
patients with mild to moderate essential hypertension. Medical databases and review articles were
screened with prespecified criteria for randomized controlled trials that reported the effects of and
adverse reactions to Candesartan and other antihypertensive drugs in treating of Chinese patients with
mild to moderate essential hypertension. The quality of included studies was critically evaluated. A total
of 735 articles were found and 19 articles were finally included. Heterogeneity test: efficacy analysis (Q
statistic = 4.60, p = 1.00, I2 = 0%), safety analysis (Q statistic = 12.19, p = 0.84, I2 = 0%). The results of
Meta-analysis confessed that there were no significant differences either in efficacy or safety between
Candesartan and other active antihypertensive agents. Funnel-plot displayed a symmetrical figure,
indicating there was no publication bias. In an analysis excluding the 6 low quality trials, our results
were unchanged indicating the sensitivity of the Meta-analysis was fine. The evidence currently
available shows that Candesartan has the similar efficacy and safety compared with other active
antihypertensive agents with mild to moderate essential hypertension.
Key words: Candesartan, essential hypertension, systemic review, meta-analysis.
INTRODUCTION
Hypertension is one of the most common cardiovascular
and cerebrovascular diseases and is associated with
human fatal diseases such as coronary artery disease
and cerebrovascular diseases. Data from the National
Health and Nutritional Examination Survey and from the
World Health Organization have clearly demonstrated
that, worldwide, less than 30% of hypertensive patients
are adequately controlled by our currently accepted blood
pressure goals (Papadopoulos et al., 2008). So the
important task which medical workers have to face is how
to choose a safe and effective antihypertensive drug.
Angiotensin receptor blocker (ARB) that works by
blocking the rennin angiotensin system (RAS) is a new
kind of drugs for hypertension and it was included in the
*Corresponding author. E-mail: qinling1958@yahoo.com.cn.
Tel: +86 15843073203. Fax: +86 0431 84841049.
ranks of first-line antihypertensive agents in 2005 by
Chinese Guideline for the Prevention and Treatment of
Patients with Hypertension (The Committee to Rebise,
1999; Chinese Guideline for the Prevention and
Treatment of Patients with Hypertension, 2004).
Candesartan is a new member of ARBs used in clinic
after Losartan, Valsartan and Irbesartan. Candesartan
was first used for patients with hypertension in Sweden in
1997 and the curative effects were affirmative (Belcher et
al., 1997). Candesartan has gone on the market in
Chinese for several years, however, there have been no
evidences about the efficacy and safety of which from
evidence-based medicine yet.
Though there are several studies about Candesartan in
treatment of hypertension, the conclusions of which are
not credible because of small sample size and lacks of
systemic evaluation of methodologic quality. This study
makes a systemic review about clinical random control
trials (RCTs) focused on Candesartan in treatment of mild

to moderate essential hypertension in Chinese in order to
obtain the best evidence about the efficacy and safety of
Candesartan in treating Chinese patients with mild to
moderate essential hypertension.
METHODS
Search strategy
The search strategy was made according to working handbook
4.2.7 from the Cochrane collaboration (Sackett et al., 2002). We
systematically searched Cochrane central register of controlled
trials (Issue 3, 2009), MEDLINE (1991 to May 2009), EMbase (1991
to May 2009), CBMdisc (1991 to May 2009), and CNKI (1994 to
May 2009) for randomized trials examining the efficacy and safety
of Candesartan on mild to moderate essential hypertension among
Chinese people. In addition, we conducted a manual search of
abstracts from selected conferences and we also searched by hand
the bibliographies of all relevant trials. The following search criterion
was used: (“hypertension” or “essential hypertension”) and
(“Candesartan” or “Candesartan Cilexetil”) and language is limited
to English or Chinese.
Study selection
Two reviewers independently conducted the literature search and
extraction of relevant articles. The title and abstract of potentially
relevant studies were screened for appropriateness before retrieval
of the full articles. The following selection criteria were used to
identify published studies for inclusion in this meta-analysis: (a)
study design—RCTs; (b) population—Chinese patients with mild to
moderate essential hypertension (WHO-ISH Hypertension
Guidelines Committee, 1999; Committee of guidebook on
prevention and treatment of hypertension, 2000); (c) intervention—
Candesartan versus other active antihypertensive agents as
monotherapy; (d) outcome variable—overall response rate and
adverse reaction rate; (e) efficacy criteria—recommendation on
evaluation methods of clinical trials about cardiovascular drugs in
Guiding principles for clinical research of new drugs made by
Chinese Ministry of Health in 1993 (Liu et al., 1998).
Data extraction
From each study, the following information was abstracted: author,
year of publication, study design, characteristics of the population,
simple size, treatment proposal, time of the therapy, overall
response rate and adverse reaction rate.
Assessment of study quality
Jadad score was used to assess the methodologic quality of the
trials by two reviewers (Jadad et al., 1996). Articles gained 1 to 2
points were regarded as low quality and the ones gained 3 to 5
points were regards as high quality (Moher et al., 1998).
Statistical methods
For dichotomous outcomes, we calculated a pooled odds ratio (OR)
and 95% confidence interval (CI). The OR was defined as the odds
of an outcome in those who received Candesartan compared with
the odds in those who received other active hypertensive agents.
The ORs of different RCTs were combined by using the random-
Wen-peng et al. 1013
effects model of Der Simonian and Laird (Der Simonian et al.,
1986), if true between-study heterogeneity exists or else using
Mantel and Haenszel fixed-effects model instead (Mantel et al.,
1959). Intertrial statistical heterogeneity was explored using the
Cochran Q test with calculated I 2 , indicating the percentage of the
total variability in effect estimates among trials that is, due to
heterogeneity rather than to chance (Higgins et al., 2003). I 2 values
of 50% or more indicate a substantial level of heterogeneity. We
evaluated the presence of publication bias by means of visual
inspection of the funnel plot (whether it was symmetrical or not). To
exclude the possibility that any one study was exerting excessive
influence on the results, we conducted a sensitivity analysis by
excluding those studies with low quality and then rerunning the
analysis to assess the change in Ors. All p values were two-sided
with statistical significance set at an α level of 0.05. We followed the
“quality of reporting meta-analysis guidelines” for reporting and
discussing these Meta-analytical results (Moher et al., 1999).
All the statistical analysis was carried out by the Cochrane
collaboration’s RevMan 4.2 software.
RESULTS
Characteristics of trials
There were 735 articles relevant to the search term and
19 articles (Chen, 2007; Gao and Jiang, 2008; Liu, 2008;
Lv, 2008; Xu, 2008; Zhang et al., 2006, 2007; Huang,
2007; Wang et al., 2007; Fu et al., 2006; Geng et al.,
2006; Hu et al., 2006; He and Yi, 2006; Chen et al., 2005;
Qian et al., 2005; Xu et al., 2005; Chang et al., 2004;
Huang et al., 2004; Sun et al., 2003) involving 1587
Chinese patients with mild to moderate essential
hypertension (group Candesartan: 794 patients, group
control: 793 patients) were included in this Meta-analysis
finally. Ages, sex ratio and initial blood pressure were
similar in each group, respectively. The flow chart for the
selection of RCTs to be included in our analysis is shown
in Figure 1. The characteristics of these trials were
showed in Table 1.
Methodologic quality assessment
All the trials included in this Meta-analysis mentioned the
term ‘random’, but the detail method was illuminated in 1
article only. There were 13 trials mentioned the term
‘double blind’, but only 9 articles explained the detail
method. All the 19 trials described the data of the patients
who withdrew during the treatment. According to the
Jadad score, 13 articles and 6 articles were regarded as
high quality literature and low quality literature, respectively
(Table 1).
Heterogeneity test
We choose fixed-effect model to make Meta-analysis
because there were no significant heterogeneities
between studies in both efficacy analysis (Q statistic =
4.60, p = 1.00, I 2 = 0%) and safety analysis (Q statistic =

1014 Afr. J. Pharm. Pharmacol.
Potentially relevant reports (n=735)
Potentially relevant reports(n=735)
CBMdisc (n=110)
CBMdisc (n=110)
CNKI (n=98)
CNKI (n=98)
Cochrane Library (n=1)
Cochrane Labrary(n=1)
Labrary(n=1)
MEDLINE MEDLINE (n=242)
EMbase EMbase (n=264)
RCT retrieved (n=423)
RCT retrieved (n=22)
RCT finally included (n=19)
Figure 1. Flow chart of article selection.
Table 1. Characteristics of important studies admitted.
Studies Groups
Chen (2008)
Gao (2008)
Liu (2008)
Lv (2008)
Xu (2008)
Treatment
proposal (mg/d)
Time of therapy
(weeks)
Duplicates removed(n=312)
RCT removed by reading titles and abstracts:
Animal experiment(n=32)
Review(n=26)
NOT focus on Chinese patients(n=193)
Focus on old people only (n=24)
Not focus on blood pressure (n=44)
Not RCT with positive grugs (n=15)
Not mild to moderate essential hypertension(n=39)
Not use Candesartan only(n=28)
RCT removed by reading full texts:
repetat publication(n=1)
data not adequate (n=2)
Sample
size
Overall response
rate (%)
Adverse reaction
rate (%)
Candesartan 4 8 38 94.3 10.8
Perindopril 4 8 42 89.7 22.5
Candesartan 8 4 50 76.0 2.0
Amlodipine 5 4 50 74.0 0
Candesartan 4 8 45 84.4 8.9
Valstartan 80 8 45 82.2 11.1
Candesartan 4 8 33 87.9 10.0
Enalapril 10 8 32 75.0 12.5
Candesartan 8 8 28 89.3 0
Enalapril 10 8 28 85.7 14.3
Candesartan 4 8 36 75.0 8.3
Losartan 50 8 36 71.4 11.1
Jadad
score
2
2
2
2
2

Table 1. Contd.
Huang (2007)
Wang et al.
(2007)
Fu (2006)
Geng (2006)
Hu (2006)
He (2006)
Zhang et al.
(2006)
Chen et al.
(2005)
Qian et al.
(2005)
Xu et al. (2005)
Chang et al.
(2004)
Huang et al.
(2004)
Sun (2003)
Candesartan 4 8 23 82.6 8.7
Eanlapril 5 8 24 87.5 29.2
Candesartan 4 8 40 85.0 5.0
Valsartan 80 8 40 87.2 5.0
Candesartan 8 8 30 80.0 13.3
Irbesartan 150 8 30 76.7 16.7
Candesartan 8 8 24 81.8 11.7
Losartan 50 8 24 81.0 0
Candesartan 8 4 29 89.7 6.9
Irbesartan 150 4 29 79.3 0
Candesartan 4 20 66 74.2 7.6
Fosinopril 10 20 64 73.4 9.4
Candesartan 4 8 32 87.5 9.4
Valsartan 80 8 30 86.7 6.7
Candesartan 8 8 24 91.7 8.3
Losartan 50 8 24 91.7 8.3
Candesartan 8 8 127 82.2 3.9
Losartan 50 8 127 84.2 1.6
Candesartan 8 8 110 81.9 12.7
Enalapril 10 8 108 77.8 16.5
Candesartan 8 8 20 75.0 5.0
Losartan 50 8 20 70.0 10.0
Candesartan 8 8 30 82.1 3.1
Losartan 50 8 31 76.7 6.5
Candesartan 8 8 18 77.8 5.6
Losartan 50 8 18 72.2 11.1
12.19, p = 0.84, I 2 = 0%) in our primary analysis.
Meta-analysis of efficacy
Overall response rates of both group: Candesartan and
group control were recorded in all the 19 trials finally
included. Active antihypertensive agents involved in this
analysis were Losartan, valsartan, Irbesartan, Enalapril,
Fosinopril, Perindopril and Amlodipine. The results of
Meta-analysis confessed that there were no significant
differences in efficacy between Candesartan and control
Wen-peng et al. 1015
group in treating Chinese patients with mild to moderate
essential hypertension (Figure 2).
Meta-analysis of safety
Adverse reaction rates of both Candesartan and control
group were recorded in all the 19 trials finally included.
Main adverse reactions of Candesartan group were
headache and dizziness. Otherwise, chief adverse reactions
of control group were cough, headache, dizziness
and gastrointestinal symptoms. The results of Meta-analysis
2
4
4
3
4
3
4
4
4
4
4
4
3

1016 Afr. J. Pharm. Pharmacol.
Figure 2. OR estimates with the corresponding 95% CI for the efficacy. The OR estimate of each study is marked with a ■.The size of the
square represents the weight that the corresponding study exerts in the meta-analysis. The CIs of pooled estimates are displayed as a horizontal
line through the diamond, this line might be contained within the diamond if the confidence interval is narrow.
analysis confessed that there were no significant
differences in safety between Candesartan and control
group in treating Chinese patients with mild to moderate
essential hypertension (Figure 3).
Publication bias
An analysis of publication bias was conducted. No
evidence of publication bias was found since the funnel
plots was symmetrical based on a visual analysis (Figure
4).
Sensitivity analyses
In an analysis excluding the 6 low quality trials, our
results were consistent with those found in our main
analysis described earlier: in the efficacy analysis, there
was no difference in overall response rates between
Candesartan and control group [Z = 0.79 (p = 0.43), OR =
1.13, 95% CL (0.84~1.51)], furthermore, no difference
was found in adverse reaction rates between
Candesartan and control group in the safety analysis [Z =
0.05 (p = 0.96), OR = 0.99, 95% CL (0.65~1.51)].
DISCUSSION
Summary of the literature quality
A total of 19 literatures were finally included in this
systemic review. All these articles, including a sample
size of 1587 totally were RCTs. Jadad score in 13 out of
the 19 articles were more than two points and the results
were not changed significantly after removing the other 6
articles with Jadad score less than three points.
Moreover, no evidence of publication bias was found and
there were no significant heterogeneities between studies
in both efficacy analysis and safety analysis, too. It was
suggested that the overall quality of this systemic review
was high.
However, there was still methodological insufficiency:
(a) Randomization method may not be rigorous because
the specific program of randomization was inferred in
only one literature.
(b) Selection bias may exist for allocation concealment
was not described in all of these articles included.
(c) Selection bias, measuring bias and implementation
bias may exist because 7 studies did not describe
whether blind method was used or not.

Wen-peng et al. 1017
Figure 3. OR estimates with the corresponding 95% CI for the safety. The OR estimate of each study is marked with a ■.The
size of the square represents the weight that the corresponding study exerts in the meta-analysis. The CIs of pooled
estimates are displayed as a horizontal line through the diamond, this line might be contained within the diamond if the
confidence interval is narrow.
Figure 4. Funnel plot.
Analysis of efficacy and safety
Candesartan, a new non-peptide ARBs, takes antihypertensive
role by selectively combining the AT1 receptor,
subtype of angiotensin Ⅱ receptor, inhibiting RAS system
and then blocking vascular smooth muscle contraction
mediated, sympathetic nerves excitation and aldosterone
release (Perrone-Filardi et al., 2009). Since AT1 receptor

1018 Afr. J. Pharm. Pharmacol.
subtype is combined with Candesartan, angiotensin
Ⅱ has to combine and stimulate with AT2 receptor
subtype which may induce a further step of antihypertension.
Compared with other ARBs, Candesartan has
the characteristics of stronger affinity to AT1 receptor
subtype and slower dissociation rate (Unger, 2000), so it
has the smallest application dose in the current listing of
ARBs. The results of this systemic review showed that
there were no significant differences in efficacy in treating
Chinese patients with mild to moderate essential
hypertension between Candesartan and control group.
Thus we can conclude that Candesarntan has the same
antihypertensive effect compared with other first-line
antihypertensive drugs. Because of the specificity of
reacting in RAS system, Candesartan may void the
complication of cough and angioneurotic edema (Kim-
Mitsuvama, 2009) while using ACEI in the treatment of
hypertension. The adverse effects, including dizziness
and headache mainly, of Candesartan in treating
essential hypertension referred in this study were less
likely to happen and tolerated, moreover, it was not
necessary to stop administrating. The results of this
systemic review showed that there were no significant
differences in safety in treating Chinese patients with mild
to moderate essential hypertension between
Candesartan and control group.
It suggests that Candesartan has the similar safety
compared with other positive antihypertensive agents.
CONCLUSION
In summary, the evidence currently available shows that
Candesartan has the similar efficacy and safety
compared with other active antihypertensive agents in
treatment of Chinese patients with mild to moderate
essential hypertension. However, as the methodological
insufficiency more literatures with high quality are needed
to obtain more rigorous and objective clinical evidence.
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Wen-peng et al. 1019

African Journal of Pharmacy and Pharmacology Vol. 5(8). pp. 1020-1026, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.015
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Reducing queues in a Nigerian hospital pharmacy
Ndukwe H. C. 1 *, Omale S. 1 and Opanuga O. O. 2
1 Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, University of Jos, PMB 2084, Jos, Plateau
State, Nigeria.
2 Outpatient Pharmacy Department, Lagos University Teaching Hospital, PMB 12003, Idiaraba, Lagos State, Nigeria.
Accepted 8 April, 2011
Queues are characterized structures formed to maintain order and create a hold on time, money and
human contribution towards development and efficient performance of any system. The aim of this
work was to characterize the queue, describe the queue discipline of the outpatient pharmacy, to
institute a cross-sectional intervention by streamlining queue behaviour and to measure the impact of
streamlining queue characteristics and queue discipline on waiting time of patients. Results showed
that queue characteristics existing at the pharmacy during the situation analysis was a single servermultiple
queue model. However, after the intervention was done involving staff re-orientation, the
streamlined process reduced waiting time from 167.0 to 55.1 min. Queue discipline was strictly
instituted by designed tally cards that were serially numbered. The characterization and discipline that
was instituted handled and/or eliminated the challenge of shunting, balking or jockeying and reduced
reneging. The workflow chart was sketched and drawn to scale, aiding the collation of baseline data
and for proposed structural modifications. Other results obtained include the waiting area to pharmacy
space ratio, which gave a good result of 1:9. Effort should therefore be intensified by hospital
pharmacists to reduce patient queues and improve efficiency of services, following the results of
snapshots from this work.
Key words: Queue characteristics, queue discipline, outpatient pharmacy.
INTRODUCTION
The effect of queuing during hospital visits in relation to
the time spent for patients to access treatment in
hospitals is increasingly becoming a major source of
concern to a modern society that is currently exposed to
great strides in technological advancement and speed
(Stakutis and Boyle, 2009). Internal operational factors
majorly determine outpatient waiting times and include;
arrival pattern of prescriptions, sequencing of work,
percentage of staff at work, interaction between the
pharmacy service providers that is assessor and
technician interaction or technician and counselor
interaction (Moss, 1987).
A queue is a waiting line, whether of people, signals or
things (Ashley, 2000). Queuing time is the amount of time
a person, signal or thing spends before being attended
to, or before value adding work is performed to or on it
*Corresponding author. E-mail: chuksemail@yahoo.com,
ndukwe.henry@gmail.com.
(Customer Management IQ, 2011). In many factories,
queue time constitutes about 90% of the total lead time
(Crabtree, 2008). Queue time can be extended to
hospitals pharmacies where patients complain about the
amount of time spent before treatment can be received
(Bunday, 1996; Zhang et al., 2000). Queues deal with
problems which involve waiting for a product or service.
Typical examples might be;
hospital/pharmacies/banks/supermarkets waiting for
service, computers used in hospital management
information system (HMIS) waiting for response, power
failure situation that involves waiting for electric power to
be restored to enable a piece of machinery used for
compounding a prescription medication function. Another
example is the public transport system- waiting for a train
or a bus to convey a consultant pharmacist to the hospital
in the advent of an accident, poisoning or hypertensive
emergency. Queuing models have mainly been used to
study congestion for interrupted traffic flows at signalized
and unsignalized intersections (Woensel and Cruz,
2009). However, it has been shown that they can also be

Arrivals
Queue
Figure 1. Single server single queue model.
usefully applied to describe and analyze congestion for
uninterrupted traffic flows in spite of the absence of the
development of a formal queue. Queuing models applied
to traffic situations provide an adequate description of the
complex dynamic and stochastic environment under
study (Woensel and Cruz, 2009).
In designing queuing systems we need to aim for a
balance in services rendered to clients. In essence, all
queuing systems can be broken down into individual subsystems,
consisting of entities queuing for some activity
e.g. dispensing and counseling. Queue characteristics
determines how, from the set of clients waiting for
service, we choose the one to be served next (e.g. FIFO
(first-in-first-out); LIFO (last-in-first-out); randomly). This
order instituted for waiting is often called the queue
discipline (David, 2005). Queue discipline can be
examined so as to determine the queue characteristics to
implore over a given period in providing pharmaceutical
services from the outpatient section or from any other
outlet (David, 2005; Noesk and Wilson, 2001). The queue
discipline can include balking (clients or patients deciding
not to join the queue if it is too long), reneging (clients or
patients leave the queue if they have waited too long for
service), jockeying (clients or patients switch between
queues if they think they will get served faster by so
doing).
Moreover, control can be attained using an electronic
or manual tally to select a client to be served, a queue of
finite capacity or of infinite capacity. Changing the queue
discipline can often reduce congestion. The queue
discipline often requires we choose the customer with the
shortest service time, which will result in the lowest
average value of time a client spends queuing. For example,
attending to all outpatients with single-drug
prescription orders before those with multiple-drug
prescription orders will result in lower monetary yield that
can influence a functional drug revolving scheme and
ultimately the effectiveness of work hours for a pharmacy.
It is important to appreciate the fact that the integral to
queuing situations is the idea of uncertainty in inter-arrival
times and service times (Willig, 1999).
Analysis of queuing situations and questions of interest,
are those typically concerned with measures of system
performance and include how long a customer expects to
wait in the queue before being served or how long it will
take to wait before service is complete. There are factors
Service facility
(Dispensing &
Counseling)
Room)
Ndukwe et al. 1021
Patient/client Leaves
that need to be defined and controlled by health
practitioners so that management can evaluate
alternatives in an attempt to control/improve queue
situations. Some of the problems that can often be
investigated in practice include the effort invested in
reducing the consultation, counseling or service time
employed in practice. In addition, the number of
pharmacists, pharmacy technicians, nurses, laboratory
scientists or physician servers that should be employed
to optimize efficiency or the introduction of priorities for
certain types of clients, could serve as indicators. The
adequacy of the waiting area for patients is subject to
constant review by the hospital management board. The
two basic approaches to solve these challenges involve
the use of analytic methods or queuing theory for simple
queues and simulated (computer based) application for
complex queues. Queuing notations are commonly
denoted by symbols; λ (lambda) being the mean arrival
rate or average number of arrivals per time period, and µ
(mu) the mean service rate or average number of
clients/patients that are served (dispensing and
counseling) per time period. Waiting in lines or “queues”
seems to be a patient’s pastime. The frustration
associated with those waits whether in line at the
stoplight, or in the pharmacy, “waiting our turn” is part of
everyday life. According to David (2005) and Zhang et al.
(2000), the types of ordered queues, to institute
discipline, are represented diagrammatically as shown in
Figures 1 to 6).
There are standard notation systems to classify
queuing models these are shown in Figures 1 to 6
(Kolobe, 2006). The server in this case is the pharmacist;
Figure 1 represents the single server- single queue
model (either being served or waiting for service), Figure
2 represents single server – multiple queue model, Figure
3 represents multiple (parallel) servers single queue
model, Figure 4 represents the multiple (parallel) servers
multiple queues model and Figure 5 represents the
Multiple servers in a series to meet specialized needs
(e.g. in an HIV/AIDS centre). Figure 6 represents multiple
servers in parallel to meet specialized needs and cases
(e.g. in a DOTs centre for tuberculosis patients). Based
on the earlier queuing characteristics, the hospital
pharmacy queuing system can then be classified
following the conventional system employed globally
(Munro et al., 2007).

1022 Afr. J. Pharm. Pharmacol.
Arrivals
Queues
Fingure 2. Single server – multiple queue model.
Arrivals
Arrivals
Queue
Figure 3. Multiple (parallel) servers single queue model
Arrivals
Queues
Figure 4. Multiple (parallel) servers multiple queues model.
Service facility
(Dispensing &
Counseling
Room)
Dispensing and counseling rooms
Dispensing and counseling rooms
Dispensing
room
Queue Patients move in
Figure 5. Multiple servers in a series to meet specialized needs (e.g. in an HIV/AIDS centre).
Arrivals
Queues
Patient Leaves
Counseling
room
Dispensing rooms Counseling rooms
Patients
Leave
Patients
leave
Patients
leave
Figure 6. Multiple servers in parallel to meet specialized needs and cases (e.g. in a DOTs centre for tuberculosis
patients).

Queuing theory and its application has gotten very little
attention from pharmacy operations management.
However, pharmacy practice could benefit by
understanding and applying some of the theory’s
concept. Donehew et al. (1978), used queuing theory to
address prescription queues and work measurement
assessment of prescription fill times (WHO/EDM, 1999).
Vemuri (1984) used computer simulation with a queuing
model to assess patient waiting time in the outpatient
pharmacy at the medical college of Virginia. This study
concluded that the most significant factor contributing to
patient waiting times was the interaction between
pharmacy service providers, specifically the pharmacist
and the technician. In a study by Moss (1987), queuing
theory was used to assess the relationships among the
number of pharmacy staff members, prescription
dispensing processes and outpatient waiting times. He
used a mathematical queuing model to estimate the
probability of waiting time exceeding a given value when
prescription arrival, service rates and number of servers
are known. The study revealed that the major factors
determining outpatient waiting time were the arrival
pattern of prescriptions at the pharmacy, sequencing of
work and percentage of staff at work.
Noesk and Wilson (2001) briefly discussed queuing
theory using advanced mathematical models from queuing
theory and customer wait-time on operations
management for pharmacists. However, the only
suggestion offered by the authors for managing perceived
waiting time was to distract the client by providing
entertainment, refreshments or comfortable conditions,
such as television and coffee in the waiting area
(WHO/EDM, 1993).
Similarly, Boyce and colleagues sought to determine
the impact of a computerized waiting time program on
order-turnaround time in a hospital pharmacy
(WHO/EDM, 2002). Perhaps the most common and
useful application of queuing theory in pharmacy
operations is to reduce patient waiting time and maximize
staff effectiveness. Lin et al. (1999) used workflow
analysis and time study to identify factors leading to
excessive waiting times in an ambulatory pharmacy at
the University Hospital Inc., Cincinnati, Ohio. In another
study by Lin, work measurement and computer simulation
were used to assess the re-engineering of
community pharmacies to facilitate patient counselling.
Though queuing theory was never mentioned in these
articles, the authors used many concepts similar to
queuing theory’s and their results could be instrumental
in designing queuing applications for reducing patient
waiting time and improving staff utilization. Analytic
queuing network models often assume infinite capacity
queues due to the difficulty of grasping the betweenqueue
correlation. This correlation can help to explain the
formation and spread of congestion. Osorio and Bierlaire
(2009) presented an analytic queuing network model for
finite capacity of queues and used structural parameters
Ndukwe et al. 1023
to comprehend the between-queue correlation. This
novel model maintained the network topology and the
queue capacities exogenous.
In addition, congestion was directly modeled via a
novel formulation of the state space of the queues which
explicitly captured the blocking phase, revealing the
sources and effects of congestion. Automated queuing
technology (AQT) is primarily utilized in the federal sector
and includes numerous pharmacies like the Department
of Veterans Affairs (VA), and the Department of Defence
(DoD) of Navy hospital pharmacies in the United states.
However, several prominent foreign pharmacy organizations
utilize AQT, including the University of North
Carolina, Medical College of Virginia, Jewish Hospital in
Cincinnati and Parkland Hospital in Dallas (David, 2005).
Both the DoD and the VA operate very busy outpatient
pharmacy departments, some filling in excess of 2,500
outpatient prescriptions and servicing over 1,000 patients
daily (David, 2005).
Automated queuing systems are typically PC based
systems that can track a multitude of useful information
that was previously very difficult to quantify for pharmacy
managers. Pharmacies utilizing AQT can easily track
variables such as customer arrival and departure time,
patterns of arrival, prescription-fill time, waiting time and
individual staff member productivity. In addition, AQT can
track numerous points of service and different service
categories (that is, certain patients may get priority
service or can be used to track patient counselling) if
desired. AQT can also provide clients with information
that can directly improve their queuing experience, such
as having a ticket with a unique number and the
estimated wait time. This makes for a less confusing,
more relaxed, and much more positive waiting environment
for the patient.
At the time of this study, QMatic Corporation was
identified as an example of a corporate body that
distributes automated queuing technology systems
(David, 2005). Many pharmacies do not experience
problems with queues; however, there are many that
experience difficulties with queue formation. For example,
pharmacies that experience high-volume prescription
workload frequently have difficulty in managing workflow
and waiting times. This could also be true in pharmacies
that offer their clients multiple points of service (that is,
bank teller design type). Pharmacies such as those in
large managed care organizations and university health
systems typically fit this description. It is safe to say that
the traditional methods employed by pharmacies to
distract clients (e.g. comfortable waiting area, coffee and
television) would be of limited benefit in pharmacies that
fill in excess of 1,000 prescriptions per day and have
patient waiting times that commonly exceed one to two
hours. Automated queuing technology has been
successfully developed and applied in areas of pharmacy
practice that specifically address customer waiting times.
Prior to this innovation, the most advanced queuing

1024 Afr. J. Pharm. Pharmacol.
applications to manage customer waiting times in
pharmacies was a consecutive number ticketing system
commonly found in shops and stores.
The aim of this work includes characterizing the queue,
to describe the queue discipline of the outpatient
pharmacy, to institute a cross-sectional intervention by
streamlining queue behaviour and pattern as well as to
measure the impact of streamlining queue characteristics
and queue discipline on waiting time of patients.
METHOD
Queuing discipline was strictly instituted by designing tally cards
that were serially numbered. Workflow analysis that aids
characterization of queues in the outpatient pharmacy involves the
sketching of a schematic flowchart as shown in Appendix 1,
representing the system of activities in the pharmacy that served
the patients. The workflow highlighted on the sequence of activities
involved in the dispensing procedure and dealt with what could be
done, how fast it could be done, and how well it fitted into our
present needs in the queue forms. The SPO (structure, process
and outcome) model was applied to analyze the current situation of
workflow present in the outpatient pharmacy (Marc, 2004).
RESULTS
Queue characteristics and queue discipline practiced
in the outpatient pharmacy
The queue characteristics existing at the pharmacy
during the situational analysis was single server-multiple
queue model. However, after staff re-orientation and
interaction, the intervention to streamline process and
reduce waiting time was done and the queue characterristics
adopted by consensus and practiced was that of
multiple servers, single queue model as adopted and
modified (David, 2005; Zhang et al., 2000).
Queuing discipline was strictly instituted by designed
tally cards that were serially numbered. The characterization
and discipline that was instituted handled and/or
eliminated the challenge of shunting, balking or jockeying
and reduced reneging. The patient waiting time was
reduced from 167.0 to 55.1 min which indicated a 67%
reduction in time.
Other observed results
Other results obtained and identified include the waiting
area to pharmacy space ratio that gave an approximate
result of a 1:9 (21.72:195.16 (m 2 )). The workflow chart
was sketched and drawn to scale with suggested inputs
as seen in Appendix 1.
DISCUSSION
Process control techniques was used to identify extremes
in waiting time; root-cause analysis was employed to
identify specific delay causes and minimize the contribution
of the root causes which led to an improvement in
queue formations and overall system performance.
During data collection, decrease in the occurrence of
excessive clinic delays caused a large and sustained
decrease in queues and in patient waiting times which
was consistent with results of the work done (Groome
and Mayeaux, 2010). Simple and compound waiting
times are implicated in an attempt to access treatment
through queues from the perspective of the patient
(David, 2005).
In a study carried out, patients experienced difficulty in
accessing treatment at healthcare facilities because of
inconvenient opening hours and provider absenteeism as
reported (Munro et al., 2007). About 60% of the respondents
reported spending four hours and above at the
clinic with some cases where participants spent the
whole day on queues after coming two hours earlier than
opening time.
However, various methods have been adopted to
reduce queues to the barest minimum and this has led to
several techniques employed by health care facilities to
queuing and its characteristics on queuing systems,
service or server efficiency, service space and service
point(s) provided (Kolobe, 2006). The queue characteristics
existing at the pharmacy during the situational analysis was
that of a single server multiple queue model as
characterized (Zhang et al., 2000). However, after staff reorientation
in strategy, technical and operational factors, and
after proper intra-professional interaction, an intervention
to streamline the queue patterns and reduce waiting time
was implemented. The queue characteristics adopted,
guided by a Delphi-technique consensus involving staff,
was that of multiple severs single queue model.
Considering the insufficient pharmacy staff strength in the
health facility, the adoption was immediately
implemented.
Analyzing the results of a previous work done by Green
et al. (2006), timely access to a provider was observed as
a critical dimension for quality performance in emergency
department of hospitals. In an understaffed environment,
analyses of arrival patterns and the use of queuing
models was useful in identifying the most effective allocation
of staff. Queuing discipline was strictly instituted,
using a time monitoring card in a first come-first serve
(FCFS) order. The queue characterization and discipline
instituted, controlled and/or eliminated the challenge of
shunting, balking and jockeying. Having determined the
waiting area to waiting space ratio to be 1:9
(21.72:195.16 (m 2 )), the recommended queuing
characteristics best for the outpatient pharmacy would
have been multiple (parallel) servers multiple queues
model. The streamlined processes reduced overall
patient waiting time by 67%; from 167.0 to 55.1 min. The
workflow chart was drawn to scale with suggested
structural modifications recommended to improve on the
existing outpatient pharmacy design to improve on

workflow as seen in Appendix 1. A similar study
conducted in a large VA hospital reported that pharmacy
redesign improved patient satisfaction because of a 50%
decrease in patient waiting time (Noesk and Wilson,
2001). Finally, another article described the relationship
between waiting time and satisfaction in the context of
social justice or injustice, as the case may be (Higby,
2002).
Conclusion
Queue characterization of a hospital’s outpatient
pharmacy is now possible for its queue characteristics
and discipline. A cross-sectional intervention that
involved streamlining queue behaviour and pattern was
implemented. The queue characterization was that of
multiple severs single queue model. Queuing discipline
was strictly instituted, using a time monitoring card in a
first come-first serve (FCFS) order.
The impact of streamlining workflow pattern on queue
characteristics and discipline was measured and found to
reduce waiting time by up to 67% from baseline data.
Problems manifesting, specifically at the health facility
under study included long waiting times, queues, lack of
privacy, inconvenient appointment times and the poor
upkeep of clinic appointments by patients.
Studies carried out elsewhere, were consistent with
results from this work, the major issue that came strongly
from the participants was the amount of time they spent
waiting for service (Madaki, 2000). Therefore, effort
should be geared towards developing appropriate techniques
by which hospital pharmacists can reduce patient
queues and improve efficiency of services rendered.
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1026 Afr. J. Pharm. Pharmacol.
Appendix 1. Workflow pattern depicting patient flow in and out of the outpatient pharmacy for LUTH.
Compounding
room for
special cases
Staff
Pharmacy
Unit
9.32 m
Cashier room
Compounding section
Stacking section
13.97 m
WAITING AREA
Chairs and Benches
≈ 18.63 m
0.69m
benches
≈4.66 m
Client
leaves
Validation/billing shutter Dispensing/Counseling Shutter for pharmacists
Prescription filling section – with pharmacy attendants
Controlled drugs section
Arrival from
consultation room
Counseling room
Counseling room
Counseling room
Counseling room
STAFF ENTRY
0.69 m
Black writings: existing structure; red writings: proposed (suggested) structural inputs; arrows: direction of patient queue formation pattern in and out of the pharmacy.
13.63 m

African Journal of Pharmacy and Pharmacology Vol. 5(8), pp. 1027-1033, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.026
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
The effect of gallic acid on kidney and liver after
experimental renal ischemia/reperfusion injury in the
rats
Mediha Canbek 1 , Mehmet Cengiz Ustüner 2 , Sahin Kabay 3 , Onur Uysal 4 , Hilmi Ozden 5 *,
Gökhan Bayramoğlu 1 , Hakan Sentürk 1 , Cansu Ozbayar 2 , Aysegül Bayramoglu 2 , Derya
Ustüner 6 and Irfan Degirmenci 2
1 Department of Biology, Faculty of Arts and Science, Eskisehir Osmangazi University, Turkey.
2 Department of Medical Biology, Faculty of Medicine, Eskisehir Osmangazi University, Turkey.
3 Department of Urology, Treating and Research Hospital, Dumlupınar University Kütahya, Turkey.
4 Vocational School of Health Services, Eskisehir Osmangazi University, Turkey.
5 Department of Anatomy, Faculty of Medicine, Eskisehir Osmangazi University, Turkey.
6 Department of Medical Genetic, Faculty of Medicine, Eskisehir Osmangazi University, Turkey.
Accepted 24 June, 2011
To investigate the effect of gallic acid (GA) on kidney and liver against oxidative stress during renal
ischemia-reperfusion (I/R) injury, by determining biochemical parameters and evaluating histological
examinations.
Key words: Gallic acid, ischemia-reperfusion, kidney, liver, antioxidant enzyme.
INTRODUCTION
Many urological surgeries such as partial nephrectomy,
traumatic kidney repair, renal vessels repair, and renal
transplantation use clamping of the renal arteries as a
common procedure. Ischemia and reperfusion (I/R)
induce oxidative stress and free radical formation. I/R
injury is a complex pathophysiological process, which can
eventually lead to cell damage, cell death, increased
vascular permeability, tissue necrosis, and multi organ
dysfunction (Salehipour et al., 2010). Restoration of blood
flow to ischemic tissues can result in recovery of cells if
the injury is not permanent. However, depending on the
intensity and duration of the ischemia, the variable
number of cells may still die after blood flow began
(Yurdakul et al., 2010). Oxidative stress is a relative
excess of oxidants caused by increased free radical
production and/or decreased antioxidant defense
systems that impairs cellular function and contributes to
the pathophysiology of many diseases (Zhao, 2005). The
protective effects of antioxidants on kidney tissue, including
*Corresponding author E-mail: hilmi_ozden@yahoo.com.
renal I/R injury, have yielded a reduction of lipid
peroxidation and positive effects on the antioxidant
system status (Aktoz et al., 2007). Reactive oxygen
species (ROS) produced in oxidative stress are
scavenged by superoxide dismutase (SOD), glutathione
peroxidase (GSH-Px) and catalase (CAT). It has been
demonstrated in numerous studies that ROS are directly
involved in oxidative damage of cellular macromolecules
such as lipids, proteins, and nucleic acids in tissues.
Malondialdehyde (MDA) is the breakdown product of the
major chain reactions leading to oxidation of
polyunsaturated fatty acids and thus serves as a reliable
marker of oxidative stress-mediated lipid peroxidation
(LPO) (Serel et al., 2004; Ozguner et al., 2005). The most
widely recognized properties of tea polyphenols are their
antioxidative properties due to their ability to sequester
metal ions and to scavenge reactive oxygen species.
One of the polyphenols identified in tea is gallic acid (GA,
3, 4, 5-trihydroxybenzoicacid). Yang et al. (2001)
reported that antioxidant activity of tea polyphenols including
gallic acid has protection of carcinogenesis. GA is
a polyhydroxyphenolic compound which is widely used in
the traditional medicine, found in various natural products

1028 Afr. J. Pharm. Pharmacol.
such as gallnuts, tea leaves, bark, green tea, applepeels,
grapes, strawberries, pineapples, bananas,
lemons, and in red and white wine. It has been shown to
be an antiallergic, antimutagenic, anti-inflammatory and
anticarcinogenic agent and strong natural antioxidant. GA
inhibits melanogenesis which may be related to its ability
to scavenge reactive oxygen species (Jadon et al., 2007;
Niho et al., 2001). Multiple organ dysfunctions caused by
hepatic (Yoshidome et al., 1999) mesenteric (Aytekin et
al., 2005) or lower limb I/R have been reported previously
(Yassin et al., 2002). Wang et al. (2009) showed that
renal I/R can cause hepatotoxicity in rats. Another study
revealed in kidney transplantation model that, the liver
was affected during I/R process as evidenced by
antioxidant enzymes in pigs (Gulec et al., 2008). In this
study, we aimed to investigate the effect of GA on kidney
and liver against oxidative stress during I/R injury of the
kidney.
MATERIALS and METHODS
Animals
The experimental protocols were approved by the institutional
animal ethics committee. Twenty-eight adult male Sprague-Dawley
rats weighting 250 to 280 g were obtained from Medical and
Surgical Experimental Research Center (Eskisehir-Turkey) and
housed in polycarbonate cages in a room with controlled
temperature (22±2°C), humidity (50±5%), and a 12 h cycle of light
and dark (07:00 AM to 07:00 PM). Rats were fed laboratory
pellet chows and given water ad libitum. The animals were
divided into four groups (7 animals each); Group I (Control; nonischemic
animals); Group II (renal I/R injury+Saline), Group III
(renal I/R injury+ GA 50 mg per kg), Group IV (renal I/R injury+ GA
100 mg per kg).
Experimental protocol
Under anesthesia (100 mg/kg ketamine and 0.75 mg/kg
chlorpromazine), a right nephrectomy was performed and the rats
were allowed to recover for 15 days before they were subjected to
I/R injury. On the 15th day following nephrectomy, rats were fasted
overnight. The renal pedicle was occluded for 45 min to induce
ischemia and then subjected for 24 h of reperfusion (I/R groups).
GA (Matheson Coleman and Bell Manufacturing Chemists, USA)
was dissolved in saline as 2 cc/kg and was given as 50 and 100
mg/kg; I/R+ GA groups. GA or saline (I/R group) was administered
intraperitoneally 15 min before ischemia and 12 h after reperfusion.
The animals were decapitated after 24 h of reperfusion period. After
induction of I/R injury, on left kidney and liver histopathological
examinations were performed.
Histopathological evaluation
Left nephrectomy specimens and livers were processed routinely in
10% formalin solution, and embedded in paraffin. Tissue sections of
5 µm were obtained, and stained with hematoxylin and eosin
(H&E). All histopathological examinations were performed under a
light microscope (NIKON, Japan) by the histologist of the institute,
who was blinded to all tissue specimens regarding their group. A
minimum of 10 fields for each kidney and liver slides with minimum
x50 magnification were examined to assign the severity of the
morphological changes; tubular structure, glomerulus, cortex and
medulla were evaluated in renal histological examination. For the
evaluation of tubular structure; epithelial desquamation and
degeneration, loss of cilia, epithelial cell swelling, picnosis,
vacuolization, cast accumulation in the lumen and focal necrotic
areas were assessed. Glomerular congestion and necrosis were
examined. Congestion, polimorphonuclear leukocytes (PMNL) and
mononuclear leucocytes (MNL) infiltration were evaluated in cortex
and medulla. In addition, fibrosis in cortex was examined.
In examination of hepatic damage; congestion in central vein,
portal area and sinusoids, severity and location of hepatic bubbling
degeneration, parenchymal focal necrotic areas, PMNL and MNL
infiltration, karyolysis in hepatic nucleus, picnosis, loss of
intercellular border, damage in hepatic cords were evaluated using
scores.
Biochemical analysis
Homogenate preparation
After sacrificing the animals, kidneys and liver were quickly
removed and perfused immediately with ice-cold normal saline, and
homogenized in chilled potassium chloride (1.17%). The homogenate
was centrifuged at 800 × g for 5 min at 4°C to separate the
nuclear debris. The supernatant so obtained was centrifuged at
10,500 × g for 20 min at 4°C to get the homogenate which was
used to assay MDA, CAT, and SOD activity.
Determination of lipid peroxidation
MDA production was an end product of lipid peroxidation reacts
with thiobarbituric acid to form a red colored complex. 0.1 ml of
homogenate, 3 ml of 1% phosphoric acid, 0.5 ml of distilled water
and 1.0 ml of 0.6% 2-thiobarbituric acid were added. The mixture
was boiled in water bath for 45 min, followed by cooling in an ice,
and addition of 4.0 ml of n-butanol to extract the cold thiobarbituric
acid reactants. The optical density of the n-butanol layer was
determined at 532 nm after centrifugation at 1,000 g for five
minutes and expressed as nmol MDA/g of wet tissue (Mihara and
Uchiyama, 1978).
Assay of SOD activity
SOD activity was spectrophotometrically assayed with commercial
kits (Fluka SOD kit USA). It is an indirect assay method based on
xanthine oxidase and a novel color reagent. SOD activity in the
homogenate was determined by inhibition of Formosan dye (450
nm) employing the xanthin-xanthin oxidase enzymatic method to
generate superoxide radicals and calculated the active SOD
concentration according to inhibition curve graphic expressed as
U/g of wet tissue.
Assay of CAT activity
One unit (1U) of CAT equals the enzyme activity that recognized 1
µmol of hydrogen peroxide in 60 s at 37°C. CAT activity was
measured with determination of absorbance of three blank samples
at 405 nm. CAT activity (kU/L) was calculated as = [ (Absblank1-
Absblanksample) / Absblank 2- Absblank 3) ] x 271 (Goth, 1991).
Statistical analysis
All statistical analysis was performed with the computer program

“SPSS for Windows’’ (SPSS Inc; Release 11.5; Sep 6, 2002). All of
the data were expressed as means ± SD. Differences between
groups were evaluated by one-way analysis of variance (ANOVA)
followed by Tukey’s multiple comparison tests. The significance
was tested at p>0.05, p < 0.05, p< 0.01 and p

1030 Afr. J. Pharm. Pharmacol.
Table 1a. Histopathological findings in the kidney.
Group
Epithelial
desquamation
and
degeneration
Loss
of
brush
border
Epithelial
cell
swelling
Tubular structures Glomerulus Cortex Medulla
Epithelial
cell
picnosis
Epithelial
cell
vacuolization
Cast
cumulation
in the
lumen
Focal
necrotic
areas
Congestion Necrosis Congestion
MNL
infiltr.
PMNL
infiltr.
Fibrosis Congestion
Group II +++ +++ +++ +++ + +++ - + - +++ +++ - - +++ +++ -
Group III +++ +++ ++ ++ + ++ - - - +++ +++ - - +++ +++ -
Group IV +++ +++ ++ ++ + +++ - + - ++ ++ - - +++ ++ -
Interpretation n.s n.s Decrease Decrease n.s n.s - n.s Decrease Decrease - - n.s Decrease -
- : none, ++: moderate, n.s: not significant, +: mild, +++: severe.
Table 1b. Histopathological findings in the liver.
Group
Congestion in
Central vein Portal area Sinusoid
Hepatic
bubbling
degeneration
Focal
necrosis
Central
vein
PMNL infiltration in MNL infiltration in
Karyolysis
and
picnosis
Loss of
intercellular
border
Group II ++ +++ ++ - - - + + + +++ ++ ++ ++ ++
Group III + ++ ++ MZ,Z2 ++ - - + - + ++ ++ + + +
Group IV + ++ ++ MZ,Z2 + - - + + + + + + + +
Interpretation Decrease Decrease n.s Decrease - - n.s n.s n.s Decrease Decrease Decrease Decrease Decrease
Portal
area
Sinusoid
-: none, ++: moderate, Z2: Midzonal area (Zone-2), n.s: no difference, +: mild, +++: severe, MZ: Microvesicular bubbling degeneration.
in MDA levels administration after I/R injury. MDA
levels in our study were similar to Yurdakul et al.
(2010) and Bi et al. (2009) studies. GA being an
antioxidant reduces the stress to a considerable
extent thereby reducing the demand of excess
sugar and thus recoups the glycogen content in
kidney and liver (Jadon et al., 2007).
Hepatic and renal cells participate in a variety of
metabolic activities and contain a host of enzymes
(Jadon et al., 2007). Niho et al. (2001)
investigated the toxicity of GA in different doses
on liver and kidney. They revealed some weak
pathological changes. Li et al. (2005) studied
Central
vein
antioxidant activities of GA in senescence
accelerated mice and they observed increase in
CAT levels compared to controls similar to our
study. Liao et al. (2010) mentioned that
augmenter of liver regeneration (ALR), which is
expressed in both the liver and kidney, and
recently, was also found to be an important
intracellular survival factor for hepatocytes,
(Thirunavukkarasu et al., 2008) effectively
reduces tubular injury and ameliorates the
impairment of renal function. The protective effect
of ALR is associated with enhancement of renal
tubular cell regeneration (Liao et al., 2010). Ozer
Portal
area
Sinusoid
MNL
infiltr
PMNL
infiltr
Damage in
hepatic
cords
et al. (2009) observed severe vascular
congestion, degeneration of the tubular
epithelium, glomerular and Bowman’s space
structures, and severe inflammatory cell infiltration
in the IR group. These degenerations were clearly
improved when the animals were treated by
Pycnogenol. Rahman et al. (2009) indicated
histopathologically lower damage in Manganese
Superoxide Dismutase (MnSOD) administered
group compared with the I/R group. The studies
examine the effects of different antioxidants on I/R
injury revealed different results for enzymatic
activities and severe histopathological findings. In

Table 2. The MDA levels, SOD and CAT enzyme activities in kidney and liver.
Canbek et al. 1031
Group SOD kidney SOD liver MDA kidney MDA liver CAT kidney CAT liver
Group I (G1) 55.68±6.48 54.12±5.01 1.79±0.19 2.52±0.24 308.30±62.81 149.13±41.56
Group II (G2) 67.53±5.77 38.71±1.50 3.18±0.31 7.3139±0.76 755.55±336.14 297.18±168.83
Group III (G3) 53.83±2.17 43.28±9.62 1.88±0.31 5.7627±1.21 602.85±380.68 461.72±108.43
Group IV (G4) 52.86±6.77 21.58±4.80 1.86±0.35 5.3560±1.28 715.93±63.23 709.63±174.84
p-values and multiple
comparison of the
groups
0.001 0.001 0.000 0.000 0.017 0.000
0.902 0.023 0.885 0.000 0.170 0.000
0.756 0.000 0.915 0.000 0.032 0.000
0.000 0.492 0.000 0.031 0.688 0.031
0.000 0.000 0.000 0.005 0.991 0.005
0.990 0.000 0.936 0.863 0.844 0.863
The significance was tested at n.s p>0.05, p < 0.05, p< 0.01 and p

1032 Afr. J. Pharm. Pharmacol.
Figure 2. Light micrographs of liver sections. (A) Liver of an untreated control. (B) Congestion in
central vein, portal area and sinusoids, damage in hepatic cords, (C) PMNL and MNL infiltration
in sinusoids, (D) midzonal (Zone-2) microvesicular hepatic bubbling degeneration in liver. Scale
bar 100 and 50 µm.
addition, antioxidants improved most of the damages.
Conclusions
The results of this study have demonstrated that GA may
have protective effect on renal I/R injury-induced renal
tubular and hepatic damages in the rat. GA in 50 mg/kg
was observed to be enough to significantly prevent renal
injury. According to these findings, it is necessary to
make more experimental studies for the evaluation of GA
effects.
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African Journal of Pharmacy and Pharmacology Vol. 5(8), pp. 1034-1037, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.104
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Effect of roasting, boiling and microwaving cooking
method on Doxycline residues in edible tissues of
poultry by microbial method
Javadi A.
Food Hygiene Department, Veterinary Science Faculty, Tabriz Branch, Islamic Azad University, Tabriz, Iran,
5157944533. E-mail: Javadi@iaut.ac.ir. Tel: +0914-116-5059.
Accepted 31 March, 2011
The purpose of this study was to determine the effects of different cooking processes like boiling,
roasting and microwaving on Doxycline residues in chicken muscle, liver and gizzard tissues of broiler
chickens. Each chick was fed by water and food with 0.1% of Doxycline in their drinking water for 5
consecutive days. Then, three locations were sampled aseptically from each carcass: Breast muscle,
liver and gizzard. Doxcycline residue was analysed using microbial inhibition method by plates seeded
with Escherichia coli. After doing different phases of the test on raw samples, the positive raw samples
were cooked by various cooking procedures and we surveyed the cooked samples with a similar
method again for the detection of residue. The results showed a reduction in the concentration of
Doxycline residue after different cooking processes and a part of the residue in the boiling process
were excreted from the tissue to the cooking fluid. Between the various agents affecting antibiotics
residue after the cooking process, cooking time and temperature can play a major role in antibiotic
residue reduction while cooking food. Regarding the results of this study, we can conclude that
cooking processes do not guarantee a full elimination of these drugs present in condemned animals
and it can only decrease its amounts.
Key words: Cooking, Doxycline, residue, poultry, edible, tissue.
INTRODUCTION
Tetracycline antibiotics (TCAs) have a broad range of
activity against variety of Gram -positive and Gramnegative
bacteria and have low cost. For these reasons,
TCAs are widely used in veterinary medicine for
preventing and treating several diseases and for
promoting growth in cattle and poultry (Bogialli and Di,
2009). Furthermore, they are easy to administer and are
effective through oral dosing via water and feed.
Tetracycline (TC), oxytetracycline (OTC),
chlortetracycline (CTC) and doxycycline (DOX) are four
members of this antibiotic group. In recent years, the
abundant and in some cases improper use of TCs has
resulted in the presence of residues in edible animal
tissues, which has harmful effects on consumer’s health,
such as allergic reactions, liver damage, yellowing ofteeth
and gastrointestinal disturbance (Debuf, 1998; Joint
FAO/WHO Expert Committee on Food Additives, 2002;
Jing et al., 2009). Doxycycline (DOX) is a broad-spectrum
antibiotic from tetracycline group that is widely used
in the treatment of respiratory tract infections and
infectious diseases caused by “rickettsiae”,
“mycoplasmas” and “Chlamydia” in various species
(Hardman et al., 2001; Kirbiš, 2006). The detection of
antibacterial residues in food requires screening methods
sensitive at antibiotic concentrations close to the
maximum residue limit (MRL). The European union (EU)
legislation on veterinary drug residues established in
1997, provisional maximum residue limits (MRL) for DOX
in bovine, porcine and poultry at 600 µg kg-1 in kidney,
300 µg kg-1 in liver, skin and fat and 100 µg kg-1 in
muscle (Croubels et al., 1998; Fuselier et al., 1999).
An efficient screening method for the detection of
antibacterial residues in food needs to be low-cost and
high-throughput, able to effectively identify potential
noncompliant samples from a large set of negative
samples. Microbial inhibitions assays were the earliest
methods used for the detection of antibiotic residues and
they are still widely used. They are very cost-effective

and in contrast to, for example, immunological or
receptor-based tests and allow detection of a wide
spectrum of antibiotics (Aerts et al., 1995; Haasnoot et
al., 1999; Pikkemaat, 2009). Their other advantages are
the option to analyze a large number of samples
simultaneously and the relatively short time needed for
preparation of samples as no purification procedures are
required. A positive result should be confirmed with
chemical or physical methods (Ferrini et al., 2006; Kirbiš,
2006). A plate test consists of a layer of inoculated
nutrient agar, with samples applied on top of the layer, or
in wells in the agar. Bacterial growth will turn the agar into
an opaque layer, which yields a clear growth-inhibited
area around the sample if it contains antimicrobial
substances. In Europe this has been the main test format
since screening of slaughter animals for the presence of
antibiotics started (Pikkemaat, 2009). On the basis of
other researches, the plate seeded with Bacillus subtilis
is suitable for detection of tetracycline residues (Kirbiš,
2006; Chang et al., 2000). Between 1995 and 1999,
Rose et al. (1995, 1999) demonstrated that residues of a
range of veterinary drugs have varying degrees of
stability during cooking and therefore, the cooking
influences the level of risk posed by such residues (Rose
et al., 1999).
Since the most of foods-producing animals are always
cooked before consumption and the variations in
doxycycline levels in the tissue are dependant on type of
cooking, more findings about the effect of cooking on
doxycycline residue are needed to accurately determine
consumer exposure to this drug.
MATERIALS AND METHODS
Chickens and drug administration
Sixty broiler chickens (aged 20 days) were randomly divided into 2
groups; control group and case group; each containing 30 chicks in
order to remove any probable antibiotic residues from chicken’s
body; they were fed by feed and water free of antibiotics for around
10 days. Each of chicks in case groups were fed by water and feed
with 0.1% of doxycycline in their drinking water for 5 consecutive
days and chicks in control group were fed by similar water and feed
but without doxycycline for similar period.
Preparation of samples
After the 5th day of the drug administration, chickens were
slaughtered and breast muscles; livers and gizzards were sampled
aseptically from each carcass. After notation of samples
characteristics; they were placed in sterile polyethylene containers.
Cooking operation
Boiling
A 20 g sample was placed into a strainer, immersed in 10 ml of
water bath preheated to 100°C and cooked for the specified time (9
min for liver samples; 24 min for muscle samples and 85 min for
gizzard samples), removed and allowed to cool.
Roasting
Javadi 1035
A 20 g sample was placed on a metal baking tray and cooked well
in an electric oven (Memmert, Germany) at 200°C for the specified
time of 25, 40 and 60 min for liver, muscle and gizzard samples,
respectively, after which it was removed and allowed to cool. No
juice, which drained from the samples as they were cooked, was
collected. The cooked muscle had a ‘‘well cooked’ appearance.
Microwaving
A 20 g sample was placed on a turned table. The sample was
cooked under full power (900 W) for the specified time (3 min for all
samples), removed and allowed to cool. No juice was collected.
Test procedure for raw and cooked samples
Test organism used in this study were Bacillus subtilis (PTCC 1365)
and the used agar “medium” was Muller Hinton agar (Quelab,
England) and the pH of the this medium were adjusted to pH = 6
with sodium hydroxide and acid acidic and autoclaved as indicated
by the manufacturers. Sterile Petri dishes (diameter 90 mm) were
filled with 25 ml of the prepared culture medium then we seeded B.
subtilis in plates. Raw samples disks (diameter 2 mm) were put on
each plates also we put a paper disk as negative control. A positive
raw sample is indicated by a complete inhibition of growth in an
annular zone not less than 2 mm wide around the disc. Less than 2
mm of inhibitory zone indicated negative result (Myllyniemi, 2001).
Results of inhibition zones diameter was read by digital caliper. The
positive raw samples were selected for cooking processes (boiling;
roasting and microwaving) then we performed the test for cooked
samples just like raw samples after complete cooking of them. Also,
we placed 0.01 ml of boiling fluid on plates after boiling process of
samples. After all samples were put onto the plates, plates were
incubated at 37°C for 24 h.
Analytical method
Comparison between the mean diameter of inhibition zones around
raw and cooked samples analyzed by ANOVA test and SPSS
software version 15.
RESULTS
Comparison of the effects of different cooking methods
on the mean diameter of inhibition zones (mean±SE)
around raw and cooked samples are showed in Table 1.
We see that all cooking processes can lead to a
reduction (p

1036 Afr. J. Pharm. Pharmacol.
Table 1. Comparison of mean inhibition zones diameter (mean±SE) between raw and cooked
samples in different cooking procedures.
Muscle Liver Gizzard
Raw 12.3 ±0.47 d 11.3± 1.49 c 8.4 ± 2.24 b
Boiled 8 ± 0.42 c 0.0 ±0.0 a 0.0 ±0.0 a
Boiling fluid 6.6 ±1.37 c 0.0 ±0.0 a 0.8 ± 0.8 a
Micro waved 0.0 ±0.0 a 0.0 ±0.0 a 3.14 ±1.03 a
Roasted 3.1 ± 0.69 b 3.9 ±0.46 b 1.3 ± 0.8 a
a, b, c and d: Differences between the means that have common letters are significant (p

products. J. Chromatogr. B. Biomed. Sci. Appl., 667(1): 1-40.
Al-Ghamdi MS, Al-Mustafa ZH, El-Morsy F, Al-Faky A, Haidr I, Essa H
(2000). Residues of tetracycline compounds in poultry products in the
eastern province of Saudi Arabia. Public Health, 114: 300-304.
Bogialli S, Di Corcia A (2009). Recent applications of liquid
chromatography–mass spectrometry to residue analysis of
antimicrobials in food of animal origin, Anal. Bioanal. Chem., 395:
947–966.
Chang CS, Tai TF, Li HP (2000). Evaluating the applicability of the
modified four-plate test on the determination of antimicrobial agent
residues in pork. J. Food Drug Anal., 8(1): 25-34.
Croubels S, Baert K, De Busser J, De Backer P (1998). Residue study
of doxycycline and 4-epidoxycycline in pigs medicated via drinking
water, Analyst, 123: 2733–2736.
Debuf Y (1998). The veterinary formulary. Pharm. London, p. 97.
Ferrini AM, Mannoni V, Aureli P (2006). Combined plate microbial assay
(CPMA): A 6 – plate method for simultaneous first and second level
screening of antibacterial residues in meat. Food Addict. Contam.,
23(1): 16-24.
Fuselier R, Cadieu N, Maris P (1999). S.T.A.R.: screening test for
antibiotic residues in muscle results of a European collaborative
study, A.F.S.S.A. community reference laboratory for residues of
veterinary drugs, France.
Hardman JG, Limbird LE, Gilman AG (2001). Goodman and Gilman’s
the Pharmacological Basis of Therapeutics, 10th edition McGraw-Hill,
New York.
Haasnoot W, Stouten P, Cazemier G, Lommen A, Nouws FM, Keukens
HJ (1999). Immunochemical detection of aminoglycosides in milk and
kidney, Analyst, 124: 301-305.
Hassani M, Lazaro R, Perez C, Condon S, Pagan R (2008).
Thermostability of oxytetracycline, tetracycline, and doxycycline at
ultrahigh temperatures, J. Agric. Food Chem., 56(8): 2676-2680
Javadi 1037
Jing T, Gao XD, Wang P, Wang Y, Lin YF, Hu XZ, Hao QL, Zhou YK,
Mei SR (2009). Determination of trace tetracycline antibiotics in
foodstuffs by liquid chromatography–tandem mass spectrometry
coupled with selective molecular-imprinted Solid-phase extraction,
Anal. Bioanal. Chem., 393: 2009–2018
Joint FAO/WHO Expert Committee on Food Additives (2002).
Evaluation of certain veterinary drug residues in food: fifty eighth
report of the Joint FAO/WHO Expert Committee on Food Additives,
WHO technical report series, No. 911. FAO, Rome, p. 33
Kirbiš A (2006). Microbilogical 5-plate screening method for detection of
tetracyclines, aminoglycosides, cephalosporines and macrolides in
milk, Slovenian Vet. Res., 43(4): 161-8
Myllyniemi AL, Nuotio L, Lindfors E, Rannikko R, Niemi A, Backman C
(2001). A microbiological six-plate method for identification of certain
antibiotic groups in incurred kidney and muscle samples. Analyst,
126: 641-646
Pikkemaat MG (2009). Microbial screening methods for detection of
antibiotic residues in slaughter animals, Anal. Bioanal. Chem., 395:
877–891
Rose MD, Bygrave J, Sharman M (1999). Effect of cooking on
veterinary drug residues in food part 9: Nitroimidazoles. Analyst, 124:
289–294
Van Egmond HJ, Nouws JFM, Schilt R, Van Lankveld-Driessen WDM,
Streutjens-van Neer EPM, Simons FGH (2000). Stability of antibiotics
in meat during a stimulated high temperature destruction process,
The Euro Residue conference IV, May 08 - 10, Veldhoven,
Netherlands, pp. 430-438

African Journal of Pharmacy and Pharmacology Vol. 5(8), pp. 1038-1045, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.152
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Anti atherosclerotic effects of verjuice on
hypocholesterolemic rabbits
Mahbubeh Setorki 1 *, Bahar Nazari 2 , Sedighe Asgary 3 , Leila Azadbakht 4,5 and Mahmoud
Rafieian-Kopaei 6
1 Department of Biology, Izeh Branch, Islamic Azad University, Izeh, Iran.
2 Isfahan Cardiovascular Research Center, Isfahan University of Medical Sciences, Isfahan, Iran.
3 Isfahan Cardiovascular Research Center, Applied Physiology Research Center, Isfahan University of Medical Sciences,
Isfahan, Iran.
4 Food Security Research Center, Isfahan University of Medical Sciences, Isfahan, Iran.
5 Department of Nutrition, School of Health, Isfahan University of Medical Sciences, Isfahan Iran.
6 Medical Plants Research Center, Shahrekord University of Medical Sciences, Sharekord, Iran.
Accepted 22 June, 2011
The aim of the present study was to evaluate the effects of verjuice on atherosclerosis risk factors in
rabbits fed a cholesterol-rich diet for how long. Two New Zealand rabbits were used for this study
which lasted 8 weeks. The rabbits were divided to four groups and treated as follows normal diet,
cholesterol-rich diet, cholesterol-rich diet supplemented with 5 ml verjuice and cholesterol-rich diet
supplemented with 10 ml verjuice. The low density lipoprotein-cholesterol (LDL-C), malondialdehyde
(MDA), oxidized LDL (ox-LDL), nitrite, nitrate; factor VII, fibrinogen and C-reactive protein (CRP) were
measured before and after experimental feeding. In all groups, fatty streak formation in right and left
coronary arteries were determined at the end of the study. Administration of low (5 ml) and high (10 ml)
dose of verjuice significantly lowered the levels of fibrinogen (p

2008; Iriti and Faoro, 2009; Setorki et al., 2009).
Evidence is provided that procyanidins, derived from
grape seed, may decrease the development of foam cell
formation by reducing cholesterol accumulation and
modulating the expression of genes interferes in
inflammation (Terra et al., 2009). Wines, manufactured
from grapes, also contain a large amount of antioxidants,
including resveratrol, catechin, epicatechin, and proanthocyanidins
(Bertelli and Das, 2009). Vinegar, one of
grape derives, has some acute effects on biochemical
risk factors of atherosclerosis (Setorki et al., 2010a).
Verjuice is an acidic juice made from unripe fruit
particularly grapes. This ancient product's culinary and
medical uses date back to medieval times. It is used
extensively in Iranian cuisine as a popular ingredient.
Grape juice and verjuice are very similar in structure and
both have flavonoid components such as catechin and
anthocyanin (Kishi et al., 1999). Some beneficial effects
of verjuice have been mentioned in Iranian medicine.
Also, in previous study we had shown that verjuice had
acute effects on some atherosclerotic factors such as
fibrinogen, ox-LDL and Malondialdehyde (MDA) (Setorki
et al., 2010b). This study was performed to determine the
chronic effects of verjuice on lipid and other risk factors of
atherosclerosis including CRP, fibrinogen, factor VII,
nitrite, nitrate and development of fatty streak formation.
MATERIALS AND METHODS
METHODS
A botanist identified the species of the grape (Vitis Sylvestris,
herbarium number 15810) from the Research Center of Isfahan
Province Natural Resources. The grapes were collected from the
Aminabad region of Isfahan. The anthocyanin, pH, density, vitamin
C, acetic acid and flavonoids contents of the verjuice were
measured.
Measurement physiochemical factors in verjuice
pH meter was used for measuring pH. Densitometer also was
utilized, vitamin C (Mccormick and Greene, 1994), flavonoids
(Kumar et al., 2008) total anthocyanin (Schutz et al., 2006) were
assayed by spectrophotometry (ShimadzoUV-3100 (Japanese) and
acetic acid was quantified by titration (Scharf and Malerich).
Treatment of rabbits
Thirty two male New Zealand white rabbits weighting 1930±287 g
were obtained from Razi Institute of Iran. They were acclimatized in
an air conditioned room (State the temperature) for two weeks and
provided with free access to water and a normal rabbit chow
(Dampars Co, Iran) which consisted of 10% protein, 40 to 50%
carbohydrates, 2% vegetable fat and 15 to 25% fiber. At the end of
this period, rabbits were randomly divided into four groups of diets:
no cholesterol, diet containing 1% cholesterol (Merck), diet
containing 1% cholesterol with 5 ml verjuice, diet containing 1%
cholesterol with 10 ml verjuice. Animals were fasted for 12 to 15 h
and venous blood samples were taken pre-experiment and end of
study (2 months). Cholesterol-rich diet was prepared by adding 1 g
Setorki et al. 1039
cholesterol (Merck,Germany) in 4 ml olive oil to 0.1 kg of
commercial rabbit chow. Verjuice was given with force feeding
(Singer et al., 1995; Boger et al., 1997). The study was reviewed
and approved by the Research council and the ethics committee of
Isfahan University of medical sciences.
Biochemical factors in rabbit measurement
Blood samples were centrifuged at 3500 rpm for 20 min to obtain
serum and plasma. The serum that was used to measure the levels
of low density lipoprotein-cholesterol (LDL-C) [were determined
using standard enzymatic kits (Pars Azmoon Co, Iran) and auto
analyzer (Hitachi 902, Japan)], oxidized LDL (ox-LDL) and CRP
[(Promokine Co, Germany) and (Kamiya Biomedical Co, USA),
were assessed with enzyme-linked immunosorbent assay kit in
accord with manufacturer’s guidelines], nitrite and nitrate [were
measured with a colorimetric assay kit (R&D Systems, USA) that
includes the Griess reaction] and plasma was used to determine
levels of MDA, fibrinogen and factor VII. MDA was quantified by
spectrophotometric way (Kostner et al., 1997), factor VII was
quantified using clotting time, in the presence of the STA-
Neoplastine reagent of a system in which all the factors are
present, constant and in excess except factor VII which is derived
from the sample being tested (Diagnostic Stago, French) and
fibrinogen was determined using coagulation kit (Mahsayaran Co,
Iran).
Assessment of the severity of atherosclerotic lesions
At the end of study, the blood sampling and storage were repeated.
All animals in groups euthanized by an overdose of sodium
pentobarbital and exsanguinated. The animal’s aortas were
harvested for pathological investigation. The entire aorta, from the
aortic arch to the external iliac arteries, was dissected out and
cleaned of excess adventitious tissue. The aortas were fixed in
buffered 10% formalin for 24 h and then embedded in paraffin. The
paraffin embedded specimens were sectioned at 5 µm (20 sections
in succession). Atherosclerotic layer, gained from slicing, was
determined in hematoxylin stained sections on an arbitrary scale 1-
4.
Trace: Minimal thickness of subintimal with little injury to aorta
artery.
Grade 1: Atherosclerotic thickness was less than half as thick as
the media with some form of endothelial dysfunction, macrophages
and isolated foam cell inside the endothelium.
Grade 2: Atherosclerotic thickness was half as thick as the media
with accumulation of intracellular lipid, macrophage and smooth
muscle cells.
Grade 3: Atherosclerotic thickness as thick as the media with an
abundance of macrophages, smooth muscle cells and connective
tissue.
Grade 4: Atherosclerotic thickness more that as thick as the media
with a large extracellular intimal lipid core that appears as a large
nucleus from the endothelial surface (Chekanov, 2003).
Statistical analysis
Results are given as Mean ± SD. Data were analyzed statistically
using One-Way-ANOVA test followed by LSD post test. The
differences between the baseline values and the two months values
of all measured parameters calculated and were used in statistical
analysis. In all instances, p value less than 0.05 was considered
significant. For histological data, SPSS software was used to
compare mean values between the groups. One-Way ANOVA and

1040 Afr. J. Pharm. Pharmacol.
Table 1. Comparison of cardiovascular risk factors among rabbits before (Baseline) and after (End of 2 months) experimental diet.
Biochemical factors
LDL-C (mg/dl)
MDA (M)
ox-LDL (ng/ml)
Nitrite (μmol/l)
Nitrate (μmol/l)
Fibrinogen (mg/dl)
VII (% activity)
CRP(μg/ml)
Groups
Cholesterolemic diet 5 ml verjuice with 1% chol 10 ml verjuice with 1% chol. Normal diet
Baseline 32.50 ±12.24 42.67±15.72 31.83±19.10 49.25± 22.47
End of 2 months 711.50 ±102.97 498.83±262.70 477.33±235.66* 50.75± 31.64*
Baseline 1.5 ± 0.24 1.54 ± 0.14 1.5 ± 0.26 1.38 ± 0.20
End of 2 months 2.85 ± 0.88 2.34±0.59 2.11 ± 0.33* 1.32 ± 0.27*
Baseline 32.99 ± 11.2 18.77± 7.25 18.12± 6.16 24.08 ± 6.6
End of 2 months 73.6 ±6.24 46.61 ± 14.32 37.67 ± 18.91* 20.98± 8.21*
Baseline 21.3 ± 7.20 22.37±8.13 16.25±4.90 31.9 ± 16.10
End of 2 months 35.61± 10.64 53.67±9.13.90* 58.18±18.15* 27.17 11.01*
Baseline 15.49 ± 2.92 12.62±4.34 17.32±8.84 8.11± 0.84
End of 2 months 22.54 ± 5.88 26.71± 4.63* 31.22±2.7* 8.2 ± 1.63*
Baseline 205.17±20.81 233.33±39.85 207.33±23.69 240.75±16.8
End of 2 months 293.67±35.10 252.67±41.52* 219.83±44.93* 244.75±13.96*
Baseline 200.17±29.54 214.33±50.60 157.83±33.49 230 ±2 2.06
End of 2 months 338.83±78.62 286.17±72.94 254.5±59.48 231.5 ± 31.46*
Baseline 2.53 ± 0.35 2.42±0.27 2.22±0.27 2.45 ± 0.12
End of 2 months 3.9 ± 0.21 3.62±0.43 3.48±0.09 2.63 ± 0.21*
Mean LDL-C (low density lipoprotein), MDA (malondialdehyde), ox-LDL (oxidized LDL), nitrite, nitrate, fibrinogen, factor VII, and CRP(C-reactive protein) ± SD, (n=8 for each
experimental group). *: p

c
a
Figure 1. Right Coronary artery intima cross-section in the studied groups at the end of 2 months. (a)
Normal diet(X40); (b) hypercholesterolemic diet (X40); (c) 5 ml verjuice +%1hypercholesterolemic
diet(X40); (d) 10 ml ver juice +%1 hypercholesterolemic diet(X40).
regarding LDL-C index. Serum nitrite level in the normaldiet
group was significantly decreased compared with the
high-cholesterol diet one (p

1042 Afr. J. Pharm. Pharmacol.
a
a
c d
Figure 2. Left Coronary artery intima cross-section in the studied groups at the end of 2 months. (a)
Normal diet(X40); c (b) hypercholesterolemic diet(X40); (c) 5 ml verjuice +%1 hypercholesterolemic
diet(X40); (d) 10 ml verjuice +%1 hypercholesterolemic diet (X40). d
diet group (Figures 1a and 2a), whereas in the intimal
surface of the coronary arteries from high-cholesterol diet
group were seen many fat-laden macrophages. The
cytoplasm of the macrophages filled with lipid droplets
(foam cell) as the result of lipid digestion by the
macrophage. Plaque thickness was also increased to
more than half of media thickness, equal to degree 3 of
Chekanov scale (Figures 1b and 2b). In the verjuice
groups some endothelial dysfunction along a few foam
cell and macrophages were seen in the intimal surface of
the coronary arteries and plaque degree were 1 (Figures
1c and 2d). Atherosclerotic thickness grade in the
verjuice groups decreased significantly compared to the
high-cholesterol group (1.8±0.45, 0.75±0.25 in right
coronary) (1.98±0.65, 0.82±0.42 in left coronary)
(p

atherosclerotic thickness
grade of right coronary
(micron)
atherosclerotic thickness
grad of left coronary(micron)
Atherosclerotic thickness grade
Atherosclerotic thickness grade
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
a
a
%1cho norm.diet VRJ 5+%1cho VRJ10+%1cho
a
Setorki et al. 1043
%1cho norm.diet VRJ 5+%1cho VRJ10+%1cho
Figure 3. Mean of atherosclerotic thickness grade in the studied groups (right and left coronary arteries) at the end of 2 months. a:
p

1044 Afr. J. Pharm. Pharmacol.
reduction in fibrinogen and factor VII (Mukamal et al.,
2001). Intake of high and low-dose verjuice did not
change serum CRP level significantly in contrast with
hypercholesterolemic diet. Following concurrent use of 10
ml verjuice, cause significant reduction in the level of
LDL-C, MDA and ox-LDL compared to hypercholesterolemic
diet group. Several studied have reported the
polyphones activities on inhibiting the enthrohepatic
cycling of cholesterol or bile acid (Osada et al., 1997).
That is not clear yet that by which mechanism flavonoids
inhibit LDL-C exactly. Decreasing the formation of free
radicals, or separating the contributing metal ions in
oxidation reaction are some possible mechanisms (Yan
et al., 1995).
Studies of Decorde et al. (2008) shown that polyphenols
components exist in grape and apple extracts can
prevent the process of atherosclerosis in rats and the
suppression rate of fatty streak formation was 93% in
grape extracts, 78% in red grape, 60% in apple extract
and 40% in apple. In previous study, we had shown that
intake of 10 ml verjuice had significant acute effect on
reduction of ox-LDL, MDA and nitrite but it did not have
effect on CRP, LDL-C (Setorki et al., 2010b). In this study
long term consumption of 10 and 5 ml verjuic intake
significantly lowered the levels of fibrinogen and
atherosclerotic lesion in right and left coronary arteries.
However, the serum level of nitrite and nitrate increased
in both verjuice supplemented groups. Consumption of
10 ml verjuice could meaningfully reduce the amount of
MDA, ox-LDL, LDL-C (p

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African Journal of Pharmacy and Pharmacology Vol. 5(8), pp. 1046-1053, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.152
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Purified alkaloid extract of Scutellaria barbata inhibits
proliferation of hepatoma HepG-2 cells by inducing
apoptosis and cell cycle arrest at G2/M phase
Tie-shan Wang 1,2# , Li-jing Chen 2# , Zhao-yu Wang 3# , Sheng-tan Zhang 1 and Jing-ming Lin 1*
1 Department of Pharmacy, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China.
2 Department of Pharmacy, Guangdong Women and Children's Hospital and Health Institute, Guangzhou 510010, China.
3 College of Life Science and Bio-Pharmaceutical, Guangdong Pharmaceutical University, Guangzhou 510006, China.
Accepted 16 June, 2011
Scutellaria barbata was widely used as an antitumor agent in traditional Chinese medicine. However, its
antitumor components and mechanism remained unclear. In the present study, the effects of purified
alkaloid extract of Scutellaria barbata (PAESB) on the cultured hepatoma HepG-2 cells were
investigated. 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay showed that
PAESB could inhibit the proliferation of HepG-2 cells in a dose- and time-dependent manner. Annexin
V-FITC and propidium iodide (PI) staining showed that PAESB had a positive effect on apoptosis of
HepG-2 cells. After treatment by PAESB (0.75 mg/ml) for 48 h, the HepG-2 cells apoptosis rate
(25.12±0.91%) had statistical significance as compared with the negative group (2.97±0.49%) (P

constituents of ESB for antitumor. Its antitumor
mechanism still remains unclear. Therefore, in the course
of our search for new and safe anti-cancer agents, the
present study was carried out to evaluate the anti-cancer
activity of PAESB in cultured hepatoma HepG-2 cells.
Our study clearly demonstrated that PAESB exhibited
potential anticancer activity and could inhibit proliferation
of hepatoma HepG-2 cells via induction of apoptosis and
G2/M cell cycle arrest.
MATERIALS AND METHODS
Plant material
S. barbata was collected in Henan Province, China, in September,
2009 and identified by Prof. Shou-yao Zhang, Department of
Pharmacy, Zhujiang Hospital, Southern Medical University,
Guangzhou, China. A voucher specimen, number 090912, was
deposited there. The collected sample was air-dried, pulverized
using a mill hammer and stored in polythene bags for use.
Preparation of PAESB
The air-dried aerial parts of S. barbata (3.59 kg) was finely cut and
soaked in acid ethanol (12 mol/L HCl-95% EtOH, 2:100, v/v) for 72
h at room temperature, then extracted three times in an ultrasonic
bath with acid ethanol for 45 min. After evaporating the solvent
under reduced pressure, the extract was dissolved and suspended
in 2% hydrochloric acid (HCl) solution, stood overnight and filtrated.
The acidic solution was basified to pH=10 with ammonium
hydroxide (NH4OH) solution and exhaustively extracted with
chloromethane (CH3Cl). The chloroform phase was combined and
dried using a speed vacuum centrifuge to yield the alkaloidal extract
which was dissolved in 2% HCl solution again and basified to
pH=10 with NH4OH solution, exhaustively extracted with CH3Cl.
The chloroform phase was combined and dried using a speed
vacuum centrifuge to yield PAESB. 300 mg PAESB was dissolved
in 2% HCl solution and basified to pH=7.4 with sodium hydroxide
(NaOH) solution, then diluted with saline to obtain PAESB solution
(30 mg/ml) which was sterilized using a 0.22 μm filter and stored at
4°C until use. The final concentrations of PAESB which were
freshly diluted with culture medium for each experiment were 0.75,
1.0, 1.5 and 2.0 mg/ml, respectively.
Cell lines and culture
Human hepatoma HepG-2 cells were kindly given by Prof. Shengjun
Liu, Nanfang Hospital, Southern Medical University,
Guangzhou, China. The cell culture was maintained in 90% RPMI-
1640 medium supplemented with 10% fetal bovine serum, 100 U/ml
penicillin and 100 U/ml streptomycin in 25 cm 2 culture flasks at
37°C in a humidified incubator with 5% carbon dioxide supplementation.
The cells were fed every two days and subcultured once
they reached 70-80% confluence.
3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide
(MTT) assay measured HepG-2 cells proliferation inhibition
rate
HepG-2 cells (1×10 5 cells/ml) in exponential growth stage were
suspended in medium and seeded in 96-well plates with100 μl/well.
After culturing for 24 h to obtain adherent monolayer cells, the
Wang et al. 1047
medium was discarded. Then cells were washed with phosphatebuffered
saline (PBS) twice and incubated in the fresh medium
containing various concentrations of PAESB solution (the final
concentrations were 0.75, 1.0, 1.5 and 2.0 mg/ml, respectively) for
24, 48, 72 and 96 h, respectively. Positive group was treated with 5-
FU solution (the final concentration was 250 μg/ml ). Wells added
the same volumes of sterilized saline were set as negative group.
At the end of each time point, the drug-containing medium was
replaced by fresh medium. Then MTT was added to each well. The
plates were incubated for additional 4 h at 37°C. After removing the
supernatant solution, dimethylsulfoxide (DMSO) solution was added
to each well, gently vibrated for 10 min. The absorbency at a
wavelength of 570 nm of the dissolved solution was measured with
a microplate reader. Results are expressed as the percentage
growth inhibition with respect to the untreated cells. The growth
inhibition rate was determined using the following formula. Growth
inhibition rate (%) = (negative group’s OD-test group’s OD)/negative
group’s OD×100%. The morphologic changes of HepG-2 cells were
observed with an inverted microscope at 200×.
Annexin V-FITC/PI double staining measured HepG-2 cells
apoptosis rate
HepG-2 cells (2×10 5 cells/ml) in exponential growth stage were
suspended in medium and seeded in 6-well plates with 2 ml/well.
After cultured for 24 h to obtain adherent monolayer cells, the
medium was removed and washed with PBS twice. Then the fresh
medium containing PAESB (0.75 mg/ml) was added. Positive group
was treated with 5-FU solution (the final concentration was 250
μg/ml). Wells added the same volumes of sterilized saline were set
as negative group. After incubating for 48 h, the detached and
attached cells were harvested and washed with PBS twice and
centrifuged at 1000 rpm for 5 min to remove PBS. Then the cells
were treated according to the Annexin V-FITC/PI apoptosis
detection kit. The early apoptosis rate of cells was analysis by flow
cytometer. Cells positive for Annexin V but negative for PI
fluorescence were identified as apoptotic.
Flow cytometer analyzed HepG-2 cells cycle distribution
HepG-2 cells (2×10 5 cells/ml) in exponential growth stage were
suspended in medium and seeded in 6-well plates with 2 ml/well.
After cultured for 24 h to obtain adherent monolayer cells, the
medium was removed and washed with PBS twice. Then the fresh
medium containing PAESB (0.75 mg/ml) was added. Positive group
was treated with 5-FU solution (the final concentration was 250
μg/ml). Wells added the same volumes of sterilized saline were set
as negative group. After incubating for 48 h, the detached and
attached cells were harvested, then washed with PBS twice and
fixed in 70% ice-cold ethanol at -20°C overnight. After fixation, the
ethanol was removed. Cells were washed with PBS twice and
resuspended in 0.5 ml PBS. Then 50 μl RNase and 450 μl PI (50
μg /ml) was added. After incubated at 37°C for 15 min in the dark,
the samples of cells were then analyzed for DNA content by flow
cytometer. The cell cycle distributions were analyzed by ModFit LT
2.0 software.
Statistical analysis
Experimental data were expressed with mean±standard deviation
( X S ). Statistical analysis was performed with analysis of variance
of factorial design and One-Way ANOVA using the statistical
software SPSS 13.0. Significant level α=0.05, P

1048 Afr. J. Pharm. Pharmacol.
Table 1. Inhibition rate of HepG-2 cells after treated with different concentrations of PAESB for 24 to 96 h by MTT assay.
Groups
(mg/ml)
24 h
Inhibition rate (%)
48 h 72 h 96 h
F P
0.75 32.88±3.37 40.50±2.28 53.35 ±1.88 51.92±6.91 16.764 0.001
1.0 40.08±5.41 61.52±2.59 70.09 ±1.04 66.39±8.34 20.310 0.000
1.5 47.85±9.07 81.79±2.05 86.69 ±1.17 78.55±5.86 30.324 0.000
2.0 54.34±1.72 81.94±0.92 87.99±0.43 81.09±0.40 656.346 0.000
F 8.283 276.590 515.382 14.100 F = 3.654 a
P = 0.003 a
P 0.008 0.000 0.000 0.001
A : interaction effect. The values were expressed as mean ± standard deviation of triplicate measurements.
RESULTS
I nhi bi t i on r at e( %)
Inhibition rate (%)
100. 00
80. 00
60. 00
40. 00
20. 00
0. 00
24 48 72 96
t ( h)
0. 0.75 75 mg/ml mg/ ml
1. 1.0 0 mg/ml mg/ ml
1. 1.5 5 mg/ml mg/ ml
2. 2.0 0 mg/ mg/ml ml
Figure 1. Inhibition of HepG-2 cells proliferation by PAESB. Cells were treated with different concentrations of
PAESB. The optical densities of cells were determined at 24, 48, 72 and 96 h, respectively by MTT assay.
Analysis of variance of factorial design showed that the growth of HepG-2 cells was inhibited by PAESB in a
dose- and time- dependent manner (P < 0.05).
Inhibition of HepG-2 cells proliferation by PAESB
The growth inhibition of PAESB in the cultured HepG-2
cells was evaluated by MTT assay. It was shown that a
shorter period of treatment with 0.75 μg/ml PAESB
reduced growth inhibition only slightly. Further increases
in the concentration of PAESB resulted in greater
increases in the proliferation inhibition rate of HepG-2
cells. The growth inhibition of cells in high concentration
of PAESB (1.5 and 2.0 mg/ml) treatment groups was not
significantly higher than that in positive group (Table 1).
MTT assay showed that PAESB could inhibits growth of
HepG-2 cells in a time and concentration dependence
manner (Figure 1).
Morphological monitoring
The number of HepG-2 cells was decreased after treatment
with PAESB when compared with negative group.
The higher the concentration of PAESB used to treat
HepG-2 cells, the fewer cells were observed. Cellular
morphology progressively changed with increasing
duration of exposure to PAESB. Figure 2 showed the
representative photos of cells treated with either 0.75 or
1.5 mg/ml PAESB for 48 h.
Effect of PAESB on HepG-2 cells apoptosis
To further confirm that PAESB induced cell apoptosis,
HepG-2 cells were treated with PAESB to study the
apoptosis by staining with Annexin V-FITC and PI and
subsequently analyzed by flow cytometer. The dual
parameter fluorescent dot plots showed the viable cell
population in the lower left quadrant (annexin V-PI-). The
cells at the early apoptosis are in the lower right quadrant
(annexin V+PI-). As indicated in Table 2, after 0.75 mg/ml
PAESB treating HepG-2 cells for 48 h, the cells apoptosis
rate (25.12±0.91%) had statistical significance as
compared with the negative group (2.97±0.49%)(P

A B
C D
Figure 2. Morphological changes of HepG-2 cells after treatment with PAESB for 48 h. (A) cells
untreated; (B) cells treated with 0.75 mg/ml PAESB; (C) cells treated with 1.5 mg/ml PAESB; (D)
cells treated with 250 μg/ml 5-FU. Cell photos were observed by an inverted microscope at 200×.
Table 2. Apoptosis rate of HepG-2 cells treated with
PAESB for 48 h.
Group Apoptosis rate (%)
Negative 2.97±0.49
Positive 1.28±0.31
Test 25.12±0.91 a
F 1358.991
P 0.000
a P< 0.01 VS negative group; Apoptosis rates of negative
group, positive group and 0.75 mg/ml PAESB-treated
cells group were determined by Annexin V-FITC/PI
staining and flow cytometric analysis. The values were
expressed as mean ± standard deviation of triplicate
measurements.
positive effects on apoptosis of HepG-2 cells (Figure 3).
Effect of PAESB on cell cycle distributions of HepG-2
cells
To determine if cell growth inhibition involves cell cycle
changes, we examined cell cycle phase distribution by
flow cytometer. As indicated in Table 3, when cells were
Wang et al. 1049
treated with 0.75 mg/ml PAESB for 48 h, significant
increase in the percentage of G2/M phase occurred by
comparison with the corresponding values of negative
group (P0.05). These results showed that PAESB (0.75
mg/ml) could arrest cell cycle progression in G2/M phase
(Figure 4).
DISCUSSION
Hepatocellular carcinoma is one of the most common
causes of malignancy-related deaths worldwide.
Currently the main treatment of hepatocellular carcinoma
is still chemotherapy. And the development of new
anticancer drugs has been a key issue in cancer
chemotherapy because more cancer cells can resist to
current chemotherapy and the chemotherapy will
eventually dominate the normal cell population, resulting
in much higher mortality (Kang et al., 2005). Recently,
there is a growing interest in the use of plant materials for
the treatment of various human diseases including
cancer. Natural products or herbal recorded in Chinese
pharmacopoeia have been prescribed in many diseases

1050 Afr. J. Pharm. Pharmacol.
A1 A2 A3
B1 B2 B3
C1 C2 C3
Figure 3. The apoptosis of HepG-2 cells after treated with PAESB for 48 h. (A) negative; (B) positive; (C) cells treated with 0.75 mg/ml
PAESB.
Table 3. The cycle distributions of HepG-2 cells after treated with PAESB for 48 h.
Group
G0/G1
Cell cycle distribution (%)
S G2/M
Negative 58.19±1.01 33.57±1.94 8.24±0.95
Positive 66.02±6.01 a 33.28±2.39 0.70±1.14 a
Test 43.69± 1.76 41.46±7.52 14.85±2.24 a
F 28.743 2.935 62.558
P 0.001 0.129 0.000
a P

Wang et al. 1051
A1 A2 A3
B1 B2 B3
C1 C2 C3
Figure 4. Flow cytometric analysis of HepG-2 cells cycle distribution after treated with PAESB for 48 h. (A) negative; (B)
positive; (C) cells treated with 0.75 mg/ml PAESB.
for a long time. Many of them have also been considered
as a valuable source for anticancer drug discovery
(Schwartsmann et al., 2002). Due to the different
components in a herb, mixtures or extracts of herbs may
have synergistic activities or buffering toxic effects, more
therapeutic or preventive activity than alone (Vickers.,
2002). From this point of view, it is important to study the
anticancer activity of a whole extract of herbal and its
mechanism.
S. barbata is a perennial herb which is natively
distributed throughout Korea and southern China. This
herb is known in traditional Chinese medicine as “Ban-
Zhi-Lian” and traditional Korean medicine as “Ban-Ji-
Ryun”, respectively. S. barbata has been traditionally
used as an antitumor agent, an anti-inflammatory agent
and a diuretic in China. In the clinic, it often can be used
to treat kinds of tumors in combination with other
traditional Chinese medicines. This plant contains a large
number of flavonoids, volatile oil, unique neo-clerodane
type diterpenoids, alkaloids as well as other organic
acids, sterides and polysaccharides. Recent studies
indicate that its extract have growth inhibitory effect on a
number of human cancer cells, including tumor, leukemia,
colon cancer, hepatoma, lung cancer, skin cancer and so
on. However, the chemical constituents of S. barbata for
antitumor activity have not been fully determined. The
underlying mechanism of anti-cancer remains unclear.
Thus, the present study investigated the effect of PAESB
on the proliferation, apoptosis or cell cycle of cultured
hepatoma HepG-2 cells. The MTT assay showed that

1052 Afr. J. Pharm. Pharmacol.
PAESB could inhibit the proliferation of HepG-2 cells in a
dose- and time-dependent manner. The Cellular
morphology progressively changed with increasing
duration of exposure to PAESB had also shown it had
potent cytotoxicity on the HepG-2 cells.
Apoptosis or programmed cell death is a normal
physiological process serving to eliminate unwanted cells
and maintain homeostasis in healthy tissue. Tumor growth
is regulated by the balance between cell proliferation and
apoptosis. Induction of apoptosis, programmed cell death
is one approach to cancer therapy (Los et al., 2003).
AnnexinV-FITC and PI staining showed that after 0.75
mg/ml PAESB treating HepG-2 cells for 48 h, the cells
apoptosis rate (25.12±0.91%) had statistical significance
as compared with the negative group (2.97±0.49%).This
finding suggested that it may have potential as a cancerprevention
agent because the therapeutic application of
apoptosis had been regarded as a model for developing
anti-tumour drugs (Hong et al., 2003). Cancer is
characterized by proliferation disorders and apoptosis
obstacles. Inhibiting cell proliferation and increasing
apoptosis in tumors are effective tactics for preventing
tumor growth. What’s more, many anti-cancer agents
arrest the cell cycle at the G0/G1, S or G2/M phase and
then induce apoptotic cell death (Tian et al., 2006). In the
present study we found that significant increase in the
G2/M phase of the cell cycle occurred after 0.75 mg/ml
PAESB treatment HepG-2 cells for 48 h. This suggested
that the anti-proliferative effect of PAESB on human
hepatoma HepG-2 cells was related to arrest at G2/M
phase of the cell cycle.
The present study did not identify or characterize the
active chemical components existing in PAESB. Therefore,
we had not known whether the antitumor activity of
the PAESB was due to the effect of an individual active
compound or the combined effects of multiple
compounds contained in the extract. But our qualitative
chemical analysis indicated that alkaloids might the most
possible ingredients responsible for the growth inhibition,
apoptosis induction and cell cycle arrest of HepG-2 cells.
Conclusion
We had demonstrated that PAESB could inhibit the
growth of the HepG-2 cells, induce apoptosis and cell
cycle arrest at G2/M phase. These results suggested that
the PAESB had anticancer activity in vitro. It could be
considered as a valuable source for anticancer drug
discovery. However, it had potential for further
investigations including elucidation of active compounds
and evaluating its anticancer activity in vivo. The specific
molecular signaling pathways for induction of apoptosis
remained to be identified.
ACKNOWLEDGEMENTS
This research was supported by Guangdong natural
science fund (Project No.10151051501000029), Tie-shan
Wang, Li-jing Chen and Zhao-yu Wang.
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African Journal of Pharmacy and Pharmacology Vol. 5(8). pp. 1054-1062, August, 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.226
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Protection of -tocopherol and selenium against acute
effects of malathion on liver and kidney of rats
Abdulaziz M. Al-Othman 1 , Khaled S. Al-Numair 1 , Gaber E. El-Desoky 2,3 , Kareem Yusuf 2 , Zeid
A. Al Othman 2 , Mourad A. M. Aboul-Soud 3,4 * and John P. Giesy 5,6,7,8
1 Department of Community Health Sciences, College of College of Applied Medical Science, King Saud University,
Kingdom of Saudi Arabia.
2 Department of Chemistry, College of Science, King Saud University, Kingdom of Saudi Arabia.
3 Biochemistry Department, Faculty of Agriculture, Cairo University, 12613 Giza, Egypt.
4 College of Science, King Saud University, P. O. BOX 2245, Riyadh 11451, Kingdom of Saudi Arabia.
5 Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, Saskatoon,
Saskatchewan, Canada.
6 Department of Zoology, and Center for Integrative Toxicology, Michigan State University, East Lansing, MI, USA.
7 School of Biological Sciences, University of Hong Kong, Hong Kong, SAR, China.
8 Zoology Department, College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia.
Accepted 22 July, 2011
Protection from effects of the organophosphate insecticide, malathion on the liver and kidney of male
Wistar albino rats by -tocopherol and selenium was investigated. Significantly greater (P

system (Desi et al., 1998), pancreas (Gokalp et al., 2005)
and liver (Kalender et al., 2006).
Malathion [O,O-dimethyl-S-(1,2-dicarbethoxyethyl)
phosphorodithioate] is one of the most widely used OP
insecticides for agriculture and public health programs
(Maroni et al., 2000). Malathion causes toxicity through
hyperexcitation of the nervous system through its
bioactivated analog, malaoxon (Hazarika et al., 2003).
Malathion is soluble in lipids and is stored in liver and
other lipophilic tissues (Garcia-Repetto et al., 1995).
Malathion has been found to exhibit rapid but
asymmetrical transmembrane uptake by the liver.
Therefore, the liver which is the most important organ in
glucose and lipid homeostasis and production of related
enzymes can be a target for malathion toxicity (Yang et
al., 2000).
More recently, it has been postulated that OPs produce
oxidative stress in different tissues through formation of
reactive oxygen species (ROS) (Banerjee et al., 1999;
Ahmed et al., 2000; Akhgari et al., 2003; Abdollahi et al.,
2004). ROS such as hydrogen peroxide, super oxide
anion and hydroxyl radical are produced in a number of
cellular reactions by enzymes such as lipooxygenases,
peroxidases and dehydrogenases (Halliwell and
Gutteridge, 1999; Dal-Pizzol et al., 2001). Oxidative
stress is also related to xenobiotic exposure and different
levels of environmental contamination (Halliwell and
Gutteridge, 1999). Peroxidation of membrane lipids
seems to be an unavoidable process in tissue injury,
which can impair antioxidant defenses, those results in
oxidative damage by changing the balance between
oxidants and antioxidants (Banerjee et al., 1988; Halliwell
and Gutteridge, 1999; Torres et al., 2004).
The basis of OP toxicity in production of oxidative
stress might be due to either: (a) their "redox-cycling"
activity, where they readily accept an electron to form
free radicals and then transfer them to oxygen to
generate superoxide anions and hence hydrogen
peroxide through dismutation reactions, or (b) to ROS
generation via changes in normal antioxidant
homeostasis that results in depletion of antioxidants
(Banerjee et al., 1999; Kovacic, 2003; Vidyasagar et al.,
2004). Regardless of OPs type and primary mode of toxic
action, oxidative stress seems to be attenuated by nonenzymatic
nutritional antioxidants such as -tocopherol
and C (Ahmed et al., 2000). OP insecticides containing
the P=S bond (called "thion") are converted to P=O
(called "oxon") by microsomal enzymes called mixedfunction
oxidase (MFO), among which the enzymes
cytochrome P450 (CYP450) plays a major role (Giri et al.,
2002). The oxons account for the cytotoxic effects of
OPs.
Malathion is genotoxic in mice, and able to induce a
variety of chromosomal aberrations in bone marrow cells
(Rupa et al., 1991). The mutagenic activity of malathion
might be due to the existence of electrophilic sites in the
parent molecule or its metabolic intermediates, which are
Al-Othman et al. 1055
capable of binding to nucleophilic sites of numerous
biomolecules. In the malathion molecule, there are two
potential electrophilic sites, alkyl group(s) and a
phosphoryl groups, these might explain, at least in part,
the occurrence of oxidative damage in rat liver subchronically
exposed to malathion, because the liver is a
primary site of oxidative metabolism and with CYP450
activity related to biotransformation of xenobiotics
(Halliwell and Guttridg, 1999).
The antioxidant enzymes, superoxide dismutase
(SOD), catalase (CAT) and glutathione peroxidase (GPx)
are the main enzymes that act as defenses and act in
concert with non-enzymatic reduced glutathion (GSH)
antioxidants and other antioxidants, such as -tocopherol
and selenium (Halliwell and Gutteridge, 1999) protect
against the adverse effects of ROS. -tocopherol
(Vitamin E) is the major lipid soluble antioxidant and is
known to protect cellular membranes and lipoproteins
from peroxidation (Yavuz et al., 2004). -Tocopherol
inhibits free radical formation (Kalender et al., 2004) and
can effectively minimize lipid peroxidation (Traber and
Atkinson, 2007). To date, several studies have examined
the protective effects of -tocopherol on pesticide
induced oxidative stress (Aldana et al., 2001; John et al.,
2001; El-Demerdash et al., 2004; Kalender et al., 2005;
Durak et al., 2008; Sodhi et al., 2008). Selenium is a
constituent of the cystosolic enzyme glutathione
peroxidase and facilitates reduction peroxy radicals by -
tocopherol (Kaneko, 1989). Absorption of -tocopherol is
impaired by severe selenium deficiency and selenium
alleviates -tocopherol deficiencies by permitting more -
tocopherol to be absorbed (Machlin, 1991). The present
study was conducted to: (1) Evaluate the effect of
malathion on lipid peroxidation on production of MDA,
and depletion of GSH and activities of the antioxidant
enzymes such as SOD, CAT, and GPx, as well as
evaluate its effect on total proteins in liver and kidney,
and (2) determine the potency of the antioxidants, -
tocopherol and selenium to modulate hepatic damage
induced by malathion.
MATERIALS AND METHODS
Chemicals
A commercial formulation of malathion (agrothion 57% EC 500 g L -
1 ) was purchased from a local market in Saudi Arabia and was used
in this study. All chemicals were of analytical reagent grade and
chemicals required for all biochemical assays were obtained from
Sigma-Aldrich Chemicals Co. (St.louis, Mo, USA) and Merck
(Darmstadt, Germany).
Animals
Male Wister albino rats, weighing 200 to 250 g, were obtained from
the animal rearing facility of the College of Medicine at King Saud
University. Rats were housed throughout the experiment in
polypropylene cages, with 10 animals per cage, and allowed to

1056 Afr. J. Pharm. Pharmacol.
acclimatize to the laboratory environment for 10 days. Animals were
maintained under controlled conditions of temperature at 25±2ºC,
relative humidity of 50±15% and normal photoperiod (12 to 12 h
light-dark cycle). The animals were allowed free access to standard
dry pellet diet and water ad libitum.
Experiments reported here complied with current laws and
regulations of Saudi Arabia on the care and handling of
experimental animals and the animal ethical committee of King
Saud University, College of Medicine, Saudi Arabia.
Experimental design
Rats were divided into five groups: control (Group I) (n=10),
malathion-treatment (Group II) (n=10), malathion plus -tocopherol
and selenium treatment (Group III) (n=10), malathion plus -
tocopherol treatment (Group IV) (n=10), malation plus selenium
treatment (Group V) (n=10). The substances were administrated via
gavage in the morning (between 09:00AM and 10:00AM) to nonfasting
animals.
Normal control group (Group I)
Corn oil at a dose of 0.5 ml per animal was given via gavage, once
a day for the treatment period of 45 days.
Malathion-treated group (group II)
Once a day, the rats were given malathion at a dose of 27 mg/kg
(1/50 LD50) body weight per day in 0.5 ml corn oil via gavage.
Malathion plus -tocopherol and selenium-treated group
(Group III)
Selenium and -tocopherol were dissolved in water and corn oil,
respectively. Once a day, the rats were treated, via gavage, first
with selenium (0.1 mg Se/kg b.w. per day) and then 100 mg -
tocopherol/kg b.w. per day, and 30 min later, malathion dissolved in
corn oil (10 mg/kg b.w. per day) was administrated via gavage.
Malation plus -tocopherol treated group (Group IV)
Once a day, rats were given -tocopherol dissolved in corn oil (100
mg/kg b.w. per day) by gavage and 30 min later, malathion
dissolved in corn oil (10 mg/kg b.w. per day) was administrated via
gavage.
Malathion plus selenium treated group (Group V)
Once a day, rats were given selenium as sodium selenite
(Na2SeO4) dissolved in water (0.1 mg/kg b.w. per day) by gavage
and 30 min later, malathion dissolved in corn oil (10 mg/kg b.w. per
day) was administrated via gavage.
The selected dose of malathion was based on previous studies in
which 1/50 LD50 (27 mg/kg b.w.) of malathion induced biochemical
alteration in rat without morbidity (Uzun et al., 2009; Aboul-Soud et
al., 2011), while that of -tocopherol and selenium was according to
previous reports (Aksoy et al., 2005; Sodhi et al., 2008). At the end
of the exposure period, rats were killed by cervical dislocation and
their liver and kidney were removed, weighed and treated for further
measurements.
Biochemical analysis
Lipid peroxidation (malondialdehyde MDA) and reduced
glutathione
The method described by Ohkawa et al. (1979) was used to
quantify concentrations of MDA in tissue. In brief, liver or kidney
tissues (200 mg) were homogenized in aqueous 0.15 M KCl
solution to give 10% homogenate. One milliliter of homogenate was
mixed with 1 ml of 10% trichloroacetic acid (TAC) and centrifuged at
3,000 rpm for 15 min. Then, 1 ml supernatant was suspended in 1
ml of 0.67% 2-thiobarbituric acid. Samples were then placed in a
boiling water bath for 15 min. Samples were allowed to cool to room
temperature and then centrifuged at 3000 rpm for 15 min. Optical
density of the clear pink supernatant was measured at 532 nm on a
spectrophotometer (APEL, PD-303 uv, Japan). Glutathione (GSH)
in tissues was measured according to the method described by
Sedlák and Lindsay (1968). A cross section of liver or kidney
tissues (200 mg) was dissected and homogenized in ice-cold 0.02
M ethylenediaminetetraacetic acid (EDTA). Aliquots of 0.5 ml of
tissues homogenates were mixed with 0.2 M Tris buffer, pH 8.2 and
0.1 ml of Ellman’s reagent, [5,5 -dithio-bis-(2-nitro-benzoic acid)]
(DTNB). Sample tubes were centrifuged at 3000 rpm at room
temperature for 15 min. Absorbance of the clear supernatant was
recorded using a spectrophotometer (APEL, PD-303 uv, Japan) at
412 nm in one centimeter quartz cell. The quantification of reduced
glutathione was done by using a standard curve.
Total proteins
Total concentrations proteins in liver and kidney were measured by
use of a modified Lowry method of Schacter and Pollack (1973).
Bovine plasma albumin was used as standard.
Antioxidant enzymes activity
Superoxide dismutase (SOD) activity: Superoxide dismutase
(SOD) activity in liver and kidney was measured
spectrophotometrically (560 nm) by the method described by
Kakkar et al. (1984) by use of nitroblue tetrazolium as the indicator
reagent. Briefly, the kidney tissues (200 mg) were homogenized
with 10 times (w/v) 0.1 M sodium phosphate buffer (pH 7.4). The
reagents: sodium pyrophosphate buffer 1.2 ml (0.052 M) pH 8.3,
0.1 ml phenazine metho-sulphate (186 µM), 0.3 ml nitro blue
tetrazolium (300 µM) and 0.2 ml NADH (780 µM) were added to 0.1
ml of processed tissue sample. The mixture was then incubated for
90 min at 30ºC. Four milliters of n-butanol and 1 ml of acetic acid
were then added. The mixture was shaken vigorously. Following
centrifugation at 4000 rpm for 10 min, the organic layer was
withdrawn and absorbance was measured at 560 nm using a
spectrophotometer (LKB-pharmacia, Mark II, Ireland).
Catalase (CAT) activity: Catalase (CAT) activity in liver and kidney
was measured by the method of Aebi (1983) using hydrogen
peroxide as the substrate. The disappearance of H2O2 was followed
at 240 nm. The activity was expressed as µmole -1 min -1 mg -1 protein
using the extension coefficient of 0.0436 mM -1 mg -1 . CAT exerts a
dual-function decomposition of H2O2 to give H2O and O2 and
oxidation of H donors. In the ultraviolet range, H2O2 shows a
continual increase in absorption with decreasing wavelength. The
decomposition of H2O2 can be followed directly by measuring the
decrease in absorbance at 240 nm. The difference in absorbance
per unit time is a measure of CAT activity. Liver or kidney tissues
(200 mg) were homogenized in 8 ml of 0.05 M phosphate buffer at
pH 7.0. The tissue homogenates were centrifuged at 4ºC for 15 min

Table 1. Effect of -tocopherol and selenium on MDA, GSH and proteins content in liver and kidney of malathion- treated rats.
Treatment
GSH
(nmole/100 mg wet tissue)
Liver Kidney
MDA
(nmole/g wet tissue)
Proteins
(mg/100 mg tissue)
GSH
(nmole/100 mg wet tissue)
MDA
(nmole/g wet tissue)
Al-Othman et al. 1057
Proteins
(mg/100 mg tissue)
Group I 381.64±10.6 238.60±5.9 17.15±0.4 95.13±4.1 268.20±14.8 17.24±0.6
Group II 238.76±36.0** 384.92±7.2** 10.44±0.6** 25.19±6.1** 377.12±14.0** 11.48±0.8**
Group III 378.91±4.3 234.02±2.9 16.75±0.9 92.98±2.4 274.54±7.5 16.78±1.0
Group IV 367.90±19.5 256.44±25.7 14.85±2.6 91.07±0.9 245.51±28.9 14.99±2.5
Group V 367.78±21.9 251.98±23.3 15.41±2.9 91.67±1.3 249.05±33.9 15.83±1.5
Values are expressed as mean ± S.D; ** P < 0.01 relative to Group I. Group I: Control; Group II: Malathion; Group III: Malathion+ -tocopherol + Selenium; Group IV: Malathion+ -tocopherol; Group V -
Malathion+ Selenium.
at 1500 g. The supernatants were removed into separate
test tubes and kept on ice until the enzymes assay.
Sample was measured against a blank containing 2.8 ml
(1:500 v/v) phosphate buffer instead of H2O2 (30 mM
hydrogen peroxide) and 0.2 ml enzyme solution. The
reaction was started by addition of H2O2. The initial
absorbance should be A=0.500 followed by the decrease
in absorbance for about 30 s.
Estimation of glutathione peroxidase (GPx) activity in
tissues: Glutathione peroxidase (GPx) activity was
measured by use of the method developed by Paglia and
Valentine (1967). The reaction mixture contained 2.6 ml of
100 mmol/L phosphate buffer (pH 7.0) with 3 mmol/L
EDTA, 0.05 ml of 10 mg/ml GSH solution, 0.1 ml GR (10
mg/ml), 0.05 (10 mg/ml) NADPH-Na salt, 0.1 ml 90 mmol/L
hydrogen peroxide solution and 0.1 ml of tissue
supernatant. The GPx activity was monitored by the
decrease in absorbance due to the consumption of
NADPH, which absorbs at 340 nm on spectrophotometer
(LKB-pharmacia, Mark II, Irland).
Statistical analyses
All data are expressed as mean ± standard deviation (SD);
data were analyzed by ANOVA test using SPSS version
17.0 for Windows» statistical program and the individual
comparison of groups were obtained by using Scheffe’s
multiple comparison procedure. Pearson correlation test
was used to determine the significant correlation between
variable. P values of less than 0.05 and 0.01 were selected
as the levels of significance.
RESULTS
Effects of -tocopherol and selenium on, MDA,
GSH, and total protein
The concentration of MDA was significantly
(p

1058 Afr. J. Pharm. Pharmacol.
Table 2. Effect of -tocopherol and selenium on SOD, CAT and GPx activities in liver and kidney of malathion-treated rats.
Treatment
SOD
(U/mg of protein)
Liver Kidney
CAT
(µmole/mg of protein)
GPx
(µmole/mg of protein)
SOD
(U/mg of protein)
CAT
(µmole/mg of protein)
GPx
(µmole/mg of protein)
Group I 6.07±0.6 595.58±16.8 183.09±15.0 5.54±0.4 598.30±50.9 239.13±19.3
Group II 4.39±0.3** 447.04±24.4** 135.56±2.6** 3.41±0.1** 411.96±5.6** 128.99±9.8**
Group III 5.61±0.2 597.03±19.6 190.25±3.8 5.92±0.3 583.27±36.2 262.88±18.5
Group IV 5.62±0.1 594.89±11.3* 166.76±16.8 5.43±0.2 578.282±0.1* 228.85±11.7
Group V 5.87±0.1* 570.43±20.3 191.22±6.2* 5.85±0.2* 548.99±8.8 261.93±27.4*
Values are expressed as mean ± S.D; ** P < 0.01 relative to Group I. Group I: Control; Group II: Malathion; Group III: Malathion+ -tocopherol + Selenium; Group IV: Malathion+ -
tocopherol; Group V - Malathion+ Selenium.
Table 3. Correlation coefficient (r) among oxidative stress and the antioxidant enzymes activities in
kidney of malathion treated rats.
Antioxidant enzymes SOD GPx CAT
Pearson correlation -0.735- -0.426- -0.728-
MDA
Sig. (2-tailed) 0.096 0.400 0.101
N 6 6 6
GSH
were consider together, there was a statistically
significant correlation between the concentration
of MDA and level of antioxidant enzymes of liver
and kidney injury. These associations indicated
that lipid peroxidation was involved in toxicity of
malathion to both liver and kidney. Indicators of
toxicity in both liver and kidney were inversely
correlated with activities of antioxidants enzymes,
which is consistent with the importance of
P
Pearson correlation 0.929** 0.713 0.934**
Sig. (2-tailed) 0.007 0.112 0.006
N 6 6 6
Pearson correlation 0.919** 0.727 0.939**
Sig. (2-tailed) 0.010 0102 0.005
N 6 6 6
*, Correlation is significant at the 0.05 level (2-tailed); **, correlation is significant at the 0.01 level (2-tailed).
antioxidants in protecting against the effects of
ROS (Tables 3 and 4).
DISCUSSION
Malathion causes both acute and chronic toxicity
to non-target animals. Malathion not only has toxic
effects on mammals, it also has toxic effects on
fish, chicks, and non-target invertebrates (Senger
et al., 2005; Sodhi et al., 2008). The oral LD50 of
malathion for male rats is1350 mg/kg (John et al.,
2001). Several case-control studies have
associated parenteral exposure to insecticides or
insecticide use in the home with childhood brain
tumors, leukemia, lymphomas, testicular cancer
and other types of cancer (Eskenazi et al., 1999).
Birth defects reproductive abnormalities and

Table 4. Correlation coefficient (r) among oxidative stress and the antioxidant enzymes activities in
liver of malathion treated rats.
Antioxidant enzymes SOD GPx CAT
MDA
GSH
P
Pearson correlation -0.945-** -0.938-** -0.974-**
Sig. (2-tailed) 0.004 0.006 0.001
N 6 6 6
Pearson correlation 0.954** 0.907* 0.984**
Sig. (2-tailed) 0.003 0.013 0.000
N 6 6 6
Pearson correlation 0.931** 0.935** 0.951**
Sig. (2-tailed) 0.007 0.006 0.004
N 6 6 6
*, Correlation is significant at the 0.05 level (2-tailed); **, Correlation is significant at the 0.01 level (2tailed).
genetic damage have also been linked to exposure of
humans and laboratory animals to malathion. Disorders
of vision, behavioral changes, impaired learning and
sensitization of skin have also been associated with
exposures to malathion (Brenner, 1992).
The objective of the study upon which is reported here,
was to evaluate the effects of malathion on lipid
peroxidation, antioxidant defense mechanisms and
histological changes in rat liver and kidney. Alleviation of
the effects of malathion by -tocopherol and selenium
were investigated. Lipid peroxidation, which is caused by
reactive oxygen species, has been implicated in the
pathogenesis of various liver and kidney injuries. The
most widely used marker of lipid peroxidation is formation
of MDA, which is assessed by measuring the
thiobarbituric acid assay. The significantly (p

1060 Afr. J. Pharm. Pharmacol.
resulting in the formation of glutathione disulphide (Paglia
and Valentine, 1967). In the present study, malathion
caused a significant (p

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African Journal of Pharmacy and Pharmacology Vol. 5(8). pp. 1063-1069, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.292
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Preparation of ambroxol hydrochloride carboxymethyl
chitosan micropheres without burst release
Hongfei Liu 1 *, Yan He 2 , Zhaoying 3 and Peng Ke 4
1 College of Pharmacy, Jiangsu University, Zhenjiang, 212013, China.
2 School of Chinese Material Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, China.
3 Shenyang Entry-Exit Inspection and Quarantine Bureau, Shenyang, 110016, China.
4 School of Pharmacy, University of London, London, WC1N 1AX, UK.
Accepted 22 June, 2011
The aim of this article was to fabricate novel ambroxol hydrochloride carboxymethyl chitosan
microspheres without burst release. Firstly the blank carboxymethyl chitosan microspheres were
fabricated by the emulsion chemical cross-linking method and the blank microspheres showed a
controllable biodegradation in vitro in lysozyme solution. Then the ambroxol hydrochloride
microspheres were prepared by the column method. The type of bonding between ambroxol
hydrochloride and carboxymethyl chitosan microspheres was investigated by X-ray diffraction; the
results showed that the drug chemically bonds to the ion exchangeable structure of the microspheres.
The evaluation of the microspheres was investigated by dynamic light scattering, scanning electron
microscopy (SEM) and UV spectrophotometer. The microspheres were spherical and consistent, and
had an average diameter of 7.4 m, the drug content of the microspheres was 15.3±0.7% (w/w). Finally
the in vitro drug release was tested in different ionic concentration dissolution mediums. The results
showed that the microspheres had a sustained-release profile for 8 h in vitro without obvious burst
release.
Key words: Ambroxol hydrochloride, carboxymethyl chitosan, lung-targeting; microsphere, in vitro.
INTRODUCTION
The development of injectable drug biodegrable
microsphere has received considerable attention over the
past few years (Langer, 1990; Saito et al., 2001;
Ravikumar and Kumar, 2001). This interest has been
sparked by the advantages of these delivery systems
possess, which include ease of application, localized
delivery for a site-specific action, prolonged delivery
periods, and improved patient compliance and comfort
(Sultana et al., 2009; Hollister, 1989; Levy et al., 1996).
Though there are many advantages for the injection
biodegradable microspheres, there are still some
shortcomings for this dosage form, such as burst,
incomplete or uncontrollable drug release (Kumar et al.,
*Corresponding author. E-mail: liuhongfei2000@163. Com. Tel:
+86-0511-88797078. Fax: +86-0511-85038451-806.
2001). Burst release is especially of concern due to its
potential to increase side affects which raises a major
obstacle for microsphere therapeutic potential.
Considerable efforts have been made to moderate this
burst release profile (Soriano et al., 1996; Nahata and
Saini, 2008), however very few microspheres without
burst release have been successfully reported.
Chitosan, a natural linear biopolyaminosaccharide, is
obtained by alkaline deacetylation of chitin, which is the
second most abundant polysaccharide next to cellulose.
Properties such as biodegradability, low toxicity and good
biocompatibility make it ideal for use in biomedical and
pharmaceutical formulations (Lu et al., 2008).
Carboxymethyl chitosan (MCC) is one of the most
abundant and familiar kinds of chitosan derivatives, it has
been applied in several drug delivery systems (Chen et
al., 2004; Ubaidulla et al., 2009).
Recently ion exchange polymers have been used in

1064 Afr. J. Pharm. Pharmacol.
Figure 1. Schemetic diagram of drug loading process in
column method. 1, Saturated section; 2, commutative section;
3, uncommutative section.
drug delivery systems (Sriwongjanya and Bodmeier,
1998; Rao et al., 2004). They have a number of attractive
properties including increased stability, taste masking,
few side effects, and a more uniform absorption and
sustained release profile. One of the most impressive
advantages of the ion exchange polymers is that
theoretically the drug release from the ion exchange
polymers is only influenced by the ion concentration in
the surrounding medium. Thus when the ion exchange
polymers are introduced into the body, the body’s natural
counter ion concentration stabilizes drug release relieving
the concern of a burst release profile. So if we can
combine the biodegradable polymer and the ion
exchange technology to make the biodegradable and ion
exchangeable microspheres that may thoroughly
overcome the “burst release” problems. Carboxymethyl
chitosan was used in this article as biodegradable
polymer because of the carboxymethyl group in it may
combine the ionic drug by the ionic bond through the ion
exchange process.
Ambroxol hydrochloride (AH) is a new type of
expectorant which is widely used clinically in many
countries. It is an active metabolite of mucolytic in vivo,
but currently very few drug delivery systems are available
or being investigated which contain AH (Wang and Pei,
2001). Because of the pharmacological actions of the AH,
a targeted and sustained release of AH to the lungs
would greatly benefit patients.
In this article, we combined biodegadable polymer
(MCC) and ion exchange technology to prepare the AH
lung targeting microspheres. Firstly the degradability of
blank carboxymethyl chitosan microspheres in vitro was
investigated. The particle size, morphology and drug
content of the microspheres were investigated. The type
of bonding between AH and carboxymethyl chitosan
microspheres was investigated by X-ray diffraction and in
vitro drug release. Finally, the drug release from the
microspheres in vitro was tested in different ionic
concentration dissolution mediums.
MATERIALS AND METHODS
AH was obtained from the Sigma-Aldrich corporation (St. Louis,
USA). Carboxymethyl chitosan, (Mol. wt. 50KD) was obtained from
Honghai biotechnology company (Qing Dao, China).
Glutaraldehyde was obtained from the Sigma-Aldrich corporation
(St. Louis, USA). All reagents were of analytical grade.
Preparation of cross-linked MCC microspheres
Twelve milliliter of MCC aqueous solution (0.25 g/ml) was added to
60 ml liquid paraffin to form a W/O emulsion with 2 g
Span80(Weerakody et al., 2008; Sayın et al., 2008). The dispersion
was stirred at 800 rpm for 40 min after the addition 2 g
glutaraldehyde. The product was filtered and washed with
chloroform several times and finally with water and dried at 40°C.
In vitro blank MCC microspheres degradation
The degradability of blank MCC microspheres in vitro was
investigated in lysozyme solution (Lu et al., 2007). 100 mg blank
MCC microspheres (n=3) were immersed in 10 ml lysozyme
solution (4 mg·ml -1 ) in PBS (pH 7.4) at 37°C. The degrading
solution was replaced with fresh lysozyme solution every 3 days. At
3, 6 and 9 days, the blank microspheres were taken out from the
lysozyme solution, rinsed with distilled water, freeze-dried and
weighed. The extent of in vitro degradation was expressed as the
percentage of the weight loss of the blank microspheres after
lysozyme treatment.
Preparation of the AH microspheres
The AH microspheres were prepared by a column processes
(Jeong and Park, 2008a, b). For the column process, a glass
column (size: 1.0 × 20 cm, bed volume: 15 ml) was used. 1 g of
MCC microspheres was then slurred with water and transferred to
the glass column equipped with a coarse-fritted glass disk at the
bottom. To stabilize the packing, the MCC microspheres were
backwashed with water using a peristaltic pump and then 8 mg/ml
drug solution was pumped upward at a rate of 60 ml/h until there is
drug detected in the eluate (time point C in Figure 1). The AH
microspheres were then washed off the physical absorption AH with
deionized water and dried at 40°C. The diagram of drug loading
process is in Figure 1.
Particle size analysis by dynamic light scattering
The mean particle size of the microspheres was measured using a
laser light scattering particle size analyzer (LS230, Beckman
Coulter, Miami, USA) according to user’s manual.
Morphology of the microspheres
The surface of the microspheres was examined by scanning
electron microscopy (SEM) (Jeol JSM-6400, Tokyo, Japan).
Samples were gold sputter coated (SCD 004 Coater; Bal-Tec,
Balzers, Lichtenstein) for 165 s at 15 mA in an atmosphere of
argon.

Table 1. The degradation percentage of the blank MCC
microspheres in vitro.
Days 0 3 6 9
W (mg) 101.7±0.3 85.2±0.6 40.1±0.7 22.6±0.8
Weight
loss (%)
0 16.2 60.6 77.8
Figure 2. The particle size distribution of AH microspheres.
Determination of drug content in the microspheres
The AH amount in the microspheres was determined by suspending
10 mg AH microspheres in 100 ml NaCl solution (1 mol/L) under
magnetic stirring for 10 h at 65°C. The solution was then filtered,
and the amount of AH in the filtrate was determined using UV
spectrophotometer at 244 nm.
Powder X-ray diffraction properties
Blank microspheres, AH, AH microspheres, physical mixture of AH
and microspheres were investigated. Wide-angle-X-ray diffraction
was recorded by a X-ray Diffractometer (PANalytical, X'Pert PRO
MPD, The Netherlands) using Cu kalpha radiation at 40 KV/40 mA
with a secondary nickel beater filter (Jenquin and McGinity, 2008).
In vitro drug release
In vitro drug release investigation was carried out following the USP
paddle (apparatus II) method at a paddle speed of 50 rpm (Chuong
et al., 2009; Patel et al., 2009). Deionized water, 0.15, 0.5, 1 mol/L
NaCl at 37±0.1°C were used as the dissolution mediums.
Microspheres containing 8 mg AH were weighed according to drug
content and were placed in the mini dialysis kits (MWCO 6-8 kDa)
(GEBA, Gene Bio-Application, Israel), and were immersed in 100 ml
dissolution medium. 2 ml dissolution medium was then withdrawn at
different intervals (1, 2, 3, 4, 6 and 8 h). The amount of drug
released in the filtrate was measured by UV spectrophotometer at
244 nm. And the drug release data in 0.15 mol/L NaCl at 37±0.1°C
Liu et al. 1065
was analysis according to the Zero-order, First-order and Higuchi
equation.
Statistical analysis
Statistical analysis for the determination of differences in the
measured properties between groups was accomplished using oneway
analysis of variance and determination of confidence intervals,
performed with a computer statistical program (Statistical Analysis
System, Version 6.08, SAS Institute, Cary, NC, USA). All data were
presented as a mean value with its standard deviation indicated
(mean±S.D.).
RESULTS AND DISCUSSION
In vitro blank MCC microspheres degradation
A critical requirement for polymeric matrices in
biodegradable injection drug delivery is controllable
biodegradation over time. To assess the degradation
behavior of blank microspheres, we incubated them in
lysozyme solution and monitored change of weight, which
is the most relevant characteristic of implanted materials.
The results clearly demonstrated that the blank MCC
microsphere degraded over time as seen in Table 1 and
could be a suitable material for biodegradable injection
drug delivery.
Particle size analysis of AH microspheres by dynamic
light scattering
The particle size distribution result is in Figure 2. The
average particle size of the microspheres was 7.4 m.
Many research groups have demonstrated the
micropshere’s particle size and the surface properties as
the main influential parameters for the microspheres
distribution in vivo (Huo et al., 2005; Harsha and Rani,
2009), and most microspheres with particle size in the
range of 5 to 25 m would target to the lungs. Because of
the pharmacological actions of the AH, a targeted and
sustained release of AH to the lungs would greatly benefit
patients.
Morphology of the AH microspheres
The scanning electron micrograph of the AH
microshpheres is shown in Figure 3. The results showed
that AH microspheres were spherical and consistent.
Determination of drug content in AH microspheres
and the drug loading efficency
The drug content in AH microspheres was 15.3 ± 0.7%
(n=6), as determined by UV absorption at 244 nm. And
because the preparation process stopped when the drug

1066 Afr. J. Pharm. Pharmacol.
Intensity
�����
�����
�����
����
�
Figure 3. The SEM of AH microspheres.
�� �� �� �� �� �� �� ��
Figure 4. X-ray of MCC microspheres.
was detected in the eluate; the drug loading efficency is
100%. So this microsphere fabrication method would
greatly benefit the prospective protein microsphere’s
fabrication. Most current studies report production of
protein microspheres with approximately 30 to 70% drug
loading efficiency, leaving the residual protein to go to
waste (Hu et al., 2000; Coppi et al., 2001).
Powder X-ray diffraction properties
The type of bonding between AH and carboxymethyl
2-Thata� O 2-Thata �(°)
chitosan microspheres was investigated by powder X-ray
diffraction analysis. The blank microspheres were
amorphous in nature, so were devoid of sharp peaks in
Figure 4. The crystallinity of AH was clearly demonstrated
by it is unique X-ray diffraction patterns shown in Figure
5, respectively. The diffraction patterns from a physical
mixture of 15.3% drug with pure polymer contained sharp
diffraction peaks corresponding to the crystalline drug
molecules present in the mixture, as displayed in Figure
6. The presence of diffraction peaks in a physical mixture
of 15.3% drug with the blank microspheres demonstrated
that the presence of undissolved, crystalline drug

Intensity
�����
�����
�����
����
�
�� �� �� �� �� �� �� ��
Figure 5. X-ray of the AH.
Intensity
�����
�����
�����
����
2-Thata (°)
�
�� �� �� �� �� �� �� ��
Figure 6. X-ray of the physical mixture.
dispersed in the matrix would exhibit diffraction peaks
when exposed to X-rays. The diffraction patterns from AH
microspheres containing 15.3% of AH were displayed in
Figure 7 respectively, and did not contain any peaks
associated with crystalline drug molecules. These
diffraction patterns were identical to those of the pure
polymer, shown in Figure 5, suggesting the drug
presented in an amorphous state within the polymer
matrix. It showed that the AH is chemically bonded to
MCC microspheres.
In vitro drug release
The in vitro drug release results are shown in Figure 8.
2-Thata (°)
Liu et al. 1067
The results showed that the microspheres released
minimum AH in water, and with the increase of ionic
strength in the dissolution mediums, the drug released
faster. The results showed that the drug release process
was mediated by the ion exchange functionality from the
microspheres. In addition, the microspheres did not
demonstrate the “burst release” at the initial stages. The
drug was released in a sustained manner for 8 h in
physiological isoosmolar 0.15 mol/L NaCl, and more than
90% of loaded drug was released after 8 h. The results
also showed that the AH was combine with the MCC
microspheres with chemical bond. And the drug release
data’s analysis results are in Table 2. The results showed
that the Zero-order equation fit for the drug release
process.

1068 Afr. J. Pharm. Pharmacol.
Conclusion
Intensity
�����
�����
�����
����
�
�� �� �� �� �� �� �� ��
Figure 7. X-ray of AH microspheres.
Drug released (%)
2-Thata (°)
Time (h)
Figure 8. Effects of ion concentration on drug release from the AH
microsphere.
Table 2. Results of mathematical study for drug release from the microsphere.
Functions Expression Equation r
Zero-order Q=Bt+A Q=15.33t+0.82 0.994
First-order In(100-Q)=Bt+A In(100-Q)=-0.46t+4.17 0.815
Higuchi Q=Bt 1/2 +A Q=37.93t 1/2 -11.13 0.903
Novel lung-targeting sustained-release AH microspheres
were prepared and characterized. The results clearly
demonstrated that the blank MCC microspheres had a
good degradation character in vitro. The AH
microsphere’s particle size was appropriate for a lungtargeting
purpose. The X-ray diffraction and in vitro drug

dissolution results indicated the drug chemically bonded
to the ion exchangeable structure of the microspheres.
The microspheres had a sustained-release profile in-vitro
without the burst release phenomenon. These
microspheres containing AH thus demonstrated a great
potential therapy option with appropriate biocompatility,
sustained release profiles and appropriate particle size
for lung targeting.
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African Journal of Pharmacy and Pharmacology Vol. 5(8), pp. 1070-1079, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.157
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Effect of absorption enhancers on nasal delivery of
basic fibroblast growth factor
Feng Chengcheng, Shao Xiayan, Zhang Chi, Liu Qingfeng, Chen Jie, Shen Yehong, Zhang
Qizhi* and Jiang Xinguo
Department of Pharmaceutics, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai, 201203,
People's Republic of China.
Key Laboratory of Smart Drug Delivery, Ministry of Education and PLA, 826 Zhangheng Road, Shanghai, 201203,
People's Republic of China
Accepted 18 July, 2011
The present study was to screen an optimal absorption enhancer for enhancing the nasal absorption of
basic fibroblast growth factor (bFGF), a promising therapeutic agent to neurodegenerative diseases. In
this study, four absorption enhancers including chitosan, sodium caprate, poly-L-arginine (poly-L-Arg,
92.0 kDa) and dimethyl- -cyclodextrin were chosen to evaluate their toxicity using in situ toad palate
model and in vitro Calu-3 cell model. Transport study of bFGF across Calu-3 cell monolayers in the
absence or presence of chitosan was performed to determine the optimal concentration of chitosan.
Pharmacokinetics study was conducted to monitor changes in the blood concentration of bFGF
following nasal administration of bFGF solution with or without 0.5% (w/v) chitoson, in comparison with
intravenous administration of bFGF alone. Of the absorption enhancers tested, chitosan at the
concentration of 0.25 and 0.5% showed little toxicity to nasal cilia and Calu-3 cells, and exerted
reversible effect on the reduction of transepithelial electrical resistance. Transport study showed that
the apparent permeability coefficient (Papp) value was increased by about 16- and 2-fold, respectively
with addition of 0.5 and 0.25% chitosan, compared with bFGF solution alone. Following nasal
administration to rats, the formulation containing 0.5% chitosan significantly enhanced the absorption of
bFGF with an area under curve (AUC0-120min) nearly 1.4-fold that of bFGF solution (p

peptides and proteins, like nerve growth factor (Zhao et al.,
2004; Vaka et al., 2009), cholera toxin B subunit-nerve
growth factor (Zhang et al., 2008), insulin (Benedict et al.,
2004; Djupesland, 2008; Henkin, 2010) and Insulin-like
growth factor-1 (Liu et al., 2001; Thorne et al., 2004), has
been demonstrated previously. However, the total amount
of these macromolecules accessing the brain was
reported to be low, because of their low membrane
permeability, rapid mucociliary clearance from nasal
cavity and susceptibility to degradation either within the
lumen of nasal cavity or during passage across the
epithelial barrier. Hence, it is necessary to explore ways to
improve the efficacy of drug targeting to the brain
following nasal administration.
The factors limiting drug uptake across the nasal
epithelium could be counteracted with the use of
absorption enhancers. Several absorption enhancers
such as chitosan (Illum et al., 1994; Sinswat and
Tengamnuay, 2003; Vaka et al., 2009, Nisha and Pramod,
2007), dimethyl- -cyclodextrin (DM- -CD) (Shao et al.,
1992; Merkus et al., 1999; Yang et al., 2004),
poly-L-arginine (poly-L-Arg) (Natsume et al., 1999;
Ohtake et al., 2002; Bertram et al., 2010) and sodium
caprate (Mishima et al., 1987; Greimel et al., 2007) have
been proved effective in intranasal delivery of proteins
and peptides. Consequently, these absorption enhancers
were chosen as potential candidates to enhance the nasal
absorption of bFGF in this study. The conventional
concentration of absorption enhancers was selected
according to their enhancing efficacy profiles (Ohtake et
al., 2002; Sinswat and Tengamnuay, 2003; Yang et al.,
2004; Greimel et al., 2007; Khan et al., 2009).
In the present work, firstly, the nasal ciliotoxicity and
cytotoxicity study were conducted for the purpose of
evaluating the safety of the absorption enhancers, using
in situ toad palate model (Jiang et al., 1995) and Calu-3
cell model. Calu-3, the human lung adenocarcinoma cell
line, has properties similar to the serous cells of the upper
airway and has been used as an in vitro nasal platform to
investigate microparticles and polymer gels for protein
delivery (Witschi and Mrsny, 1999; Chemuturi et al., 2005;
Seki et al., 2007). Secondly, the transport of bFGF across
Calu-3 cell monolayers in the absence or presence of
chitosan was investigated to determine the optimal
concentration of chitosan. Finally, pharmacokinetics study
was conducted to monitor changes in the blood
concentration of bFGF using enzyme linked
immunosorbent assay (ELISA) method following nasal
administration of bFGF solution with or without 0.5%
chitosan, in comparison with intravenous administration of
bFGF alone.
MATERIALS AND METHODS
Materials
Calu-3 cells were purchased from American Type Culture Collection
(ATCC, Manassas, VA, USA). Recombinant human basic fibroblast
Chengcheng et al. 1071
growth factor (bFGF) was obtained from Beijing SL Pharmaceutical
Co., Ltd (Beijing, China). Lactate dehydrogenase (LDH) assay kit
was purchased from Jiancheng Bioengineering Institute (Nanjing,
China). Poly-L-Arg hydrochloride (MW 92.0 kDa), collagen solutions
(Type I from rat tail), (3-[4, 5-dimethylthiazol-2-yl] -2, 5-
diphenyltetrazolium) bromide (MTT), N-[2-hydroxyethyl]
piperazine-N'-[2-ethanesulfonic acid] (HEPES), sodium caparate
and Triton ® X-100 were obtained from Sigma-Aldrich Chemical Co.
(St. Louis, MO, US). Non-essential amino acid solution 100×,
penicillin streptomycin solution 100×, Dulbecco’s Modified Eagle’s
Medium-Ham’s F-12 nutrient (DMEM/F-12 1:1), fetal bovine serum
and trypsin/EDTA (0.25%/0.03% in phosphate buffer solution) were
purchased from Invitrogen (CA, USA). DM- -CD was obtained from
Waker Chemie AG (Munich, Germany). Chitosan in the form of
hydrochloride salt (83% N-deacetylated, viscosity 46 mPa⋅s, Mw
~300 kDa) was a gift of Golden-Shell Biochemical Co. (Nanjing,
China). Human bFGF ELISA development kit was purchased from
Peprotech (NJ, USA). All other reagents were of analytical grade.
Animals
Male Sprague–Dawley (SD) rats weighing 180 to 230 g were
obtained from Shanghai Sino-British Sippr/BK Lab Animal Ltd.
(Shanghai, China) and housed in animal holdings with fixed dark
and light cycle of 12 h at a constant temperature (25 ± 1°C). Toads
weighing 20 to 30 g (male and female) were obtained from Nanjing
Experimental Animal Center. The studies were approved by the
Animal Ethics Committee of Fudan University, and every effort was
made to reduce the stress of animals.
Nasal ciliotoxicity studies
In situ toad palate model was employed to evaluate the nasal
ciliotoxicity of four absorption enhancers at test concentrations
(Jiang et al., 1995). Specific methods are as follows: 0.5 and 0.25%
(w/v) chitosan, 5% (w/v) DM- -CD, 0.5% (w/v) poly-L-Arg and 0.5%
(w/v) sodium caprate were dissolved in saline respectively and used
as test solutions. The upper palate of toads was exposed and
treated with test solutions for 240 min (ensuring that the maxillary
mucosa of toad was fully submerged in solution). Then the test
solution was washed away with saline, and one piece of mucosa
about 3 × 3 mm was dissected out from the palate. The mucocilia
was examined with a light microscope (UFX-DX, Nikon, Japan) at
enlargements of 400 ×, and the duration of the ciliary movement (the
time from drug administration to the ending of ciliary movement was
recorded. Meanwhile, 1% (w/v) sodium deoxycholate and saline
were severed as positive and negative controls, respectively (n = 4).
Cell experiments
Calu-3 cells culture
Calu-3 cells were maintained in 10 cm tissue culture dishes in
DMEM/F12 medium supplemented with 10% fetal bovine serum, 1%
non-essential amino acids, penicillin (100 U/ml) and streptomycin
(100 mg/ml). Each week, cells were passaged and cultured in a
humidified atmosphere of 5% CO2 /95% air at 37°C. For transport
studies, the cells were seeded at an initial density of 5 × 105
cells/cm 2 on collagen-coated Transwell ® polyester inserts (pore size
0.4 m, surface area 0.33 cm 2 , Corning, NY, USA). After 48 h, an
air-interface was created and the cells were maintained with 0.6 ml
of culture medium in the basolateral chambers of Transwell ® . The
air-interface conditions stimulated differentiation of the cell
monolayer to form polarized, bioelectrically ‘‘tight’’ epithelial
monolayer. The passages used for the following experiments were

1072 Afr. J. Pharm. Pharmacol.
25 to 32. Transepithelial electrical resistance (TEER) values were
measured with a Millicell ERS (Millipore, MA, USA).
MTT assay and lactate dehydrogenase (LDH) assay
To evaluate the cytotoxicity of the absorption enhancers, cell viability
was determined by MTT assay. The absorption enhancers at
concentrations mentioned previously were aseptically dissolved in
Hank’s balanced salt solution with 30 mM HEPES (HBSS/HEPES)
of pH 7.4 to be used as test solutions. Calu-3 cells were seeded into
96-well cell culture plates at a density of 5 × 10 4 cells/well. On the
second day, the medium was replaced with 100 l of pre-warmed
test solutions (n = 4). After exposure for 4 h, the cells were
incubated with 100 l of MTT solution (1.25 mg/ml in phosphate
buffer solution) for 4 h. Following the treatment, the medium was
then removed and the formazan crystal, the metabolite of MTT,
formed during these procedure was dissolved in sodium
dodecylsulfate (SDS) solution (20% w/v) prepared in N, N-dimethyl
formamide (DMF)/water (1:1 V:V) at pH 4.7 (pH adjusted with 1 M
hydrochloric acid: acetic acid: Water 10:8:2 V:V:V) (Grenha et al.,
2007; Matilainen et al., 2008). After thoroughly mixing, the plate was
read at 570 nm using a SpectraMax multi-plate reader (Thermo,
Shanghai, China) for optical density that is directly correlated with
cell quantity. Survival rate was calculated from the relative
absorbance at 570 nm and expressed as the percentage of control.
In the MTT test, HBSS/HEPES and 5% SDS solution were used as
negative and positive controls, respectively. The relative cell viability
was then calculated as a percentage of the negative control from the
absorbance values.
The LDH test was performed using a commercial LDH assay kit to
monitor the cell membrane damage (Lin et al., 2006). Calu-3 cells
were seeded into a 96-well plate at a density of 5 × 10 4 cells per well
overnight. After 4-h exposure to test solutions, 100 l of the cell
culture supernatants were withdrawn. HBSS/HEPES and 1% (v/v)
Triton ® X-100 were used as negative and positive controls,
respectively. LDH leakage was then determined according to the
manufacturer’s protocol and calculated as a percentage of the
positive control.
TEER studies
Cell monolayer was initially washed twice and allowed to be
equilibrated for 30 min with HBSS/HEPES buffer. Then the apical
medium was replaced by test solutions mentioned previously in MTT
assay and lactate dehydrogenase (LDH) assay. TEER value was
measured 30 min before test solutions exposure and at 0, 15, 30, 60,
90, 120, 180 and 240 min after the administration. At the end of the
experiment, the cells were rinsed two times with HBSS/HEPES,
then replaced by fresh pre-warmed culture medium, and kept 48 h in
the incubator to determine the recovery of the monolayer integrity
(Florea et al., 2006; Salem et al., 2009). Background TEER value
due to the differences among filters and inter-groups, were deducted
by normalizing the measurement of 0 min to 100%, and all
experiments were performed in triplicates.
In vitro permeation studies
The test solutions were prepared by dissolving 0.25 or 0.5%
chitosan in HBSS/HEPES solution with moderate stirring, and then
mixed well with bFGF. The end concentration of bFGF was 50 g /ml.
bFGF alone in HBSS/HEPES (50 g/ml) was used as control. The
osmotic pressure of all solutions was 290 to 340 mOsm.
In transport studies, Calu-3 cell monolayers were equilibrated with
100 l HBSS/HEPES in the apical medium and 1 ml HBSS/HEPES
in the basolateral chamber for 30 min. Afterwards, the apical
medium was removed, and the cells were exposed to 100 l test
formulations as well as control solution (n = 6). At 15, 30, 60, 90, 120,
180 and 240 min after administration, 110 l samples were
withdrawn from the basolateral chamber, and were replaced by
fresh HBSS/HEPES (Florea et al., 2006). Samples were assayed by
using a human bFGF ELISA development kit according to the
manufacturer’s instruction.
Apparent permeability coefficients (Papp) were calculated by
Equation (1), where dQ/dt is the flux of bFGF across the monolayers
(ng•ml -1 ), C0 is the initial concentration in donor chamber, and A is
surface area of Transwell ® polyester inserts.
Papp
dQ 1
×
dt AC
= (1)
0
Pharmacokinetics studies of the bFGF formulations
Fifteen SD rats weighing 180 to 230 g were divided into three groups
(n = 5/group). All of the animals were anesthetized by chloral
hydrate (5% w/v, 350 mg/kg, intraperitoneal) and fixed in a supine
position. For intravenous (i.v.) administration, bFGF (1 g/kg) diluted
in saline was injected into the femoral vein rapidly. For intranasal
(i.n.) administration, test formulations contained bFGF and 1% BSA
(to reduce non-specific adsorption) were prepared without or with
0.5% chitosan. Then, 20 l of dosing solution were given to the
nostril of each rat (40 g/kg) via a polyethylene 10 (PE 10) tube
attached to a microlitre syringe. Blood was sampled from the tail
vein at predetermined point, that is, 0.033, 0.083, 0.25, 0.5, 0.75, 1,
1.5, 2, 4, 8 and 12 h after administration. The serum samples,
separated by centrifugation at 10,000 rpm for 5 min at 4°C, were
analyzed by ELISA method.
Data analysis
Area under the blood concentration of bFGF versus time curve
(AUC0-t) was calculated by the trapezoidal method. The in vivo
pharmacokinetic parameters was obtained using DAS version 2.0
(Bontz Inc., Beijing, China). All the data were expressed as mean ±
standard deviation (SD). For multiple-group comparison, one-way
ANOVA was used followed by Fisher's least significant difference
post hoc test. Specific comparison between groups was carried out
with an unpaired Student’s t-test (two tailed). p

Chengcheng et al. 1073
Figure 1. Optical microscopic images of (A) negative control, (B) 0.25% chitosan, (C) 0.5% chitosan and (D)
positive control. Cilia indicated by arrow (10 × 40 magnification, n = 4).
peptide drugs accompanied by severe nasal membrane
damage (Marttin et al., 1998; Merkus et al., 1999;
Natsume et al., 1999). Therefore, in this study, we
investigated the toxicity of four candidate absorption
enhancers at test concentrations at first.
Nasal ciliotoxicity
Nasal Cilia are mobile fingerlike appendages extending
from the surface of the nasal epithelial cells which move in
a well-organized and coordinated way to propel the
overlying mucus layer toward the throat. They contribute
to the body’s primary nonspecific defense mechanism by
removing potentially hazardous substances. Since the
ciliary movement is a major indicator for mucociliary
clearance in the upper airways, it is important to evaluate
the possible effects of the absorption enhancers on ciliary
morphology (Hermens et al., 1990; Ugwoke et al., 2000).
For the present study, the toad palate model is considered
to be the optimal choice for the study of cilia toxicity, as
the results can give nice reproducibility and the
experimental technique is easy to be mastered (Jiang et
al., 1995). Optical microscopic observation of toad palate
showed that there were a great number of cilia with a fast
beating rate on the edge of the mucosa treated with 0.25
and 0.5% chitosan solution for 4 h (Figures 1B and C).
However, for toads treated with 0.5% sodium caprate,
0.5% poly-L-Arg or 5% DM- -CD, it was found that partial
cilia fell off from the edge of the mucosa and partial cilia
were stasis (data not shown). For all the mucosa treated
with test solutions, the ciliary movement lasted for more
than 11.7 h, slightly lower than that of the negative control
(14.57 h) and notablely longer than that of the positive
control (only 15 min). It is worth noting that the
percentages of duration of ciliary movement compared
with the negative control after treated with 0.25 and 0.5%
chitosan solution were above 90%, indicating no
ciliotoxicity of chitosan. However, significant differences
can be found between the other three absorption
enhancers and the negative control (Figure 2), suggesting
that 0.5% sodium caprate, 0.5% poly-L-Arg and 5%
DM- -CD cause mild to moderate nasal ciliotoxicity
Cytotoxicity
The vitality of Calu-3 cells after exposure to different

1074 Afr. J. Pharm. Pharmacol.
Figure 2. Percentage of duration of ciliary movement for mucosa treated with saline (as negative control),
0.25 and 0.5% chitosan, 5% DM- -CD, 0.5% poly-L-Arg, 0.5% sodium caprate (SC) and 1% sodium
deoxycholate (SDC, as positive control) (mean±SD, n = 4). *p

Figure 4. The TEER values of absorption enhancers across Calu-3 cell monolayers in apical to
basolateral direction (mean±SD, n = 6). The TEER value for every monolayer at time zero was
normalised to 100%. (A) Changes in TEER values measured before and after the
administration; (B) the TEER recovery of Calu-3 monolayers after the test solutions were
removed.
cytoplasm of normal cells, can be released in the event of
cell membrane damage. As a result, the degree of cell
damage can be determined by detecting the LDH level in
cell culture medium (Lin et al., 2006). Both 0.25 and 0.5%
chitosan solution resulted in a little LDH leakage from the
Calu-3 cells, without significant differences from the effect
of HBSS/HEPES. However, after exposure to 0.5%
poly-L-Arg, 5% DM- -CD and 0.5% sodium caprate for 4
h, LDH releases were increased to 21.22±3.09,
27.07±8.42 and 37.59±1.87%, respectively, significantly
higher than the negative control (p

1076 Afr. J. Pharm. Pharmacol.
monitored the TEER value of cell monolayers till 48 h after
washing out the dosing solution. After 48 h recovery
procedure, diminished TEER value of 0.25 and 0.5%
chitosan group nearly recovered to baseline level
(approximately 90% of the initial) (Figure 4B), indicating
the chitosan treatment caused only a transient disruption
of the cell monolayer’s barrier property and this effect was
reversible. However, TEER of cell monolayers treated
with other three absorption enhancers remained at low
level during the entire recovery period, and some cells
were found floating at the end of the experiment,
suggesting that sodium caprate, poly-L-Arg and DM- -CD
at test concentrations resulted in severe damage on
monolayer integrity and the effects were irreversible.
In the present study, the in situ toad palate model and in
vitro cell model were employed to find safe absorption
enhancers for intranasal delivery of bFGF. According to
the results from these two models, among four absorption
enhancers, chitosan was the safest, while 0.5% sodium
caprate, 0.5% poly-L-Arg and 5% DM- -CD showed mild
to moderate nasal ciliotoxicity and severe cytotoxicity.
More severe toxic effects in vitro could be attributed to the
fact that in MTT experiment, the cells were totally
immersed in absorption enhancer solutions, while the
ciliated epithelium in situ is protected by the mucus barrier
and mucociliary clearance, and was exposed to the test
solutions only apically. In addition, the epithelial cells of
the nasal mucosa were constantly replaced by cells from
the basement membrane in vivo, a situation which could
not be mimicked in vitro (Dimova et al., 2005).
In consideration of nasal cilitoxicity and cytotoxicity,
chitosan at concentrations of 0.25 and 0.5% were finally
chosen as absorption enhancer in the subsequent cell
transport experiment.
bFGF transport across Calu-3 cells
Paracellular transport experiments with marker
fluorescein sodium which could permeate across Calu-3
cell monolayers have been previously performed in our
lab. Apparent permeability coefficient (Papp) value was
2.46 × 10 -7 cm/s, similar to the previous report (Forbes,
2000), indicating Calu-3 cells had formed a confluent
sheet.
In this study, the Papp values of bFGF in the absence or
presence of chitosan were shown in Table 1. The Papp of
control group was 0.67±0.18×10 −8 cm/s, and cumulative
transport amount at 4 h was only 0.039% (w/w) of the
applied dose. The addition of 0.25 and 0.5% chitosan
dramatically enhanced bFGF permeation across the
Calu-3 cell layer. Especially, compared with solution alone,
the Papp value and the cumulative transport amount had
been increased nearly 16-fold and 12-fold with 0.5%
chitosan, respectively.
Based on the transport results, 0.5% chitosan exhibited
stronger permeation facilitation on bFGF than 0.25%
chitosan. These findings are in agreement with the results
previously published (Artursson et al., 1994), so chitosan
at the concentration of 0.5% was used in subsequent
pharmacokinetic study.
Pharmacokinetics studies of the bFGF formulations
The potency and efficacy of chitosan in enhancing nasal
absorption of bFGF were investigated by formulating
bFGF with 0.5% chitosan. The preparation supplemented
with 1% BSA to minimize bFGF loss by non-specific
absorption. The concentrations of bFGF with time in blood
following Intravenous (i.v.) and Intranasal (i.n.)
administration to rats are given in Figure 5 and the main
pharmacokinetic parameters were illustrated in Table 2.
The i.v. injection of bFGF solution resulted in immediate
appearance of the peptide in blood with a rapid decline
showing no detected concentration 1 h after the
administration. On the other hand, nasal administration of
bFGF solution alone resulted in a much lower blood bFGF
level. The peak concentration was reached at about 40
min. Inclusion of 0.5% chitosan in the formulation caused
higher Cmax and extended tmax (about 60 min). The
AUC0-120h of the bFGF formulation following i.n.
administration was 335.34±22.10 ng�min/ml, increased to
472.2±52.36 ng min/ml as a result of 0.5% chitosan
added. The absolute bioavailability was 5.35 and 7.53%
for intranasal bFGF in the absence and presence of 0.5%
chitosan, respectively. Although the absolute nasal
bioavailability seemed to be low when compared to i.v.
administration, the AUC0-120h values of 0.5% chitosan
group was significantly greater than the control intranasal
group (p< 0.05, Table 2).
It was reported that the mechanism of chitosan on
absorption enhancement may include the properties of
mucoadhesion and reversible opening of tight junctions
among cells, as the result of the interaction between its
positively charged amino groups with the negatively
charged sialic acid residues in mucus, which lengthened
nasal residence time of drugs and led to improvement in
the transport of large hydrophilic compounds across the
epithelium (Illum et al., 1994).
In this study, addition of chitosan in bFGF solution
increased the bioavailability of bFGF by 1.4-fold after
intranasal administration, which was not as significant as
other reports. Sinswat and Tengamnuay (2003) proved
that chitosan elevated the bioavailability of calcitonin from
1.22% to 2.45% (2-fold) and Dyer et al. (2002)
demonstrated that the bioavailability of insulin was
promoted from 0.5 to 3.6% (7.2-fold) in the presence of
0.5% chitosan. The relatively lower enhancement in nasal
absorption is probably because the higher bioavailability
of bFGF alone (5.35%) than that of insulin and calcitonin.
bFGF is a cationic peptide, which may interact with
anionic cell membrane and therefore improve its
permeability through nasal mucosa and transport to the

Figure 5. Concentration-time profiles of bFGF in blood following intranasal (i.n.) administration of
bFGF solution with or without 0.5% chitosan, in comparison with intravenous (i.v.) administration of
bFGF solution to rats (mean±SD, n = 5). bFGF-CS, bFGF solution with 0.5% chitosan.
Table 1. Effect of chitosan on bFGF transport across Calu-3 cell monolayers (mean±SD, n = 6).
Formulation Papp ×××× 10 -8 (cm/s) ER a bFGF transport (%) c ER b
Control 0.67±0.18 − 0.039±0.005 −
0.25% chitosan 1.71±0.51* 2.57 0.088±0.023* 2.24
0.5% chitosan 10.09±3.54* 16.31 0.465±0.123* 11.83
Chengcheng et al. 1077
a Papp enhancement ratio= Papp test solution/ Papp control. b Transport (%) enhancement ratio = bFGF transport (%) test solution /
bFGF transport (%)control. c Cumulative transport amount of the applied dose. *p

1078 Afr. J. Pharm. Pharmacol.
Technology Major Project (2009ZX09310-006), National
Natural Science Foundation of China (30772657) and
National Basic Research Program of China
(2007CB935800).
Abbreviations: bFGF, Basic fibroblast growth factor; CNS,
central nervous system; Poly-L-Arg, poly-L-arginine; DM- -CD,
dimethyl- -cyclodextrin; MTT, (3-[4, 5-dimethylthiazol-2-yl]-2,
5-diphenyltetrazolium) bromide; LDH, lactate dehydrogenase;
ELISA, enzyme linked immunosorbent assay; TEER,
transepithelial electrical resistance; HEPES, N-[2-hydroxyethyl]
piperazine- N' -[2-ethanesulfonic acid]; HBSS/HEPES, Hank’s
balanced salt solution with 30 mM HEPES; Papp, apparent
permeability coefficients; AUC, area under curve; SD,
standard deviation; bFGF-CS, bFGF solution with 0.5%
chitosan;
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African Journal of Pharmacy and Pharmacology Vol. 5(8), pp.1080-1085, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.299
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Modulation of monoamines and amino-acids
neurotransmitters in cerebral cortex and hippocampus
of female senile rats by ginger and lipoic acid
Hoda G. Hegazy 1 and Elham H. A. Ali 2 *
1 Faculty of Science, Ain Shams University, Cairo, Egypt.
2 Women College for Arts, Science and Education, Ain Shams University, Cairo, Egypt.
Accepted 07 July, 2011
Brain aging is the major risk factor for common neurodegenerative diseases. Free radicals are involved
in neurodegenerative disorders such as aging. Several neural systems are affected in aging.
Neurotransmitters exhibited a marked alteration in different regions of the brain as part of the normal
aging process. In the present study, age-related changes in the levels of monoamines and amino-acids
neurotransmitters in normal female senile rats and the effect of oral administration of two antioxidants
lipoic acid (LA) and ginger for 30 days accumulation on the neurotransmitters in the brain areas
(hippocampus and cerebral cortex) is investigated. The levels of hippocampal monoamines
[norepinephrine (NE) and serotonin 5-HT] and cortical dopamine (DA) and 5-HT were increased after
administration of LA while, no significant differences were found in cortical monoamines compared
with senile female rats. On the other hand, the levels of hippocampal DA, 5-HT and NE were increased
after administration of ginger while, no statistical difference were found in cortical monoamines after
administration of ginger compared with the adult ones. Furthermore, the effect of LA and ginger on the
amino-acid profile in female senile rats was impressive. There were significant and remarkable
increases in hippocampal glutamic, aspartic, GABA and cortical glutamic, aspartic, glycine and alanine
compared with the levels of senile female rats. Ginger supplementation showed increased the amino -
acids in the hippocampus (glutamic, aspartic, GABA and alanine) significantly as well as cortical
aspartic, glycine, GABA and alanine compared with the normal senile female rats. In conclusion, LA and
ginger may have shown a significant ameliorative value in counteracting age induced deficiency in
some brain areas of female aged rats via modulating the investigating monoamines and amino acids in
the brain cortex and hippocampus.
Key words: Ginger, lipoic acid, senile, monoamines neurotransmitters, amino acids, cortex, hippocampus.
INTRODUCTION
Aging is genetically programmed deterioration of all
physiological function with age. Multiple studies
demonstrated that aging increased excitability of principle
hippocampal neurons (Bekenstein and Lothman, 1993;
Barnes, 1994; Papa-theodoropoulos and Kestopoulos,
1996) and signified a diminution in the number and
function of inhibitory interneurons with aging. Therefore, it
is imperative to determine the extent of alternations in
*Corresponding author. E-mail: elhamhassan2006@yahoo.com.
Tel: 02-010-5530-704.
function of inhibitory gamma- amino butyric acid (GABAergic)
interneurons in different regions of brain as a
function of age (Stanly and Shetty, 2004).
Previous research showed that metabolism of
monoamines neurotransmitters significantly changed with
aging such as reduction of dopamine (DA) and
norepinephrine (NE) in cerebrum and brain stem of aged
rats (Barili et al., 1998; Lee et al., 2001). The levels of
DA, NE and 5-hydroxytryptamine (5-HT) decreased
throughout the brain of aged rats as compared with the
brain of adult ones, display higher monoamines levels in
striatum, entrohinal cortex, hippocampus and frontal
cortex (Levine et al., 1990; Miguez et al., 1999).

Moreover, Zambrzycka et al. (2002) indicated that
aging processes play a major role in inhibition of choline
acetyltransferase ChAT activity and this enzyme in
striatum is selectively for β amyloid peptides, as well as,
5-HT or its metabolites are likely to enhance plasma lipid
peroxidation via changes in the redox potential and lipid
peroxidation chain reaction (Aviram et al., 1991).
Ginger extract and its biological active compounds
caused some pharmacological activities, including antiinflammatory
(Lantz et al., 2007), anti-emesis (Sharma et
al., 1997), anti-tumor (Surh, 2002) and analgesic effects
(Aktan et al., 2006; Lantz et al., 2007) in numerous
diseases. Recently, it had reported that zingerone and 6shogaol
(active compounds of ginger) prevented 6hydroxy-dopamine
induced dopamine depression
(Kabuto et al., 2005) and apoptotic neural cell death
(Kyung et al., 2006).
On the other hand, lipoic acid or its reduced form,
dihydro-lipoic acid, is a physiological constituent of the
mitochondrial membranes and is an essential cofactor for
dehydrogenase. Biologically, alpha lipoic acid functions
as cofactor of oxidative decarboxylation reactions in
glucose metabolism to yield energy (Zulkhairi et al.,
2008). Moreover, lipoic acid acts as an effective
antioxidant in protecting the rat brain against reperfusion
injury following cerebral ischemia (Panigrahi et al., 1996).
Furthermore, Lykkesfeldt et al. (1998) discovered that the
level of lipoate is lowered during the process of aging.
Recent studies proved that lipoic acid acts as a potent
antioxidant by inhibiting lipid peroxidation and thereby
enhances the antioxidant status in aged rats (Bilska et
al., 2008).
The present study amid to evaluate the age-related
changes in the levels of monoamines and amino-acids
neurotransmitters in normal female senile rats and the
effect of orally administration of two antioxidants lipoic
acid (LA) and ginger for accumulative 30 days on the
neurotransmitters in the brain areas (hippocampus and
cerebral cortex).
MATERIALS AND METHODS
Animals
Adult female albino rats weighing approximately 130-150 gm (3-4
months old) and senile (24 months old) weighing 280-300 gm were
used. Rats were maintained in plastic cages and housed for 10
days prior to the initiation of the experiments, for adaptation to
laboratory conditions. Animals were fed with commercial standard
rat-pellet and tap water was provided ad libitum. Handling and
usage of animals agreed strictly with the regulations and guidelines
set by the research Ethics Committee of the Faculty of Science, Ain
Shams University.
Drugs
Ginger was purchased from MEPACO (Arab Company for
Pharmaceuticals and Medicinal Plants), Egypt and alpha lipoic acid
(thiotacid) was purchased from EVA Company, Egypt. All other
Hegazy and Ali 1081
chemicals and solvents used were of the high performance liquid
chromatography (HPLC) and analytical grade.
Experimental design
The animals were divided into four groups each of six rats as
follows: the first was the control young rats and received orally 0.5%
carboxy-methyl cellulose (CMC) sodium salt (0.1 ml/100 gm body
weight), the second was the control senile rats and received the
same amount of CMC. The third group, senile rats administrated
ginger at a dose of 250 mg/kg body weight dissolved in CMC
vehicle. The fourth group was senile rats administrated alpha-lipoic
acid (ALA) (65 mg/kg body weight CMC). All groups received
treatments for four consecutive weeks. Doses were calculated
related to the human therapeutic dose according to Reagan-Shaw
et al. (2007).
Following the completion of the experiments, the rats sacrificed
after 12 hours from the last dose by rapid decapitation. Brain was
excised for the determination of frontal cortex and hippocampus.
The frontal cortex and hippocampus was homogenized in 75%
methanol HPLC grade for determination of monoamines and free
amino-acids using the precolumn phenylisothiocyanate (PTC)
derivatization technique according to the method of Heinrikson and
Meredith (1984).
Biochemical assay
The Agilent HPLC system used with Rheodyne injector 20µl loop
and an ultraviolet (UV) variable wavelength detector was used for
monoamine assays where the samples were injected directly into
an AQUA column C18, purchased from Phenomenex, USA under
the following conditions: mobile phase 97/3 20Mm potassium
phosphate, pH 3.0/ methanol, flow rate 1.5ml/min, UV 270 nm. NE,
DA, and 5-HT were separated after 10 minutes. The resulting
chromatogram identified each monoamine position and
concentration from the sample as compared to that of the standard,
and finally, the calculation of the content of each monoamine as g
per gram brain tissue was made according to Pagel et al. (2000).
As regard to amino acids assay the derivatization started by
drying and re-drying the sample under test using re-drying solution
consisted of 2:2:1 mixture (by volume) of methanol: 1M sodium
acetate trihydrate: triethylamine (TEA). The drying solution was
added to the dry sample, shook well and then put under vacuum till
complete dryness. The derivatizing agent consisted of 7:1:1:1
mixture (by volume) of methanol: TEA: water: PITC
(Phenylisothiocyanate). The derivatizing solution was added to the
re-dried sample, shook well and left to stand at room temperature
for 20 min, then applied to vacuum (70 millitore) till dryness. The dry
sample was then reconstituted by sample diluent composed of 0.71
g disodium-hydrogen phosphate adjusted to a pH of 7.4 by 10%
phosphoric acid. Acetonitrile was then mixed, as 5% by volume with
the resulting solution. PICO- TAG column (Waters) was used for
free-amino acid analysis 3.9 × 30 cm. The assay conditions were as
follows: temperature: 46°C; wave-length: 254 nm; flow rate:
1ml/min. Standards and eluents are Waters chemistry package for
free amino acids.
Statistical analysis
Reported values represent means ± SE. Statistical analysis was
evaluated by one-way analysis of variance (ANOVA). Once a
significant F test was obtained, LSD comparisons were performed
to assess the significance of differences among various treatment
groups. Statistical Processor System Support "SPSS" for Windows
software, Release 12.0 (SPSS, Chicago, IL) was used.

1082 Afr. J. Pharm. Pharmacol.
RESULTS
Figure 1. Effect of ginger and lipoic acid on norepinephrine (ug/g), dopamine (ug/g) and serotonin 5HT (ng/g) in
hippocampus (Hippo) and cortex (Cor) of senile female rats. Values are means of 6 rats± SE. A = significant from
control, B = significant from senile rats and C = significant from senile treated with ginger at p 0.05.
Figure 1 shows the effects of ginger and -lipoic acid
administration on monoamines neurotransmitter levels in
two brain regions, hippocampus and frontal cortex in rat
groups. The data demonstrate that NE, DA and 5-HT
levels in hippocampus and frontal cortex decreased
significantly in senile female rats compared with that of
the adult groups. The levels of NE, and 5-HT increased
and DA decreased significantly in hippocampus of ginger
treated senile group in comparison with senile non
treated group. On the other hand orally administered
lipoic acid group exhibited significant increase in NE and
5-HT levels in hippocampus when compared to the senile
female rats. There were significant decreases in lipoic
acid treated group in cortical DA and 5-HT levels at
p 0.05 as compared with the senile female group.
The effect of the daily oral administration of ginger and
lipoic acid on the concentration of amino acids
(glutamic acid, aspartic acid, glycine, GABA and alanine)
in the hippocampus and frontal cortex of senile female
rats are presented in Figure 2. The senile female rats
showed significant decreases in hippocampal glutamate
and aspartate compared with adult female rats. In frontal
cortex area of the senile group, significant decrease in
levels of all amino acids investigated were found (p 0.05)
as compared with the corresponding adult group. The
daily oral administration of ginger induced significant
increases in hippocampal glutamic, aspartic, GABA and
alanine amino acids levels compared with the senile non
treated female rats levels. As well as, in frontal cortex,
administration of ginger showed significant increase in
glutamic, glycine, GABA and alanine levels compared
with senile female ones.
On the other hand, Figure 2 depicts the effect of -
lipoic acid on amino acid neurotransmitter levels. The
levels of all amino acids investigated were significantly
elevated in hippocampus and frontal cortex except in the
hippocampal glycine and alanine and cortical GABA
comparing with the senile female group (p 0.05).
DISCUSSION
Aging is an inherently complex process that is regulated
with multiple levels including genetic, molecular, and
cellular and system levels (Kregel and Zhang, 2007).
There are various structural, chemical and genetic
changes that occur in brain of old age (Hedden and
Gabrieli, 2004). Compared to other tissues in the body,
the brain is deemed abnormally sensitive to oxidative
damage (Keller et al., 2005). Recently, experimental
evidence supports the existence of relationship between
aging and various alternations of neurotransmitters in
different areas of central nervous system (Ota et al.,
2006; Lee et al., 2010).
Monoamine neurotransmitters are known to play an
important role in the cognitive functions (Arnsten et al.,
1994). The experimental results showed that the levels of
DA, NE and 5-HT in the brain hippocampus and DA and
5-HT in cortical area of naturally senile female rats were
remarkably lower than adult group. The present data
indicated that monoamine neurotransmitters changed a
lot during the aging process and orally administration of

Hegazy and Ali 1083
Figure 2. Effect of ginger and lipoic acid on glutamic (ug/g), aspartic (ug/g), glycine (ug/g), GABA (ug/g) and alanine
(ug/g) in hippocampus (hippo) and cortex (cor) of senile female rats. Values are means of 6 rats± SE. A= significant
from control, B= significant from senile rats and C= significant from senile treated with ginger at p 0.05.
ginger and – lipoic acid for 30 days can enhance the
levels of hippocampal NE and 5-HT and cortical DA and
5-HT in case of – lipoic acid only. In line with previous
results of Aviram et al. (1991) about alternation in 5-HT
level, which played a major role in brain function,
supported the formation of free radicals by aging.
Furthermore, Jiang et al. (2009) reported that the levels
of DA, NE and 5-HT in the brain tissues of naturally aged
mice were remarkedly lower than adult mice. Hof and
Mobbs (2009) reported the age relation to changes in
dopamine synthesis, binding sites, and number of
receptors. Recently, Santos et al. (2010) reported that
significant age - and region – dependant impairments in
monoamines modulatory neurotransmitter system that
correspond well with the motor phenotype observed in
the brain of methyl CpG binding protein 2 (a protein
gene) in mice.
The result of the current study demonstrated that the
levels of brain amino acids (hippocampal glutamic and
aspartic acids) and cortical glutamic, aspartic, glycine
GABA and alanine were significant declined in senile
female rats compared with the adult female rats.
Glutamate neurotransmitter decrease with age in living
human brain in the motor cortex (Kaiser et al., 2005;
Saliasuta et al., 2008; Chang et al., 2009). A significant
age-related decline especially in the parietal gray matter,
basal ganglia and to a lesser degree, the frontal white
matter in human brain has also been noted (Kaiser et al.,
2005; Saliasuta et al., 2008). On the other hand, Stanly
and Shetty (2004) demonstrated that age- related decline
in the functional GABA-ergic interneuron numbers may
underlie some of the hippocampus related behavioral
alterations observed in aged animals. In addition, Lee et
al. (2010) discussed the decreased protein level and the
age related functional decline and alterations of inhibitory
function in the hippocampus.
Regarding ginger, the present result revealed that there
were a remarkable increase in monoamine
neurotransmitters (NE, DA and 5-HT) and amino acids
neurotransmitters (glutamic, aspartic, GABA and alanine)
in hippocampal area while no significant changes in the
same monoamines in cortical regions, whereas there
were significant increases in amino-acids (glutamic,
glycine, GABA and alanine) neurotransmitters compared
with the levels of the same areas in adult female rats.
Kabuto et al. (2005) reported that zingerone and 6shogaol
(biological active compounds) prevent 6-hydroxy
dopamine – induced dopamine depression. In addition,
Geiger (2005) showed that Ginger exhibits 5HT3 receptor
antagonism which effectively antagonizes serotonin at 5-
HT3 receptors. This effect is mediated by galanolactone
(one of the ginger constituents). On the other hand,
Abdel-Aziz et al. (2006) found that the anti-emetic effect
of ginger and some of its constituents is not mediated by
competitive antagonism on the 5-HT3 receptor, since the
constituents were not able to displace the specific
radioligand from its binding site. Their action, therefore,
may be mediated by binding to a modulatory site distinct
from that of serotonin. Furthermore, Kyung et al. (2006)
found that zingerone and 6-shogaol reduced apoptotic
neuronal cell death and restores motor function in rat
spinal cord injury. Qiang et al. (2009) showed that oil
from ginger rhizome did not alter chronic unpredictable
mild stress-induced reduction on 5-HT levels in prefrontal
cortex, hippocampus and striatum in rats.
On the other hand, the effect of ginger on monoamines
contents may be through androgenic activity (Chrubasik
et al. 2005; Kamtchouing et al., 2002). Furthermore, Lin

1084 Afr. J. Pharm. Pharmacol.
et al. (2006) indicated that 1-(3, 4-dimethoxyphenyl)-3, 5dodecenedione
(I(6)), a derivative of gingerdione
mediated neuroprotective effect. This effect may be due
to increasing phosphorylation levels of extracellular
signal-regulated kinases (Waggas, 2009). Another
possible mechanism for Ginger, it shares with
nonsteroidal anti-inflammatory drugs (NSAIDs) the
property of inhibiting prostaglandin synthesis. Some
ginger constituents are dual inhibitors of COX and LOX,
and thereby reduce the biosynthesis of both
prostaglandins and LTs. This remarkable property
distinguishes ginger from conventional NSAIDs and may
account for its lack of gastrointestinal and renal side
effects (Grzanna et al., 2005).
The results of the present study demonstrated that the
levels of monoamines (hippocampal NE, 5-HT and
cortical NE) were significantly increased after
administration of lipoic acid in aged normal female rats
compared with the senile ones. Alpha lipoic acid or its
reduced form proposed as an effective antioxidant in
protecting the rat brain against reperfusion injury
following cerebral ischemia (Panigrahi et al., 1996) and
the level of lipoate was lowered during the process of
aging (Lykkesfeldt et al., 1998). The present results can
be explained by de Sales et al. (2010) who showed, for
the first time, the effects of LA on monoamines levels, in
the CNS. They results were consistent with the
hypothesis that LA stimulates the release and/or
syntheses or reduces the metabolization rate of
endogenous monoamines. LA increased the dopamine
and norepinephrine levels in normal rat hippocampus.
Moreover, serotonin levels were decreased. Together,
these results are of interest, considering that some
neurodegenerative diseases are related to the imbalance
of these monoamines levels in the central nervous
system (CNS). The present results support the same
hypothesis of modulation of investigated brain
neurotransmitters. DA has a modulator effect on the
dopaminergic system, in which increase of DA in the
hippocampus is correlated with decreased glutamate
levels de Sales Santos et al. (2010). Furthermore, the
increased dopamine and norepinephrine levels can be
produced by three mechanisms: 1) by increasing their
synthesis and or release; 2) by reduction in their
metabolization rate; and/or 3) by decreasing its reuptake
brain. In another set of experiments previous study
showed that ventral tegmental area stimulation releases
endogenous dopamine from the axonal terminals of
dopaminergic neurons in the accumbens, and de Sales
Santos results suggested that LA induces the release of
dopamine in hippocampus (Tao et al., 1996). Inhibition of
the GABA receptor decreases hippocampal 5-HT level,
while application of GABA agonists to hippocampus
decreases 5-HT release (Stanzione et al., 1984). This
reciprocal modulation of 5-HT and GABA in the
hippocampus appears incompatible with the simplistic
hypotheses of decreased 5-HT and GABA levels in
neurodegenerative diseases. GABA prevented over firing
of the nerve cells by blocking the transmission of an
impulse from one cell to another in the central nervous
system. Which produce anti-aging effects in brain mice
(Bist and Bhatt, 2009).
On the other hand, Zhang et al. (2001) found that
treatment with LA protected primary neurons of rat
cerebral cortex against cytotoxicity induced by both -
amyloid (A ) and hydrogen peroxide. In a similar study,
Lovell et al. (2003) reported that pretreatment of neurons
(hipocampal cultures) with LA significantly protected
against A and Fe/H2O2 toxicity, whereas concomitant
treatment of cultures with LA potentiated the toxicity of
Fe/H2O2. Furthermore, Zambrzycka et al. (2002) indicated
significant inhibitory effect of aging on
acetylecholinestrase (AChE) in brain cortex and striatum.
Moreover, Han et al. (1997) demonstrated that LA can
protect cells from both excitotoxicity and cystine-inhibtion
oxidative stress aspects of glutamate. They concluded
that LA has remarkable therapeutic potential in protecting
against neurological injuries involving glutamate and
oxidative stress.
Conclusion
From the present results ginger and LA have shown a
significant ameliorative effect in counteracting age
induced deficiency in some brain areas of female aged
rats through increasing the investigating monoamines
and amino acids in the brain cortex and hippocampus of
senile female rats.
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African Journal of Pharmacy and Pharmacology Vol. 5(8), pp. 1086-1091, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.305
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Induction of apoptosis by Rhizoma Paridis saponins in
MCF-7 human breast cancer cells
Chuan Lu 1† , ChunJiang Li 1,2† , Dongmei Wu 1 , JingMei Lu 1 *, Fan Tu 1 , Lijuan Wang 3
1 School of Life Sciences, Northeast Normal University, Renmin Street, Changchun, Jilin Province 130024, P. R. China.
2 College of Basic Medicine, Jiamusi University, 148 Xuefu Street, Jiamusi, Heilongjiang Province 154007, P. R. China.
3 Depatment of Ophthalmology, Eye Hospital, 151 East Street, Harbin, Heilongjiang Province 150001, P. R. China.
Accepted 11 July, 2011
This study investigates the relationship between the induction of MCF-7 human breast cancer cell
apoptosis by Rhizoma Paridis saponins (RPS) and the mitochondrial apoptotic pathway. We treated
MCF-7 cells with RPS at various concentrations and examined the inhibitory effect of RPS on the
proliferation of MCF-7 cells using the 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay, the change in mitochondrial membrane potential (MMP) using JC-1 staining, and the expression
levels of related proteins using western blot. The results showed that the inhibitory effect of RPS
against the growth of MCF-7 human breast cancer cells might be related to the apoptosis induced
caspase family and the caspase-3-dependent pathway. The results suggest that RPS has the potential
to be a valuable anticancer agent.
Key words: Rhizoma paridis saponins, MCF-7 cells, apoptosis, mitochondria
INTRODUCTION
Breast cancer is the second most serious malignant
tumor in women worldwide, especially in Europe and
America (Parkin et al., 2005). The treatment of breast
cancer has usually included surgery, radiotherapy,
chemotherapy, immunotherapy, and traditional Chinese
medicine therapy. However, a significant number of
patients experience severe side effects (Group EBCTC,
1998). Thus, it is important to find a new alternative for
breast cancer treatment that has high efficiency and low
toxicity.
Rhizoma Paridis refers to the roots and rhizomes of
Paris polyphylla var. yunnanensis. It has been used
widely in traditional Chinese medicine for cancer
treatment for a thousand years. Recent studies have
shown that Rhizoma Paridis has significant antipyretic,
alexipharmic, detumescent, demulcent, and hemostatic
effects and is useful in the treatment of hepatopathy
*Corresponding author. E-mail: jmsljm@yahoo.com.cn. Tel:
+86-431-85099629. Fax: +86-431-85099629.
Abbreviations: RPS, Rhizoma Paridis saponins; PARP, poly
ADP-ribose polymerase; FBS, fetal bovine serum; MMP,
mitochondrial membrane potential; MTT, 4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium
bromide.
(Chen et al., 1995; Li, 1984; Matsuda et al., 2003; Wang
et al., 1990, 1996). Studies have also shown that steroid
saponins are the main components in Rhizoma Paridis
(Cheunga et al., 2005; Lee et al., 2005; Wu et al., 2004).
Although many studies have shown that Rhizoma
Paridis saponins (RPS) have strong antitumor activity,
few studies have reported on the mechanism of RPS
against human cancer cell lines. In this study, we treated
MCF-7 cells with RPS at various concentrations. The
potential of RPS to inhibit cell proliferation and induce
apoptosis in MCF-7 cells was evaluated by measuring
cytotoxicity using the MTT assay, cell apoptosis, the
change in MMP, and expression levels of Bax, Bcl-2,
cytochrome C, caspase-3/9, cleaved caspase-3/9, poly
ADP-ribose polymerase (PARP), and cleaved PARP
associated with the apoptosis signaling pathway.
MATERIALS AND METHODS
Chemicals and reagents
Growth medium RPMI-1640 and fetal bovine serum (FBS) were
purchased from Gibco (Grand Island, NY). Annexin V-FITC/PI
Apoptosis Assay Kit and JC-1 dye were purchased from Molecular
Probes, Inc. (Eugene, OR). Antibodies of Bax, Bcl-2, caspase-3/9,
cleaved caspase-3/9, PARP cleaved PARP���� -actin and
cytochrome C were purchased from Cell Signaling Technology Inc.

(Danvers, MA). The ECL Western Blotting kit was purchased from
Pierce (Rockford, IL). 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium
bromide (MTT) was purchased from Sigma-
Aldrich (St. Louis, MO). Cell Mitochondria Isolation Kit was
purchased from Beyotime (Suzhou, China).
Cell lines
Human breast cancer cell lines MCF-7 (ER+, HER2/neu-) was
purchased from Cancer Institute and Hospital, Chinese Academy of
Medical Sciences.
Preparation of RPS
The crushed Rhizoma Paridis (100 g) were extracted with 70%
ethanol (800 ml) for 3 times, 2 h under reflux. The combined 70%
ethanol extracts were filtered and centrifuged after being
concentrated. The supernatant dissolved in water was then eluted
by 65% ethanol on macroporous adsorptive resin D101. The eluent
was finally condensed with a vacuum rotary evaporator to give a
gray, viscous extract, which was RPS. Then RPS was lyophilized
and stored at -20°C for further studies.
Cell culture and cytotoxicity assay
Human breast cancer MCF-7 cells were cultured in RPMI-1640
medium supplemented with 10% FBS under the condition of 37°C
in a humidified atmosphere with 5% CO2. The effect of RPS on
MCF-7 cells viability was determined using the MTT assay. MCF-7
cells were treated with RPS at various concentrations (0, 20, 40,
80, and 160 g/ml). 48 h later, 50 l of MTT stock solution (2 mg/ml)
was added and after incubation for 4 h, the absorbance at 490 nm
was then measured on a scanning multi-well spectrophotometer.
The cytotoxicity was evaluated with reference to the IC50 value. The
tests were performed 3 independent times.
Apoptosis assay
Apoptosis induced by RPS treatment for 48 h were determined with
an Annexin V-FITC/PI Apoptosis Assay Kit according to
manufacturer’s instructions. Analyses were performed on a
FACSCalibur flow cytometer (BD Biosciences; Mountain View, CA).
The cells in the FITC-positive and PI-negative fraction were
regarded as apoptotic cells.
MMP assay
Treated MCF-7 cells were washed twice and incubated in medium
containing 10 g JC-1 dye for 30 min at 37°C in the dark. Stained
cells were harvested, washed, resuspended, and subjected to flow
cytometry analysis according to manufacturer's instructions. JC-1
was selectively accumulated in intact mitochondria and formed
multimer J-aggregates that fluoresced red (590 nm) at a high
membrane potential. Monomeric JC-1 fluoresced green (527 nm) at
a low membrane potential. Thus, the different colors of fluorescence
of JC-1 represented different MMP, which could be analyzed by flow
cytometer.
Western blot assay
Treated cells were lysed and then total protein was extracted.
Lu et al. 1087
Samples were separated in a 10% polyacrylamide gel and then
transferred onto polyvinylidene fluoride (PVDF). The membranes
were blocked in 5% nonfat milk in Tris Buffer Saline Tween20
(TBST) buffer for 1 h and hybridized with antibodies specific for
Bax, Bcl-2, caspase-3/9, cleaved caspase-3/9 and -actin
respectively, and then with appropriate HRP-conjugated secondary
antibodies. Signal was developed using an ECL kit, and the relative
photographic density was analysed by Quantity one software.
Cytosolic cytochrome C assay
Treated MCF-7 cells were processed by Cell Mitochondria Isolation
Kit according to manufacturer's instructions. Cytosolic extracts were
resolved in a 12.5% polyacrylamide gel and transferred, and the
blot was hybridized with cytochrome C antibody. Signal was
developed using an ECL kit, and the relative photographic density
was analysed by Quantity one software.
Statistical analysis
The data were presented as mean±SD ( x ±s). Differences between
the means of the individual groups were assessed by one-way
ANOVA with Duncan's multiple range tests. Differences were
considered significant at p

1088 Afr. J. Pharm. Pharmacol.
Figure 1. Cytotoxic effects of RPS in MCF-7 cells. Cell viability was
assessed by MTT assay. Data are expressed as mean ± SD (n = 3).
Triplicate measurements were performed. * P < 0.05. ** P < 0.01
compared with control.
Figure 2. Flow cytometer analysis of apoptotic cells by annexin V-FITC/PI expression. RPS induced apoptosis in MCF-7 cells: (A) MCF-7
cells not treated with RPS; (B) MCF-7 cells treated with 20 g/ml RPS; (C) MCF-7 cells treated with 40 g/ml RPS; (D) MCF-7 cells treated
with 80 g/ml RPS; (E) MCF-7 cells treated with 160 g/ml RPS. Early apoptotic cells were defined as annexin V-positive, PI- negative
cells.

Figure 3. RPS induced the loss of MMP in MCF-7 cells. Cells were treated with various
concentrations of RPS for 48 h. MMP was measured by flow cytometry after JC-1 staining. Triplicate
measurements were performed. * P < 0.05. ** P < 0.01 compared with control.
Figure 4. Bax, Bcl-2, caspase-3/9, cleaved caspase-3/9,
PARP, and cleaved PARP were determined by western blot
analysis. -Actin was used as an internal control.
Western blot analysis
To investigate the change in protein levels involved in
RPS-induced apoptosis, total cell lysates from treated
Lu et al. 1089
cells were prepared, and Bax, Bcl-2, caspase-3/9,
cleaved caspase-3/9, PARP, and cleaved PARP levels
were determined by western blot analysis. Results
showed that the 48-h RPS treatment activated PARP,
caspase-3, and caspase-9 and increased the ratio of Bax
and Bcl-2 (Figures 4 and 5).
RPS induced the release of cytochrome C in MCF-7
cells
The disruption of the mitochondrial membrane is known
to result in the release of cytochrome C into the cytosol,
and this was detected by western blot analysis (Figure 6).
These results showed that the treatment of MCF-7 cells
with RPS induced the release of cytochrome C from the
mitochondria in a dose-dependent manner.
DISCUSSION
In recent years, natural compounds have shown
significant antitumor effects. To balance cell proliferation
and cell death to maintain homeostasis in normal tissues
is an important mechanism. Cell death occurs via 2
pathways; apoptosis and necrosis. Apoptosis, also called
programmed cell death, is characterized by the
maintenance of intact cell membranes during the cell
suicide process (Hetz et al., 2005). Therefore, apoptosis
is considered to play a role in cancer prevention. Some
studies have shown that RPS has the ability to reduce

1090 Afr. J. Pharm. Pharmacol.
Figure 5. The change in Bax/Bcl-2 levels. The relative photographic density was analyzed by
Quantity One software. Triplicate measurements were performed. * P < 0.05. ** P < 0.01 compared
with control.
Figure 6. The release of cytochrome C from mitochondria was detected by
western blot analysis. -Actin was used as an internal control.
the viability ratio of cancer cells and induce apoptosis and
that it has a strongly cytotoxic effect on MCF-7 human
breast cancer cells, but the molecular mechanisms
underlying these effects are not clear.
Apoptosis is the most potent defense mechanism
against cancer (Sun et al., 2004). Mitochondrial apoptosis
pathway is the main procedure in cell apoptosis. MMP is
important in maintaining the stability of the environment
of the inner mitochondria and the oxidative
phosphorylation pathway (Jack et al., 2005).
The role of the Bcl-2 family proteins in apoptosis
regulation has been extensively studied. These proteins
are widely distributed in the mitochondrial outer
membrane, nuclear membrane, and endoplasm. The
family is divided into anti-apoptosis proteins like Bcl-2
and Bcl-xL, and apoptosis-promoting proteins like Bax,
which are the most important regulators for characterizing
apoptosis (Deveraux and Reed, 1999; Roy et al., 1997).
Bcl-2 is a kind of mitochondrial membrane protein that
can inhibit apoptosis and increase cell viablity. Bax is an
apoptosis-promoting protein in the mitochondrial
apoptosis pathway. In the mitochondrial-dependent
pathway, the major function of the Bcl-2 family proteins is
to regulate the permeabilization of the mitochondrial
membrane. The ratio of Bax/Bcl-2 is the key factor in
determining whether apoptosis occurs after cells receive
the apoptotic signal. When the ratio of Bax/Bcl-2
increased, the loss of MMP was promoted, the

permeabilization of mitochondrial membrane was
increased, and more cytochrome C was released (Kakkar
and Singh, 2007; Sheridan et al., 2008). As an apoptosis
induction factor, cytochrome C mediates the binding of
Apaf-1 to pro-caspase-9, resulting in the activation of
caspase-9 and initiation of the caspase cascade (Katoh
et al., 2008; Zhao et al., 2007), and then it cleaves PARP
and induces apoptosis (Egger et al., 2007). Cleavage of
caspase-3 and caspase-9 are characteristics of apoptosis
(Kok et al., 2005; Sunaga et al., 2004).
In the present study, RPS treatment resulted in the loss
of MMP and induced the down-regulation of antiapoptotic
Bcl-2 levels as well as the up-regulation of proapoptotic
Bax levels. The expression level of cleaved
caspase-3/9 and cleaved PARP and the concentration of
cytochrome C in the cytoplasm increased. These results
demonstrated that RPS induced apoptosis in MCF-7
human breast cancer cells via the mitochondrial
apoptosis pathway, involving the release of cytochrome
C, activation of Bax, inhibition of Bcl-2, activation of the
caspase cascade, and activation of PARP.
Conclusion
This study demonstrated that RPS has a potent inhibitory
effect on the proliferation of MCF-7 human breast cancer
cells. Induction of apoptosis by RPS is confirmed via the
loss of MMP, release of cytochrome C, increase of
Bax/Bcl-2 ratio, activation of the caspase cascade, and
cleavage of PARP. These results suggest that the RPSinduced
loss of MMP has an important role in the
apoptosis mechanism of MCF-7 human breast cancer
cells and that RPS may be potentially used as an
anticancer agent against human breast cancer.
ACKNOWLEDGMENT
†Chuan Lu and Chunjiang Li contributed equally to this
work as co-first authors
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African Journal of Pharmacy and Pharmacology Vol. 5(8). pp. 1092-1095, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.346
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
In-vivo study of stratum corneum water content and
transepideramal water loss using a newly formulated
topical cream of hippophae rhamnoides fruit extract
*Barkat Ali Khan 1 *, Naveed Akhtar 1 , Tariq Mahmood 1 , Haji M.Shoaib Khan 1 , Shahiq-Uz-
Zaman 1 , Akhtar Rasul 1 , Muhammad Iqbal 1 , Atif Ali 1 , Salimullah Khan 2 and Mughal Qayum 3
1 Department of Pharmacy, Faculty of Pharmacy and Alternative Medicine, the Islamia University of Bahawalpur,
Pakistan.
2 Department of Pharmacy, Anbar Campus, Abdul Wali Khan University, Mardan, Pakistan.
3 Department of Pharmacy, University of Peshawar, Peshawar, Pakistan.
Accepted 7 June, 2011
This study was purposed to determine the effects of newly formulated topical skin-care cream (w/o
emulsion) of Hippophae rhmanoides versus its vehicle (Base) as control on stratum corneum (SC)
water content and transepidermal water loss (TEWL). Concentrated H. rhamnoids (sea Buckthorn) fruit
extract was entrapped in the inner aqueous phase w/o emulsion. Newly formulated and previously
evaluated base (containing no active material) and a formulation (containing 1% concentrated extarct of
H. rhmanoides) were applied. Both the base and formulation were applied to the cheeks of 21 healthy
human volunteers for a period of 8 weeks. SC water content and TEWL were monitored every week to
measure any effect produced by these topical creams. The vehicle (base) showed insignificant (p 0.05)
effects whereas the formulation showed statistically significant (p 0.05) decrease in TEWL. SC water
content was significantly (p 0.05) increased by the formulation. The newly formulated cream of H.
rhmanoides fruit extract, applied is suitable for improvement and quantitative monitoring of skin
hydration level (SC water content/ moisturising effects) and reducing TEWL in people with dry skin.
Key words: Sea Buckthorn, skin, Stratum corneum (SC), water content and transepidermal water loss (TEWL).
INTRODUCTION
Hippophae rhmanoides (Sea Buckthorn) is a deciduous,
plant with numerous greenish-yellow flowers and bright
orang, globular, ellipsoid fruit which belongs to family
Elaeagnaceae (Heber, 2007). It is native to Europe, India,
Nepal, Bhutan, Pakistan and Afghanistan. H. rhmanoides
shrub is 2 m tall with 2 to 6 cm long leaves (Rizvi et al.,
2007). H. rhmanoides juice is an important source of
some valuable chemicals such as vitamin C, toco-
pherolmacrotrients, organic acids and polyunsaturated
fatty acids (Zeb, 2006). H. rhmanoides has been used for
the treatment of radiation damage, inflammation and
*Corresponding author. E-mail: barki.gold@gmail.com or
aq_nuzhat@yahoo.com Tel: 0092-333-9732578. Fax:
0092629255243.
burns in Chinese folk medicines (Negi et al., 2005). H.
rhmanoides oil extracts have also been used in the
treatment of skin disorders such as eczema,psoriasis,
lupus erythematosus and dermatosus (Guliyev et al.,
2004).
The main advantage of applying topical emulsions is
that they increase the solubility and bioavailability of
therapeutic drugs as well as the ability to favor the topical
transport of hydrophilic solute. Topical emulsions also
avoid gastrointestinal environment and first pass effect
(Marti-Mestres et al., 2002).
The epidermis especially the Stratum corneum is
concerned with environmental protection of organism, the
dermis together with hypodermis is considered essential
for protecting the skin from mechanical stress (Escoffier
et al., 1989). Exposure to ultraviolet (UV) radiations
results in skin damage through several mechanisms such

Table 1. Score given by volunteer to base and formulation on the basis of itching/irritation a .
No of volunteer
Score 0 1 2 3
Base 16 3 2 0
Formulation 14 4 3 0
Khan et al. 1093
a No severe erythema occurred in any of volunteer; mild erythema occurred in 2 and 3 volunteers; moderate erythema occurred in 3 and
4 volunteers, whereas no erythema occurred in 16 and 14 volunteers for both base and formulation, respectively.
as collegenase production, thymine dimer formation and
enhancing inflammatory reaction. Antioxidants protect
human skin from free radicals produced by UV radiations
in the untimely stages of revelation (Bauman, 2005).
MATERIALS AND METHODS
Materials
For the formulation of emulsions /Creams (Applied in the study) H.
rhamnoides berries were purchased from Pak Sea Buckthorn
International Skardu, Pakistan. ABIL-EM90 was purchased from
Franken Chemical (Germany) and Methanol, n. Hexane and
paraffin oil were purchased from Merk KGaA Darmstadt (Germany).
Ethanol was taken from BDH England.
Apparatus
The apparatus used includes Corneometer MPA 5 and TEWA
Meter MPA 5 (Courage + Khazaka, Germany), SPSS 12.
Corneometer was used for SC water content measurement while
TEWA meter was used for TEWL measurement.
Emulsions (Creams)
In this study the products studied were newly formulated W/O
emulsions (base and formulation) which were found stable after
evaluating for pH, electrical conductivity, centrifugation, phase
separation, temperature stability tests at 8 ± 0.1°C (in refrigerator),
25 ± 0.1°C, 40 ± 0.1°C and 40 ± 0.1°C with 75% RH (in incubator)
and Physical characteristics, that is, color, creaming and
liquefaction.
Study design for product evaluation on skin
One-sided blind study was designed with placebo control in the
month of August to September. 21 healthy human volunteers who
signed the informed consent, with age range of 20-35 years were
selected. Male volunteers were included in this work as they were
easily available with regular under control observations. All the skin
tests were performed at 21±01°C and 40±2% relative humidity
conditions. The experiments were carried out on the cheeks of
volunteers as cheeks are uniformly and more prone to UV
radiations. On the first day, patch test (Burchard test) was
performed on the forearms of each volunteer to determine any
possible reactions to the emulsions. Each volunteer was provided
with two creams. One cream was Base and the other one was
formulation containing the active ingredients. Each cream was
marked with “right” or “left” indicating application of that cream to
the respective cheek. The creams were applied by the volunteers
themselves as instructed for 60 days. Every individual was
instructed to come on 1 st , 2 nd , 3 rd , 4 th , 6 th and 8 th week for the skin
measurements.
Burchard tests (Patch tests)
On the first day of skin testing, patch tests were performed on the
both forearms of each volunteer. A 5 X 4 cm region was marked on
the forearms. The patch (Bandage disc) for the right forearm was
saturated with 1.0 g of Base while the patch for left forearm was
saturated with 1.0 g of formulation. Each was applied to the 5 X 4
cm marked regions separately on each forearm. The regions were
covered with the surgical dressing after application. The patches
were removed after 48 h and the forearms were washed with
physiological saline (Hachem et al., 2002). After 48 h, scores were
recorded for the presence of erythema (skin redness) using a scale
with 4 points from 0 to 3.0 stands for absence of erythema, 1 for
mild erythema, 2 for moderate erythema while 3 stands for severe
erythema.Each volunteer was asked to note their irritation/itching
towards the patches and then assign a score from the same scale.
Average score with respect to volunteers is given in Table 1.
Panel test
Every individual was provided with a Performa prepared previously
to test the sensory values of creams. This Performa consisted of
seven parameters to be evaluated and every parameter was
assigned 11 values from –5 to +5 indicating very bad to very good,
respectively. This Performa was asked to be completed independently
by each individual at the end of study period. From the
average reply of volunteers it was concluded that base and
formulation were felt well on the skin, produced pleasant feeling on
application to skin and no irritation on the skin in both cases i.e.
base and formulation, as these were assigned 0.00 point for
irritation by all the volunteers. Shine on skin was more for
formulation. This was expected since the formulation contained
essential fatty acids. Similarly, the formulation led to more softness
of the skin than base.
It was found from paired sample t-test that there was an
insignificant difference between the average points of sensitivity for
base and formulation. It was concluded that there was no immense
variation between base and formulation regarding the sensory
evaluation. Both of the creams have similar performance from the
sensory of view.
Mathematical analysis
The percentage changes for the individual values of different
parameters, taken every week, of volunteers were calculated by the
following formula;

1094 Afr. J. Pharm. Pharmacol.
Table 2. *Percentage of change in SC water content after application of base and formulation*.
Time (week) 1 st 2 nd
Values of SC water content (Mean ± SEM)
3 rd
4 th
6 th 8 th
Base -0.88±1.57 -3.38±2.03 -6.57±3.56 -2.15±2.56 -3.55±3.90 -2.29±6.63
Formulation 13.16±3.07 22.36±4.17 31.02±4.70 59.69±10.68 61.62±9.43 62.79±12.72
*Percent change values are the average of 21 volunteers calculated by using the formula; Percentage Change = [(A – B) / B]*100. Where, A =
Individual value of any parameter of 1 st , 2 nd , 3 rd , 4 th , 6 th or 8 th week; B = Zero hour value of that parameter.
Table 3. *Percentage of Change in Values of transepidermal water loss (TEWL) after application of base and formulation*.
Time (week) 1 st 2 nd
Values of TEWL (Mean ± SEM)
3 rd
4 th
6 th 8 th
Base -5.33±12.25 9.38±13.02 10.68±10.52 15.07±13.65 -0.52±9.17 11.24±8.13
Formulation -9.66±4.94 -18.89±5.59 -28.68±6.62 -39.61±8.22 -46.31±8.75 -52.03±6.39
*Percent change values are the average of 21 volunteers calculated by using the formula; Percentage Change = [(A – B) / B]*100. Where, A =
Individual value of any parameter of 1 st , 2 nd , 3 rd , 4 th , 6 th or 8 th week; B = Zero hour value of that parameter.
2Percentage Change = [(A – B) / B]*100
where A = Individual value of any parameter of 1st, 2nd, 3rd, 4 th,
6th, or 8th week; B = Zero hour value of that parameter.
Statistical analysis
The measured values obtained for SC water content and TEWL
were analyzed using SPSS 12.0 on the personal computer (paired
samples t-test for variation between the two preparations; two-way
analysis of variance (ANOVA) for variation between different time
intervals while using a 5% level of significance for both skin
parameters.
RESULTS
Stratum corneum (SC)
The percent change occurred in the SC water content
before and after applications of base and formulation
have been presented in Table 2.
Transepidermal water loss (TEWL)
The percent changes occurred in the values of TEWL
before and after applications of base and formulation
have been given in Table 3.
DISCUSSION
Stratum corneum (SC) water content
In this study, it was found that there was an irregular
decline in SC water content values throughout the study
period after the application of base samples. In case of
formulation samples, it was found that there was gradual
increase in SC water content from the 1st week up to the
8th week of study.
With the help of ANOVA test, it was found that the base
produced insignificant (p 0.05) effects on SC water
content with respect to time while formulation produced
significant (p 0.05) effects on moisture contents with
respect to time.
With the help of paired sample t-test, significant
(p 0.05) differences were observed between the SC
water content of base and the formulation from the 2nd
week of study period.
Vitamin C has the advantage of stimulating dermal
fibroblasts for the synthesis of collagen. As the collagen
level is increased, the hydration level also improved
(Sharma et al., 2008; Colven and Pinnell, 1996). The
vitamin C concentration in H. rhmanoides fruit ranges
from 28 to 2500 mg/ 100 g (Zeb, 2006) so the formulation
produced a significant (p 0.05) increase in SC water
content.
Transepidermal water loss (TEWL)
In this study, it was found that there were variations in
TEWL values after the application of base. On the 1st
and 6th week it was decreased while on the 2 nd , 3 rd , 4 th
and 8 th week it was increased. In case of formulation
there was gradual decrease in TEWL throughout the
study period.
With the help of ANOVA test, it was found that changes
in TEWL values produced by base were insignificant (p
0.05) with respect to time. Whereas applying ANOVA test

to the formulation it was concluded that the changes in
TEWL values were significant (p 0.05) with respect to
time.
With the help of paired sample t-test it was found that
there was insignificant (p 0.05) variation in TEWL with
respect to base and formulation for the 1st three weeks
while from 4 th to the 8 th week of study significant (p 0.05)
differences were observed between the TEWL values of
base and the formulation.
Oils having linoleic acid are considered good for
reducing TEWL and restoring skin barrier function. H.
rhmanoides oils made up of about 12.4% linoliec acid
(POINT OF INTEREST, 2009). So the formulation
reduces TEWL significantly.
Conclusion
In conclusion, a stable topical cream (W/O emulsion)
containing H. rhamnoids fruit extract can produced a
pronounced increase in moisture content of the skin
showing that the formulation has skin moisturizing
effects. The formulation was observed to decrease TEWL
significantly which shows that the formulation has antiwrinkle
affects. Since both of the aforementioned
parameters are involved in aging so this formulation can
be used as anti-aging product.
ACKNOWLEDGEMENTS
The authors express their thanks to Higher Education
Commission of Pakistan for financial support and The
Department of Pharmacy, The Islamia University of
Bahawalpur for moral support.
REFERENCES
Khan et al. 1095
Bauman L (2005). How to Prevent Photoaging?. J. Invest. Dermatol.,
125: XII-XIII.
Colven RM, Pinnell SR (1996). Topical vitamin C in aging. Clinic.
Dermatol., 14: 227-234.
Escoffier C, Rigal JD, Rochefort A, Vasselet R, Leveque JL, Agache PG
(1989). Age-Related Mechanical Properties of Human Skin: An In
Vivo Study. J. Invest. Dermatol., 93: 353-357.
Guliyev VB, Gul M, Yildiri A (2004). Hippophae rhamnoides L:
chromatographic methods to determine chemical composition, use in
traditional medicine and pharmacological effect. J. Chromatogr. B.,
812: 291-307.
Hachem JP, Paepe KD, Vanpee E (2002). The effect of two
moisturisers on skin barrier damage in allergic contact dermatitis.
Euro. J. Dermatol., 12: 136-138.
Heber D (2007). PDR for Herbal Medicines. Montvale: Thomson
Healthcare, pp. 740-741.
Marti-Mestres G, Nielloud F (2002). Emelsion in Health care
applications-An Overview. J. Disp. Sci. Technol., 23(1-3): 419-439
Negi PS, Chauhan AS, Sadia GA, Rohinishree YS (2005). Antioxidant
and antibacterial activities of various Sea Buckthorn (Hippophae
rhamnoides L.) seed extracts. Food. Chem., 92: 119-124.
POINT OF INTEREST!: Sea Buckthorn OIL. 2009. [Cited 2009 Dec 20].
Available from http://swiftcraftymonkey.blogspot.com/2009/12/seabuckthorn-oil.html
Rizvi MA, Saeed A, Zubairy N (2007). Medicinal plants History,
Cultivation and Uses. Karachi: Hamdard Institut. Adv. Studies Res.,
p. 85-87.
Sharma SR, Poddar R, Sen P, Andrews JT (2008). Effect of vitamin C
on collagen biosynthesis and degree of birefringence in polarization
sensitive optical coherence tomography (PS-OCT). Afr. J.
Biotechnol., 7: 2049-2054.
Zeb A (2006). Chemical and Nutritional constituents of sea Buckthorn
juice. Pak. J. Nutr., 3(2): 99-106.

African Journal of Pharmacy and Pharmacology Vol. 5(8). pp. 1096-1105, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.307
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Ameliorative effects of ginger and -lipoic acid on
oxidative stress and inflammation in senile female rats
Hoda G. Hegazy
Zoology Department, Faculty of Science, Ain Shams University, Cairo, Egypt. E-mail: hodahegazy@hotmail.com. Tel:
20 173042042
Accepted 18 July 2011
Oxidative stress is recognized as an important environmental factor in aging. The reactive oxygen
species and related free radicals are normally produced both intra and extracellular and air-breathing
organisms cannot avoid the risk of oxidative stress. Moreover, recent studies have advanced the notion
of chronic inflammation as a major risk factor underlying aging and age-related diseases. In the present
study, the evaluation of the protective effects of ginger and -lipoic acid (ALA) supplementation on
senile female rats is evaluated during inflammation. The results showed a significant increase in lipid
peroxidation but a significant reduction in the reduced glutathione level (GSH), the activities of
superoxide dismutase (SOD), catalase (CAT) and cytochrome P450 (CytoP450) in hepatic aged female
rats. In addition, this study revealed a significant increase in the inflammatory mediators interlukin-1(IL-
1), interlukin-6 (IL-6), tumor necrosis factor alpha (TNF- ) as well as the activity of cyclooxygenase
enzyme (COX-2). Furthermore, there was a significant decrease in serum nitric oxide (NO) in aged
female rats. Ginger and ALA were effective in minimizing aged-related oxidative burden through
decreasing lipid peroxidation, increasing GSH content and promoting antioxidant enzymes. Moreover,
the compounds under investigation reduced the levels of the pro-inflammatory cytokines IL-1, IL-6 and
TNF- , in addition to inhibiting the activity of COX-2. The levels of serum NO was also increased by the
treatments.
Key words: Ginger- -lipoic acid- oxidative stress- inflammatory mediators' factors- cyclooxygenase-2.
INTRODUCTION
Modern science has made tremendous attempts to
understand the phenomenon of the aging process.
Several theories have been postulated, but at present,
the most popular and widely tested one is the free radical
theory of aging. This theory proposes that aging occurs
as a consequence of the deleterious effects of radicals
produced during the course of cellular metabolism.
According to this theory, the primary cause which initiates
the processes leading to the aging of an organism and its
ensuing death is the uncontrolled production of free
radicals. The free radicals have a direct influence on the
genetic and molecular mechanisms that determine the
life span of the organisms. Reactive oxygen species can
attack vital cell components like polyunsaturated fatty
acids, proteins and nucleic acids (Arivazhagan et al.,
2002). Moreover, free radicals-induced cellular stress
response mechanism is a major contributing factor to cell
and tissue decline with age. Susceptibility versus
resistance to exogenous and endogenous stresses is
now recognized as a key determinant of successful aging
(Shay and Hagen, 2009). Today many botanicals natural
products are used in therapy of different disease
(Ogungle and Lawal, 2006). Ginger is an example of
botanicals which is gaining popularity amongst modern
physicians. The underground rhizomes of ginger are the
medicinally useful part (Ahmed et al., 2008). Among the
pharmacological effects demonstrated are anti-platelets,
antioxidant, anti-tumor, anti-rhino viral, anti- hepatotoxicity,
anti-arthritic and anti-inflammatory effects (Lantz
et al., 2007; El-Sharakyet al., 2009). The strong anti-
oxidant action of ginger has been proposed as one of the
major possible mechanisms for the protective actions of
the plant against toxicity and lethality of radiation (Jagetia
et al., 2003; Haksar et al., 2006). Ginger counteracts the
toxic effects of carbon tetrachloride and cisplatin (Amin
and Hamza, 2006; Yemitan and Izegbu, 2006), and is
effective as an antiulcer agent (Siddaraju and Dharmesh,
2007).

Recently, it has been shown that ginger has strong antiinflammatory
and anti-apoptotic actions (Kim et al.,
2007). It is well known that T-helper (Th)-lymphocytes
play a key role in the regulation of immune and
inflammatory reactions through the release of cytokines
(Ahui et al., 2008). The anti-inflammatory properties of
ginger have been known for a long time (Grzanna et al.,
2005; Ali et al., 2008). Kiuchiet al. (1982) reported, for the
first time, that the anti-inflammatory action was the result
of its inhibitory effects on prostaglandins synthesis.
Further, Kiuchiet al. (1992) demonstrated gingerols (the
major bioactive compound present in ginger) were more
potent inhibitors of leukotrienes synthesis than
prostaglandins synthesis in vitro. More recently, it has
been shown that ginger as a whole or some of its
constituents are effective against cytokines synthesized
and secreted at sites of inflammations (Grzanna et al.,
2005). -Lipoic acid (ALA), a disulphide derivative of
octonic acid, and its reduced form dihydrolipoicacid
(DHLA) are natural compounds widely distributed in
plants and animals.They are synthesized through a
reaction catalyzed by lipoic acid sysnthase within the
mitochondria (Wollin and Jones, 2003). The therapeutic
actions of ALA are based on unique
antioxidantALA/DHLA system. Thus DHLA is able to
reduce not only reactive oxygen species (ROS) but also
oxidized forms of other antioxidants. ALA regenerates
other antioxidants and for this reason it is called an
antioxidant of antioxidants (Bilska et al., 2008).
Therefore, dietary lipoic acid is effective in attenuating
oxidative stress induced by drugs (Amudha et al., 2006)
and aging (Arivazhagan et al., 2002).
Previous studies showed that ALA acted as a potent
antioxidant by inhibiting lipid peroxidation and revitalizing
antioxidants in the liver and kidney of aged rats
(Arivazhagan et al., 2000). Unfortunatelly, the level of
ALA has been found to decrease gradually by aging
(Lykkesfeldt et al., 1998).
ALA is also used as modulator in several liver disorders
such as alcohol induced liver damage, mushroom
poisoning, metal intoxication and chloroform poisoning
(Basamante et al., 1998). ALA elevates the hepatic GSH
levels due to the presence of thiol groups (Packer et al.,
1995). Free thiols represent essential precursors or
intermediates in GSH synthesis and degrading pathways
as well as in the metabolism of several agents used in
medical treatments (Lilling and Holmgren, 2007).
In the light of these studies, the present work was
designed to scrutinize the hepatoprotective and
antioxidant potentials of ginger and ALA against aging
oxidative stress in senile female rat liver.
MATERIAL AND METHODS
Ginger was purchased from MEPACO (Arab Company for
Pharmaceuticals and Medicinal Plants), Egypt and thiotacidalpha
Hegazy 1097
lipoic acid (thiotacid) was purchased from EVA Company, Egypt. All
other chemicals and solvents used were of the highest purity and
analytical grade. Adult female albino rats weighing approximately
130 to 150 g (3 to 4 months old) and senile (24 months old)
weighing 280 to 300 g were used. Rats were maintained in plastic
cages (five per cage at 24 ± 2°C and 40± 10 humidity) and housed
for ten days prior to the initiation of the experiments, for adaptation
to laboratory conditions. Animals were fed with commercial
standard rat-pellet and tap water was provided ad libitum. Handling
and usage of animals agreed strictly with the regulations and
guidelines set by the research Ethics Committee of the Faculty of
Science, Ain Shams University.
The animals were divided into four groups each of five rats as
follows: The first was the control adult rats and received orally (by
means of stomach tube) 0.5% carboxymethyl cellulose (CMC)
sodium salt (0.1 ml/ 100 g body weight), the second was the control
aged rats and received the same amount of CMC. The third group,
aged rats administrated gingerat a dose of 250 mg/kg body weight
dissolved in CMC vehicle. The fourth group was aged rats
administrated ALA (65 mg/kg body weight CMC). All groups
received the different treatments for four consecutive weeks. The
doses were calculated from the human therapeutic dose (Regan-
Shaw et al., 2007).
At the end of the experimental period, all the animals were
sacrificed by cervical decapitation. Liver tissues were immediately
excised and rinsed in ice cold physiological saline. Blood was
collected and serum was separated for the assessment of total
nitric oxide (NO) using commercial ELISA kits specific for rat assays
(Assay Designs, Inc- Germany), the levels of rat interleukin-1 and
interleukin-6 (IL-1 and IL-6) and tumor necrosis factor (TNF- ) were
determined using enzyme immunoassay (EIA) techniques (IBL
Gesellschaft, Hamburg, Germany). The activity of cyclooxygenase2
(COX-2) was assayed by ELISA (sandwich) using commercial kits
(IBL-Hamburg Co., Germany).Liver tissue was removed, cleared of
blood, and rinsed in cold saline and used for the biochemical
analyses. The levels of reduced glutathione (GSH) were
determined according to Ellman (1959), the activity of cytochrome
P450 (CytoP450)in S-9 fractions according to the method modified
by McLean and Day (1974), superoxide dismutase (SOD) activity
according to the method of Oyanagui (1984) and catalase (CAT)
activity by the method of Sinha (1972). The levels of lipid
peroxidation (LP) were determined by measuring the content of the
thiobarbituric acid reactive substances (TBARS) in the tissue
homogenates following the procedure of Hogberg et al. (1974).
Statistical analysis
The results were presented as the mean ± standard error (SE) for
five animals in each group. Statistically significant differences
between groups were calculated using one- way analysis of
variance (ANOVA) followed by Snedecor and Cochran (1982). The
criterion for significance was set at p< 0.05.
RESULTS
Effect of ginger and -lipoic acid on endogenous
antioxidant defense system
Table 1 depicts the effect of ginger and lipoic acid on
reduced glutathione content (GSH) and lipid peroxidation
(LP) of adult and aged female rats. The activities of
superoxide dismutase (SOD), catalase (CAT) and cyto-

1098 Afr. J. Pharm. Pharmacol.
Table
1. Effect of ginger and -lipoic acid on oxi-redox system in liver senile female rats.
Parameter
GSH
(mg/g tissue)
SOD
(U/mg tissue)
CAT
(u/mg tissue)
CYTO P450
(P mol/ 100 mg)
LP
(nmol/ 100 mg tissue)
Treatment
Adult
female rats
Senile
female rats
Senile female rats
treated with ginger
Senile female rats treated
with -lipoic acid
Mean 20.54 A
14.11 B
16.56 C
18.89 D
±SE ± 0.22 ± 0.32 ± 0.29 ± 0.28
% of change (-31.30) (-19.38) (-8.03)
Mean 7.61 A
5.13 B
5.98 C
6.61 C
±SE ± 0.21 ± 0.18 ± 0.64 ± 0.11
% of change (-32.59) (-21.42) (-13.27)
Mean 61.63 A
40.62 B
47.56 C
52.69 D
±SE ± 0.17 ± 0.32 ± 0.59 ± 0.73
% of change (-34.09) (-22.83) (-14.51)
Mean 69.39 A
44.54 B
52.53 C
56.31 D
±SE ± 0.34 ± 0.17 ± 0.35 ± 0.31
% of change (-35.81) (-24.30) (-18.85)
Mean 0.35 A
0.64 B
0.56 C
0.44 D
±SE ± 0.01 ± 0.02 ± 0.01 ± 0.01
% of change 82.86 60.00 25.71
Values are expressed as mean±SE. A, B, C, D values with different superscripts within the same row are significantly different at P

0.30 0.40 0.5 0.6 0.70
Hegazy 1099
Figure 1. Simple correlation between the hepatic lipid peroxidation content (LP) and GSH level, and activities of antioxidant
enzymes (SOD, CAT, CYTO P450) in the liver of female senile rats.
due to accumulative oxidative damage to cells and
molecules (Castillo et al., 2006). Several authors believe
that the reactive species, oxygen (ROS) and nitrogen
(RNS) are the primary causal factor underlying aging –
associated declines in physiological function (Gonzalo-
Calvo et al., 2010; Castillo et al., 2006; Kregel and
Zhang, 2006). An impairment in mitochondrial function
with age has been also reported (Van Remmen and
Richardsin, 2001), which may be a major factor
underlying the increase in the rate of ROS production in
mitochondria as well as in the reduction of the energy
supply in old cells.The results of this work support the
importance of oxidative stress in the aging process and
emphasize the role of lipid peroxidation-induced damage.
After LP initiation by free radicals, it becomes a self
perpetuating chain reaction which could induce the
peroxidation of bivotal lipid molecules of the cell, reduce
membrane fluidity and alter closely situated proteins.
These perpetuations would result in deterioration of the
functions of cell membrane and other biological
membranes inside the cell (VanRemmen and
Richardson, 2001).In the present study, lipid peroxidation
has been found to significantly increase in old female
rats.Endogenous antioxidant defenses include a network
of compartmentalized antioxidant enzymes that are
usually distributed within the cytoplasm and among
various organelles in cells. Several ubiquitous primary
antioxidant enzymes such as SOD, CAT and different

1100 Afr. J. Pharm. Pharmacol.
Table 2. Effect of ginger and -lipoic acid on pro- inflammatory mediators in the serum of senile female rats.
Treatment
Parameter
Adult female rats Senile female rats
Senile female rats
treated with ginger
Senile female rats
treated with -lipoic acid
IL-1
(Pg/ml)
Mean 3.72 A
5.78 B
5.17 C
4.6 D
±SE
% of change
± 0.02 ± 0.04
55.38
± 0.04
38.98
± 0.59
25.54
IL-6
(Pg/ml)
TNF-
(Pg/ml)
TNO
(N mol /L)
COX-2
(Ng/ml)
Mean 10.53 A
19.32 B
16.58 C 14.72 D
±SE ± 0.04 ± 0.17 ± 0.24 ± 0.28
% of change 83.48
57.45 39.79
Mean 5.15 A
8.29 B 6.76 C 6.02 D
±SE ± 0.03 ± 0.05 ± 0.86 ± 0.09
% of change 60.97 31.26 16.89
Mean 58.35 A
37.91 B 44.62 C 47.68 D
±SE ± 0.34 ± 0.38 ± 0.27 ± 0.29
% of change -35.03 -23.53 -18.29
Mean 61.59 A
87.82 B 83.21 C 79.62 D
±SE ± 0.73 ± 0.45 ± 0.49 ± 0.79
% of change 42.59 35.10 29.27
Values are expressed as mean ±SE. A, B, C, D values with different superscripts within the same row are significantly different at P

0.30 0.40 0.5 0.6 0.7
Hegazy 1101
Figure 2. Simple correlation between serum tumor necrosis factor – alpha and IL1, IL6, NO and COX-2 in the serum of senile female rats.
tein kinase (MAPK) pathways and the NF-kB signaling
pathways. Moreover, Kim et al. (2000) reported that the
up-regulation of NF-kB activity is accompanied by
increased ROS production during aging. Furthermore,
Gonzalo-Calvo et al. (2010) showed that TNF- was
significantly increased in the aged population, implying
that aging is accompanied by a gradual increase in this
inflammatory biomarker which is triggered by oxidative
stress induced by decrease in antioxidant defenses in the
elderly population.The change in antioxidant system due
to aging process might be due to the changes of the
cellular structure and function which result in redox
deregulation during aging. The redox deregulation implycated
with NF-KB activation in central to inflammation
process during aging because of the sensitivity of NF-KB
to oxidative stress (Chung et al., 2001). It is possible
to suggest that the anti-inflammatory properties of ginger
are crucial in its antioxidant effects. Ginger was reported
to suppress inflammatory actions of macrophages and
the release of monocycte chemo-attractant protein-1 from

1102 Afr. J. Pharm. Pharmacol.
adipocytes (Woo et al., 2007). Furthermore, Kim et al.
(2010) indicated that the beneficial efficacy of short- term
ginger supplementation at the molecular levels is through
its ability to blunt age-related oxidative stress and
suppress age-related inflammatory actions via NF-KB
activation.
On the other hand, xanthine oxidase is a source of
oxygen free radicals. In the reperfusion phase (that is,
reoxygenation), xanthine oxidase reacts with molecular
oxygen, thereby releasing superoxide free radicals
(Dugasani et al., 2010). Based on the report (Cos et al.,
1998) that the phenolic compounds inhibited xanthine
oxidase and/ or scavenged superoxide, the inhibition of
superoxide production by ginger extract in this study
might have resulted from the combined effects of
scavenging superoxide and inhibition xanthine oxide
activity.
Furthermore, in addition to cytokines, metabolites of
arachidonic acid also participate in the inflammatory
process. Products such as PGE2 are representative of
one of the pathways that initiate polymorphonuclear
leukocytes recruitment and change in vascular tone and
blood flow. Increased production of prostaglandins during
an inflammatory response is achieved by induction of
cyclooxygenase 2. COX-2 is not normally present, but is
inducible in certain cells in response to inflammation
stimuli and control of cell growth (Dewick, 2002). Thus,
compounds that inhibit the activity of COX-2 are crucial
for anti-inflammation.In the present study, ginger extract
showed a remarkable ability to inhibit Cox-2 activity.
Lantz et al. (2007) demonstrated that organic extracts of
ginger and compounds found in ginger are highly
effective at inhibiting lipo-polysaccharides induced
production of prostaglandinE2. These compounds appear
to not only inhibit COX-2 enzyme activity, but are also
able to alter COX-2 mRNA levels, suggesting at least two
sites of action. Moreover, ginger was found to reduce
thromboxane-B2 and prostaglandin-E2 production in rats
(Thomson et al., 2002).
In the present work, the decrease in hepatic lysosomal
cytochrome P450 enzyme activities was observed in
aged rats after ginger treatment. Moreover, Arkene et al.
(2006) suggested that this herb can inhibit the release of
lysosomal enzymes by its stabilizing action and showed
that ginger caused significant reduction in total
leucocytes migration as well as in lymphocytes and
monocytes/macrophages migration from the blood into
the synovital cavity. These inflammatory cells are the
major contributors to the initiation and maintenance of
inflammation response (Weyand, 2000). A recent study
revealed that 6-gingerol isolated from ginger plays a vital
role in suppressing ROS and RNS generation, and
inhibits the expression of inducible pro-inflammatory
genes in macrophages (Pan et al., 2008). These results
are supported in the present study by the depleted TNF-
level, IL-1and IL-6in ginger treated group when compared
to the senile rats. Moreover, Aktan et al. (2006) reported
that ZTX42 (a closely related gingerol analog)
suppressed NO production in murine macrophages by
partially inhibiting inducible nitric oxide synthase (iNOS)
enzymatic activity and reducing iNOS protein production.
This effect was through attenuation of NF-Kappa Bmediated
iNOS gene expression, which provides a
possible mechanism for the anti-inflammatory activity
reported for this class of compounds. It has been also
shown that ginger and its constituents are effective at the
sites of inflammation by suppression of pro-inflammatory
cytokines, and chemokines produced by chondrocytes,
synoviocytes and leucocytes (Phan et al., 2005). In this
regard, Tripathi et al. (2009) demonstrated that whole
ginger extract has a global inhibitory effect on
macrophages function in vitro and that this accounts for
its reputed anti-inflammatory effect in vivo. They also
hypothesized that the active constituent in ginger, 6gingerol,
is an effective anti-inflammatory substance
because of its inhibition of macrophages activation, more
specifically by its inhibition of pro- inflammatory cytokines
and antigen presentation by lipopolysaccharide-activated
macrophages. One advantage of 6-gingerol is the fact
that it does not affect the antigen presenting cells
functions and this may be useful to reduce inflammation
without interfering with antigen presenting function of
macrophages. Antigen presentation by the macrophages
is an integral step in the initiation of the adaptive
immunity by lymphocytes. Furthermore, 6-gingerol has
been shown to suppress the production of proinflammatory
cytokines (TNF- , IL-1 and IL-12) from
macrophages (Williams et al., 2007). Recently, Kim et al.
(2010) showed that zingerone, a major component found
in ginger root, had not only antioxidant effects by
constitutive suppression of ROS, but also antiinflammatory
effects through interference with NF-kB
activation in aged rats. In addition, zingerone treatment
suppressed gene activation of pro- inflammatory
enzymes, COX-2 and iNO, which were upregulated with
aging through NF-kB activation and IkB kinase/mitogenactivated
protein kinase signaling pathway. These
findings strongly indicate that zingerone treatment exerts
a beneficial efficacy by suppressing both oxidative stress
and age-related inflammation through the modulation of
several key pro-inflammatory genes and transcription
factors. When the female senile rats were treated with
ALA in the present study, there was a significant
reduction in LP level and a significant elevation in the
GSH level and the activities of SOD, CAT and
cytochrome P450 as compared with the senile female
rats. In this regard, it was demonstrated that ALA
prevented inflammation induced LP production in the
plasma, liver and brain. The reduction in the level of LP
was ascribed to the potent free radical scavenging
capacity of ALA. As thiols play apivotal role in protecting
cells against LP, it was postulated that the dithiol nature

of DHLA is responsible for the suppression of LP levels in
specify the nature of the experiment (Jesudason et al.,
2008). Moreover, Lapenna et al. (2003) reported that
DHLA, at therapeutical concentrations, can counteract
15-lipoxgenase–dependant lipid peroxidation. The
authors showed that its effect stems primarily from the
reduction of the active ferric 15-lipoxgenase form to the
inactive ferrous state after DHLA-enzyme hydrophobic
interaction. Other possible interventions of DHLA include
scavenging of fatty acid peroxyl radicals formed during
lipoperoxidative processes and inhibition of 15lipoxigenase
oxidative activity.
CAT activity is the key component of the enzymatic
antioxidant defense system and the inhibition of
protective mechanism of defense system leads to free
radicals-induced cellular damage (Zaidi and Banu, 2004).
The observed decrease in the activity of CAT in aged rats
is consistent with earlier studies. It was reported that ALA
increases the glucose uptake in vitro and this enhanced
cellular glucose uptake which serves as the fuel for both
the pentose phosphate shunt and oxidative
phosphorylation, thus bringing up the cellular levels of
NADPH and NADH, thereby enhancing the activity of
CAT under stressful conditions (Arivazhagan et al.,
2000).SOD is an enzyme extensively used as
biochemical indicator of pathological states associated
with oxidative stress. This antioxidant eliminates
superoxide anions thereby preventing free radical chain
reaction. Decreased SOD activity may result from a
suppression of SOD synthesis due to a genetic defect,
leak of SOD out of cell due to increased production of
oxygen radical causing cell membrane damage and
inactivation of SOD by increased intracellular H2O2 level
(Suzuki et al., 1991). Increased superoxide anions may
be one of the factors responsible for decreased activation
of CAT (Meister and Anderson, 1983). The restoration of
SOD and CAT activities in the liver with ALA treatment as
the present results indicate that in liver tissue this rescue
agent can protect the cells against maintaining the
functions of these enzymes (Jesudason et al., 2008). It
has been shown that the diminution in the concentration
of GSH level in senile female rats leads to fast
accumulation of lipid peroxide in cells and the GPx which
is coupled with GSH, together with CAT functions as a
major cellular reducer of hydrogen peroxide and
subsequent lipid peroxidation (Arteel and Sies, 2001).
Studies have also shown that ALA indirectly influences
the activities of SOD, increases intercellular GSH content
that might also activate the GSH-dependent enzymes,
GPx and increases the activity of CAT (Shanmugarajan
et al., 2008). Moreover, Kokilavani et al. (2005) showed
that ALA increases the levels of these enzymes by
directly reacting with various reactive oxygen species and
nullify the oxidation processes in lipids and inter cellular
components. More recently, Shay and Hagen (2009)
demonstrated that Akt is a highly regulated
Hegazy 1103
serine/threonine kinase involved in stress response and
cell survival. Stress response pathways must cope with
increasing chronic stress susceptibility with age and an
age-related lesion in Akt activity via loss of
phosphorylation on Ser473. In hepatocytes from old rats,
the basal phosphor-Ser473Akt was 30% lower when
compared to the adult animals. Treatment with
physiologically relevant doses of ALA provided a 30%
increase in phosphor-Ser-473; thus, ALA appears to
induce a compensation for the constitutive decline in Akt
activity, thereby maintaining this enzyme's critical function
which otherwise declines with age. Some emerging
evidence strongly supports the notion that the molecular
inflammatory process plays a central role in the aging
process and age-related diseases (Chung et al., 2006).
COX-derived reactive species generation as well as gene
expressions of IL-1, IL-6, TNF- , COX-2 and iNO are
enhanced during aging (Chung et al., 2006; Kim et al.,
2007). COX activity and the production of
thromboxaneA2 and prostaglandins are also increased
during aging. The present work is consistent with these
findings. The treatment with ALA significantly inhibited IL-
1, IL-6, TNF- , COX-2 and increased NO hepatocytes in
aged female rats. In this regard, it was reported that ALA
significantly inhibited IL-1 induced oesteoclast formation
in cocultures of mouse osteoblasts and bone marrow
cells. In addition, ALA inhibited COX-2activity,PGE2
production, and sustained receptor activator of NFkappaB
ligand (RANKL) expression, thereby inhibiting
oesteoclast formation and bone loss in inflammatory
conditions associated with the aging process(Ha et al.,
2006).In conclusion, ginger and α-lipoic acid have shown
a significant ameliorative value in counteracting ageinduced
oxidative stress in liver of female aged rats via
scavenging free radicals as well as enhancing the
antioxidant system and can efficiently reduce systemic
inflammation.
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African Journal of Pharmacy and Pharmacology Vol. 5(8). pp. 1106-1114, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.308
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Modeling a murine model of immunoglobulin-E (IgE)mediated
qingkailing injection anaphylaxis
Guoping Liao, Wenshi Li, Shuai He and Zhongyi Zhang*
Department of Pharmacy, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, P. R. China.
Accepted 19 July, 2011
Qingkailing injection (QKLI) is a classic compound traditional Chinese medical injection (TCMI). It can
cause anaphylatic shock and death in clinic. Modeling a murine model of qingkailing injection
anaphylaxis appears to be essential. In this study, for the first time, we used several strategies to
prepare cationic bovine serum albumin (cBSA) and QKLI-cBSA conjugate. The native bovine serum
albumin (nBSA), cBSA and QKLI-cBSA conjugate were separated by cationic exchange method and
also identified by ultraviolet-visible (UV-Vis) and fourier transform infrared (FT-IR) spectrometry. Balb/c
mice were then treated with subcutaneous injection of QKLI-cBSA plus alum. Serum was analyzed for
total IgE and histamine levels by enzyme-linked immunosorbent assays (ELISAs), respectively. Lung
reactions were evaluated by analyzing lung pathologic changes. QKLI-cBSA group mice were
significantly and successfully sensitized. QKLI-cBSA conjugate significantly increased total IgE and
Histamine in Balb/c mice serum. Moreover, histological examination of QKLI-cBSA conjugate treated
mice showed acute injury with pulmonary alveoli and injury of trachea, as well as neutrophilic cell
infiltration. In conclusion, we successfully established a murine model of immunoglobulin-E (IgE)mediated
qingkailing injection anaphylaxis. This model should provide a useful tool for detecting
allergens in QKLI and for exploring new allergens-detecting approaches of other TCMIs.
Key words: Qingkailing injection, murine, immunoglobulin-E (IgE)-mediated anaphylaxis, Qingkailing injection
(QKLI) - cationic bovine serum albumin (cBSA) conjugate.
INTRODUCTION
Qingkailing injection (QKLI) is a modified formulation in a
new dosage from a well-known classic formulation “An
Gong Niu Huang Wan”, which contains four plant species
(Fructus gardeniae, Radixisatidis, Radix scutellaria, and
Flos lonicerae), a number of animal products (Beijing
College of TCM, 1975). The QKLI is not only cheap, but
also can be used extensively by Chinese doctors to cure
cerebral ischemia, intracerebral hemorrhage (ICH),
neurotoxic damage, bacterial meningitis and vascular
dementia (Liu et al., 2006; Hua et al., 2008; Yue et al.,
2006; Hu et al., 1992; Tian, 1998). In 1992, QKLI was
specified as a "must-have" preparation for TCM
emergency clinics by the State Administration of
Traditional Chinese Medicine of China (SATCM) (Lee et
al., 2000). However, with the wide application of QKLI,
*Corresponding author. E-mail: zhang43499@sohu.com Tel/fax:
+86 20 61643499.
the reports of adverse reactions related to QKLI appear
to have increased significantly in China over the past ten
years. From 2001 to 2007, the Chinese National Adverse
Drug Reaction Monitoring Center (CNARMC) received
notices of 580 serious adverse drug reaction (ADR)
cases caused by QKLI, including 20 (3.45%) fatalities
(Huang et al., 2007). Adverse drug reactions (ADRs)
reported for QKLI from 2001 through 2007 mainly
included anaphylactic shock (Niu and Zhou 2000) (33%),
dyspnoea (23%), hypotension (10%), cataphora (5%),
pulmonary edema (5%), laryngeal edema (2%), and
convulsions (2%). Since the explosion of adverse event,
CNARMC posted a bulletin in which it warned of the
potential for severe ADRs to QKLI (SFDA, 2009b). Those
safety concerns caused a reduced usage of QKLI and
increasingly questioned its rationality, and thus ultimately
lead to the "crisis of confidence", to the whole traditional
Chinese medicine industry. Qingkailing injection, the most
common and most complicated of TCMIs, became the
focus of governments and scientists throughout China
due to the strong potential hypersensibility shown

Figure 1. The fluidogram of cBSA preparation.
in clinic applications. Therefore, there are strong
economic and safety reasons for establishing methods
for detecting potential allergens in QKLI.
Immediate anaphylaxis, generally being considered as
the most frequent and severe adverse reaction induced
by QKLI. Taking the mechanism of immediate
anaphylaxis into consideration, obtaining QKLI-specific
antibodies is one of the key steps in establishing methods
for detecting allergy. However, there is no time to draw
blood from autopathes during salvages. Thus, we have to
modeling an anaphylaxis animal model to obtain QKLIspecific
antibodies. At present, the anaphylaxis animal
models of macromolecule weight compound (MMWC),
such as, cow's milk (Xiu et al., 1999), peanut (Xiu et al.,
2005) and latex (Ramos et al., 2007), had been widely
used for detecting allergens. However, the anaphylaxis
animal models of traditional Chinese medicine injection
are still scarce. In our previous work (Xu et al., 2010), we
found that QKLI include no MMWC but low weight
molecule compound (LWMC, 1.0-1.5 KD). Nevertheless,
LMWC cannot directly induce immunological response,
unless coupled with carrier protein (Ilkka and Seppälä,
2004). As it has no MMWC, the QKLI has significant
differences from conventional MMWC in establishing
anaphylaxis animal models. Obviously, establishing an
animal anaphylaxis model for QKLI will be a challenging
work.
In the current study, for the first time, we used several
strategies to prepare QKLI-cBSA conjugate based on a
Mannich-type method, in which carrier protein (native
BSA, nBSA) is cationized by ethylendiamine, then the
QKLI-cBSA conjugate is prepared by condensing the
ethylendiamine group in the cationic protein (cationic
BSA, cBSA) with formaldehyde and the -hydrogen
adjacent carbonyl in QKLI. The cBSA and QKLI-cBSA
conjugate were also firstly separated and purified by
Liao et al. 1107
cationic ion exchange methods. After purification, the
cBSA and QKLI-cBSA conjugate were then identified by
FT-IR spectrometry. BALB/c mice were then treated with
subcutaneous injections of QKLI-cBSA plus alum. Serum
was analyzed for total IgE and histamine levels by
ELISAs. Lung reactions were evaluated by analyzing lung
pathologic changes.
MATERIALS AND METHODS
Materials
Qingkailing injection was purchased from HeBei ShineWay
Pharmaceutical Company (ShiJiazhuang, China). 1-Ethyl-3-[3dimethylaminopropyl]-
carbodiimide hydrochloride (EDC), 37%
formaldehyde, ovalbumin (OVA), 2-(N-morpholino)-ethane sulfonic
acid (MES) and alum were obtained from Pierce (Rockford, IL,
USA). Native bovine serum albumin (nBSA) was purchased from
Merck (Darmstadt, Germany). Mouse IgE ELISA KIT was obtained
from BioLegend (San Diego, CA, USA). Mouse Histamine ELISA
KIT was obtained from Rapid Bio Lab (California, USA).
Ethylenediamine (EDA), common reagents and other organic
solvents were purchased from Guangzhou Chemical Reagent
Company (Guangzhou, China).
Animals
Female, 6-week-old, BALB/c mice were purchased from The
Laboratory Animal Research Center of the Southern Medical
University (Guangzhou, China). The animals used for the
experiment were treated according to protocols evaluated and
approved by the ethical committee of Southern Medical University.
Preparation of cBSA
The fluidogram of cBSA synthesis (Jean et al., 1999) is shown in
Figure 1. Briefly, 200 mg of crystallized native bovine serum
albumin (nBSA) was dissolved in 2 ml of conjugation buffer (0.1 M
MES pH 4.7). Anhydrous ethylenediamine (EDA) solution was pre-

1108 Afr. J. Pharm. Pharmacol.
pared by mixing 2.68 ml of EDA and 20 ml of conjugation buffer in
an ice bath; the pH value was readjusted to 4.7 with 6 N HCl and
the solution cooled to room temperature (RT). This EDA solution
was added slowly to the nBSA solution followed by 150 mg of 1ethyl-3-[(3-dimethylaminopropyl)-carbodiimide
hydrochloride] (EDC)
and incubated at RT for 4 h with continuous magnetic stirring.
The reaction solution was then added into 20 ml cationic ion-
exchange column (BioLogic DUO FLOW system, Bio-Rad,
USA)which was filled with SP sepharose fast flow base. The
separation for cBSA was described (Chen et al., 2004) previously.
After loading samples, the column was washed with 100 ml of pH
6.8 phosphate buffer (10 mM) to remove the non-reacted nBSA.
Subsequently, the cationic BSA (cBSA) was eluted with 100 ml of
pH 10.6 phosphate buffer (10 mM) and collected by BioLogic
Fraction Collector (Bio-Rad, USA). The collections were monitored
by UV-Vis spectrometry. The fractions which showed highest
absorbance were pooled and concentrated to 4 ml by using
JumboSep TM centrifugal device (Pall, Mexico). Then the
concentrated cBSA were dialyzed exhaustively against deionized
water for 72 h. Finally, the salt-free cBSA was lyophilized, identified
by UV-Vis and FT-IR spectrometry and stored at -20°C.
Preparation of QKLI-cBSA conjugate for immunization
The preparation of QKLI-cBSA conjugate was based on the
Mannich reaction (Gerg, 1996). Briefly, 4 mg of QKLI freeze-dried
powder was dissolved into 0.4 ml of deionized water. Then 2 mg of
cBSA was dissolved in 0.2 ml of conjugation buffer (0.1 M MES pH
4.7). After dropwise addition of QKLI solution into the cBSA
solution, the mixture was stirred gently, then 50 l of conjugation
reagent (37% formaldehyde) were added into the mixture and
immediately incubated for 24 h at 37°C.
The reaction solution was then added into 20 ml cationic ionexchange
column which was filled with SP sepharose fast flow
base. Subsequently, the conjugate was eluted with buffer A (10 mM
PBS, pH = 7.2), and the non-conjugated cBSA was eluted by buffer
B (10 mM PBS, 0.5 M NaCl, pH = 7.2). All collections were
collected by BioLogic Fraction Collector, respectively. The
collections were monitored by UV-Vis spectrometry. The fractions
which showed highest absorbance were pooled and concentrated
to 4 ml by using JumboSep TM centrifugal device. Then the
concentrated products were dialyzed exhaustively against
deionized water for 72 h. Finally, the salt-free conjugate was
lyophilized, identified by UV-Vis and FT-IR spectrometry and stored
at -20°C.
Identification of cBSA and QKLI-cBSA conjugate
Ultraviolet-visible (UV-Vis) analysis of nBSA, cBSA, QKLI-cBSA
conjugates
The nBSA, cBSA and QKLI-cBSA conjugate were analyzed
spectrometically in a quartz cuvette with a 1-cm light path,
employing a double bundled UV-Vis spectrometer, mode Lambda
35 (PerkinElmer, USA). Absorption spectra of all samples in PBS
were scanned from 200 to 400 nm at 1-nm intervals. The scanning
speed was 12 nm·s -1 and the bandwidth was 1 nm.
Investigation of coupling by Fourier-transform infrared
spectrometry
FT-IR spectra of nBSA, cBSA and QKLI–cBSA conjugate were
recorded in the region of 400 to 3500 cm −1 at room temperature on
a FT-IR spectrometer, model Avatar 380 (Thermo Nicolet, USA).
The spectral resolution was set at 4 cm −1 and 32 scans; the test
sample was dispersed into a potassium bromide pellet and the
Thermo Electron software, OMNIC 6.0, was used to analyze the
results.
Animal immunization and serum collection
Groups of 30 6-week-old male Balb/c mice if weight 18 to 22 g
received subcutaneous injection of 50 g of QKLI-cBSA, OVA,
QKLI, cBSA or NS in 0.15 ml of sterile saline plus 0.15 ml of
aluminum hydroxide as adjuvant, respectively. Booster injections
were carried out on the 14th, 21st, 28th and 35th day after the
primary doses. Mice were bled from the retro-orbital plexus just
before first immunization (day 0) and at day 14, 21, 28 and 35 days
after first injection. Posterior blood collections were performed 2 min
after their skin was scratched. After blood collection, serums were
allowed to repose for 30 min at RT and thus slightly centrifuged in a
bench centrifuge for 10 min at RT, the clean samples finally stored
at -20°C until use.
Determination of total IgE level in mice serum
The IgE ELISA kit from BioLegend, Inc. was used to determine
the total IgE content in mice serum. The assay was performed
according to the protocol provided by the supplier. Briefly, ELISA
plates (96-well EIA/RIA plate, 96-well easy wash TM , high binding,
Corning, New York) were coated with capture antibody diluted in
carbonate buffer (0.05 M, pH 9.6) and incubated at 4°C, over night.
Unbound extract was discarded and the plates were blocked with
dilution buffer (0.1% BSA in PBS) at 37°C. After washing (0.05%
Tween-20 in PBS), IgE standards and serum samples were added.
Following incubation, plates were washed and biotin-labeled antimouse
IgE antibody added. After incubation, plates were washed
and streptavidin alkaline phosphatase conjugate added.
Subsequently, plates were washed and TMB substrate added.
Reactions were allowed to develop at room temperature in the dark
and stopped by 2 N H2SO4 solution. Finally, the absorbances were
measured in a micro-plate reader (Model 580, Bio-Rad, USA) at
450 nm.
Determination of histamine level in mice serum
The histamine ELISA kit from Rapid. Bio. Laboratories, Inc. was
used to determine the histamine content in mice serum. The assay
was performed according to the protocol provided by the supplier.
Briefly, histamine standards and serum samples were added to
ELISA plates which had been pre-coated with anti-mouse histamine
antibody. Following incubation, plates were washed and biotinlabeled
anti-mouse histamine antibody added. After incubation,
plates were washed and streptavidin alkaline phosphatase
conjugate added. Subsequently, plates were washed and
added. Reactions were allowed to develop at room temperature in
the dark and stopped by adding 2 N H2SO4 solution. Finally, the
absorbance was measured in a micro-plate reader at 450 nm.
Histological examination
For assessment of pathologic alterations, dissected lung tissues
were washed with normal saline (5 ml) and then placed in 10%
neutral-buffered formaldehyde for 1 week. After fixation, lung
specimens were embedded in paraffin wax, and five-micrometer
sections were cut and stained with hematoxylin and eosin dye for
morphology. Images of selected sections were captured at X100
magnification using a zoom digital camera (Kodak Company,
Rochester, NY, USA). One pathologist blind to the study groups

A
1.000
0.95
0.90
0.85
0.80
0.75
0.70
0.65
0.60
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
Figure 2. The UV-Vis spectra of nBSA (A), cBSA (B) and QKLI-cBSA conjugate (C) in MES buffer.
performed all the histological examinations.
Statistical analysis
All experiments were repeated at least three times. Experiment data
were expressed as mean ± SD and statistical differences between
groups were performed by one-way analysis of variance (ANOVA)
followed by Student Newman-Keuls test. Differences were
considered significant at P

1110 Afr. J. Pharm. Pharmacol.
Wavelength (cm -1 )
Wavelength (cm -1 )
Wavelength (cm -1 )
Figure 3. The FT-IR spectra of nBSA (A), cBSA (B) and QKLI-cBSA conjugate (C) were
obtained after dispersion into potassium bromide.
the bands’ intensity, in the range of 1090 to 950 cm -1 ; it was (Figure 3C) due to reactions between QKLI and

Figure 4. Effect of treatments on total IgE and histamine levels in mice serum. (A) Total IgE in serum.
(B) Histamine in serum *(P0.05), have no significant difference for groups compared with negative control.
primary amine, which depressed vibration of R-CH2-NH2.
Total IgE and histamine in mice serum
The levels of total IgE in mice serum were measured by
ELISA. QKLI-cBSA and OVA treatments significantly
Liao et al. 1111
increased total IgE levels in serum compared to the
negative control (NS group) at different times (P0.05) (Figure 4A).
The levels of histamine in mice serum were measured
by ELISA. QKLI-cBSA and OVA treatment significantly

1112 Afr. J. Pharm. Pharmacol.
Figure 5. Histological evaluation of mice lungs treated with NS and QKLI-cBSA with hematoxylin
and eosin staining (H&E × 200). (A) and (B) Negative control lung tissue. (C) QKLI-cBSA treated
mice lung showed fusion of pulmonary alveoli. (D) QKLI-cBSA treated mice lung showed
hyperemia. (E) QKLI-cBSA treated mice lung showed injury of trachea. (F) QKLI-cBSA treated
mice lung showed neutrophilic cell infiltration.
increased histamine levels in serum compared to
negative control (NS group) at different times (P0.05) (Figure 4B).
These results suggest that total IgE and histamine are
two of the major mediators involved in the anaphylaxis in
this model.
Histology
QKLI-cBSA conjugate induced significantly acute injury in
mice lung, as seen with hematoxylin and eosin staining.
Blank lung tissue was seen in Figure 5A and B. QKLIcBSA
showed a considerable change in tissue structure
due to acute injury, which can be demonstrated by fusion
of pulmonary alveoli (Figure 5C), hyperemia of lung

(Figure 5D), injury of trachea (Figure 5E) and neutrophilic
cell infiltration (Figure 5F).
Conclusion
Hapten is a class of LMWC and its immunogenicity is
obtained only by coupling with some carrier protein (Roitt,
2001). However, the haptens in QKLI were unknown and
might have no available common functional groups
(amines, carboxylates, sulfhydryls, etc.) except active
hydrogens. Therefore, choosing a suitable carrier protein
seems to be extremely important. Taking the
aforementioned into consideration, we prepared an ideal
carrier protein, cBSA. An active hydrogen-containing
compound can be condensed with formaldehyde and an
amine of cBSA in the Manncih reaction (Aime et al.,
2004; Nobles and Potti, 1968), resulting in a stable
conjugate. In this work, we found cBSA was an ideal
carrier for unknown haptens in QKLI, indicating that it
might be suitable for detecting unknown allergens in
compound preparations and TCMIs.
The former preparation (Chu et al., 1982; Youxiang et
al., 2007; Yi et al., 2009; Feng et al., 2010) for conjugate
did not have any strategies of separation or purification.
Furthermore, we firstly separated and purified the QKLIcBSA
conjugate by cationic ion exchange method, which
was beneficial to eliminate the interference of impurities.
The routine method of identifying the cationization of
carrier protein was isoelectric focusing-polyacrylamide
gel electrophoresis (IEF-PAGE) (Jean et al., 1999). In our
work, the cationized carrier protein and the QKLI-cBSA
conjugate were monitored by FT-IR spectroscopy, which
was an effective tool to inspect the structural
modifications in molecules by interpreting the position of
wavenumbers and the variable transmittance in
characteristic waveband. FT-IR spectrometry, an effective
and convenient method, might be an alternative method
to verify the cationization of protein.
The Balb/c mice were selected as such mice are high
IgE responders, equivalent to an atopic phenotype
(Baeza and Zubeldia, 2007; Nierkens and Pieters, 2005).
On the one hand, Ian et al. (2004) measured the total and
Ag-specific IgE in OVA-sensitized Balb/c mice serum by
using ELISAs, discovered a strong and consistent
relationship between them. On the other hand, IgE
specific ELISAs require extensive optimization for each
antigen examined and generally yield data expressed in
arbitrary units unless monoclonal antibody (mAb) of
similar affinity (that is, those that exhibit slopes parallel to
that of polyclonal responses throughout the titration
curve) are generated and made available in sufficient
quantity to act as a standard. As soon as allergen binds
to the receptor-bound IgE antibodies, stimulation of mast
cells and release inflammatory mediators (Histamine and
tryptase, etc) occur with minutes and results in the
immediate development of clinical signs of
anaphylaxis (Janos, 2005; Kinet, 1999).
Liao et al. 1113
Therefore, we measured the total IgE and histamine in
Balb/c mice serum to judge whether the mice were
sensitized by QKLI-cBSA or not.
In this study, we successfully cationized the nBSA and
prepared QKLI-cBSA conjugate. We immunized the
BALB/c mice with subcutaneous injection of QKLI-cBSA
plus alum. After immunization, we verified the model by
analyzing the total IgE and histamine levels in serum. In
addition, we evaluated the lung reactions by analyzing
lung pathologic changes. Finally, we successfully
established a murine model of IgE-mediated Qingkailing
injection anaphylaxis. The model might be enlightening
allergen researchers on detecting allergens in QKLI and
other Traditional Chinese Medical Injections (TCMIs). On
the one hand, the protocol is expected to be applied in
preparing QKLI-specific antibodies and detecting
allergens in QKLI. On the other hand, the protocol can
also be applied in establishing anapylaxis animal models
of other TCMIs.
ACKNOWLEDGEMENTS
The authors are grateful to Prof. Zhang for his assistance
with IR spectra analysis. Financial support for Zhang
Zhongyi was provided by National Nature Science
Foundation of China (NSFC 90709046) and Science and
Technology Planning Project of Guangdong Province,
China (2007B031402006).
.
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African Journal of Pharmacy and Pharmacology Vol. 5(8). pp. 1115-1119, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.349
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Comparison of the effect of acupressure, fish oil
capsules and ibuprofen on treatment of primary
dysmenorrheal
Zafari, M. 1 *, Tofighi, M.1, Aghamohammady, A. 1 , Behmanesh, F. 2 and Rakhshaee, Z. 3
1 Department of Midwifery, Islamic Azad University, Sari Branch, Sari, Iran.
2 Department of Midwifery, Babol University of Medical Science. Iran.
3 Department of Midwifery, Islamic Azad University Rasht Branch, Rasht, Iran.
Accepted 12 June, 2011
Primary dysmenorrhea is one of the common causes of absence from workplace and decline in the
quality of life of women. Because of the importance and prevalence of dysmenorrhea among women
and girls of childbearing age, and due to the publication of reports concerning the effectiveness of fish
oil and acupressure in curing primary dysmenorrhea, this study was conducted with the purpose of
comparing the effectiveness of acupressure, fish oil capsules, and ibuprofen in treating primary
dysmenorrheal. This empirical study was carried out on students of the University of Medical Sciences
of Mazandaran in 2010. Students afflicted with primary dysmenorrhea were randomly divided into three
groups. The first group (60 students) received 1000 mg of fish oil capsule every day for the duration of
two successive cycles; the second group (76 students) was given 400 mg ibuprofen pills, as soon as
the pain started, for two months; and in the acupressure group (60 students), the Saninjao point was
pressed, at the start of the pain, with a thumb for 20 min. After the information was gathered, the SPSS
software, Fisher s test, Duncan s test, Friedman s test, and analysis of variance ( + 0.05) were used to
test the data. Results obtained showed that there was a significant difference with respect to pain
before and after the use of the medicines and acupressure (P = 0.000). Moreover, all three groups
needed the same quantity of extra painkillers during the treatment duration (P = 0.295); and participants
were most satisfied with ibuprofen, with acupressure and fish oil ranking second and third, in this
respect (P = 0.000). Acupressure, fish oil capsules, and ibuprofen had similar effects in curing
dysmenorrheal.
Key words: Primary dysmenorrhea, ibuprofen, fish oil capsule, acupressure.
INTRODUCTION
Dysmenorrhea, or painful menstruation, is a common
gynecological disorder experienced by at least 50% of
women during their fertility period. Ten percent of these
women have such severe pain that it keeps them from
attending their school or from going to their workplace.
Therefore, women afflicted with dysmenorrhea are more
often absent from their workplace and do worse in their
studies than women who do not suffer from this disorder.
Dysmenorrhea has two forms: Primary and secondary.
From the accounts given by patients, and in clinical
*Corresponding author. E-mail: mandanazafari@iausari.ac.ir.
Tel: 00989112513051.
studies and tests, no clear background reasons have
been found for primary dysmenorrhea, but secondary
dysmenorrhea is accompanied by a background disorder
(Yaghmaee et al., 2004).
The prevalence of primary dysmenorrhea varies from
50 to 90% in different communities. In Iran, its prevalence
has been reported to be from 74 to 86.1%. Although
primary dysmenorrhea does not threaten lives and does
not cause any disfigurement, it can influence the quality
of life of women and, in severe cases, it can so disable
them and make them inefficient that they cannot attend
their school or go to their workplace (Dolatian and
Jaafari, 2004). Several reasons have been given for
primary dysmenorrhea, the most agreed upon of which is
the increase in the production of prostaglandins, and the

1116 Afr. J. Pharm. Pharmacol.
role these compounds have in causing pain. The
common treatment of primary dysmenorrhea is based on
the prevention of the production of prostaglandins
through the use of nonsteroidal anti-inflammatory drugs
(NSAIDs), or on taking oral contraceptives so that
ovulation and the entrance of the endometrium into the
luteal stage are prevented, and the level of
prostaglandins in the body is reduced. Other
recommended treatments include prescription of tocolytic
drugs, inhibitors of the calcium canal, progesterones,
magnesium, calcium, vitamin B1; quitting alcohol and
cigarettes, TENS, changing the lifestyle, sleep therapy,
psychotherapy, and consumption of herbal medicines
and fish oil (Yaghmaee et al., 2004).
Fish oil supplements are dietary supplements that
contain oil from the flesh of cold water fish such as
mackerel, salmon, black cod, albacore tuna, sardines,
and herring. The active ingredients in fish oil supplements
are essential fatty acids known as omega-3 fatty acids.
They typically include eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA). Most fish oil capsules or
pills are obtained from the flesh of fish. In contrast, fish
liver oils are derived from the livers of white fish, such as
cod and halibut. Fish liver oils contain vitamins A and D
and may have a different concentration of EPA and DHA
than fish oils derived from flesh.
The chemical composition of fish oil is, 120 mg DHA and
180 mg EPA per fish oil softgel (www.fishoil-s.com).
According to studies conducted, consumption of fish oil
causes the production of prostacyclin and reduces the
intensity of dysmenorrhea. It seems that the main effect
of using the Omega-3 fatty acid supplements is in the
production of weaker prostaglandins and leukotrienes.
Increasing the Omega-3 fatty acids content of the diet
brings about an increase in the inclusion of these fatty
acids into the structure of cellular membranes. As one of
the results obtained in this study, it can be said that more
of series three prostaglandins (PGE2, PGI3, and TXA2)
are produced during menstruation; and that the use of
fish oil causes the production of prostaglandins belonging
to the prostacyclin class in the uterus, which brings about
a reduction in the contraction of myomeres and in the
contraction of the vessels of the uterus, which, in turn,
decreases ischemia, and hence reduces pain (Zamani et
al., 2005).
In acupressure, the technique of touching the body is
used to balance the flow of energy in the body. The
Saninjiao, the intersection of the three canals between
the spleen, the kidneys, and the liver, is one of the most
important points used in acupuncture. This point is
situated 3 Kans (four fingers) above the inside ankle of
the foot behind the hind edge of the tibia; and it is
extensively used in curing gynecological, genitourinary,
and digestive disorders, in the treatment of weakness
and low blood pressure, in creating anesthesia during
operations in the pelvic area, and in painless childbirth
(Pooresmaili and Ibrahimzadeh, 2002).
Although the nonsteroidal anti-inflammatory drugs are the
drugs of choice for curing primary dysmenorrhea, and
despite the fact that they are widely available without
prescription, adolescents do not take advantage of
effective treatment programs. Because of fears
concerning the side effects of nonsteroidal antiinflammatory
drugs and oral contraceptive pills, and
based on a number of false beliefs, these two types of
drugs are not regularly used. The purpose of this study is
to compare the effects of fish oil and acupressure on
painful menstrual cramps with that of ibuprofen pills; so
that we can substitute fish oil, if it is effective, for
ibuprofen; because fish oil has few side effects, while
ibuprofen has many.
METHODS
Prepration of plant extracts
The criteria for being accepted as participants in this study were
that the students had to be 18 to 22 years old, single, have regular
menstrual periods (every 26 to 30 days), have had menstrual pains
in most of the menstrual cycles of the past six months, and have
had severe and moderate pains, according to the standard criterion
of multi-dimensional speech. The criteria for omitting student
candidates for the study included sensitivity to nonsteroidal antiinflammatory
drugs, use of medicinal and non-medicinal methods of
relieving pain, having special diets (those undergoing hydrotherapy,
vegetarians, uncooked-food eaters), doing any kind of regular
sports activities or attending special classes (sports classes,
physical fitness classes, etc.), having used body relaxation
techniques during the past six months , having any kind of
diagnosed physical and mental illnesses or any type of genital
diseases, having a history of surgery of the stomach or of the pelvis
,addiction to cigarettes or alcohol, taking hormonal drugs or
contraceptive pills, or having intense psychological or mental
stresses in the duration of the study.
Experimental groups
The first group (60 students) received the daily dosage of one gram
of fish oil. This capsule is ghelatin form and yellow colour. It
contains polyunsaturated fat (1 g), saturated fat (1 g), EPA (180
mg) and DHA (120 mg). The company produces these drugs in the
American 21 st Century. According to the study that was done in
animals by Jacoby et al. (1992), there was no specific side effect
during the ordinary dose of fish oil and its authorization number was
91-460 (issued in 5 June). The second group (76 students) was
prescribed ibuprofen, and for the third group (60 students)
acupressure was used.
Experimental design
This study was conducted as a random clinical test of effectiveness.
Among the medical students of the University of Medical Sciences
of the province of Mazandaran, 196 students were afflicted with
moderate and severe primary dysmenorrhea. They were randomly
divided into three groups.
Study outcomes
The primary outcomes were to compare the severity and duration of
pain before and after treatment. The secondary outcomes need
more sedative with an amount of satisfaction.

Experimental
The entire participant was studied for the duration of three
menstrual cycles. For the first cycle (the control cycle) no treatment
was offered, and the participants were only asked to write down the
features of the menstruation period with respect to the intensity and
the duration of pain: They had to determine the intensity of their
pain using the grading system of multi-dimensional speech. In this
grading system, those having the very slight form of painful
menstruation (grade zero) are not hindered in their normal activities
and feel a very slight pain. Those who suffer from the slight form of
the menstruation pain (Grade 1) feel a very slight pain and their
normal activities are rarely limited. In those afflicted with the
moderate form (Grade 2) daily activities are affected, they feel a
moderate pain, a few bodily symptoms appear, and they need
painkillers. Those with the severe form (Grade 3) have their daily
activities greatly limited, painkillers give them little relief, they have
intense pain, and there are physical and somatic symptoms like
fatigue, nausea, vomiting, and diarrhea. The participants calculated
the duration of pain, from the time it started till the time it ended, in
hours. Then the participants were treated, using the three methods,
for the duration of the second and the third menstruation periods.
Treatment of pain
The 60 students of the first group received 100 mg/day of fish oil
capsules for two months; the 76 students in group two were
prescribed 400 mg ibuprofen pills, to be taken at the start of the
pain, and be repeated 8 h later if the pain persisted; the 60 students
in group three were trained to press the Saninjiao point, at the start
of the pain, with their thumb for 5 min (press the point for 6 s,
release the pressure for 2 s, press for another 6 s, release the
pressure for two s, and so on). After the 5 min, they had to change
feet and repeat this process is on the same point for 5 min.
Altogether; this point was pressed for 20 min (2 to 5 min on each
foot). It must be added that each student was interviewed once a
week to check upon the correct use of the medicines and the
proper way of performing the acupressure. The medicine packages
were monthly given to the students; the possibility of the occurrence
of side effects was explained to them, and they were asked to
report any side effects they observed. At the end of the two months,
both groups receiving medicines were studied and investigated with
respect to the intensity and the duration of pain.
Data collection
Patients received drug boxes each month, and we described for
three groups how they use drug and do acupressure technique. We
asked them to refer us if mentioned complications occurred.
Treatment of three groups was studied on intensity and duration of
pain, and amount of satisfaction.
Statistical analysis
After gathering the information, it was coded and analyzed using
the statistical software SPSS and Fisher s test, Friedman s test,
Duncan s test, and the analysis of the variance.
Ethical issues
This study was approved by the research and ethics committee of
Medical University of Babol, and it was registered in a clinical trial
center.
RESULTS
General observations
Zafari et al. 1117
There were no statistically significant differences among
the 196 student participants with respect to the average
age, the age at which the first menstruation happened,
the age at which dysmenorrhea started, the intensity of
pain before and after the treatment, and the duration of
pain: The average age, the age at which the first
menstruation happened, the age at which dysmenorrhea
started, the pain before and after the treatment, and the
duration of pain in the group receiving fish oil capsules
were respectively 20.13, 11.96, 14.2, 2.33, and 48.8; in
the group taking ibuprofen were, 20.13, 12.69, 14.77,
2.39, 36.31; and in the acupressure group were 21.16,
12.13, 14.88, 2.28, and 35.1.
Severity and duration of pain before and after
treatment
As for the intensity of pain before and after the
intervention, there was a statistically significant difference
in the group using fish oil capsules (P = 0.000), the group
taking ibuprofen pills (P = 0.000), and the acupressure
group (P = 0.000).
Regarding the duration of pain before and after the
treatment, there was a statistically significant difference in
the group taking ibuprofen pills (P = 0.000), the group
using fish oil capsules (P = 0.000), and the acupressure
group (P = 0.000). The intensity of pain after treatment
was not the same in the three groups, and the average
intensity of pain was the same only for the acupressure
and the fish oil groups (P = 0.000) (Table 1).
The duration of pain after treatment was not the same
for the three groups either (P = 0.000) (Table 2).
The three groups had the same need for extra
painkillers (P = 0.295), but there was a statistically
significant difference in the degree of satisfaction in the
three groups (P = 0.000): The group taking ibuprofen was
the most satisfied, followed by the acupressure and the
fish oil groups.
DISCUSSION
In this study, the average age, the age at which the first
menstruation happened, the age at which dysmenorrhea
started, and the intensity and the duration of pain before
intervention were the same for all three groups. However,
after the treatments, the intensity and the duration of pain
decreased in the fish oil capsule group. Therefore, it can
be concluded that fish oil has a considerable effect in
reducing the intensity of pain in primary dysmenorrhea. In
this respect, the results of this study are similar to those
obtained by Harel et al. (1996), Wilkinson and Muphy

1118 Afr. J. Pharm. Pharmacol.
Table 1. Comparison of average and standard deviation of menses intensity before and after receiving treatment.
Group pain
intensity
Ibubrofen (M+SD )
Acupressure
(M+SD )
Fish oil capsulD
(M+SD )
p-Value
Before use 2/39±0/49 2/28±0/45 2/33±0/47 0/000
After use 1/24±0/49 1/63±0/54 1/75±0/47
Table 2. Comparison of average and standard deviation of menses pain duration before and after receiving treatment.
Group menses pain
duration
Ibubrofen (M±SD)
Acupressure
(M±SD)
Fish oil capsulD
(M±SD )
Before use 36/31±0/66 35/10±12/13 48/80±21/96
After use 14/46±0/68 29/9±11/20 43/30±22/09
(2001), Dolatian and Jaafari (2004), Yaghmaee et al. (2004),
and Zamani et al. (2005). In the study conducted by Wilkinson
and Muphy (2001), fish oil (at 4 g/day) was more effective than
the placebo. Fish oil has a significant influence in reducing the
duration of pain in primary dysmenorrhea. We obtained these
result with 1 g/day. Increasing the side effects can accrue in
high dose intake (more than 3 g/day), such as: Vomiting and
diarrhea; reduce the absorption of vitamin A. D.K.E,
acceleration of glucose in diabetic person and hemorrhagic
infarcts (www.fishoil-s.com).
In the study conducted by Deutch et al. (2000) the
effectiveness of treatment with fish oil and seal oil was
compared with that of a combination of fish oil and vitamin B12,
and it was found that the curative effect of fish oil and vitamin
B12 was more stable. Fish oil is effective for healthy brain and
effective on alzheimers disorder, anxiety, bipolar disorder,
eyesight, heart disease, inflammation and painful menses
(www.fishoil-s.com).
Due to the relatively severe digestive side effects of
nonsteroidal anti-inflammatory drugs (that cure 80 to 90% of the
cases of dysmenorrhea), these drugs, despite their
considerable curative effects, enjoy a low level of acceptability;
and although they have been the most widely used cure for
dysmenorrhea so far, numerous studies have been carried out
to find drugs with similar effects, but with fewer side effects.
Playing various sports together with sauna, using hot water
bottles, quitting cigarettes and alcohol, including supplements
such as calcium and sodium in the diet, and recently,
acupuncture, somewhat cure dysmenorrheal (Pooresmaili and
Ibrahimzadeh, 2002).
In this study, the intensity and the duration of pain decreased
when acupressure was used. In this respect, these results were
consistent with those obtained by Habek and Mehmet (2007),
Bostani et al. (2010), and Kidney et al. (2001). After the
intervention, the intensity of pain in the acupressure group was
significantly reduced.
Pooresmaili and Ibrahimzadeh (2002), who carried out a
study in this area, concluded that the three methods of,
acupressure, the use of placebo, and taking ibuprofen were
effective in reducing primary dysmenorrhea, but that
acupressure and ibuprofen had very similar effects, which were
considerably greater than those of the placebo.
Chen and Chen (2004) also conducted a study in the same
area and stated that in the two groups of acupressure and
ibuprofen, there was a significant difference in the intensity of
pain before and after the treatments. Of course, these
p-Value
0/000
differences did not considerably vary in the three consecutive
months, but were very noticeable and important compared to
the pain before the treatments. They also found that both the
acupressure method and the use of painkillers were very
effective in reducing pain, and reduced it equally.
In another study carried out on 216 female students of 14 to
18 years of age, in 50% of those in the acupressure group, and
in 18% of those in the placebo group, the intensity of pain after
thetreatments was reduced to zero (Lee et al., 1999).
Conclusion
Acupressure and fish oil capsules (which have few side
effects and are very acceptable and tolerable) can be
substituted for the nonsteroidal anti-inflammatory drugs (which
have many side effects) in curing those afflicted with
dysmenorrhea. It is also recommended that different levels of
fish oil should be used in future studies.
ACKNOWLEDGEMENTS
The authors are grateful to the students who used drugs and
did the acupressure technique, according to order.
REFERENCES
Bostani Z, Abedinzadeh M, Safari A (2010). Comparison of acupressure
effect on sanyinjiao point with that of vitamin E on primary
Dysmenorrhea. J. Gonabad Uni. Med. Sci., 14(3): 35-42.
Chen H, Chen C (2004). Effect of acupressure at the sanyinjaiao point
on primary dysmenorrhoea. J. Adv. Nursing, 48(4): 380-387.
Deutch B, Jorgensen EB, Hansen JC (2000). Menstrual disorder in
Danish women reduced by diatery supplements of Omega – 3 PUFA
and B12 (fish oil or seal oil capsules). Nutr. Res., 20: 621-631.
Dolatian M, Jaafari H (2004). Study of the effects of fish oil capsules on
primary dysmenorrheal. J. Zanjan Uni. Med. Sci., 12(47): 7-13.
Habek D, Mehmet H (2007). Acupuncture treatment for prevention of
early dysmenorrheal. Anesth. Analg., 95(3): 632-635.
Harel Z, Biro FM, Kottenhan RK, Rosenthal SL (1996). Supplementation
with omega – 3 polyunsaturated fatty acids in the management of
dysmenorrheal in adolescents. Am.. J. Obstet. Gynecol. 174(4):
1335-1338.http:// www.fishoil-s.com
Jacoby L, Smythe C, Phetteplace H, Tabares A (1992). Adaptation to a
fish oil diet before indusing sepsis in rats preventing fatty infiltration of
the liver. Am. Soc. Parenter. Enteral Nutr., 16(4): 353-358.

Kidney M, Slade P, Randals CM (2001). Social support in women
reporting dysmenorrheal. J Psycosom. Res., 24:79-84.
Lee A, Boehler M, Taerum T (1999). The use of nonpharmacologic
techniques to treatment of dysmenorrheal. Am. J Pharmacol., 88:
1326-1329.
Pooresmaili Z, Ibrahimzadeh R (2002). Effect of acupressure and
Ibubrofen on The severty primary Dysmenoeehea. J. Trad. Chinese
Med., 22(3): 205-210.
Wilkinson ML, Muphy P (2001). A herbal and dietary therapies for
primary and Secoundary dysmenorrhea. Am. Obs. Gyn. May, 3: 804-
806.
Zafari et al. 1119
Yaghmaee M, Moradi A, Hosseini R (2004). Comparison of the effects
of mefenamic acid with mefenamic acid and fish oil in the reduction of
pain in primary dysmenorrheal. J. Gilan Uni. Med. Sci., 13(49): 68-72.
Zamani M, Nasrollahi S, Kashani K (2005). The curative effects of fish
oil on primary dysmenorrhoea. J. Gorgan Uni. Med. Sci., 17(1): 39-
42.

African Journal of Pharmacy and Pharmacology Vol. 5(8). pp. 1120-1124, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.421
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Correlation between clinicopathology and expression
of heat shock protein 72 in human primary lung
carcinomas
Yan Fang, Xiao-Ping Wang*, Huan-Ping Lin and Qiao-Xia Wang
1 Laboratory of Molecular Biology and Pathology, State Administration of Traditional Chinese Medicine, Shaanxi
University of Chinese Medicine, Xianyang, Shaanxi 712046, P. R. China.
Accepted 20 July, 2011
Heat shock protein 72 (HSP72) is highly expressed in cancer tissues. Recent studies indicate the
possible roles of HSP72 in the development and progression of different carcinomas, but detailed
information is still ambiguous. The aim of the study is to investigate the correlation between
clinicopathology and immunolocalization of HSP72 in human primary lung carcinoma. The expression
of HSP72 was studied in 96 human primary lung carcinomas with or without metastasis, as well as in
tissues adjacent to cancer by way of immunohistochemistry. The distribution of HSP72 immunoreactive
was examined by continuous cell counting in restricted fields by pathology photograph analysis.
HSP72 immunoreactivities were detected in 90 of 96 primary tumors (93.7%). The expression of HSP72
has a correlation with the differentiation of primary lung carcinoma. HSP72 expression in lung
carcinomas with lymph node and organ metastasis was significantly higher than those with nonmetastasis.
The results indicate that there exists a significant correlation between the expression of
HSP72 and the progression of primary lung carcinomas. HSP72 expression were significantly
associated with the presence of tumor infiltration, lymph node and remote metastasis. The expression
characters of HSP72 in primary lung carcinoma may contribute to study the pathogenesis and
progression of lung carcinoma.
Key words: Heat shock protein 72, primary lung carcinoma, clinicopathology, prognosis.
INTRODUCTION
The heat shock protein (HSP) family is a highly
conserved group of cellular proteins and is up-regulated
under stress conditions, such as heat, hypoxia, serum
deprivation, neoplasia and virus infection (Argon and
Simen, 1999; Morimoto, 1993; Schlesinger, 1990). It
functions as molecular chaperone and biochemical
regulator to mediate cell growth, apoptosis, protein
homeostasis and cellular targets of peptides (Morimoto,
*Corresponding author. E-mail: wxpphd@yahoo.com.cn. Tel:
+86-029-38185359.
Abbreviations: HSP72, Heat shock protein72; HSP70, heat
shock protein70; HSP, heat shock protein; gps, glycoproteins;
PBS, phosphate buffered saline; H2O2, hydrogen peroxide;
DAB, 3,3’-diaminobenzedine solution.
1993). Aside from their response to heat shock and
chemical or physical stress stimuli, HSPs have been
reported to be over expressed in a wide range of human
tumors including breast, endometrial, ovarian, colon, lung
and prostate (Ciocca and Calderwood, 2005). Studies
have also shown that HSP expression have a close
relationship with carcinoma prognosis (Ciocca and
Calderwood, 2005; Lebret et al., 2003). They may
combine with oncogene products to form complexes and
transport them into intracellular special sites and promote
cancer cell proliferation and heterogeneous differentiation
(Dorsey and Tchounwou, 2003; Villaseca et al., 1997).
Recent studies have also shown that heat shock protein
72 (HSP72) is highly expressed in cancer tissues and
have been used as prognostic markers in some tumors
(Bausero et al., 2004; Gabai et al., 2005; Wang et al.,
2002; Wang et al., 2007; Wang et al., 2008).
Study indicates the possible roles of HSP72 in the

development and progression of lung carcinomas but
detailed information is still ambiguous (Maehara et al.,
2000). Primary lung carcinoma is one of the most
malignant cancers, and there may be a correlation
between the progression of lung carcinoma and overexpression
of HSP72. The present study aimed to
estimate the extent of the expression of HSP72 proteins
in tumoral specimens obtained from lung cancer patients.
We also aimed to evaluate the association between the
extent of expression of HSP72 and various
clinicopathological parameters, tumor proliferative capacity.
The results showed that there exists a significant
correlation between the expression of HSP72 and the
progression in primary lung carcinoma.
MATERIALS AND METHODS
Immunochemistry reagents
Mouse anti-human HSP72 monoclonal antibody was obtained from
StressGen Biotechnologies (Victoria, British Columbia, Canada).
EnVisionTM kits were purchased from Dako Corp (Carpinteria, CA,
USA).
Tissue samples
This investigation was approved by the ethics committee on human
study at Shaanxi university of Chinese medicine (2004-4B). Paraffin
specimens of primary lung carcinoma from 96 patients undergoing
lung resection were collected from the affiliated hospital, Shaanxi
university of Chinese medicine, Xianyang, China from 2002 to
2008. None of the patients received any kind of anti-cancer
treatment or other therapies prior to surgery. The patients consisted
of 46 male and 26 female, with a mean age of 58.2 ± 4.6 years,
ranging from 42 to 79 years. Routine pathological diagnosis
showed that all cases were primary lung carcinoma. Tumors were
categorized as: squamous carcinoma type in 61 (63.5%),
adenocarcinoma type in 23 (24.0%), and small cell carcinoma type
in 12 (12.5%) out of 96 cases. Among the cases, 49 cases had
regional lymph node metastases, and 38 cases had remote
metastases. The specimens were fixed in 10% buffered formalin
and embedded in paraffin. Serial sections, 5-µm-thick, were cut and
placed on poly-lysine coated glass slides.
Immunohistochemical staining methods
All sections were deparaffinized and rehydrated with graded
alcohols. Endogenous peroxidase was then blocked with 3 mL/L
hydrogen peroxide (H2O2) diluted in methanol for 30 min at room
temperature. Antigen retrieval was performed by treating the slides
in citrate buffer in a microwave for 10 min. The slides were
incubated in a moist chamber with HSP72 mouse monoclonal
antibody (1:100) at 4°C overnight. After a complete wash in
phosphate buffered saline (PBS), the slides were incubated with
horseradish peroxidase labeled goat anti-mouse antibody (1:100)
for 45 min at 37°C. After a complete wash in PBS, the slides were
developed in 0.5 g/L freshly prepared 3,3’-diaminobenzedine
solution (DAB) (DAB, Sigma Co, St.Louis, Mo, USA) for 8 min, and
then counterstained with hematoxylin, dehydrated, air dried, and
mounted. Normal mouse IgG was used to substitute for the primary
antibody as a negative control. No specific immunoreactivity was
detected in these tissue sections. Two of the authors initially
Fang et al. 1121
determined the fields simultaneously using a double-headed light
microscope. The evaluation of HSP72 positive cells was performed
on high-power fields (×400) using a standard light microscope. Only
distinctive intranuclear or intra-cytoplasm immunoreactivity was
considered positive. In each case, more than 1000 cells were
counted and the percentage of immunoreactivity was independently
determined. When interobserver differences were greater than 5%,
the immunostained slides were re-examined simultaneously using a
double-headed light microscope and the percentage of positive
cells were determined. When interobserver differences were less
than 5%, the mean value was obtained as the positive rate.
Statistical analysis
HSP72 expression differences between lung carcinomas and
tissues adjacent to cancer were analyzed statistically using u test.
The relationship between expression of HSP72 in primary
carcinoma tissue with or without metastasis was analyzed
2
statistically using test. P < 0.05 was considered statistically
significant.
RESULTS
Immunolocalization of HSP72 in primary lung
carcinomas and adjacent tissues to cancer
The results of immunohistochemistry of HSP72 were
summarized in Table 1. HSP72 immunoreactivities were
detected in 90 of 96 primary tumors (93.7%) and in 16 of
96 mucous membranes adjacent to cancers (16.7%).
HSP72 was mainly stained in the nuclei of cancer cells.
HSP72 positive rates in lung carcinoma group were
significantly higher than that in adjacent tissues to cancer
(P < 0.05, Table 1).
Relationship between clinicopathology and
expression of HSP72 in primary lung carcinomas
Results showed that HSP72 expressed higher in low
differentiation of lung carcinomas than that in tissues
adjacent to cancers (P < 0.05). There were significant
differences of HSP72 expression between metastasis
groups and non-metastasis groups (P < 0.05). To assess
if HSP72 bear a pronounced prognostic effect in patient
subgroups, we conducted an extensive analysis of
HSP72 protein expressions. We stratified by nodal status
(absence vs presence of lymph node metastases),
presence of organ metastases and histopathological type
(squamous and adenocarcinoma vs small cell
carcinoma). In cross-tables, HSP72 expression was
significantly associated with the presence of organ
metastases and lymph node positivity (Table 1). These
results suggest that there exists a significant correlation
between expression of HSP72 and progression of lung
carcinomas.
DISCUSSION
In this study, we examined the expressions of HSP72 in

1122 Afr. J. Pharm. Pharmacol.
Table 1. Relationship between clinicopathology and immunoreactivity of HSP72 in lung
carcinomas.
Pathologic type
HSP72
n - (%) + (%)
Tissues adjacent to cancers 96 80 (83.3) 16 (16.7)
Lung carcinomas a 96 6(6.3) 90 (93.7)
Tumor pathologic type b :
Squamous carcinoma 61 4 (14.0) 57 (86.0)
Adenocarcinoma 23 2(8.7) 21(91.3)
Small cell carcinoma 12 0 (0) 12 (100)
Lymph node metastasis c :
Yes 49 0 (0) 49 (100)
No 41 5(12.2) 36 (87.8)
Remote metastasis d :
Yes 38 0 (0) 39 (100)
No 52 6(11.5) 46(88.5)
a P < 0.05, b P < 0.05, vs tissues adjacent to cancers; c P < 0.05, d P < 0.05, vs non-metastasis groups.
72 lung carcinoma samples by immunohistochemistry.
The results showed that almost all of the detected lung
carcinomas expressed HSP72, which had significant
differences compared with that in tissues adjacent to
cancers. By way of immunohistochemistry, we found that
there was a definite correlation between expression of
HSP72 and development of lung carcinomas.
The HSP family is group of highly conserved proteins
synthesized after heat induction or other stressors (Argon
and Simen, 1999; Morimoto, 1993; Schlesinger, 1990). In
mammalian cells, this system is divided into two
predominant categories, which appear to be structurally
and functionally related: the HSPs and the glycoproteins
(gps) (Schlesinger, 1990). During the growth and
development of normal cells, heat shock protein70
(HSP70) is constitutively expressed at low levels but the
expression was dramatically enhanced by stressful
conditions (Morimoto, 1993). HSP72, belonging to the
family of HSP70, is a highly conserved protein
synthesized under various stresses (Mohanty et al.,
2011). In non-transformed cells at normal conditions,
HSP72 is expressed at very low levels. It is, however,
present at elevated levels in the major fraction of tumors
and in many transformed cell lines (López-Cotarelo et al.,
2000; Kato et al., 2000; Volloch and Sherman, 1999). It is
commonly assumed that in tumor cells the expression of
HSP72 at elevated levels is the consequence of
oncogenic transformation and enhanced expression of
HSP72 has a close relationship with epithelial carcinoma
cells growth (Hwang et al., 2003; López-Cotarelo et al.,
2000; Volloch and Sherman, 1999). Up-regulated
expression of the HSP70 family in tumor cells may be a
requirement to serve as molecular chaperones in
regulating and stabilizing oncofetal protein and mutant
oncogene products during tumor growth process (Hwang
et al., 2003; Lee, 2001; Volloch and Sherman, 1999).
Recent studies have shown that HSP72 is highly
expressed in cancer tissues and have been used as
prognostic markers in some tumors, such as gastric
cancers, colonic tumors, breast cancers, lung cancers
and so on, which have also been verified to be
associated with the development and progression of the
above-mentioned carcinomas (Bausero et al., 2004;
Gabai et al., 2005; Wang et al., 2002; Wang et al., 2007;
Wang et al., 2008). In this experiment, we found that
HSP72 was highly expressed when lung carcinomas
progressed, but their roles in lung carcinoma are not
clear. It is reasonable to propose that HSP72 upregulation
in these tumor cells are closely related with
tumor cell survival and proliferation. Recent studies have
suggested that HSPs take part in cell growth and
proliferation in several ways such as signal transduction
and cell cycle regulation through combining certain protooncogene
products. This indicates that these proliferating
cells need higher level of HSPs to maintain the stability of
tumor proteome (Fisher et al., 2000; Liu et al., 1999). It is
believed that tumor cells are a group of highly
proliferative heterogeneous cells which progress gradually
through mutant oncogene products (King et al.,
2001; Renan, 1990). Continuous expression of HSP in
tumor cells may be required to serve as molecular
chaperones in regulating and stabilizing these products
during tumor growth process. At the same time, the
existence of mutant or oncogene products may stimulate
HSP synthesis (Dorsey and Tchounwou, 2003; Villaseca
et al., 1997).
The present study further supports the clinical signifycance
of HSP72 expression in the progression of lung

carcinoma. In the study, HSP72 expression was found to
be associated with important clinicopathological characteristics
for patients' management. Consistently, HSP72
expression was significantly associated with the presence
of tumor size, lymph node and organ metastasis. Our
results showed that not only the expres-sion of both
HSP72 in lung carcinoma was higher than that in tissues
adjacent to cancer, but also the expression of HSP72 in
lung carcinomas with metastasis was definitely higher
than that of lung carcinomas without metastasis. The
expression of HSP72 in lung carcinoma was related to
the pathologic type of lung cancer. The results indicate
that up-regulation of HSP72 is likely to have some
relationship with progression, invasion and metastasis of
lung carcinomas.
Studies revealed that considerable expression of HSPs
was found in tumor cells, showing that HSPs may be
induced by other stresses and participate in broader
array of defenses during cell growth and cell
differentiation of tumors (Dorsey and Tchounwou, 2003;
Lebret et al., 2003; Villaseca et al., 1997). Thus, it may
be presumed that under various stimuli and stressful
conditions, in order to avoid the damage caused by
deleterious factors such as nitrosamines, 3,4-
Benzopyrene-oncogenesis evocator, bronchopulmonary
epithelial cells have to transcribe and translate high levels
of HSPs in order to sustain normal metabolism and
functions of cells. Under these conditions,
bronchopulmonary epithelial cells should synthesize
HSPs rapidly to exert a protective role for themselves.
The progression of lung carcinoma is a gradual process
under the long-term influence of various stimuli. During
the process, inducible HSP synthesis increases gradually
(Wang et al, 2010). This viewpoint was confirmed by our
results, in that HSP72 was expressed at a higher level in
lung carcinoma than that in tissues adjacent to cancer.
The expression levels of HSP72 may be useful
prognostic markers for lung carcinoma.
Conclusion
In this study, we examined the expressions of HSP72 in
primary lung carcinoma samples by way of immunehistochemistry.
Human lung carcinomas existed with high
level of expression of HSP72. HSP72 expression was
significantly associated with the presence of tumor lymph
node and remote metastasis. There is a close correlation
between the overexpression of HSP72 and progression
of lung carcinomas. The expression characters of HSP72
in lung carcinoma may be useful to study the
pathogenesis and progression of lung carcinoma.
ACKNOWLEDGMENT
This study was financially supported by the Research
Fang et al. 1123
Program of Shaanxi Education Committee (07KJ233,
10KJ484).
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African Journal of Pharmacy and Pharmacology Vol. 5(8). pp. 1125-1131, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.396
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Synergy of daptomycin with fusidin against invasive
systemic infection and septic arthritis induced by type
VI group B streptococci in mice
Adel Galal El-Shemi 1,3 * and Hani S. Faidah 2
1 Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Umm Al-Qura University, Saudi Arabia.
2 Department of Microbiology, Faculty of Medicine, Umm Al-Qura University, Saudi Arabia.
3 Department of Pharmacology, Faculty of Medicine, Assiut University, Egypt.
Accepted 2 August, 2011
In view of the emergence of multidrug-resistant group B streptococci (GBS), and its significant clinical
impact, there is a necessary need for the development of more effective therapeutic alternatives. Here,
we assessed the therapeutic efficacy of daptomycin, a novel lipopeptide antibiotic, in the treatment of
type IV GBS-induced invasive systemic infection and septic arthritis in mice. We also evaluated the
possible synergy between daptomycin and fusidin to combat GBS disease. Mice infected with type IV
GBS and left without drug treatment displayed high incidence of deaths and severe diffuse septic
arthritis, associated with excessive production of proinflammatory cytokines (tumor necrosis factor
alpha (TNF- ), interleukin-1beta (IL-1 ) and interleukin-6 (IL-6)), cyclooxygeanse-2 (COX-2) and
prostaglandin E2 (PGE2) in their blood and joints. However, treatment of these GBS-infected mice with
daptomycin significantly inhibited the inoculated bacteria to grow in the blood and joints. Daptomycintreated
mice had significantly showed lower mortality rates, less frequent arthritis and lower levels of
TNF- , IL-1 , IL-6, COX-2 and PGE2 than infected untreated animals. More interestingly, a marked in
vivo synergy between daptomycin and fusidin that completely protected the mice from GBS infection
and its associated mortality and serious sequels was clearly observed. In summary, the present study
showed that daptomycin is a welcome newcomer antibacterial arsenal to eradicate GBS invasive
infection and septic arthritis, in particular when given in combination with other antibacterial agents
such as fusidin.
Key words: Group B streptococci, septic arthritis, daptomycin, fusidin, proinflammatory cytokines,
cylooxygenase-2, prostaglandin E2.
INTRODUCTION
Daptomycin is a novel lipopeptide antibiotic with unique
mechanism of action and excellent bactericidal activity
against a wide range of pathogenic gram-positive
bacteria, including multidrug-resistant gram-positive cocci
(LaPlante and Rybak, 2004; Brauers et al., 2007).
Antibacterial activity of daptomycin against many clinical
isolates was compared with that of -lactams,
vancomycin, linezolid and quinupristin/dalfopristin.
Overall, daptomycin showed greater bactericidal activity,
*Corresponding author. E-mail: dr_adel_elshemy2006@yahoo.
com or agshemi@uqu.edu.sa. Tel: +966-50- 9655135. Fax:
+966-2-5270000. Ext: 4234.
low incidence of bacterial resistance and more safety
profile than all other tested antibiotics (Rybak et al., 2000;
Pfaller et al., 2007). Furthermore, synergistic interactions
between daptomycin and other antimicrobials such as
aminoglycosides, -lactams and rifampicin were also
observed (Rand and Houck, 2004; Credito et al., 2007;
Figueroa et al., 2009).
Fusidic acid and its salt sodium fusidate (fusidin) are
narrow-spectrum antibiotics. The main indication for their
systemic use is in treatment of penicillin-resistant
staphylococcal infections, including osteomyelitis and
endocarditis, in combination with other antibacterials to
prevent emergence of resistance (Falck et al., 2006). In
addition to its antibacterial activity, fusidin has also
powerful immunomodulatory and anti-inflammatory

1126 Afr. J. Pharm. Pharmacol.
properties that are probably related to its capacity to
reduce inflammatory cell infiltration and down-regulate
the production of proinflammatory cytokines (e.g., TNF-α,
IFN-γ, IL-1 and IL-2) (Genovese et al., 1996; Milenkovic
et al., 2005). These unique properties of fusidin have
been reported against human immunoinflammatory
diseases (Nicoletti et al., 1999) and in several experimental
animal models of organ-specific inflammatory
diseases including sepsis and endotoxic shock
(Genovese et al., 1996), acute hepatitis (Nicoletti et al.,
1997), acute pancreatitis (Osman et al., 1998), allergic
encephalomyelitis (Di Marco et al., 2001), formalininduced
edema (Kilic et al., 2002) and myocarditis
(Milenkovic et al., 2005).
Group B streptococci (GBS), or Streptococcus
agalactiae, have long been known as the most common
leading cause of life-threatening bacterial infections in
neonates and young infants (Pettersson, 2007). Recently,
these microorganisms have also been recognized as an
ever-growing cause of serious infections and mortality
among older adults (Skoff et al., 2009). Case fatality rate
of invasive GBS infections has been reported to be
between 21 and 32% in newborns (Lukacs et al., 2004)
and can exceed 40% in immunocompromised adult
patients (Liakopoulos et al., 2004). Rapidly progressive
septic arthritis is an important clinical manifestation of
GBS infection (Nolla et al., 2003). Unfortunately, one-half
of the patients with GBS septic arthritis do not respond to
the current therapy, and thus the disease is associated
with substantial irreversible functional complications (Lee
et al., 2007). The emergence of multi-drug resistant GBS
strains has complicated the clinical scenario of GBS
disease (Simoes et al., 2004; Nagano et al., 2008).
Moreover, there are no vaccines suitable for the
prevention of GBS infections, and consequently identifycation
of alternate drug targets is essential for future
therapeutic measures (Rajagopal, 2009). Taken together,
these facts reinforce the importance of GBS as a public
health concern and raise the critical need for development
of more efficient alternative therapeutic strategies.
At the experimental level, Tissi et al. (1990) have
described a model of invasive GBS systemic infection
and septic arthritis induced in mice by type IV GBS. In
this model, the clinical features of induced GBS disease
and its associated deaths and septic arthritis greatly
mimic the human situation (Tissi et al., 1990, 1999; Puliti
et al., 2002, 2010). In the present study, we successfully
established this model and investigated the therapeutic
efficacy of daptomycin against GBS-induced high
mortalities and severe arthritis in mice. We also assessed
whether co-administration of fusidin with daptomycin
could result in an in vivo synergy against this lifethreatening
bacterial disease. Results showed that
therapy with daptomycin significantly suppressed in vivo
growth of inoculated GBS and its associated lethal trend
and septic arthritis. More interestingly, co-administration
of fusidin significantly enhanced daptomycin activity in
combating GBS infection and its serious sequels.
MATERIALS AND METHODS
Antibiotics and bacteria
Daptomycin (Cubist Pharmaceuticals, Inc., Lexington,
Massachusetts, USA) and sodium fusidate (fusidin) (Sigma, St.
Louis, MO, USA) were purchased as powder for reconstitution.
Prior to each experiment, all drug preparations were freshly made in
accordance with their manufacturers’ recommendations. Type IV
group B streptococci (GBS; reference strain CNCTC 1/82) was
supplied by Czech National Collection of Type Cultures (Prague,
Czech Republic). For experimental infection, the bacteria were
grown overnight at 37°C and 5% CO2 in Todd-Hewitt broth (Oxoid
Ltd., Basingstoke, Hampshire, England) and then washed and
suspended in RPMI 1640 medium at a concentrations of 1 × 10 7
colony forming units (CFU) per ml as previously described (Tissi et
al., 1990; Puliti et al., 2002).
Animals, experimental GBS infection and treatment schedule
This study was approved by the Local Animal Care Committee of
Umm Al-Qura University, KSA, and carried out in accordance with
the principles of laboratory animal care, formulated by the National
Academy of Sciences. A total of 60 adult male albino mice (weight,
25 to 35 g) were housed in autoclaved cages, maintained at a 12 h
light/dark cycle in a humidity and temperature-controlled
environment with autoclaved food and water ad libitum. For
induction of GSB infection, mice were intravenously inoculated with
10 7 colony-forming units (CFU) of GBS per mouse in a volume of 1
ml of RPMI 1640 medium. The animals were randomly divided into
five groups: Group 1 (n = 10); neither infected nor drug-treated
control mice, Group 2 (n = 14); GBS-infected and received no drug
treatment, Group 3 (n =12); GBS-infected and treated with
daptomycin (6 mg/kg/day; intraperitoneally; i.p., for 6 consecutive
days), Group 4 (n = 12); GBS-infected and treated with fusidin (80
mg/kg/day; i.p., for 6 sequential days) and Group 5 (n = 12); GBSinfected
and treated with daptomycin + fusidin. The used dosage
regimens of daptomycin and fusidin were in accordance with those
applied in the clinical field or had been tested in the previous
studies (Figueroa et al., 2009; Milenkovic et al., 2005). Seven days
after GBS inoculation, the survival mice of each group were
sacrificed under ether anesthesia. After that, their blood samples
were collected and their affected joints were aseptically removed,
weighed and homogenized in sterile RPMI 1640 medium (1 ml/100
mg of joint weight) as previously described (Tissi et al., 1990; Puliti
et al., 2002, 2010). Each resultant joint tissue homogenate was
divided into two portions: a portion employed for bacterial culturing
as mentioned subsequently, while the second part was centrifuged
at 2,000 ×g for 10 min and then its supernatant was collected and
stored at -20°C until used. Similarly, each collected blood sample
was divided into unequal two parts: the small part was directly
employed for bacterial culturing, while the large one was
centrifuged for 10 min at 4000 rpm and its serum was aspirated and
stored at -20°C until used.
Clinical evaluation of arthritis and mortality
All mice were daily and individually monitored for the signs of
arthritis and for mortality. Arthritis was defined as visible joint
erythema and/or swelling of at least one joint (Tanaka et al., 1996).
To evaluate the intensity of developed arthritis, a macroscopic

El-Shemi and Faidah 1127
Table 1. Effect of daptomycin and/or fusidin therapy on type IV group B streptococci (GBS)-induced deaths and arthritis in
mice.
Group n Mortality rate (%)
Arthritis
Incidence (%) Severity (mean ± SEM)
Normal control 10 0 0 0.0
GBS 14 42.9 a 78 ± 11 a 2.7 ± 0.3 a
GBS + daptomycin 12 8.3 b 17 ± 2 b 0.5 ± 0.06 b
GBS + fusidin 12 25 c 29 ± 3 c 1.2 ± 0.02 c
GBS + daptomycin + fusidin 12 0 d, * 0 d, * 0.0 d, *
Mice were infected with 10 7 colony-forming units of GBS/mouse and then treated or not-treated with daptomycin (6 mg/kg/day)
and/or fusidin (80 mg/kg/day) for 6 consecutive days. a P

1128 Afr. J. Pharm. Pharmacol.
Table 2. Effect of daptomycin and/or fusidin therapy on systemic and local bacterial growth and intra-articular production of cyclooxygenase-
2 (COX-2) and prostaglandin E2 (PGE2) in mice infected with type IV group B streptococci (GBS).
Group n
GBS (CFU/ml)
Blood Joint homogenate
COX-2(ng/ml) PGE2 (pg/ml)
Normal control 10 0.0 0.0 0.4 ± 0. 1 9.6 ± 2.3
GBS 8 2.3 × 10 5 ± 0.1 × 10 5a 4.1 × 10 6 ± 0.1 × 10 6a 35 ± 9.3 a 560 ± 93 a
GBS +daptomycin 11 0.7 × 10 1 ± 0.2 × 10 1b 1.5 × 10 1 ± 0.1 × 10 1b 2.5 ± 0.2 b 45 ± 9.5 b
GBS +fusidin 9 1.3 × 10 2 ± 0.4 × 10 2c 2.3 × 10 2 ± 0.2 × 10 2c 9.3 ± 2.7 c 135 ± 21 c
GBS + daptomycin + fusidin 12 0.0 d, * 0.1 × 10 1 ± 0.01 × 10 1d 0.5 ± 0.03 d, * 11 ± 2.6 d, *
Mice were infected with 10 7 colony-forming units of GBS/mouse and then treated or not-treated with daptomycin (6 mg/kg/day) and/or fusidin
(80 mg/kg/day) for 6 consecutive days. At day 7, all survival mice were examined. Results are expressed as the mean ± SEM. a P

El-Shemi and Faidah 1129
Table 3. Effect of daptomycin and/or fusidin therapy on production of proinflammatory cytokines in mice infected with type IV group B
streptococci (GBS).
Group n
TNF- (pg/ml) IL-6 (pg/ml) IL-1 (pg/ml)
Serum Joint Serum Joint Serum Joint
Normal control 10 5.3 ± 0.5 5.8 ± 0.7 3.7 ± 0.2 2.9 ± 0.3 6.3 ± 0.8 4.5 ± 0.4
GBS 8 57 ± 11 a 83 ± 15 a 600 ± 75 a 223 ± 43 a 260 ± 33 a 167 ± 13 a
GBS + daptomycin 11 9.5 ± 1.7 b 11 ± 2.4 b 37 ± 6.5 b 13 ± 2.6 b 20 ± 2.8 b 13.5 ± 1.2 b
GBS + fusidin 9 20 ± 3.5 c 17 ± 3.3 c 120 ± 25 c 55 ± 7.7 c 43 ± 6.3 c 47 ± 6.3 c
GBS + daptomycin + fusidin 12 5.8 ± 0.9 d, * 6.2 ± 1.1 d, * 4.0 ± 0.2 d, * 3.1 ± 0.8 d, * 7.0 ± 1.2 d, * 5.1 ± 0.5 d, *
Mice were infected with 10 7 colony-forming units of GBS/mouse and then treated or not-treated with daptomycin (6 mg/kg/day) and/or fusidin
(80 mg/kg/day) for 6 consecutive days. At day 7, all survival mice were killed and the levels of tumour necrosis factor alpha (TNF- );
interleukin-6 (IL-6) and interleukin-1beta (IL-1 ), in their sera and joint tissue homogenates were measured. Values are expressed the mean
± SEM. a P

1130 Afr. J. Pharm. Pharmacol.
and joints of GBS-infected untreated mice, and their
levels were directly correlated with the severity of GBS
disease and its associated mortalities and arthritis. By
contrast, the levels of these proinflammatory cytokines
were extremely low in infected mice simultaneously
treated with daptomycin/fusidin combination therapy.
Mechanistically, there are atleast two possibilities by
which daptomycin and fusidin had exerted their beneficial
inhibitory effects on the production of TNF- , IL-1 and
IL-6. First, they directly killed the causative agent (that is,
the inoculated type IV GBS). Second, it has been
generally accepted that fusidin possess powerful
immunomodulating and anti-inflammatory properties that
are probably related to its capacity to reduce
inflammatory cell infiltration and down regulate the
production of proinflammatory cytokines including TNF-α,
IFN-γ, IL-1 and IL-2 (Di Marco et al., 2001; Kilic et al.,
2002; Milenkovic et al., 2005).
The discovery of cyclooxygenase (COX) isoforms
(COX-1 and COX-2) led to the concepts that the
constitutive COX-1 isoform tends to be homeostatic in
function, while COX-2 is mainly induced during
inflammation and tends to facilitate the inflammatory
response (Smith et al., 2000). Prostaglandins (PGs)
produced by COX-2 (especially PGE2) are potent
inflammatory mediators and are associated with pain and
other signs of inflammation (FitzGerald, 2003). In the
present study, either COX-2 or PGE2 was intensively
expressed in the joints of GBS-infected mice and did not
receive any medication, while concurrent treatment of
these infected mice with daptomycin and/or fusidin
significantly and synergistically abrogated the intraarticular
COX-2 expression and PGE2 production. An
important question remains to be answered: how
daptomycin and fusidin did inhibit COX-2 activity and
subsequently the production of PGE2. There is evidence
that COX-2 is induced in inflamed tissues by
inflammatory chemical mediators including TNF- and IL-
1 (Smith et al., 2000). Accordingly, inhibition of these
proinflammatory cytokines, as observed here, might,
atleast in part, led to inhibition of COX-2 expression and
PGE2 production.
Conclusions
The present study clearly indicates the potential
therapeutic efficacy of daptomycin antibiotic against
invasive GBS infection, and septic arthritis, and
demonstrates its remarkable in vivo synergy with fusidin
in combating the disease. Co-administration of
daptomycin with fusidin greatly suppressed GBS infection
at the microbiological, clinical and pathologic levels; 1)
has complete eradicating effect on the inoculated
bacteria, 2) has full protective effect against GBS-induced
high mortality rates and high incidence of severe arthritis,
3) significantly down-regulated COX-2 expression and
PGE2 production in the joint tissues and 4) significantly
decreased the levels of proinflammatory cytokines ( TNF-
, IL-1 and IL-6) in the blood and joints of infected mice.
Taken together, daptomycin is a welcome newcomer
antibacterial arsenal to eradicate gram-positive cocci
including GBS, in particular when given in combination
with other antibacterial agents such as fusidin.
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African Journal of Pharmacy and Pharmacology Vol. 5(8). pp. 1132-1144, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP10.407
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Anti-nociceptive, anxiolytic and anticonvulsant effects
of an aqueous leaf extract of Leea guineensis G. Don
(Family: Leeaceae)
Eric Woode*, David Abasiwani Alagpulinsa and Wonder Kofi Mensah Abotsi
Department of Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences, College of Health Sciences, Kwame
Nkrumah University of Science and Technology, Kumasi, Ghana.
Accepted 12 June, 2011
Leea guineensis G. Don (Family: Leeaceae) is an evergreen shrub or small tree used in Ghanaian
traditional medicine to treat various ailments including epileptic fits and pain. However, little scientific
evidence exists to support its use. The present study examined the anti-nociceptive, anti-anxiety and
anticonvulsant effects of the aqueous leaf extract of L. guineensis (LGE) in murine models of pain
(formalin test), anxiety (elevated plus-maze and light/dark box tests) and convulsion
(pentylenetetrazole-, picrotoxin- and maximal electroshock-induced seizures tests). LGE (30 to 300
mg/kg, p.o.) and the positive control morphine (3 to 10 mg/kg, i.p.) exerted profound dose-dependent
anti-nociceptive activity in both phases of the formalin test. LGE (30 to 300 mg/kg, p.o.) also showed
anxiolytic effects similar to diazepam (0.1 to 1.0 mg/kg, i.p.) but contrary to pentylenetetrazole (3 to 30
mg/kg, i.p.). LGE increased the frequency and duration of open arm exploration while decreasing the
protected forms of stretch-attend postures and head-dips in the elevated plus-maze. It also decreased
the emergence latency of mice into the lit compartment of the light/dark box and increased the amount
of time spent there. LGE (30 to 300 mg/kg, p.o) also exhibited significant anticonvulsant activity by
protecting mice against pentylenetetrazole-, picrotoxin- and maximal electroshock-induced seizures. At
all the doses used, LGE produced no motor deficits in mice. Together, the results suggest that the
extract has anti-nociceptive, anxiolytic and anticonvulsant properties which support its traditional use.
Key words: Leea guineensis, seizures, maximal electroshock, formalin test, elevated plus-maze, light/dark box.
INTRODUCTION
Leea guineensis G. Don (Family: Leeaceae) is an
evergreen shrub or small tree that grows to about 6 m
high and is widespread in moister woodland and forest
areas of tropical Africa. It is commonly known as
Agyaben, Okatanini or Okatakyi by the Akans in Ghana.
The plant has been used traditionally for the treatment of
various ailments in the West African sub-region. The
leaves are used to treat toothache, rheumatism, skin
ulcer, vertigo, epileptic fits and paralysis (Burkill, 1985;
Irvine, 1961; Mshana et al., 2000). The roots and seeds
are also used as a purgative (Irvine, 1961).
Not much pharmacological studies have been done on
*Corresponding author. E-mail: ewoode.pharm@knust.edu.gh,
ericwoode@yahoo.com. Tel: +233 244 589793.
the plant. Falodun et al. (2007) have shown that the
aqueous leaf extract has anti-inflammatory activity in the
carrageenan-induced rat paw oedema test model.
Phytochemical evaluation of the woods and leaves
revealed volatile constituents including terpenoids (Op de
Beck et al., 2000) and hydrophilic flavonoids namely
quercetin-3′-sulphate-3-O-α-L-rhamnopyranoside,
quercetin-3,3′-disulphate and quercetin-3,3′,4′-trisulphate,
together with kaempferol, quercetin, quercitrin,
mearnsitrin, gallic acid and ethyl gallate (Op de Beck et
al., 2003). These flavonoids were found to have free
radical scavenging activity when evaluated in the DPPH
assay (Op de Beck et al., 2003). Our search has revealed
that there are no scientific reports available in established
literature to support the effectiveness of the plant in the
treatment of pain and epilepsy. In this study, we
investigated the anti-nociceptive, anxiolytic and anticon-

vulsant effects of the aqueous leaf extract of the L.
guineensis in mice.
MATERIALS AND METHODS
Plant collection and extraction
Fresh leaves of L. guineensis were collected from the Botanic
Gardens of Kwame Nkrumah University of Science and Technology
(KNUST), Kumasi, Ghana, in the month of September, 2007 and
authenticated by Dr. Kofi Annan of the Department of Herbal
Medicine, Faculty of Pharmacy and Pharmaceutical Sciences,
College of Health Sciences, KNUST, Kumasi, Ghana. A voucher
specimen of the plant (LEEACEAE/FP/08/26) has been deposited
at the Faculty’s Herbarium. The fresh leaves of L. guineensis were
blended and then macerated with distilled water at room
temperature for 24 h. The mixture was filtered and the extract
obtained by concentrating the filtrate to dryness using a freezedryer.
The dried aqueous extract, herein referred to as LGE or the
extract, was then kept in a refrigerator (4°C). The yield was 12%.
Animals
Cancer Research (ICR) mice (267 males, 84 females; 30±5 g) were
used. The animals were purchased from the Noguchi Memorial
Institute for Medical Research (NMIMR), University of Ghana,
Legon and housed in the animal house of the Department of
Pharmacology, KNUST, Kumasi. Animals were housed in groups of
five in stainless steel cages (34 × 47 × 18 cm) with soft wood
shavings as bedding, fed with normal pellet diet (GAFCO, Tema),
given water ad libitum and maintained under laboratory conditions
(temperature 25±2°C, relative humidity 60 to 70%, and 12 h lightdark
cycle). All behavioural experiments were carried out under dim
light. To acclimatize the animals to the test conditions, they were
brought to the laboratory a week before the experiments. All
procedures and techniques used in these studies were in
accordance with accepted principles for laboratory animal use and
care (EU directive of 1986: 86/609/EEC) and were approved by the
local Departmental Ethics Committee.
Drugs and chemicals
The following drugs and chemicals were used: Formalin,
theophylline (BDH, Poole, England); naloxone, pentylenetetrazole
(PTZ), picrotoxin (Sigma-Aldrich Inc., St. Louis, MO., USA);
diazepam (INTAS, Gujarat, India); carbamazepine (Tegretol ® ,
Novartis, Basel, Switzerland) and morphine (Phyto-Riker, Accra,
Ghana). Doses of drugs were selected based on data from
literature and preliminary experiments in our laboratory.
Phytochemical analysis
The freshly prepared aqueous extract was subjected to preliminary
phytochemical analysis using methods described by Trease and
Evans (1989) to determine the presence of alkaloids, saponins,
cardiac glycosides, flavonoids and tannins.
The formalin test
The anti-nociceptive effect of the extract was evaluated using the
formalin test. The test was carried out as described previously
Woode et al. 1133
(Hunskaar and Hole, 1987; Malmberg and Yaksh, 1992) with few
modifications. Male mice were randomly divided into seven groups
(n=5) and each animal acclimatized to the testing environment
(Perspex chamber, 15×15×15 cm) for 30 min. Mice were pretreated
with LGE (30, 100 and 300 mg/kg, p.o.), morphine (1, 3 and
10 mg/kg, i.p.) or vehicle (10 ml/kg, p.o.) 30 min (i.p.) or 1 h (p.o.)
before the induction of nociceptive behaviours in the animals by
intraplantar injection of 10 µl of 5% formalin. Animals were
immediately returned individually into the testing chamber. A mirror
angled at 45° beneath the floor of the chamber allowed an
unobstructed view of the paws. The behaviour of the animals was
then captured (1 h) for analysis by a camcorder (Everio model
GZ-MG1300, JVC, Tokyo, Japan) placed directly opposite to the
mirror and attached to a computer. Pain responses were scored for
1 h, starting immediately after formalin injection. A nociceptive
score was determined for each 5-min time block by measuring the
amount of time spent biting/licking of the injected paw (Hayashida
et al., 2003). The scoring of pain responses was done with the aid
of the public domain software JWatcher Version 1.0 (University of
California, Los Angeles, USA and Macquarie University, Sydney,
Australia available at http://www.jwatcher.ucla.edu/). Average
nociceptive score for each time block was calculated by multiplying
the frequency and time spent in biting/licking. Data were expressed
as the mean ± S.E.M. scores between 0 to 10 and 10 to 60 min
after formalin injection.
The elevated plus-maze test
This test was carried out as described previously (Lister, 1987;
Pellow et al., 1985) with modifications. The apparatus was made of
Plexiglas and consisted of two open arms (30×5×0.5 cm) and two
enclosed arms (30×5×15 cm). The arms extended from a central
platform (5×5 cm) forming a plus-sign with like arms opposite each
other. The maze was elevated 60 cm from the floor, and placed in a
lit room (~750 lux).
Male mice were randomly assigned to ten groups (n = 6): vehiclecontrol
(distilled water, 10 ml/kg, p.o.), LGE (30 to 300 mg/kg, p.o.),
diazepam (0.1 to 1.0 mg/kg, i.p.) and pentylenetetrazole (3 to 30
mg/kg, i.p.). Diazepam and pentylenetetrazole served as reference
anxiolytic and anxiogenic drugs, respectively. Thirty minutes after
i.p. injection with diazepam and pentylenetetrazole and 1 h after
oral treatment with the extract and vehicle, mice were placed
individually in succession in the central platform of the maze for 5
min and their behaviour recorded with a camcorder (Everio TM ,
model GZ-MG1300, JVC, Tokyo) placed 100 cm above the floor.
The digitized video of each 5 min trial were later scored using
JWatcher TM Version 1.0 for behavioural parameters including: (1)
Number of closed and open arm entries, absolute value and
percentage of the total number; (2) Time spent in exploring the
open and closed arms of the maze: Absolute time and percentage
of the total time of testing; (3) Number of head-dips (absolute value
and percentage of the total number): Protruding the head over the
ledge of either an open (unprotected) or closed (protected) arm and
down toward the floor; (4) Number of stretch-attend postures
(absolute value and percentage of the total number): The mouse
stretches forward and retracts to original position from a closed
(protected) or an open (unprotected) arm. An arm entry was
counted only when all four limbs of the mouse were within a given
arm.
To compute total distances travelled by the mice, the software
Behavior Collect
(http://cas.bellarmine.edu/tietjen/DownLoads/computer_programs_f
or_data_colle.htm) was used to obtain raw XY data from the videos.
These data were then exported into Microsoft® Office Excel 2007
and further analyzed. Distance between two X-Y coordinate pairs
was calculated from the formula:

1134 Afr. J. Pharm. Pharmacol.
[(X − X )
1
2
2
+ (Y −Y
)
The light/dark box test
1
2
2
]
The test was carried out as described earlier (Ardayfio and Kim,
2006) but with modifications. The apparatus was a wooden box (45
×30×30 cm) divided into two compartments by a wooden board with
a small opening (7×7 cm) connecting the compartments. The larger
compartment comprised two-thirds of the apparatus, painted white,
open and illuminated by a 60-W lamp placed 50 cm above the floor.
The smaller compartment was painted black and had a cover that
was closed during testing.
Male mice were divided into ten groups (n=6) and received
treatments similar to that described previously for the elevated plusmaze
test. One hour (p.o.) or 30 min (i.p.) after drug administration,
each mouse was placed in the dark compartment (head facing a
corner) and the compartment covered. The sessions were
videotaped and later scored to determine the latency to emerge into
the lit compartment and the percentage of time spent in the lit
compartment.
Pentylenetetrazole-induced seizure test
The test was carried out similar to that described by Swinyard and
Kupferberg (1985). Female mice were divided into seven groups
(n=6) and received the extract (30 to 300 mg/kg, p.o), diazepam
(0.1 to 1.0 mg/kg, i.p.) or vehicle (distilled water, 10 ml/kg, i.p.).
Thirty minutes (i.p.) or 1 h (p.o.) after the treatments, mice were
injected with pentylenetetrazole (85 mg/kg) subcutaneously. The
animals were placed individually in an observation chamber and
videotaped for 30 min. The videos were later scored with
JWatcher TM Version 1.0 to determine the latency to first myoclonic
jerks, the latency to tonic convulsions and the frequency and
duration of tonic convulsions for each mouse.
Picrotoxin-induced seizure test
The procedure used was the same as the one described previously
for pentylenetetrazole-induced seizures except that instead of
pentylenetetrazole, picrotoxin (10 mg/kg, i.p.) was administered to
mice (Mackenzie et al., 2002; Ngo Bum et al., 2004; Swinyard,
1969). Latency to myoclonic jerks, latency to tonic convulsions as
well as the frequency and duration of tonic convulsions were
recorded from the videos as in the pentylenetetrazole seizure test.
Maximal electroshock seizure test (MEST)
The test was performed as described by Schmutz et al. (1990) with
some modifications. Male mice were divided into seven groups
(n=10) and received LGE (30 to 300 mg/kg, p.o.), vehicle (10 ml/kg,
p.o) or carbamazepine (3 to 30 mg/kg, p.o.). One hour after the
treatments, tonic convulsions of the hind extremities of mice were
induced by passing alternating current (50 Hz, 60 mA, 0.2 s) from
an electroconvulsive therapy (ECT) apparatus (Model 7800, Ugo
Basile, Camerio, Italy) via ear electrodes. The current used was
predetermined before experimentation and was the maximal current
that caused hind limb extension in all mice in the trials. Mice were
restrained by gripping the loose skin on their back. The number of
animals protected from tonic hind limb extension seizure and the
time spent in this position were determined in each group.
Effect on motor coordination - rotarod test
The effect on motor co-ordination was assessed using rotarod
apparatus (Model 7600, Ugo Basile, Cormerio, Italy) rotating at a
speed of 25 rpm. This apparatus consists of a base platform and a
rotating rod of 3 cm diameter with a non-skid surface. The rod, 50
cm in length, is divided into five; equal sections by six disks. Five
mice were tested simultaneously.
Male mice were initially selected for their ability to remain on the
rotarod for at least two consecutive 60 s trials before the test day.
Mice were randomly divided into seven groups (n=6) and received
LGE (30 to 300 mg/kg, p.o.), diazepam (0.1 to 1.0 mg/kg, i.p.) or
vehicle (10 mg/kg, i.p.). Thirty minutes (i.p.) or 1 h (p.o.) after the
treatments, the latencies to fall from the rod were measured. Mice
that stayed on the rotarod for more than 60 s were given the
maximum score, 60 s.
Statistical analysis
Data are presented as mean±SEM. Data were analyzed using oneway
analysis of variance (ANOVA) with drug treatment as a betweensubjects
factor. Whenever ANOVA was significant, further
comparisons between vehicle- and drug- treated groups were
performed using the Newman-Keuls test. GraphPad Prism for
Window 5 (GraphPad Software, San Diego, CA, USA) was used for
all statistical analysis and P

Woode et al. 1135
Figure 1. Effect of LGE (30 to 300 mg/kg) and morphine (1 to 10 mg/kg) on the time course curves (a, c) and the total
nociceptive score (AUCs) (b, d) of formalin test in mice. Nociceptive scores are shown in 5 min time blocks up to 60 min for the
time course curves. Data are presented as mean ± SEM (n=5). ***P

1136 Afr. J. Pharm. Pharmacol.
Figure 2. Effect of LGE (30 to 300 mg/kg, p.o.), diazepam (0.1 to 1.0 mg/kg, i.p.) and pentylenetetrazole (3 to 30 mg/kg, i.p.) on open arm
entries (a, b, c), percentage open arm entries (d, e, f) and percentage time spent in open arms (g, h, i) of the EPM over a 5 min test period in
mice. Data are presented as mean±SEM (n=6). The lower and upper margins of the boxes (d-i) represent the 25 th and 75 th percentiles, with
the extended arms representing the 10 th and 90 th percentiles, respectively. The median is shown as the horizontal line within the box.
*P

Woode et al. 1137
Figure 3. Effect of LGE (30 to 300 mg/kg, p.o.), diazepam (0.1 to 1.0 mg/kg, i.p.) and pentylenetetrazole (3 to 30 mg/kg, i.p.) on
percentage protected stretch-attend postures (a-c), percentage protected head-dips(d-f) and total distance travelled (g-i) in the
EPM over a 5 min test period in mice. Data are presented as mean±SEM (n=6). The lower and upper margins of the boxes (a-f)
represent the 25 th and 75 th percentiles, with the extended arms representing the 10 th and 90 th percentiles, respectively. The
median is shown as the horizontal line within the box. Outlying points are shown individually. *P

1138 Afr. J. Pharm. Pharmacol.
Figure 4. Effect of LGE (30 to 300 mg/kg, p.o.), diazepam (0.1 to 1.0 mg/kg, i.p.) and pentylenetetrazole (PTZ; 3 to
30 mg/kg, i.p.) on the latency to emerge into lit compartment (a,c,e) and time spent in compartments (b, d, f) of the
light-dark box over a 5 min test period in the mice. Each bar represents mean ± S.E.M. (n=6). *P

Figure 5. Effect of LGE (30 to 300 mg/kg, p.o.) and diazepam (0.1 to 1.0 mg/kg, i.p.) on frequency (a, c), latency to
(a, c) and duration (b, d) of PTZ-induced tonic convulsions as well as latency to PTZ-induced myoclonic jerks (e) in
mice. Each point or column represents the mean ± S.E.M. (n=6). *P

1140 Afr. J. Pharm. Pharmacol.
Figure 6. Effect of LGE (30 to 300 mg/kg, p.o.) and diazepam (0.1 to 1.0 mg/kg, i.p.) on frequency (a, c), latency to (a, c)
and duration (b, d) of picrotoxin-induced tonic convulsions as well as latency to picrotoxin-induced myoclonic jerks (e) in
mice. Each point or column represents the mean ± S.E.M. (n=6). *P

Woode et al. 1141
Figure 7. Effect of LGE (30 to 300 mg/kg, p.o.) and carbamazepine (3 to 30 mg/kg, p.o.) on the duration of MESinduced
tonic hind limb extension. Each column represents Mean ± S.E.M. (n=10). *P

1142 Afr. J. Pharm. Pharmacol.
significant decrease in the latency to fall off the rotating rod
(P

anxiety rather than locomotor behaviour (Dawson et al.,
1995; Weiss et al., 1998), usually help to resolve these
conflicts. In this study, LGE and diazepam increased total
distance travelled in the EPM while PTZ decreased it.
However, the effect of LGE, diazepam and PTZ on risk
assessment behaviours in the EPM suggests that the
activities exhibited by these agents on anxiety were
selective and not due to non-specific changes in
locomotor activity. The results of the rotarod test also
show that LGE did not induce any significant motor
deficits in mice at the doses tested.
Inhibition of seizures induced by pentylenetetrazole and
maximal electroshock in laboratory animals is the most
common predictive screening test used for characterizing
potential anticonvulsant drugs (Krall et al., 1978; Loscher
and Schmidt, 1988; Sayyah et al., 2004). The maximal
electroshock-induced seizure test (MEST) is considered to
be a predictor of likely therapeutic efficacy against
generalized tonic-clonic seizures. Anticonvulsant effect in
MEST further indicates the ability of the testing material to
prevent seizure spread through neural tissue (Castel-
Branco et al., 2009; Piredda et al., 1985). By contrast, the
PTZ-induced seizure test represents a valid model for
human generalized myoclonic and absence seizures
(Loscher and Schmidt, 1988). Anticonvulsant activity
against PTZ seizures also identifies compounds that can
raise seizure threshold in the brain (Goodman et al., 1953;
Mandhane et al., 2007; Piredda et al., 1985). In this study
LGE inhibited MES- and PTZ-induced seizures suggesting
that L. guineensis may contain compounds that have
activity against generalized tonic-clonic as well as
generalized myoclonic and absence seizures. PTZ is
believed to exert its convulsant effect by inhibiting the
action of GABA at GABAA receptors (De Sarro et al.,
1999). Picrotoxin, a GABAA receptor antagonist, has also
been shown to elicit seizures by blocking chloride channels
linked to GABAA receptors (Meldrum and Rogawski,
2007). Since LGE showed anticonvulsant activity against
PTZ- and picrotoxin-induced seizures, it is highly probable
that GABAergic mechanisms are involved in the extract’s
anticonvulsant action. Further studies are obviously
necessary to establish the exact mechanism of action of
the extract.
Conclusion
The results from the present study have shown that the
aqueous leaf extract of L. guineensis possess antinociceptive,
anxiolytic and anticonvulsant properties.
However, further research is necessary to determine the
components involved and their mechanism of action in
bringing about the observed pharmacological effects.
ACKNOWLEDGEMENTS
The authors wish to show their sincere appreciation to
Woode et al. 1143
Mr. Thomas Ansah of the Department of Pharmacology
for his technical assistance.
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maze models of anxiety. Neurosci. Biobehav. Rev., 23: 265-271.

African Journal of Pharmacy and Pharmacology Vol. 5(8). pp. 1145-1149, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.060
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
HI-6 and obidoxime implication in oxidative stress,
antioxidants level and apoptosis
Miroslav Pohanka 1 *, Ladislav Novotny 1,2 , Josef Fusek 1 and Jiri Pikula 2
1 Faculty of Military Health Sciences, University of Defense, Trebesska 1575, 50001 Hradec Kralove, Czech Republic.
2 Faculty of Veterinary Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Palackeho 1/3,
612 42 Brno, Czech Republic.
Accepted 12 June, 2011
The oxime reactivators, obidoxime and asoxime (HI-6) are suitable for antidotal treatment after
exposure to nerve agents. Although they are considered for use in humans under emergency
situations, complete clinical studies are lacking as there were no clinical trials. We examined obidoxime
and HI-6 in laboratory rats intramuscularly exposed to 25% of the median lethal dose (210 and 780
mg/kg) of each oxime reactivator and sacrificed 40 min after exposure. Brain and liver ferric reducing
antioxidant power, reduced glutathione (GSH), glutathione reductase, thiobarbituric acid reactive
substances, acetylcholinesterase, caspase 3 and glutathione S-transferase were assessed using
standard protocols. We found significant depletion of liver and brain low molecular weight antioxidants.
On the other hand, the depletion was partially recovered by an increase in the GSH level. Obidoxime
was implicated in alteration of apoptotic processes in brain. Overall effects of oxime reactivators are
discussed in this study. The pertinent adverse effects and strong modulation of disparate parameters of
oxime reactivators, HI-6 and obidoxime are not well understood in antidotal treatment. We found strong
impact of oxime reactivators on redox homeostasis and apoptotic processes.
Key words: Oxime, sarin, acetylcholinesterase, adverse effects, apoptosis, oxidative stress.
INTRODUCTION
Oxime reactivators are a group of antidotes suitable for
causal treatment after exposure to organophosphorous
pesticides and/or nerve agents (Bajgar, 2004). Nerve
agents act as irreversible inhibitors of the enzyme,
acetylcholinesterase (AChE; EC 3.1.1.7). Interaction of
nerve agents with AChE results in alkylation of serine
hydroxyl in the enzyme active site followed by inability to
split the neurotransmitter, acetylcholine (Barthold and
Schier, 2005). Oxime reactivators are able to break AChE
- nerve agent complex providing active AChE and nerve
agent moiety bound to the oxime reactivator (Ekstrom et
al., 2009). The reactivation process is effective until
spontaneous dealkylation of the organophosphate
moiety, also called aging. The aging process is to the
individual nerve agents (Sanson et al., 2009).
Despite the known molecular mechanism of AChE
reactivation, the impact of oxime reactivators on the body
*Corresponding author. E-mail: miroslav.pohanka@gmail.com.
functions during treatment processes is not well
understood. Oxime reactivators can act as reversible
inhibitors of AChE and antagonists at acetylcholine
receptors (AChR) as reported, e.g., by Soukup et al.
(2008). In earlier experiments, we found that the
treatment process following exposure to nerve agents
had adverse effects and modulated antioxidant barriers
and oxidative stress (Pohanka et al., 2011a).
Our continuous effort is aimed at recognition of oxime
reactivator implication in oxidative stress, apoptosis and
other adverse effects. Two oxime reactivators (Figure 1),
obidoxime and asoxime (HI-6), are prospective military
purpose substances (Kassa, 2002). We chose these two
reactivators for assessment of their impact on the
cerebral cortex and liver in order to determine
neurotoxicity and hepatotoxicity respectively. The doses
of oxime reactivators were chosen as approximately ten
times of recommended therapeutic dose (Kassa and
Krejcova, 2003). This dose represents upper limit suitable
for emergency application (Kassa, 2006; Pohanka et al.,
2011a). The experiment was designed to assess

1146 Afr. J. Pharm. Pharmacol.
2Cl -
N +
O
NOH
H 2N
N +
O
HI-6
Figure 1. Structures of tested oximes.
2Cl -
N +
O
NOH
N +
NOH
Obidoxime
oxidative stress in the time interval where the oxidative
insult can be the most striking when considered in the
recent paper (Pohanka et al., 2011a).
MATERIAL AND METHODS
Chemicals
Phosphate buffered saline (PBS) in tablets, caspase 3 colorimetric
kit, 1-chloro-2,4-dinitrobezene, reduced glutathione and total protein
kit TP100 were purchased from Sigma-Aldrich (Saint Louis,
Missouri, USA). Ethanol and sodium chloride were purchased from
Penta (Prague, Czech Republic). Deionized water was prepared by
MilliQ Ultrapure Water Purification system (Millipore, Billerica,
Massachusetts, USA). All other chemicals were achieved from local
sources in the analytical purity.
Animal exposures
Female Wistar rats (190 to 210 g body weight) were purchased
from the Velaz Company (Prague, Czech Republic). The animals
were kept under standard conditions (temperature 22±2°C, humidity
50±10% and light period 12 h day -1 ). Feed and drinking water were
provided ad libitum. The experiment was permitted and supervised
by the Ethical Committee of the Faculty of Military Health Sciences,
University of Defence, Hradec Kralove, Czech Republic. In separate
experiments, the toxicity of obidoxime and HI-6 were measured
previously in our institution (Kassa and Cabal, 1999; Kassa, 2002).
The median lethal dose (LD50) for HI-6 and obidoxime, respectively,
was calculated as 780 and 210 mg/kg.
The rats were divided into three groups, n = 6 animals. Controls
were injected with 100 µl of saline only. The two experimental
groups were intramuscularly (i.m.) injected with 25% of obidoxime
or HI-6 (saline solution) LD50 in an amount 100 µl. After 40 min, the
animals were euthanized using CO2 narcosis.
Ex vivo assays
Two organs were sampled, that is, brain and liver. The frontal lobes
of cerebral cortex and left lateral hepatic lobe were excised and
then processed immediately at standard ambient temperature and
pressure (SATP) conditions. 100 mg of tissue sample was
immersed in 1 ml phosphate buffered saline (PBS) and mixed at
8,000 RPM using the Ultra-Turrax system (Ika, Werke, Staufen,
Germany). The mixing lasted one minute and crude fragments were
displaced by centrifugation at 1,000×g for five minutes.
AChE activity was assayed by the modified Ellman´s method in
compliance with the reference (Pohanka et al., 2008). Caspase 3
(Casp3) activity was evaluated by a standard colorimetric kit as
recommended by the producer. Assay was performed using a
multichannel spectrophotometer and standard disposable 96-well
microplates. Ferric reducing antioxidant power (FRAP) was carried
out in order to estimate the total level of low molecular weight
antioxidants. A standard protocol was used for this purpose. The
experimental protocol was the same as in the reference (Pohanka
et al., 2009). Glutathione-S-transferase (GST) activity was assayed
using the following protocol in a slight modification of reference
(Pohanka et al., 2011b): 10 µl 100 mM 1-chloro-2,4-dinitrobezene
was poured with 10 µl of 100 mM reduced glutathione, 980 µl of
PBS, and 50 µl of tissue homogenate. Absorbance was measured
at one minute intervals. Enzyme activity was calculated considering
the extinction coefficient 9,600 M -1 cm -1 . Reduced glutathione
(GSH), glutathione reductase (GR) and thiobarbituric acid reactive
substances (TBARS) were evaluated according to the cited protocol
(Pohanka et al., 2011a). Total protein (TP) level was assayed by
total protein kitin compliance with the protocol provided. The
assessed markers were reached for the gram of protein. All ex vivo
assays were carried out at SATP conditions.
Statistical analysis
Origin 8 SR2 (OriginLab Corporation, Northampton, MA, USA) was
used throughout for experimental data processing, descriptive as
well as inferential statistics. The significance of differences against
controls was calculated using a one-way ANOVA with Scheffe´s
test. Both probability levels of p< 0.05 and 0.01 were calculated for
the examined parameters and groups of 6 specimens.
RESULTS AND DISCUSSION
The evaluated markers of oxidative stress (Ferric
reducing antioxidant power (FRAP), thiobarbituric acid
reactive substances (TBARS), glutathione reductase
(GR) and reduced glutathione (GSH)) are clearly
depicted in Table 1. The levels of brain and liver GR and
brain TBARS were not significantly altered. However,
liver TBARS was extensively decreased. This could be
due to slight metabolic depression as the TBARS marker
can relate to the basal metabolism where residua coming
from lipid peroxidation appear as a side product of liver
oxidative metabolism (Lespine et al., 2001; Huang et al.,
2008).
The level of low molecular weight antioxidants was
markedly altered by oxime reactivators in terms of the
FRAP value and the results are matched for obidoxime
and HI-6. Liver is obviously more sensitive to the impact
of oxime reactivators in terms of the FRAP value
compared to brain. The total level of low molecular weight
antioxidants was significantly (P ≤ 0.01) decreased in the
liver by oxime reactivators. HI-6 caused a molar decrease
in low molecular weight antioxidants of 44%. Obidoxime
was more striking as the decrease in antioxidants was
55%. Changes in the total level of low molecular weight
antioxidants in the brain of exposed animals were not as
extensive as in the case of liver. The FRAP value for
cerebral cortex of HI-6 exposed animals was significantly
(P ≤ 0.01) decreased. The decrease was approximately

Table 1. Summarization of assessed oxidative stress, apoptosis and miscellaneous markers.
Pohanka et al. 1147
Marker Organ Control HI-6 Obidoxime
FRAP (µmol/g ) Brain 3.96± 0.41 2.15±0.28 ** 2.96±0.37
Liver 9.73±1.32 5.47±0.43 ** 4.45±0.22**
GSH (µmol/g) Brain 0.268±0.033 0.567±0.054** 0.904±0.043**
Liver 1.73±0.11 1.53±0.09 1.28±0.06*
GR (kat/ g) Brain 34.3±2.2 30.0±2.3 30.7±1.4
Liver 13.8±0.9 12.6±0.9 10.7±0.7
TBARS (µmol/g) Brain 32.2±2.0 30.1±3.2 28.6±1.3
Liver 47.2±5.1 28.3±3.1* 25.4±1.8 *
AChE (kat/g) Brain 218± 13 218±17 188±11
Liver 49.9±6.3 41.6±6.4 33.4±2.7
Casp3 (µkat/ g ) Brain 1.17±0.05 1.12±0.08 0.956±0.027*
Liver 1.52±0.06 1.42±0.07 1.35±0.09
GST (kat/ g) Brain 160±14 147±11 134±6
Liver 92.1±5.9 80.6±5.6 70.0±3.7*
Value/gram of protein ± standard error of mean. FRAP, Ferric reducing antioxidant power; GSH, Reduced glutathione; GR, Glutathione
Reductase; TBARS, thiobarbituric acid reactive substances; AChE, acetylcholinesterase; Casp3 - Caspase 3; GST - glutathione Stransferase;
* = p

1148 Afr. J. Pharm. Pharmacol.
and Soininen, 2010). However, these effects should be
elucidated more accurately. Slight inhibition can be
covered by expression of new AChE.
The increased activity of Casp3 indicates apoptosis of
cells. Casp3 may also be implicated in the regulation of
neurogenesis apart from its main apoptotic function
(D´Amelio et al., 2010). We proved alteration in Casp3
activity in the liver and cerebral cortex after obidoxime
and HI-6 administration. Obidoxime seems to be more
effective in Casp3 activity modulation when compared
with HI-6. It caused significant (0.01

distribution, and oxidative stress. Environ. Toxicol Pharmacol, In press,
DOI: 10.1016/j.etap.2011.03.014.
Pohanka M, Sobotka J, Stetina R (2011b). Sulfur mustard induced
oxidative stress and its alteration by epigallocatechin gallate. Toxciol
Lett., In press. DOI:10.1016/j.toxlet.2010.12.011.
Pohanka M, Sobotka J, Svobodova H, Stetina R (2011c). Investigation
of oxidative stress in blood, brain, kidney and liver after oxime
antidote HI-6 application in a mouse experimental model. Drug
Chem. Toxicol, In press.DOI: 10.3109/01480545.2010.542753.
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PAO in brain, liver, lung, and kidney. J. Chromatogr., 878: 1414-
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HM, Sussman JL, Ashani Y, Masson P, Silman I, Weik M (2009).
Crystallographic snapshots of nonaged and aged conjugates of
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aged conjugate with pralidoxime. J. Med. Chem., 52: 7593-7603
Sit R, Radic Z, Geradi V, Zhang L, Garcia E, Katalinic M, Amitai G,
Kovarik Z, Fokin V, Sharpless KB, Taylor P (2011). New structural
scaffolds for centrally acting oxime reactivators of phosphylated
cholinesterases. J. Biol. Chem., In press. DOI:
10.1074/jbc.M111.230656.
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Soukup O, Pohanka M, Tobin G, Jun D, Fusek J, Musilek K, Marek J,
Kassa J, Kuca K (2008). The effect of HI-6 on cholinesterases and on
the cholinergic system of the rat bladder. Neuro. Endocrinol. Lett., 29:
759-762.
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Lipid peroxidation was involved in the memory impairment of carbon
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Vogel T, Worek F, Kreuter J, von Briesen H (2010). Nanoparticulate
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African Journal of Pharmacy and Pharmacology Vol. 5(8). pp. 1150-1156, August, 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.183
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Triterpenoids from Vernonia auriculifera Hiern exhibit
antimicrobial activity
Joyce Jepkorir Kiplimo 1* , Neil Anthony Koorbanally 1 and Hafizah Chenia 2
1 School of Chemistry, University of KwaZulu–Natal, Durban 4000, South Africa.
2 School of Biochemistry, Genetics and Microbiology, University of KwaZulu–Natal, Durban 4000, South Africa.
Accepted 8 July, 2011
Phytochemical investigation of Vernonia auriculifera afforded farnesylamine, a sesquiterpene amine
that has not been found previously in plant species, together with lupenyl acetate, oleanolic acid, -
amyrin acetate, -amyrin, friedelanone, friedelin acetate, -amyrin and -sitosterol. The compounds
were characterized using nuclear magnetic resonace (NMR) spectroscopy and comparison with
literature values. The isolated triterpenoids exhibited moderate antibacterial activity; and -amyrin
had minimum inhibitory concentration (MIC) of 0.25 mg/ml against Staphylococcus aureus, Bacillus
subtilis, Enterococcus faecium and Staphylococcus saprophyticus while lupenyl acetate and oleanolic
acid exhibited MIC of 0.25 mg/ml against Stenotrophomonas maltophilia. Sub-MIC exposure of -amyrin
acetate was effective in decreasing adhesion of S. aureus, Klebsiella pneumonia and E. faecium while
oleanolic acid decreased adhesion of K. pneumonia and Pseudomonas aeruginosa significantly at sub-
MIC. These compounds show potential for synergistic coupling with antimicrobial agents to improve
therapeutic efficiency, in the face of rising bacterial resistance.
Key words: Vernonia auriculifera, triterpenoids, farnesylamine, antibacterial activity.
INTRODUCTION
The genus Vernonia (Asteraceae family) has more than
1000 species growing all over the world with more than
30 species growing in Kenya (Beentje, 1994; Oketch-
Rabah et al., 1997). V. auriculifera is a small tree or
woody herb that grows between 1 and 7.5 m high and is
easily recognizable by its deep purple flowers. V.
auriculifera has a wide variety of applications in traditional
medicine. A drop of the juice squeezed from the crushed
stem bark, inserted into the nostrils, is known to relieve
headache (Kusamba, 2001). The Kikuyu people of central
Kenya use the leaves of this plant as a wrap for pounded
material used as a poultice (Kokwaro, 1976). Heated
*Corresponding author. E-mail: jjkiplimo@yahoo.com Tel: +27-
720-334-548.
Abbreviations: TMS, Tetramethylsilane; MIC, minimum
inhibitory concentration; TSB, tryptone soy broth; DMSO,
dimethyl sulfoxide; LB, Luria Bertani broth; OD, optical density;
IR, Infrared; NMR, nuclear magnetic resonace; FT-IR, Fourier
transform infrared spectroscopy; ATR, attenuated total
reflection; GC-MS, gas chromatography- mass spectrometry.
crushed leaves of Aspilia mossambicensis, are tied in the
leaf of V. auriculifera, and then applied over the eyes to
treat conjunctivitis (Muthaura et al., 2007). Cold water
infusion of V. auriculifera is administered orally in Uganda
and Kenya to treat fever associated with viral and
bacterial infections (Muthaura et al., 2007; Freiburghaus
et al., 1996). In Ethiopia, the roots are used to treat
toothache (Mirutse et al., 2009) and snake poison (Mesfin
et al., 2009).
Hydroperoxides of unsaturated fatty acid methyl esters
previously isolated from V. auriculifera were found to
have lethal toxicity (Keriko et al., 1995a). Plant growth
stimulators have also been identified from this plant
(Keriko et al., 1995b). Other Vernonia species that have
received extensive phytochemical and pharmacological
research include: V. galamensis (Miserez et al., 1996), V.
brachycalyx (Oketch-Rabah et al., 1997), V. colorata
(Rabe et al., 2002), V. amagdalina (Erasto et al., 2006),
V. cinerea (Chen et al., 2006), V. mapirensis (Morales-
Escobar et al., 2007), V. cumingiana (Mao et al., 2008),
V. ferruginea (Malafronte et al., 2009) and V. scorpioides
(Buskuhl et al., 2010). Members of the genus Vernonia
are an excellent source of sesquiterpene lactones which

include vernolide, vernolepin, vernodalin and
hydroxyvernolide (Kupchan et al., 1969; Jisaka et al.,
1993; Koshimizu et al., 1994). Other compounds have
also been isolated from this genus such as triterpenoid
glycosides, flavonoids, coumarins and benzofuranones
(Miserez et al., 1996; Oketch-Rabah et al., 1997; Mao et
al., 2008).
The current study was undertaken primarily to
investigate the phytochemistry of V. auriculifera from
which only fatty acids were previously isolated and to test
the isolated compounds for antimicrobial activity since
extracts of Vernonia species have been cited as
antimicrobials in traditional medicine (Kokwaro, 1976).
MATERIALS AND METHODS
General experimental procedure
NMR spectra were recorded using a Bruker Avance III 400 MHz
spectrometer. All the spectra were recorded at room temperature
with all chemical shifts ( ) recorded against the internal standard,
tetramethylsilane (TMS). Infrared (IR) spectra were recorded on a
Perkin Elmer Spectrum 100 Fourier transform infrared spectroscopy
(FT-IR) spectrometer with universal attenuated total reflection
(ATR) sampling accessory. For gas chromatography-mass
spectrometry (GC-MS) analyses, the samples were analysed on an
Agilent GC–MSD apparatus equipped with DB-5SIL MS (30 m ×
0.25 mm i.d., 0.25 µm film thickness) fused-silica capillary column.
Helium (at 2 ml/min) was used as a carrier gas. The mass
spectrometry (MS) was operated in the EI mode at 70 eV. Optical
rotation was recorded using a PerkinElmerTM, Model 341
Polarimeter. Melting points were recorded on an Ernst Leitz Wetziar
micro-hot stage melting point apparatus.
Plant material
The leaves, stem bark and root bark of V. auriculifera were
collected in August, 2009 from Egerton University Botanical
Garden, Rift Valley Province in Kenya. The plant was identified by
taxonomist, Dr S. T. Kariuki, of the Botany Department, Egerton
University, Kenya and a voucher specimen (Kiplimo, 02) was
deposited in the Ward Herbarium, University of KwaZulu-Natal
Westville, Durban, South Africa.
Extraction and isolation
The air-dried and ground plant material of V. auriculifera (823 g
leaves, 710 g roots, 600 g stems) was sequentially extracted with
organic solvents in order of increasing polarity including; hexane,
dichloromethane, ethyl acetate and methanol, using a soxhlet
apparatus for 24 h in each case. The yields obtained for each
solvent were, hexane 66.68 g (leaves), 9.10 g (roots), 16.73 g
(stems); dichloromethane 16.77 g (leaves), 5.13 g (roots), 9.25 g
(stems); ethyl acetate 8.28 g (leaves), 0.93 g (roots), 5.02 g (stems)
and methanol, 27.01 g (leaves), 20.28 g (roots), 15.09 g (stems).
Isolation and purification of Compounds 1, 3, 4, 5, 8 and 9
The hexane extract from the leaves (30 g) was separated by
column chromatography using a step gradient of hexane:
dichloromethane: ethyl acetate gradient, starting with 100% hexane
stepped to 10, 20, 30, 50, 80 and 100% dichloromethane, followed
Kiplimo et al. 1151
by 20 and 30% ethyl acetate in dichloromethane. Twenty fractions
of 100 ml each were collected in each step. Fractions 5 to 12 were
combined and purified using 100% hexane, to produce
farnesylamine (9) (12 mg). Fractions 21 to 25 were recrystallised in
methanol to yield sitosterol (8) (78 mg). Fractions 41 to 67 were
combined and separated with 20 and 30% dichloromethane in
hexane. Lupenyl acetate (1) (52 mg) was obtained in fractions 8 to
12 while fraction 18 to 35 was further purified using 20% ethyl
acetate in hexane where fractions 5 to 9 afforded -amyrin acetate
(4) (150 mg) and fractions 11 to 18 afforded a mixture of -amyrin
(5) and -amyrin (3) (89 mg).
Isolation and purification of Compound 2
The ethyl acetate extract (0.93 g) from the roots was dissolved in
dichloromethane and separated with a mobile phase consisting of a
hexane: ethyl acetate step gradient 1:0 (fractions 1 to 10), 9:1
(fractions 11 to 20), 7:3 (fractions 21 to 38), 6:4 (fractions 52 to 64)
and 3:7 (fractions 65 to 70). Fractions 22 to 27 were further purified
with 20% ethyl acetate in hexane. Oleanolic acid (2) (25 mg) was
obtained in fractions 9 to 13.
Isolation and purification of Compounds 6 and 7
The hexane extract from the stems (16.73 g) was subjected to
column chromatography. The mobile phase consisted of a hexane:
dichloromethane step gradient; 1:0 (fractions 1 to 45), 9:1 (fractions
46 to 66), 8:2 (fractions 67 to 80), 7:3 (fractions 81 to 98) and 1:1
(fractions 99 to 121). Friedelin acetate (7) (31 mg) was eluted in
fraction 24 to 32 and the pure compound was obtained by
recrystallisation in methanol. Friedelanone (6) (120 mg) was
obtained by purification of fractions 55 to 80 using 10%
dichloromethane in hexane as the mobile phase where the
compound was eluted in fractions 7 to 15, followed by
recrystallisation in methanol.
Farnesylamine (9)
White crystals, m/z (rel%): 221 [M] + , 206 (1), 189 (3), 179 (3), 161
(3); IR spectra (Vmax cm -1 ): 3412, 2975, 1623, 968; 1 H NMR spectral
data (400 MHz, CDCl3) H 5.11(H-2, 6, 10), 2.07 (H-1), 2.05 (H-8),
2.03 (H-9), 1.69 (H-12), 1.62 (H-13, 14, 15), 1.28 (H-4); 13 C NMR
spectral data (400 MHz) 134.82 (C-3), 134.16 (C-7), 130.97 (C-11),
124.12 (C-6), 124.02 (C-10), 123.98 (C-2), 39.45 (C-1), 29.42 (C-4,
8), 27.99 (C-5), 26.48 (C-9), 25.41 (C-12), 15.71 (C-15), 17.39 (C-
13), 15.75 (C-14).
BIOLOGICAL STUDIES
Minimum inhibitory concentration (MIC)
Four strains of gram-negative bacteria (Escherichia coli ATCC
25922, Pseudomonas aeruginosa ATCC 35032, Klebsiella
pneumoniae ATCC 700603 and Stenotrophomonas maltophilia
ATCC 13637) and five Gram-positive bacteria (Staphylococcus
aureus ATCC 25923, Bacillus subtilis ATCC 6051, Enterococcus
faecium ATCC 19434, Staphylococcus epidermidis ATCC 14990
and Staphylococcus saprophyticus ATCC 35552) were selected for
the determination of antimicrobial activity.
The antibacterial activities of the compounds were determined
using the broth microdilution method as described by Andrews
(2001). Bacterial strains were cultured for 18 h at 37°C in Tryptone
Soy Broth (TSB) and standardized to a final cell density of 1.5×10 8
cfu/mL equivalent to 0.5 McFarland Standard. The 96-well plates

1152 Afr. J. Pharm. Pharmacol.
Table 1. Minimum inhibitory concentrations (MIC in mg/ml) of compounds isolated from V. auriculifera.
Microoganism MIC (mgml -1 ) of the test organisms
1 2 3 and 5 4 6 7
S. aureus 1.0 0.5 0.25 1.0 1.0 1.0
B. subtilis 1.0 0.5 0.25 1.0 0.25 1.0
E. faecium 0.5 1.0 0.25 0.5 1.0 1.0
S. epidermidis 1.0 0.25 0.5 1.0 0.5 0.5
S. saprophyticus 0.25 1.0 0.25 1.0 1.0 1.0
E. coli 0.12 1.0 0.12 0.5 1.0 0.5
K. neumoniae 1.0 0.5 0.5 1.0 1.0 1.0
P. aeruginosa 1.0 1.0 1.0 1.0 1.0 1.0
St. maltophilia 0.25 0.25 0.5 0.5 0.5 0.5
were prepared by dispensing into each well, 90 µl Muller-Hinton
(MH) broth and 10 µl of the bacterial inoculum. Test compounds
were dissolved in dimethyl sulfoxide (DMSO) to a concentration of
10 mg/ml while tetracycline (a broad-spectrum antimicrobial agent)
the positive control was dissolved in ethanol. Serial two-fold
dilutions were made in a concentration range of 0.002 to 2 mg/ml.
Wells containing MH broth only were used as a medium control and
wells containing medium and cultures without the test compound
were used as the growth control. Plates were covered to avoid
contamination and evaporation and incubated for 24 h at 37°C. The
MIC was described as the lowest concentration of the test
compounds completely inhibiting the growth of microorganisms.
The tests were done in triplicate on two separate occasions and the
results are as shown in Table 1.
Anti-biofilm activity evaluation
To determine the anti-biofilm activity of -amyrin acetate and
oleanolic acid, three strains of Gram-negative bacteria (E. coli
ATCC 25922, P. aeruginosa ATCC 35032 and K. pneumoniae
ATCC 700603) and four Gram-positive bacteria (S. aureus ATCC
25923, S. aureus ATCC 43300, E. faecium ATCC 19434, and S.
saprophyticus ATCC 35552) were used. Bacterial isolates were
cultured overnight in TSB to determine the effect of MIC, sub-MIC
(0.5×MIC) and supra-MIC (2×MIC) exposures on biofilm formation.
Cells were washed and resuspended in distilled water to a turbidity
equivalent to a 0.5 McFarland standard. Wells of sterile, 96-well Ubottomed
microtiter plates were each filled with 90 µl Luria Bertani
broth (LB) and 10 µl of cell suspension, in triplicate. Based on
individual MICs for each isolate the effect of MIC, sub-MIC and
supra-MIC of -amyrin acetate and oleanolic acid on bacterial
adhesion was investigated. Plates were incubated aerobically at
37˚C for 24 h with shaking on an Orbit P4 microtitre plate shaker
(Labnet).
The contents of each well were aspirated and then washed three
times with 250 µl of sterile distilled water. To remove all the nonadherent
bacteria, the plates were vigorously shaken and the
remaining attached cells were fixed with 200 µl of 99% methanol
per well. After 15 min, plates were left to dry and then stained for 5
min with 150 µl of 2% Hucker crystal violet. Excess stain was
washed with running tap water and plates were left to air dry
(Basson et al., 2008). The bound stain was resolubilised with 150
µl of 33% (v/v) glacial acetic acid per well. The optical density (OD)
of the contents of each well was obtained at 595 nm using the
Fluoroskan Ascent F1 (Thermolabsystems).
Tests were done in triplicate on two separate occasions and the
results were averaged (Stepanovi et al., 2000). The negative
control for both assays was un-inoculated LB, while the positive
control was tetracycline, with respective cell suspensions without -
amyrin acetate or oleanolic acid. OD595nm values of treated cells
were compared with untreated cells to investigate the
increase/decrease of biofilm formation as a result of antimicrobial
agent exposure. Treated and untreated samples were compared
statistically using paired t-tests and Wilcoxon signed rank tests if
normality failed (SigmaStat V3.5, Systat Software).
RESULTS AND DISCUSSION
The phytochemical investigation of V. auriculifera led to
the isolation of eight triterpenoids 1 to 8 and a
sesquiterpene amine (9). Extracts from the leaves were
found to contain the sesquiterpene amine along with one
lupane-type triterpenoid (lupenyl acetate 1), one ursanetype
triterpenoid ( -amyrin 5), two oleanane-type
triterpenoids ( -amyrin 3 and -amyrin acetate 4) and a
common steroid (sitosterol 8) (Figure 1). The stem bark
afforded friedelanone (6) and friedelin acetate (7)
belonging to the friedelane class. From the roots,
oleanolic acid (2), the parent oleanane type triterpene,
was isolated. Compounds 1 to 8 were identified using 2D
NMR spectral data and by comparison with literature
values, which supported the structures as lupenyl acetate
(Jamal et al., 2008), oleanolic acid (Seebacher et al.,
2003), -amyrin, -amyrin acetate, friedelin acetate and
-amyrin (Mahato and Kundu, 1994), friedelanone (Igoli
and Gray, 2008) and sitosterol (Kamboj and Saluja,
2011). Although farnesylamine (9) was previously
reported (Jones et al., 2003), here we are reporting the
complete data for the first time.
Compound 9 was isolated as a colourless oily liquid;
its molecular formula was assigned as C15H27N. The IR
spectrum showed the presence of primary amine (3412
cm -1 ) and (1623 cm -1 ). The 13 C NMR spectrum showed
the presence of six olefinic carbon resonances, 3protonated
carbons at C 124.2 and 3-non-protonated
carbons at C 131-134. The olefinic methine resonances
could also be seen at H 5.11 in the 1 H NMR spectrum.
The methylene carbon resonances were observed
between C 26.48 and C 29.42 except for the methylene
bonded to the amine group which was observed

HO
H COCO
5
3
32 31 4
23 24
1 lupenyl acetate
HO
2
3
5 α-amyrin
1
8 sitosterol
25 11
10
9
6
30
12
26
29
8
7
13
14
27
19
18
29
20
15
17
16
21
R
22
28
R 1
2 R 1 = OH; R 2 = COOH (oleanolic acid)
3 R 1 = OH; R 2 = CH 3 (β−amyrin)
4 R 1 = OAc; R 2 = CH 3 (β−amyrin acetate)
NH 2
6 f riedelanone
7 f riedelin acetate
Figure 1. Structures of compounds (1-9) isolated from Vernonia auriculifera.
downfield at C 39.47. All the methylene proton
resonances, including 2H-1 were present at H 2.05
except for one methylene resonance which appeared
upfield at H 1.27. Three of the four methyl proton
resonances overlap at H 1.62 (3H-13, 14, 15) and one is
in a different chemical environment at H 1.69 (3H-12).
This same overlapping of the methyl carbon resonances
can be seen in the 13 C NMR spectrum with methyl carbon
resonances at C 25.41, confirmed by the DEPT
spectrum. This compound has been detected in an
30
27
26
30
9 Farnesylamine
20
R = =O
R = OCOCH 3
Kiplimo et al. 1153
extract of the ant Monomorium fieldi Forel from Australia
(Jones et al., 2003) and has only now been found in a
plant species.
The triterpenic family of compounds to which all the
isolated compounds belong are reported to possess
antibacterial activity (Collins and Charles, 1987). The
sesquiterpene, farnesylamine, could not be screened for
antibacterial activity due to sample decomposition. MIC
values recorded for all tested compounds (Table 1)
suggested moderate antibacterial activity. The most
29
R2

1154 Afr. J. Pharm. Pharmacol.
A dherence (O D 595 n m)
3.0
2.5
2.0
1.5
1.0
0.5
0.0
E . coli A TC C 25922
K . pneum oniae A TC C 700603
P . aeruginosa A TC C 35032
Figure 2. Antibiofilm results for -amyrin acetate (4).
active compound was amyrin (mixture of -and -), with
MICs of 0.12 mg/ml against E. coli, 0.25 mg/ml against S.
aureus, B. subtillis, E. feacalis, S. saprophyticus, and
0.5mg/mL against S. epidermis, K. pneumonia, and S.
maltophilia. The other compounds 3-7 had MIC of 0.5
mg/mL against S. maltophilia. The least active
compounds were 6 and 7 with MIC of 1.0 mg/mL against
six microorganisms. All tested compounds had MICs of
1.0 mg/mL for P. aeruginosae and 0.5 mg/mL for S.
maltophilia. The oleanane triterpernoids (2-4) displayed
better antibacterial activity than the friedelane
triterpenoids (6-7). It is reported that the 28-COOH and
ester functionality at C-3 contributes to pharmacological
activities of pentacyclic triterpenes (Mallavadhi et al.,
2004) like lupenyl which has greater antimutagenic
activity than lupenyl acetate (Guevara et al., 1996).
These effects are observed for friedelanone and friedelin
acetate where the ketone has higher activity against B.
subtilis than the ester.
E . faecium A TC C 19434
Isolates
S . aureus A TC C 25923
S . aureus A TC C 43300
UNTREATED
SUB-MIC
MIC
SUPRA-MIC
Biofilm is a microbially derived sessile community
characterized by cells that are irreversibly attached to a
substratum or interface or to each other and are
embedded in a matrix of extracellular polymeric
substances they have produced. When planktonic
bacteria adhere to surfaces, they initiate biofilm
formation. The nature of biofilm structure and
physiological attributes of biofilm organisms confer an
inherent resistance to antimicrobial agents such as
antibiotics, disinfectants or germicides (Donlan and
Costerton, 2002).
-amyrin acetate and oleanolic acid were tested for
antibiofilm activity against seven strains of bacteria. -
amyrin acetate decreased adhesion of S. aureus (ATCC
43300), K. pneumonia and E. faecium significantly at
sub-MIC concentrations (Figure 2). For K. pneumonia,
this decreased adhesion was also seen at MIC
concentrations and in S. saphrophyticus a marked
decrease in adhesion was seen at MIC and supra MIC
S . saprophyticus A TC C 35552

A d h e re n c e (O D 5 9 5 n m )
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
E . c o li A T C C 2 5 9 2 2
K . p n e u m o n ia e A T C C 7 0 0 6 0 3
P . a e ru g in o s a A T C C 3 5 0 3 2
E . fa e c iu m A T C C 1 9 4 3 4
Isolates
Figure 3. Antibiofilm results for oleanolic acid (2).
concentrations. Sub-MIC oleanolic acid exposure also
decreased adhesion of K. pneumonia and P. aeruginosa
significantly (Figure 3), but MIC and supra-MIC
exposures of oleanolic acid increased adhesion of all
tested bacterial strains. These results suggest that
oleanolic acid and -amyrin acetate that are relatively
abundant, can be used at low concentrations to decrease
adhesion of certain bacterial strains to abiotic surfaces.
Since bacterial resistance to antibiotics and their survival
are associated with their ability to form biofilms (Donlan
and Costerton, 2002), compounds which decrease biofilm
formation would be useful in being used in conjunction
with other antibiotics to decrease bacterial resistance.
Agents that decrease adhesion of bacteria may also be
useful in improving the efficacy of antibiotics and hygiene
in hospitals that have devices such as incubation tubes,
catheters, artificial heart valves, water lines and cleaning
instruments on which bacterial biofilm have been found
(Donlan and Costerton, 2002).
S . a u re u s A T C C 2 5 9 2 3
Conclusion
UNTREATED
SUB-MIC
MIC
SUPRA-MIC
S . a u re u s A T C C 4 3 3 0 0
S . s a p ro p h y tic u s A T C C 3 5 5 5 2
Kiplimo et al. 1155
This is the first report of a phytochemical investigation of
V. auriculifera. The finding of a sesquiterpene amine in V.
auriculifera is unique as it has not been isolated from a
plant species before. Although the genus Vernonia is
known to be a rich source of sesquiterpene lactones,
none were isolated from V. auriculifera. However, eight
pentacyclic compounds with moderate antibacterial
activity were isolated. Oleanolic acid and -amyrin
acetate exhibited moderate anti-adhesion properties.
These compounds show potential for synergistic coupling
with antimicrobial agents to improve therapeutic
efficiency, in the face of rising bacterial resistance,
however this needs further investigation.
ACKNOWLEDGEMENTS
The authors are thankful to Organization for Women in

1156 Afr. J. Pharm. Pharmacol.
Science for the Developing World (OWSDW) for the
financial support and Chester Everia for organizing the
collection of the plant material.
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African Journal of Pharmacy and Pharmacology Vol. 5(8), pp. 1157-1161, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.205
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Nutritional and elemental analyses of some selected
fodder species used in traditional medicine
Ali Bahadur 1 , Zubeda Chaudhry 1 , Gul Jan 1 , Mohammad Danish 1 , Atta ur Rehman 1 , Rafiq
Ahmad 1 , Aman khan 1 , Shah Khalid 3 , Irfan ullah 4 , Zahir Shah 5 , Farman Ali 2 , Tahira Mushtaq 1
and Farzana Gul Jan 6
1 Department of Botany, Hazara University, Mansehra, Pakistan.
2 Department of Chemistry, Hazara University, Mansehra, Pakistan.
3 Department of Plant Sciences, Quaid-e-Azam University, Islamabad, Pakistan.
4 Government Post Graduate College for Boys, Abbottabad, Pakistan.
Accepted 13 May, 2011
The present study was carried out to evaluate the nutritional value and elemental analysis of some
fodder plant species. Proximate composition of proteins, crude fibers, fats and oils, moisture, ash
contents and carbohydrates and elemental composition of aerial parts have been determined by using
Atomic Absorption Spectrophotometer (AAS). A total of 16 elements; Na, Mg, Al, Si, P, S, Rb, K, Ca, Fe,
Mn, Ti, Ni, Cu, Zn and Cl have been measured. Their concentrations were found to vary in different
samples.
Key words: Nutritional and elemental analyses, fodder species, traditional uses.
INTRODUCTION
The World Health Organization (WHO) recognized
traditional medicine or herbal medicine about 20 years
ago and started exploring the possibilities to improve or
popularize the herbal medicine already used by the
people in developing countries of the world for thousands
of years (Akerle and Heywood 1991). Herbs not only
provide us chemicals of medicinal value but also provide
us nutritional and trace elements (Zafar et al., 2010).
Minerals and trace elements are chemical elements
required by our bodies for numerous biological and
physiological processes that are necessary for the
maintenance of health. Those minerals that are required
in our diets in amounts greater than 100 mg per day are
called "minerals" and those that are required in amounts
less than 100 mg per day are "trace elements." Minerals
include compounds of the elements such as calcium,
magnesium, phosphorus, sodium, potassium, sulfur and
chlorine. Trace elements that are necessary for human
health include iron, iodine, copper, manganese, zinc,
molybdenum, selenium and chromium etc (Hendler and
Sheldon, 1990).
*Corresponding author. E-mail: drguljan@yahoo.com.
All human beings require a number of complex
organic/inorganic compounds in diet to meet the need for
their activities. The important constituents of diet are
carbohydrates, fats, proteins, vitamins, minerals and
water (Indrayan et al., 2005). Every constituent plays an
important role and deficiency of any one constituent may
lead to abnormal developments in the body (Zafar et al.,
2010). Plants are the rich source of all the elements
essential for human beings. There is a relationship
between the element content of the plant and its nutriational
status. Some elements are essential for growth, for
structure formation, reproduction or as components of
biologically active molecules while others have some
other beneficial effects (Newall et al., 1996). Qualitative
or quantitative determination of mineral elements present
in plants is important because the concentration and type
of minerals present must often be stipulated on the label
of a food. The quality of many foods depends on the
concentration and type of minerals. What they contains
also play a very significant role against a variety of
degenerative diseases and processes, they may also
prevent and reduce injury from environmental pollutants
and enhance the ability to work and learn, some minerals
are essential to a healthy diet (for example calcium,
phosphorus, potassium and sodium) where as some can

1158 Afr. J. Pharm. Pharmacol.
be toxic (for example Lead, Mercury, cadmium and
aluminum). In the present study the nutritional value and
trace elements content in Amaranthus viridis, Chenopodium
album, Medicago denticulata, Setaria viridis, Sonchus
arvrnsis are investigated.
MATERIALS AND METHODS
Sources of plant materials
Five medicinal plants including; A. viridis, C. album, M. denticulata,
S. viridis, S. arvrnsis were analyzed in the form of aerial parts.
Plants were collected from the fields and identified with the help of
Flora of Pakistan (Ali and Qaiser, 2007). These plants were shadedried
at a temperature of 28 to 30°C for 14 days and powdered
mechanically with a China herb grinder. The powder was kept in
dry, clean, air tight glass jars and stored at 4°C until used.
Nutritional analysis
Ash contents, moisture contents, and crude protein were
determined by Macrokjeldahl method while fats or ether extracts,
crude fibers and carbohydrates were determined by standard
methods following AOAC (Anonymous, 2000).
Elemental analysis
Samples in powder form were used for atomic absorption
Spectrophotometer (AAS). Each plant material (0.25 g) was taken
in 50 ml flask and add 6.5 ml of mixed acid solution that is, Nitric
acid (HNO3), Sulfuric acid (H2SO4) and Perchloric acid (HClO4)
(5:1:0.5) The samples were boiled in acid solution in fume hood on
hot plate (model VWR VELP scientifical, Germany) . Thereafter, few
drops of distilled water has beethe completion of digestion in
completing indicated white fumes coming out from the flask added
and allowed to cool. Then these digested samples were transferred
in 50 ml volumetric flasks and the volume was made up raised to 50
ml by adding distilled water in them. Then the extract was filtered
with filter paper (Whatmann No. 42) and filtrate were collected in
labeled plastic bottles. The solutions were analyzed for the
elements of interest utilizing atomic absorption spectrometer
(Shimadzu AA-670) with suitable hollow cathode lamps. The
percentages of different elements in these samples were
determined by the corresponding standard calibration curves
obtained by using standard AR grade solutions of the elements, for
example K + , Mg 2+ , Ca 2+, Na +, Fe 2+, Co 3+, Mn 2+, Cu 3+, C 3r+ , Zn 2+, Ni 3+ ,
Li 1+ , Pb 4+ and. Cd 2+ ..
RESULTS AND DISCUSSION
Health treatment is based on medicinal plants are being
prescribed by doctors in the form of plant extracts,
infusion or by direct ingestion of very fine powder of plant.
Likewise, these are recommended as a nutritional
supplement for the treatment of everyday problems such
as stress and insomnia. There is a resurgence of interest
in herbal medicine for the treatment of various ailments,
chiefly because of the prohibitive cost of allopathic drugs,
chiefly because of the prohibitive cost of allopathic drugs,
their unavailability in remote areas and the popular belief
that naturally occurring products are without any adverse
side-effects (Hungard et al., 1988). Similarly Ahmad (2007)
highlighted the importance of wild medicinal plants along
road side verges (M-2) Pakistan.
From a medical point of view, the important
constituents of plants are pharmacologically active
compounds such as flavonoids, alkaloids, glycosides and
similar other organic substances. In addition, medicinal
plants contain essential and trace elements, which can
be available to the human body on consumption of herbs
and their extracts. Indeed today many, if not most,
pharmacological classes of drugs include a natural
product prototype. The search for pharmacologically
active chemicals from plant sources has continued and
many compounds have been isolated and introduced into
clinical medicine. Modern medicine is now beginning to
accept the use of standardized plant extracts. Present
study was also conducted to enhance the same
knowledge further and is focused to investigate chemical
composition including estimation of nutritional value,
trace elements / heavy metals of A. viridus, C. album, M.
denticulata, S. viridus and S. arvrnsis.
Local uses
Plants analyzed in the present work with their botanical
name, local name, part of the plant used and their
medicinal uses are shown in Table 1.
Nutritional analysis
Table 2 shows the chemical composition of aerial parts of
A. viridis, C. album, M. denticulata, S. viridis, and S.
arvrnsis.
The ash content was highest in S. arvrnsis and lowest
in M. denticulata. The crude protein content was highest
in C. album and lowest in S. viridis and S. arvrnsis. Crude
fiber content was found highest in M. denticulate and
lowest in Chenopodium album. Kononov et al. (2005)
reported that the highest dry weight yield (8.8 t/ha) was
achieved using Medicago falcata; High moisture was
found in Sonchus arvrnsis and low in Chenopodium
album. Carbohydrate was highest in Medicago
denticulata and lowest in Chenopodium album. Adetuyi
and Akpambang (2006) reported moisture, ash, crude fat,
crude fiber, protein and carbohydrate in Sorghum bicolor.
Naseem et al. (2006) reported carbohydrates 7.16%,
crude fiber 27.2%, moisture 63.10%, ash 5.67% and
crude fat 6.36%. Alfawaz (2006) reported protein value
17.1 to 0.1 g/100 g, moisture 87.8 to 93.5 g/100 g, ash
14.6 to19.6 g/100 g and lipids 3.1 to 3.8 g/100 g in
Rumex vesicarius. Khodzhaeva et al. (2002) reported the
content and composition of lipids, proteins, flavonoids
and carbohydrates in the aerial part of the Rumex
confertus.
Elemental analysis
The data obtained are cited in Tables 3 and 4. The results

Table 1. Local uses of fodder species.
S/No. Plant name Local name Family Part used Medicinal uses
1 Amaranthus viridis L. Gunhar Amaranthaceae Leaves
2 Chenopodium album L. Bathwa Chenopodiaceae Whole plant
Bahadur et al. 1159
Leaves are used as emollient; also used in
scorpion and snake bite. Cocked as
vegetable.
Laxative, anthelmintic; used in hepatic
disorder and enlarged spleen. The roots are
used in jaundice, urinary, problems and
rheumatism. Fruit and roots are known as
antidote to snake poison. Also used as pot
herb.
3 Medicago denticulata Willd Spashtary Papillionaceae Shoots Plant is used as fodder and as pot herb.
4 Setaria viridis (L.) P. Beauv. Wakha Poaceae Vegetative portion Fodder for cattle.
5 Sonchus asper L. Shawda pai Asteraceae Vegetative portion
Table 2. Nutritional analysis of the arial parts of fodder species.
The plant is diuretic, sedative, cooling,
hypnotic, diaphoretic, antiseptic and
expectorant; used in cough, bronchitis,
asthma, curing constipation. Young shoots
are eaten as salad and the leaves as
vegetables. The plant is relished by horses
and cattle.
S/No. Plant name Part used Ash (%) Crude protein (%) Crude fiber (%) Moisture (%) Carbohydrate (%)
1 Amaranthus viridis L. Arial parts 18.42 31.19 15.21 5.54 40.86
2 Chenopodium album L. Arial parts 19.23 34.31 14.82 4.53 36.55
3 Medicago denticulata Willd Arial parts 11.77 27.06 25.62 5.71 50.61
4 Setaria viridis (L.) P. Beauv. Arial parts 20.85 20.53 16.15 5.0 49.98
5 Sonchus arvrnsis L. Arial parts 21.78 20.53 15.11 6.72 44.87
showed that Amaranthes viridus, Chenopodium
album, Medicago denticulata, Setaria viridus and
Sonchus arvrnsis exhibits the highest concentration
of the elements Mg, Si, S, Ca, Cl, K
and Fe (Table 3). The highest more concentration
of Mg was found in Medicago denticulate (4.08
%), Si was in Sonchus arvrnsis (4.80%), S and K
was in Medicago denticulate (3.82%, 4.65%), Cl
was in found in Sonchus arvrnsis (3.00%) and Fe
was found in high concentration in Medicago
denticulate (4.92%). Ni and Cu were found only in
Amaranthes viridus and Medicago denticulate in

1160 Afr. J. Pharm. Pharmacol.
Table 3. Elemental analysis of the arial parts of fodder species (in high concentration).
S/No. Plant name Part used
Na
(%)
Mg
(%)
1 Amaranthus viridis L. Arial parts 0.28 3.34 0.89 2.98 1.7 2.24 1.46 4.35 3.52 1.52
2 Chenopodium album L. Arial parts 0.30 2.54 0.48 1.44 1.55 1.44 1.00 3.65 3.85 0.93
3 Medicago denticulata Willd Arial parts 1.48 4.08 1.26 3.77 1.85 3.82 3.68 4.62 3.93 4.92
4 Setaria viridis (L.) P. Beauv. Arial parts 0.93 2.28 0.55 1.65 1.39 0.91 2.48 4.11 3.16 1.16
5 Sonchus arvrnsis L. Arial parts 0.45 1.63 1.33 4.80 0.83 3.6 3.00 3.54 4.00 2.21
Al
(%)
Table 4. Elemental analysis of the arial parts of fodder species (in low concentration).
S/No. Plant name Part used Mn (%) Ti (%) Ni (%) Cu (%) Zn (%) Rb (%)
1 Amaranthus viridis L. Arial parts 0.08 0.12 0.04 0.05 0.24 0.03
2 Chenopodium album L. Arial parts 0.14 0.08 - - 0.26 0.06
3 Medicago denticulata Willd Arial parts 0.22 0.46 0.14 0.25 0.69 0.07
4 Setaria viridis (L.) P. Beauv. Arial parts 0.06 0.08 - - 0.20 0.01
5 Sonchus arvrnsis L. Arial parts 0.12 0.23 - - 0.16 0.02
low concentration (Table 4). Mn, Ti, Cu, Zn and Rb were
found in low concentration (Table 4). Kaneez et al. (2001)
stated that Mg in the plant lowers the cholesterol level but
alleviates heart diseases. Mg is being investigated in
migraine headache and attention deficit hypersensitivity
disorder. Mg plays an important role in regulating the
muscular activity of heart, maintains normal heart rhythm
and also converts blood sugar in to energy. Iron (Fe)
deficiency is associated with myocardial infection.
Calcium is needed in the development of bones and
teeth, regulates heart rhythm, helps in normal blood
clothing maintain proper nerve and muscle functions and
lowers the blood pressure. Mn is essential for normal
functioning of central nervous system and is a good antioxidant
(Bibi et al., 2006). The presence and concentrations
of various elements in different plant depend
on the composition of the soil, water and fertilizers used
as well as permissibility, selectivity and absorbability of
plants for the uptake of these elements. Hence, the
observed variations in concentration of the elements are
attributed to the nature of the plant as well as its
surroundings (Udayakumar and Begum, 2004). Trace
elements are essential for all forms of life and having
wide range of clinical applications that play a key role in
the treatment of various diseases (Kaneez et al., 2001).
The elements Fe, K, Mg, Na, Ca, Co, Mn, Zn and Cu
have been classified as essential elements, Ni, Cr are
possibly essential while Cd, Pb and Li are non essential
elements for the human body. Among the various
elements detected in different medicinal plants used in
the treatment of different diseases. It is interesting to note
that some of the medicinal plants used by local physician
and common people have high concentration in the range
of ppm of Mn, Fe, Cu, Zn etc. The concentration of K and
Si
(%)
P
(%)
S
(%)
Cl
(%)
K
(%)
Ca
(%)
Fe
(%)
Ca are in the percentage level. Zn is important in wound
healing and also functions as an antioxidant. The
researchers are trying to link the contents of the trace
elements and medicinal values of the plants (Zafar et al.,
2010). This is for the first time that such an exhaustive
work on elemental content has been carried out on the
medicinal plants. The data obtained in the present work
will be useful in synthesis of new herbal drugs with
various combinations of plants, which can be used in the
treatment of different diseases at global level generally
and in Pakistan particularly.
REFERENCES
Adetuyi AO, Akambang VOE (2006). The nutritional value of Sorghum
bicolor stem flour used for infusion drinks in Nigeria. Pak. J. Sci. Ind.
Res., 49(4): 276-280.
Ahmad SS (2007). Medicinal wild plants from Lahore-Islamabad
Motorway (M-2). Pak. J. Bot., 39(2): 355-375.
Akerle OV, Heywood HS (1991). The conservation of medicinal plants.
Cambridge University Press, Cambridge.
Alfawaz MA (2006). Chemical composition of hummayd (Rumex
vesicarius) grown in Saudi Arabia. J. Food Compos. Anal., 19(6-7):
552-555.
Ali SI, Qaisar M (2007). A phyto geographical analysis of the
Phanerogames of Pakistan and Kashmir. Proc. Royal Soc.
Edinburgh, 89(3): 89-101.
Anonymous (2000). Association of Official Analytical Chemists,
Gaithersburg, MD, USA. 17th edition.
Bibi S, Dastagir G, Hussain F, Sanaullah P (2006). Elemental
composition of Viola odorata Linn. Pak. J. Pl. Sci., 12(2): 141-143.
Hendler E, Sheldon S (1990). The Doctors' Vitamin and Mineral
Encyclopedia. New York, NY: Simon & Schuster, pp. 112-207.
Hungard BL, Goldstein DB, Villegas F, Cooper T (1988). The
ganglioside GM 1 reduces ethanol induced phospholipase activity in
synaptosomal preparation from mice. Neurochem Int., 25: 321-325.
Kaneez FA, Qadirrudin M, Kalhoro MA, Khaula S, Badar Y (2001).
Determination of major trace elements in Artemisia elegantissima and
Rhazya stricta and their uses. Pak. J. Sci. Ind. Res., 45: 291-293.

Khodzhaeva MA, Turakhozhaev MT, Saifulaev KHI, Shakhidoyatov
KHM (2002). Chemical composition of the aerial part of Rumex K-I.
Chem. Natural Compound, 38(6): 524-526.
Kononov VM, Dikanev GP, Rassadnikov VN (2005). New high protein
fodder crops in the lower Volga region. Kormoproizvodstvo., 5: 22-23.
Naseem R, Mahmud K, Arshad M (2006). Chemical composition and
antibacterial activity of Crotalaria burhia, from Cholistan Desert,
Pakistan. Hamdard Medicus, 49(4): 49-52.
Newall CA, Anderson LA, Phillipson JD (1996). Herbal medicines: A
Guide for health care professionals. London, the Pharmaceuticals
Press.
Bahadur et al. 1161
Udayakumar R, Begum VH (2004). Elemental analysis of medicinal
plants used in controlling infectious diseases, etc., Hamdard
Medicus, 47: 35-36.
Zafar M, Khan MA, Ahmad M, Jan G, Sultana S, Ullah K, Marwat SK,
Ahmad F, Jabeen A, Nazir A, Abbasi AM, ZR, Ullah Z (2010).
Elemental analysis of some medicinal plants used in traditional
medicine by atomic absorption spectrophotometer (AAS). J. Med.
Plants Res., 4(19): 1987-1990.

African Journal of Pharmacy and Pharmacology Vol. 5(8), pp. 1162-1169, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.234
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Acute toxicity and anti inflammatory effects of
supercritical extracts of Ilex paraguariensis
Tiago R. Pasquali 1 , Sandra M. D. Macedo 1 , Silvane S. Roman 1 , Valéria Dal Prá 1 , Rogério L.
Cansian 2 , Altemir J. Mossi 3 , Vladimir J. Oliveira 2 and Marcio A. Mazutti 4 *
1 Department of Clinical Pharmacy, URI - Campus de Erechim, Brazil.
2 Department of Food Engineering, URI - Campus de Erechim, Av. Sete de Setembro, 1621, Erechim, 99700-000, RS,
Brazil.
3 Department of Agronomy, Universidade Federal da Fronteira Sul, Av. Dom João Hoffmann, Erechim - RS, 99700-000 –
Brazil.
4 Department of Chemical Engineering - Federal University of Santa Maria, Av. Roraima, 1000, Santa Maria, 97105-900,
Brazil.
Accepted 4 June, 2011
This work reports the in vivo evaluation of acute toxicity and anti-inflammatory activity of supercritical
carbon dioxide (CO2) extracts of Ilex paraguariensis. For the acute toxicity study, the extracts diluted in
sunflower oil were administered to adult male wistar rats in single doses of 250 mg/kg by the
intraperitoneal route. Adverse effects and mortality were monitored along 14 days. In the case of antiinflammatory
activity, the animals were submitted to acute induced-peritonitis assays by injection of
oyster glycogen and a single dose of 500 mg/kg of supercritical I. paraguariensis extract diluted in
sunflower oil administered by subcutaneous injection. Results obtained in this work suggest the
incidence of acute toxicity due to the presence of cells with hyperchromatism, vacuolization and
tumefaction in the livers and the presence of cells with tumefaction in the cortical region of the kidneys
of the treated group. It was also experimentally observed that treatment inhibited the neutrophil
recruitment in the circulating blood, hence demonstrating the anti-inflammatory effect of the I.
paraguariensis extracts. The results obtained may be very promising since they open new perspectives
for the therapeutic use of supercritical extracts of I. paraguariensis or its preparations as antiinflammatory
agents.
Key words: Acute toxicity, Anti-inflammatory, Supercritical fluid extraction, Ilex paraguariensis.
INTRODUCTION
Mate (Ilex paraguariensis St. Hill., Aquifoliaceae) a worldfamous
tea consumed in Brazil, Paraguay, Uruguay and
Argentina, was used by the Indians of South America
before the European colonization. The mate tea leaves
has remarkable importance in the economy and in the
*Corresponding author. E-mail: marciomazutti@gmail.com Tel:
+55-55-3220-9591.
Abbreviations: CO2, Carbon dioxide; PBS, phosphatebuffered
saline; NH4Cl, ammonium chloride; ANOVA,
analysis of variance; G, glomerullus; TCD, distal
convoluted tubules; TCP, proximal convoluted tubules;
CV, centrolobular vein; C, control; T, treated.
cultural life of South America, with an average of 300,000
t produced each year (Alikaridis, 1987; Tormen, 1995). In
the southern region of Brazil, for example, one can find
more than 40 mates processing industries and about
180,000 medium and small rural properties dedicated
almost exclusively to cultivation of this raw material
(Mosele, 2002).
The mate raw material, as leaves and green stalks, is
processed as dye herb for the classical “Chimarrão”,
“Mate” or “Tereré”, as fine or soluble powder for teas, or
as essences used for different industrial purposes.
“Chimarrão” or “mate” is prepared by steeping dry leaves
and twigs of mate in hot water, while “tereré” is prepared
with cold water (Cansian et al., 2008a).
Mate is considered as a stimulant drink that eliminates
lassitude through increasing physical and mental activities.

Biological and therapeutical activities on the
cardiovascular, respiratory, muscular, gastrointestinal,
renal and neurological systems have been attributed to
the presence of xanthines, caffeine, theobromine, tannic
substances, flavonoids, vitamins, and other substances
present in mate extracts (Heck and Mejia, 2007; Cansian
et al., 2008b). In folk medicine, mate infusion has been
used for the treatment of arthritis, rheumatism and other
inflammatory diseases, constipation, hemorrhoids,
headache, obesity, fatigue, fluid retention, hypertension,
slow digestion and, hepatic and digestive disorders
(Cansian et al., 2008b; Silva et al., 2008; Gorzalczany et
al., 2001). Recent findings have shown that the aqueous
extracts of I. paraguariensis can inhibit the progression of
atherosclerosis in cholesterol-fed rabbits (Mosiman et al.,
2006).
The literature is somewhat vast regarding the extraction
and chemical characterization of I. paraguariensis
(Esmelindro et al., 2004; Esmelindro et al., 2005;
Jacques et al., 2006; Jacques et al., 2007a; Jacques et
al., 2007b; Jacques et al., 2008) and there are several
reports dealing with the major constituents of Ilex
species, like the presence of saponins (Taketa et al.,
2004; Taketa et al., 2000; Ouyang et al., 1998; Pires et
al., 1997), the occurrence of flavonoids (Martínez et al.,
1997), xanthines (Saldaña et al., 2002; Reginatto et al.,
1999; Saldaña et al., 1999), aldehydes (Don-Xu and
Zhong-Liang, 1996), hemiterpene glycosides (Fuchino et
al., 1997), triterpenes and alkanes (Vangenden and
Jaarsma, 1990), anthocyanins (Ishikura, 1975), pentyl
esters, hexyl esters, and other lipophylic compounds
(Vangederen et al., 1988).
Some phytochemical or pharmacological investigations
related to the use of alcoholic or aqueous extracts of I.
paraguariensis have also been reported in the literature
(Prediger et al., 2008; Alves et al., 2008; Silva et al.,
2008; Strassmann et al., 2008; Cansian et al., 2008b).
Nevertheless, to our knowledge, no reports are available
concerning the use of mate extracts obtained from
supercritical extraction in pharmacological studies.
The present report is part of a broader project aiming at
characterizing and developing new processes and
products from mate tea leaves. In this context, the main
objective of this work is to evaluate the acute toxicity and
the anti-inflammatory activity of the crude mate extracts
obtained from supercritical carbon dioxide extraction. The
extracts were administered via subcutaneous injections in
rats with the aim to obtain information on the safety of
extracts of I. paraguariensis and provide guidance to the
development of new pharmaceutical products from this
plant.
MATERIALS AND METHODS
Plant materials and chemicals
Samples of mate tea leaves were collected in an experiment
conducted under agronomic control cultivation system, located at
Pasquali et al. 1163
Barão de Cotegipe (Rio Grande do Sul State, Brazil, 27° 37° 15°), a
typical I. paraguariensis open plantation. The leaves were collected
from plants of about 7 years old, growing at complete sunlight
exposure and without any additional fertilization with age of leaves
of 12 months old. For each tree, fractions from the top, middle, and
bottom were sampled and homogenized. The samples were
immediately dried after collection at room temperature, triturated
and sieved, being collected 100 to 200 mesh particles. The final
moisture of all samples was around 2%. The samples were then
stored at room temperature under nitrogen atmosphere prior to the
extraction. The carbon dioxide (CO2) employed in the extractions
(99.9% in the liquid phase) was purchased from White-Martins S.A.
(Brazil) and the reagents were all of analytical grade.
Supercritical extraction procedure
The extraction was performed in a laboratory-scale unit, presented
in detail elsewhere (Esmelindro et al., 2004; Esmelindro et al.,
2005; Dariva et al., 2003; Rodrigues et al., 2004; Jacques et al.,
2007a; Jacques et al., 2007b). Basically, the apparatus consists of
a CO2 reservoir, two thermostatic baths, a syringe pump (ISCO
500D – Lincoln, USA), a 100 mL jacketed extraction vessel, an
absolute pressure transducer (Smar, LD301 - Brazil) equipped with
a portable programmer (Smar, HT 201 - Brazil) with a precision of
± 0.3 bar, a collector vessel with a glass tube, and a cold trap.
Typically, amounts of around 25 g of comminuted mate tea
leaves were charged into the extraction vessel. The CO2 was
pumped at a constant mass flow rate of 2 g.min -1 into the bed,
which was supported by two 300 mesh wire disks at both ends of
the extractor. The CO2 was kept in contact with the herbaceous
matrix for one hour to allow the system stabilization. Afterwards, the
extract was collected by opening the micrometric valve. The
extraction was accomplished until no significant mass was
extracted (about 4 h) and at the end of the process the extract was
weighed and transferred to an appropriate vessel (“bulk”, stock
extract vessel). Based on the results obtained by Jacques et al.
(2007a), all the extractions were conducted at constant temperature
and pressure of 40°C and 250 bar, respectively. The extraction runs
performed led to an overall standard deviation of the extraction
yields of about 0.05. A whole experimental run lasted in general 10
h, including all steps involved: sample weighing, temperature
stabilization (baths, extractor), depressurization, etc. Extracts
obtained were dissolved in dichloromethane prior to gas
chromatography/mass spectrometry analysis.
Extract analysis
The extracts were analyzed in a gas-chromatograph coupled with a
mass selective detector (GC/MSD, Shimadzu QP5050A – Kyoto,
Japan), using a capillary column DB5 (30 m, 0.25 mm, 25 m).
Column temperature was programmed 70°C/3 min, 4°C/min to
260°C, 2.5°C/min to 300°C/25 min. Helium was the carrier gas and
the injection port and detector temperatures were 290 and 300°C,
respectively. The sample (1 L of 40.000 mg.L -1 in CH2Cl2)
components were identified by matching their mass spectra with
those of Wiley library database. Triplicate measurements were
performed for each sample.
Animals
Forty eight adult male wistar rats (3 months old, 180-220 g) were
obtained from the breeding colony at the Department of Clinical
Pharmacy (URI - Campus de Erechim, Brazil). The animals were
kept in a room under controlled humidity (50 ± 5%) and temperature
(22 ± 2°C) and subjected to a 12 h light cycle with free access to

1164 Afr. J. Pharm. Pharmacol.
food and water. All the procedures used in the present study
comply with the guidelines on animal care of the Ethics committee
on the use of animals of the University (Register number
187/TCA/07).
Acute toxicity evaluation
For the acute toxicity evaluation of the I. paraguariensis extracts
obtained from supercritical extraction, the animals were divided into
two groups of six animals each. For the treated group, a single
dose of 250 mg/kg of the mate extract diluted in sunflower oil was
administered by the intraperitoneal route, while the control group
received the vehicle only (sunflower oil). The animals were
continuously observed for general behavior changes, signs of
toxicity, death and latency of death during 1 h after treatment, then,
at each 4 h, and thereafter at each 24 h up to 14 days. During the
whole period, the weight and the consumption of water and food by
the animals were monitored. The tests were carried out in duplicate.
To evaluate the statistical significance of the results the signs of
toxicity were expressed in terms of semi quantitative analysis as
following: 0 - absence, 2 - Low, 3 - moderate, and 4 - Intense.
After the 14 th day, the rats were anesthetized with CO2 and
sacrificed with their livers and kidneys carefully dissected out. Small
slices of these freshly harvested tissues were fixed in buffered
formaldehyde solution (10%), dehydrated by serial ethanol solution,
diaphanized with ethanol-benzene and embedded in paraffin.
Micrometer sections, cut by a microtome (Leitz 1512), were stained
with hematoxylin-eosin and examined under a light microscope,
taking photomicrographs of the samples. All the tests concerning
acute toxicity evaluation of the I. paraguariensis extracts obtained
from supercritical extraction were carried out following the
resolution of National Agency of Sanitary Vigilance of Brazil, which
regulates the pre-clinical studies of toxicity using phytotherapy
(ANVISA, 2004).
Anti-inflammatory evaluation
The anti-inflammatory activity of the I. paraguariensis extracts
obtained from supercritical extraction was evaluated using twenty
four animals, which were equally divided into two groups, control
and treated. All the animals were submitted to blood collection
(orbital plexus) the acute induced-peritonitis assay by the injection
of 10 mL of oyster glycogen (1%) dissolved in of phosphatebuffered
saline (PBS) 10 mM, 7.4 pH under light Zoletil 50 ®
anesthesia (Oktar et al., 2004). For the treated group, it was
administered a single dose of 500 mg/kg of the mate extract diluted
in sunflower oil through subcutaneous injection, while the control
group received the vehicle only (sunflower oil). 4 h later, rats were
re-anesthetized for blood collection (orbital plexus), and then
sacrificed in CO2 chamber, with cells in the peritoneum removed by
washing with 30 mL of PBS containing 1000 U.L -1 heparin. The
peritoneal exudates at 4 h contained >98% neutrophils. The
suspension was centrifuged at 2000 rpm for 10 min and the
erytrocytes were destroyed by lysis buffer containing 0.15 M
ammonium chloride (NH4Cl). Cells were re-suspended in ice-cold
PBS, and the cell counting was performed using a light microscope.
None of the treatments altered the cell viability, which was >95%.
The assays were carried out in duplicate.
Statistical analysis
The values were expressed as the mean value ± standard error.
One-way analysis of variance (ANOVA) followed by Tukey test
applied for the statistical evaluations of the results obtained in the
anti-inflammatory tests, while for the acute toxicity tests it was
applied the ANOVA followed by the Kruskal-Wallis test. Values of
p

a
Pasquali et al. 1165
Figure 1. a, Effect of acute toxicity of supercritical I. paraguariensis extracts on histopathological parameters of liver; b, kidneys
tissues of rats. C, control; T, treated. * p

1166 Afr. J. Pharm. Pharmacol.
(a)
(b)
(c)
Figure 2. a, Photomicrographs of the sections of the livers showing
normal features in control group; b, the liver of animals treated with a
single dose of 250 mg/kg of supercritical I. paraguariensis extracts
after 14 days showing the presence of hyperchromatism (black arrow)
and vacuolization (white arrow); c, the presence of tumefaction
(double black arrow) and hepatocytes with hyperchromatism (single
black arrow). CV, centrolobular vein. (Hematoxylin-eosin, 100x).
while the cells with vacuolization decreased for this
group. There are no signals of cells with hyperchromatism
for both control and treated groups (Figure
1b). The cellular alteration in the renal tissue can be
better visualized in Figure 3 through photomicrographs of
the sections of the kidneys (magnification of 100 times).
The supercritical extracts of I. paraguariensis caused an
increase in the cellular tumefaction in the cortical region
of the kidneys of treated group (Figure 3b) when
compared with control group (Figure 3a), which suggests
the presence of renal necrosis. It was not verified the
presence of cells with hyperchromatism and vacuolization,
possibly due to the advanced necrosis stage of
the renal tissue.
Anti-inflammatory evaluation
Initially, the anti-inflammatory activity of the supercritical
extracts of I. paraguariensis on acute peritonitis induced
by oyster glycogen was investigated in a single dose of
250 mg/kg, but no significant differences were verified in
the cell recruitment. However, it was verified the
tendency to inhibit the inflammatory process. Then it was
decided to inject a single dose of 500 mg/kg. The results
obtained are presented at Figure 4.
The animals presented a level of 7.5 leukocytes/mm 3
before inflammatory induction, with no variation verified
compared to the treated group after induction with
supercritical extracts of I. paraguariensis, while the
control group decreased the level of leukocytes in the
circulating blood. The statistical analysis of the results
indicated significant differences (p

(a) (b)
Pasquali et al. 1167
Figure 3. a, Photomicrographs of the sections of the kidneys showing normal features in control group; b, the kidneys of animals
treated with a single dose of 250 mg/kg of supercritical I. paraguariensis extracts after 14 days showing the presence of cellular
tumefaction in the cortical region (double black arrow). G, glomerullus; TCD, distal convoluted tubules; TCP, proximal
convoluted tubules. (Hematoxylin-eosin, 100x)
Total leukocytes Neutrophils
Lymphocytes Monocytes
Figure 4. Effect of the supercritical I. paraguariensis extracts on the cell recruitment of the circulating blood. * p

1168 Afr. J. Pharm. Pharmacol.
The mean levels of lymphocytes in the circulating blood
was around 7000 lymphocytes/mm 3 for the treated group
with supercritical extracts of I. paraguariensis, which was
very similar to that obtained for the animals before the
induced-inflammation. The control group showed around
2000 lymphocytes/mm 3 in the circulating blood after the
induced-inflammation process.
The mean levels of monocytes in the circulating blood
was around 250 monocytes/mm 3 for both treated and
control group, which is similar to the value obtained for
the animals before the induced-inflammation. This
occurred due to the fact that the blood was collected 4h
before the induced-inflammation, where the monocyte
level did not change, as previously reported by Male
(2003).
In this work it was verified an increase in the number of
segmented neutrophils in the control group (this group
received sunflower oil only), which indicates that the
experimental model used in this study to evaluate the cell
recruitment was applicable and effective. The segmented
neutrophils are responsible for the initial defense of the
body subjected to an inflammatory process, which
confirms the existence of an inflammation caused by the
oyster glycogen.
The number of cells migrated to the peritoneum of the
treated animals was equivalent to that of control animals
(8760±3301 cells/mm 3 for treated and 6280±1647
cells/mm 3 for control).
Some of the pharmacological activities of I.
paraguariensis are attributed to the high content of
caffeyol-derivatives and flavonoids (Filip et al., 2001).
Among several important biological activities exerted by
flavonoids, it may be important to highlight its inhibitory
effect on the enzyme systems involved in the initiation
and maintenance of the inflammatory and immune
response (Gorgen et al., 2005). In fact, it has been shown
that I. paraguariensis presents a higher content of
flavonoids, such as quercetine, when compared to other
assayed species (Filip et al., 2001). Although the
flavonoids content was not determined in the supercritical
extracts of I. paraguariensis obtained in this work, based
on literature results (Jacques et al., 2007a Jacques et al.,
2008, Martínez et al., 1997), it seems reasonable to
believe that this class of compounds was also extracted
in the process. In this regard, the anti-inflammatory effect
of the supercritical extracts of I. paraguariensis could be
associated with the flavonoids content.
Conclusions
Results obtained in this work suggest the incidence of
acute toxicity due to the presence of cells with
hyperchromatism, vacuolization and tumefaction in the
livers and the presence of cells with tumefaction in the
cortical region of the kidneys of the treated group.
Toxicity studies in experimental animals may not however
be directly extrapolated to humans since a reasonable
estimate of the self-administered dose may be difficult to
make. Besides, in view of the widespread use of
supercritical extracts of I. paraguariensis, additional
clinical toxicological evaluations need to be performed to
determine a safe dose and protect the population from
possible toxic effects. Regarding the anti-inflammatory
effect of the supercritical extracts of I. paraguariensis, it
was verified that 500 mg/kg inhibited the neutrophil
recruitment in the circulating blood. It is well known that
numerous medicinal plants present significant antiinflammatory
activities, evaluated in different models, and
several active metabolites which are responsible for
these actions have been identified. Therefore, it is
believed that results found in the present work may be
very promising since they open new perspectives for the
therapeutic use of supercritical extracts of I.
paraguariensis (and other plants) or its preparations as
an anti-inflammatory agents. Further studies are underway
within our working group to identify the active
compounds responsible for the reported antiinflammatory
activity as well as to elucidate their
mechanisms of action.
ACKNOWLEDGEMENTS
The authors thank CNPq and CAPES for the financial
support and scholarships.
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African Journal of Pharmacy and Pharmacology Vol. 5(8), pp. 1170-1174, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.319
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Evaluation of the stem bark of Pistacia integerrima
Stew ex Brandis for its antimicrobial and phytotoxic
activities
Shafiq ur Rahman 1,2 *, Muhammad Ismail 2 , Naveed Muhammad 2 , Farhat Ali 1 , kamran Ahmad
Chishti 1 and Muhammad Imran 3
1 Department of Pharmacy, Sarhad University of Science and Information Technology, Pakistan.
2 Department of Pharmacy, University of Peshawar-25120, Pakistan.
3 School of Pharmacy University of Lahore, 24 avenue blue area Islamabad, Pakistan.
Accepted 7 July, 2011
The antimicrobial and phytotoxic activities of the crude methanolic extract and its subsequent solvent
fractions of Pistacia integerrima bark were investigated. The outstanding activity was shown by the
ethyl acetate fraction followed by aqueous fraction against Staphylococcus aureus having zone of
inhibition 19 and 15 mm respectively. The ethyl acetate fraction was also effective againsn, hoteus
vulgaris having zone of inhibition 15 mm. The outstanding minimum inhibitory concentration (MIC) was
observed against Staphylococcus aureus by ethyl acetate (0.31 mg/ml) and Salmonella typhi by hexane
fraction (0.37 mg/ml). The crude methanolic extract and subsequent solvent fractions were also tested
against two fungal strains, that is, Candida albicans and Aspergillus niger no outstanding antifungal
activity was found. All the fractions are good herbicidal and weedicidal at high concentration, however
the considerable activity was shown by ethyl acetate (90% growth inhibition) followed by chloroform
(70% growth inhibition) and methanol (60% growth inhibition) at a concentration of 500 ppm. The ability
of crude methanol extract and its subsequent solvent fractions, specially the ethyl acetate fraction, to
inhibit the growth of microorganism and plant Lemna minor L as an indication of its antimicrobial and
phytotoxic potential. This provides a baseline for isolation and identification of new antimicrobial and
phytotoxic compounds to the world of medicine.
Key words: Pistacia integerrima bark, antimicrobial, phytotoxic, minimum inhibitory concentration.
INTRODUCTION
Traditional medicines provide treatment for about 80% of
the world population, especially in developing countries,
indicating that about 3.5 to 4 billion people in the world
rely on plants as a source of drug (Farnsworth et al.,
1985; Patwardhan et al., 2004). Infectious diseases are
the leading causes of death throughout the world,
accounting for nearly one half of all death in the tropical
countries, which is also becoming a serious problem in
developed countries (Shah et al., 2011). So far a lot of
indigenous plants have been tested against various
*Corresponding author. E-mail: Shafiq_pharma01@yahoo.com.
Abbreviation: MIC, minimum inhibitory concentration.
microorganisms (Jones et al., 2000; Omer et al., 2000;
Islam et al., 2001; Ficker et al., 2003) and showed
considerable results.
A large number of secondary metabolites have been
isolated from the medicinal plants having antimicrobial
activities, which is the increasing demand of the present
era due to the developing resistance of microorganism
against synthetic drugs (White et al., 1998). Phytotoxicity
assays has been an important approach for identifying
plants that are likely to be a source of herbicidal
compounds of interest (Ma et al., 2011). Several studies
reported the phytotoxicity of different solvent extracts of
plants such as methanol, ethanol chloroform etc (Turker
Camper, 2002; Goncalves et al., 2009). Pistacia
integerrima Stewart ex Brandis belongs to family
Anacardiaceae, commonly known as kakar singhi (Hindi)
Shnaie (Pushto).

The galls, leaves and bark of this plant are used in the
traditional medicine for the treatment of fever, cough,
asthma, diarrhea, jaundice, and snake bites (Padulosi et
al., 2002; Jan et al., 2009).The phytochemical
constituents of leaves of P. integerrima Stewart ex
Brandis has been reported (Ahmad et al., 2008).
Tetracyclic triterpenoids from galls of P. integerrima
Stewart ex Brandis have been isolated. Analgesic and
anti-inflammatory activities of gall have been reported
(Ansari and Ali, 1996). The free radical scavenger activity
of leaves of P. integerrima Stewart ex Brandis in
hyperurecimia and gout has also been reported (Ahmad
et al., 2008). Literature survey revealed that there is no
scientific reported work on the antimicrobial and
phytotoxic activities of the bark of the plant, therefore this
study was carried out to evaluate the antimicrobial and
phytotoxic effects of crude methanol and its subsequent
solvent fractions of plant bark against some selected
microorganism and plant Lemna minor L respectively, so
as to establish a scientific ground for the use of the
Pistacia integerrima bark and exploration of the plant
bark for new active compounds.
MATERIAL AND METHODS
Plant material
Fresh stem bark of P. integerrima Stew ex Brandis was collected
from district buner of Khyber pukhthunkhwa, Pakistan. The plant
was collected in the month of April 2010 and was identified by
Chairman, Department of Botany, Prof. Dr. Muhammad Ibrar
University of Peshawaing oucher sample (No.10420Bot) was kept
in the herbarium at Botany Department, University of Peshawar for
reference.
Extraction and preparation of plant extracts
The stem bark was air dried at room temperature and pulverized by
using electric grinder. The powdered plant material (5.4 kg) was
soaked in commercial grade methanol for 14 days at room
temperature and was subjected to occasional shaking. After 14
days methanol soluble materials were filtered. The filtrate were then
concentrated at 40°C using rotary evaporator and kept in air tight
container until required for use. This procedure was repeated three
times. Crude methanolic extract weighing 250 g was suspended in
1000 ml of distilled water and filtered. The filtrate was then
partitioned successively with hexane (3×600 ml), chloroform (3×600
ml), ethyl acetate (3×600 ml) and butanol (3×600 ml). The resultant
solvent extracts were concentrated using rotary evaporator and
kept in air tight container until use (Bashir et al., 2009). The crude
methanolic extract and its subsequent solvent fractions were
screened for antimicrobial and phytotoxic activities.
Antibacterial assay
The bacterial isolates were first grown in a nutrient broth for 18 h
before use. Surface viable counting technique was used for
average number of viable organism per ml of stock suspension.
Each time fresh stock suspension was prepared using about 10 6
cfuml -1 (Igbinosa et al., 2009). Standard bacterial suspension 0.6 ml
was spread on sterile nutrient agar in Petri dishes. Wells are then
Rahman et al. 1171
bored in to the agar using sterile cork borer 8 mm in diameter
through micro titer pipette. Filled the well with 0.1 ml (10 mgml -1 ) of
each extract and allow standing for 2 h at room temperature and
then incubated at 37°C. Control was set up in parallel using the
solvents that were used to reconstitute the extracts. The zone of
inhibition was observed after 24 h. The results were compared with
standard drug streptomycin at a concentration of 0.5 mg/ml
(Igbinosa et al., 2009; Shah et al., 2011).
Antifungal assay
The fungal isolates were allowed to grow on a saboraud dextrose
agar at 25°C until they sporulated. The fungal spores were
harvested, washed with sterile normal saline and were standardized
to 10 8 cfuml -1 (Shah et al., 2011).The antifungal activity was carried
out in accordance with Igbinosa et al. (2009) and Shah et al.
(2011).
Phytotoxicity assay
The crude methanol extract and its subsequent solvent fractions
were screened for phytotoxicity against Lemna minor L. The
medium was prepared by mixing various inorganic components in
900 ml of distilled water (for 1000 ml of medium) and KOH solution
was added for the adjustment of pH at 6.0 - 7.0. The medium was
autoclaved at 121°C for 15 min. Stock solution was prepared by
dissolving 15 mg of test sample 1.5 ml of in ethanol. Nine flasks
(three for each dilution) were inoculated with 1000, 100 and 10 µl of
the stock solution for 500, 50 and 5 ppm. The solvent was then
evaporated overnight under sterilized conditions. Each flask was
supplemented with 20 ml of the medium. Thereafter, 10 plants each
containing a rosette of three fronds, were added to each flask. One
other flask, supplemented with solvent as control and reference
plant growth inhibitor (Paraquat), served as a standard phytotoxic
drug. The flasks were plugged with cotton and placed in growth
cabinet for 7 days. On the 7 th day, the number of fronds per flask
was counted. Results were analyzed as growth inhibition in
percentage (Saeed et al., 2010; Ghazala and Shameel, 2005).
RESULTS AND DISCUSSION
Antibacterial assay
The antibacterial action, that is, the zone of inhibition and
minimum inhibitory concentration (MIC) is shown in Table
1 and Table 2, respectively. The methanolic extract and
its subsequent solvent fractions were tested against three
gram positive bacteria, that is, Staphylococcus aureus,
Proteus vulgaris and Bacillus subtilis and three gram
negative bacteria, that is, Pseudomonas aeruginosa,
Escherichia coli and Salmonella typhi. The antibacterial
activity of all the samples were compared with the broad
spectrum antibacterial drug, that is, Streptomycin. The
outstanding activity was shown by the ethyl acetate
followed by aqueous fraction against S. aureus having
zone of inhibition 19 and 15 mm, respectively. The ethyl
acetate was also effective against P. vulgaris having
zone of inhibition 15 mm. It is clear from Table 1 that the
plant extracts and especially the ethyl acetate fraction is
very effective against gram positive bacteria as compare
to gram negative bacteria. Methanolic extract is effective

1172 Afr. J. Pharm. Pharmacol.
Table 1. Antibacterial activity of Pistacia integerrima bark (Zone of inhibition in mm).
Solvent fraction (10 mg/ml)
Staphylococcus
aureus
Zone of inhibition(mm)
Proteus
vulgaris
Bacillus
subtilis
Pseudomonas
aeruginosa
Escherichia
coli
Salmonella
typhi
Methanol 10 ± 0.22 12 ± 0.30 10 ± 0.19 14 ± 0.21 11 ± 0.18 12 ± 0.56
Aqueous 15 ± 0.23 12 ± 0.32 11 ± 0.20 12± 0.25 10 ± 0.20 11± 0.12
Ethyl acetate 19 ± 0.13 15 ± 0.28 12 ± 0.21 13 ± 0.27 10 ± 0.22 11± 0.11
Hexane 8 ± 0.11 13 ± 0.22 10 ± 0.23 9 ± 0.11 10 ± 0.23 5± 0.24
Streptomycin (0.5 mg/ml) 17 ± 0.11 16 ± 0.09 16 ± 0.00 19 ± 0.07 9 ± 0.02 15± 0.06
Table 2. Minimum inhibitory concentration of different solvent fractions of Pistacia integerrima bark (mg/ml).
Test organism
Minimum inhibitory concentration (MIC)
Methanol Aqueous Ethyl acetate Hexane Streptomycin (Std)
Staphylococcus aureus 5.00 ± 0.12 0.63 ± 0.21 0.31 ± 0.12 10.0 ± 0.23 0.08 ± 0.03
Proteus vulgaris 10.0 ± 0.20 5.00 ± 0.26 1.25 ± 0.50 5.00 ± 0.19 0.03 ± 0.01
Bacillus subtilis 5.00 ± 0.24 9.00 ± 0.25 8.00 ± 0.32 10.0 ± 0.12 0.07 ± 0.02
Pseudomonas aeruginosa 2.50 ± 0.21 10.0 ± 0.12 0.41 ± 0.33 0.61 ± 0.10 0.05 ± 0.01
Escherichia coli 6.00 ± 0.32 6.00 ± 0.23 1.20 ± 0.32 8.00 ± 0.13 0.79 ± 0.02
Salmonella typhi 5.00 ± 0.11 9.00 ± 0.11 0.625 ± 0.31 0.37 ± 0.14 0.07 ± 0.05
Table 3. Antifungal activity of Pistacia integerrima bark (Zone of inhibition in mm).
Test organism
Methanol
(10 mg/ml)
Aqueous
(10 mg/ml)
Zone of inhibition(mm)
Ethyl acetate
(10 mg/ml)
Hexane
(10 mg/ml)
Amphotericin B
(0.5 mg/ml)
Candida albicans 8 ± 1.2 10 ± 1.2 14 ± 0.67 13 ± 1.1 21 ± 0.5
Aspergillus niger 7 ± 1.0 8 ± 1.9 8 ± 0.09 10 ± 1.5 18 ± 0.9
against the gram negative bacteria, that is, P. aeruginosa
which is common cause of nosocomial infections. The
outstanding MIC was observed against S. aureus by ethyl
acetate (0.31 mg/ml) and S. typhi by hexane fraction
(0.37 mg/ml).The growth inhibiting capability of the ethyl
acetate and aqueous fractions, is an indication for its
antibacterial potential and making the P. integerrima bark
a good candidate for antibacterial drugs. The different
fractions of the Pistacia integerrima bark do not have
significant antifungal activity as shown in Table 3.
The search for new antimicrobial agent is the need of
the modern era due the developing resistance of the
available antimicrobial agents. Majority of the available
antimicrobial agents are expensive and cannot affordable
for poor patients therefore the aim of screening crude
extract against various microbes specially bacteria and
fungi is to find such antimicrobial materials, which are
easily available and economically affordable. The larger
zone of inhibition exhibited by the methanol, ethyl acetate
and aqueous fraction of P. integerrima bark as shown in
Figure 1 may be due to the presence of active
compounds such as tannins, flavonoids, alkaloids and
saponins. The phytochemical study of theses fractions
revealed the presence of tannins, flavonoids and
saponins. The phytochemicals like alkaloids, saponins,
flavonoids are the antimicrobial principles of the plant
which are responsible for their antimicrobial activities
against different pathogens (Haiza, 2000). The results
has also supported by Hymete et al. (2005).They
reported that flavonoids compounds have antimicrobial
activities.
Phytotoxicity assay
The phytotoxic activity of all the fractions is shown in
Figure 2. It is clear from the results that all the fractions
have herbicidal activities at high concentration however;
the outstanding activity was shown by ethyl acetate 90%
growth inhibition followed by chloroform 70% growth
inhibition and methanol 60% growth inhibition at a
concentration of 500 ppm. This reflects that these fractions

zone of inhibition
25
20
15
10
5
0
Methanol
Aqueous
Ethyl acetate
Hexane
streptomycin
Figure 1. Zone of inhibition of different against selected bacteria.
Growth inhibition in percentage
100
90
80
70
60
50
40
30
20
10
0
Methanol
Aqueous
Butanol
Ethyl acetate
chloroform
Figure 2. Phytoxic activity of different fractions against lemna minor L.
have active compounds which are responsible for their
phytotoxic effect. While comparing all the fractions the
ethyl acetate has excellent activities while the hexane
fraction has the least. Lemna plants are mono-
Paraquat Std drug
Staphylococcus aureus
Proteus vulgaris
Bacillus subtilis
Rahman et al. 1173
Pseudomonas aeruginosa
Escherichia coli
Salmonella typhi
5ppm
50ppm
500ppm
3.124µg/ml
cotyledonous plants which are very sensitive to bio active
compounds. Lemna assay has been used to detect
natural antitumor and phytotoxic compounds (Rehman,
1991). The quality and quantity of agricultural crops is

1174 Afr. J. Pharm. Pharmacol.
mostly affected by the presence of extra weeds in the
crops. The world economy is strongly affecting by the
loss of agriculture crops (Piment et al., 2001). The
reduction in the growth of these weeds is the prime action
for increasing the agriculture crops. Synthetic herbicides
are commonly used for the destruction of weeds in
agricultural sectors.
However, various factors that restricted the use of
synthetic herbicides include water and soil pollution,
herbicide-resistant weed populations, and detrimental
effects on non-target (Li et al., 2003). In the modern era,
more emphasis has been laid on the natural
allelochemicals from plants, for weeds control in crops
production especially to manage with the problem of
weed resistance. It has been proved that the phytotoxicity
of plants reduces the growth of weeds without any
negative effect on the crops growth and overall yield
under normal field condition. It is therefore, assumed on
the basis of results that the phytotoxic principle(s) of the
bark of the plant could be a significant source of natural
herbicides for weeds control.
Conclusion
The result of the study provides a scientific justification to
the traditional use of the bark of P. integerrima as an
antimicrobial agent. Moreover the results showed that the
bark has some valuable antimicrobial and phytotoxic
compounds which provide a base line for isolation and
characterization of innovative compounds. This represents
the first preliminary reports on the antimicrobial and
phytotoxic activities of P. integerrima bark.
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African Journal of Pharmacy and Pharmacology Vol. 5(8). pp. 1175-1178, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.359
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Antioxidant and antibacterial activity of crude
methanolic extract of Euphorbia prostrata collected
from District Bannu (Pakistan)
Mushtaq Ahmad 1 , Abdus Saboor shah 1 , Rahmat Ali Khan 1 *, Farid Ullaha Khan 1 , Noor Aslam
Khan 1 , Mir Sadiq Shah 1 and Muhammad Rashid Khan 2
1 Department of Biotechnology, Faculty of Biological Sciences, University of Science and Technology, Bannu, Khyber
Pakhtunkhwa, Pakistan.
2 Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University Islamabad, Pakistan.
Accepted 25 July, 2011
Medicinal plants help in improving the human health that is primarily due to bioactive constituents such
as flavonoids, alkaloids, saponins, cardiac glycosides and tannins. These compounds play important
role in minimizing oxidative stress, cancer, impotency, cardiac dysfunction and microbial inhibition.
Euphorbia prostrata (L) is a small prostrate, annual herb found all over the world including
Pakistan. This study was designed to investigate the protective effects of E. prostrata against oxidative
stress and antibacterial potential. Methanolic extract of E. prostrata were used in three different
concentrations of 1, 3 and 5 mg/ml. The maximum inhibition was shown at highest concentration of 5
and at 3 mg/ml the moderate inhibition while at 1 mg/ml the minimum inhibition was shown. Similarly
concentration of methnolic extract of E. prostrata exhibited excellent scavenging activity. The %
scavenging of E. prostrata is directly proportional to the concentration. The order of % scavenging
activity is 50 < 100 < 150 < 200 < 250 g/ml. Ascorbic acid is used as negative control
Key words: Antioxidant, antibacterial, Euphorbia prostrata, dimethyl sulfoxide (DMSO), 1,1-diphenyl-2picrylhydrazyl
(DPPH), ascorbic acid.
INTRODUCTION
The imbalance between antioxidant and reactive oxygen
-
species, such as superoxide radical (O2 ), hydroxide
radical ( - OH), peroxide radical (ROO - ), and nitric oxide
radical produced due to excessive metabolism in the cell
and its ability to detoxify these reactive intermediates is
called oxidative stress. These reactive oxygen species
damage the biological system and causes different
chronic diseases like cancer and heart diseases (Aruoma
and Cuppette, 1997; Prakash et al., 2007). The modern
research claims that oxidative stress is the cause of
various disorders and diseases, therefore, the researcher
focus on the role of antioxidants in the maintenance of
biological system (human health), its remedy and treatment
(Etsuo, 2010).
The sources of naturally occurring antioxidants are
*Corresponding author. E-mail: Rahmatgul_81@yahoo.com.
Tel: 92 928633425.
primarily plant phenolic compounds which are present in
all parts of plants (Pratt and Hudson, 1990). The phenolic
compounds, especially flavonoids, import the antioxidant
and pharmacological properties to plants and herbs
(Dawidowicza et al., 2006). The biological,
pharmacological and medicinal properties of this group of
compounds have been extensively reviewed (Marchand,
2002). Medicinal plants play important role in recovery of
various diseases (Khan et al., 2009, 2010a, b, 2011a, b;
Sahreen et al., 2010, 2011), microbial inhibition (Khan et
al., 2010c) and cardio protection (Khan et al., 2011).
Medicinal plants are the best source to obtain different
drugs, about 80% of people in industrialized countries
uses traditional medicines, which are derived from
medicinal plants, so the properties and efficiency of
plants must be, investigated (Ellof, 1998). In developing
countries, more than 40% of the people are put to death
due to infection of the microorganism, besides this is also
spoiliage of food materials due its pathogenicity (Marino
et al., 2001). The food materials can be protected by use

1176 Afr. J. Pharm. Pharmacol.
% Scavenging
80
64
48
32
16
0
Plant extract Ascorbic acid
50 100 150 200 250
Concentration (µg/ml)
Figure 1. DPPH scavenging activity of E. prostrate.
of phenolic compounds (essential oils) as antibacterial
agents because some of the antimicrobial compounds
produced by plants are effective against pathogens
(Mitscher et al., 1987). Antimicrobial agents, including
food preservatives, inhibit food burn bacteria and
preserve the food. Many naturally occurring extract from
herbs, medicinal plants, are known to possess
antimicrobial activities and can serve as antimicrobial
agents, against food spoilage (Bagambula et al., 2003).
MATERIALS AND METHODS
Plant collection
Plant of Euphorbia prostrata (L) was collected from District Bannu
at its maturity during the month of December 2010, identified by
associate Professor Mr. Abdur Rehman, Govt. Post Graduate
College, Bannu. Shade dried plant was then selected for biological
study and powdered into very fine powder.
Plant extraction
700 g of plant powder of E. prostrata plant were socked in 3 L of
80% methanol, with random shaking for a period of 7 days. The
plant is extracted and filtered by using Whatman filter paper and
concentrated with the help of rotary evaporator at 37°C to obtained
crude extract which was 40 g.
Antioxidant bioassay
Solution of extract and ascorbic acid prepared
In the proceeding of antioxidant activity, three extract solutions
were prepared as stock solution. From the stock solution, another
type of solution was prepared with different concentrations. The
total volume of this solution was 500 µl. Another five solutions of
different concentrations were prepared as discussed previously;
these five solutions of 50, 100, 150, 200 and 250 µg/ml was also
reused as a stock solution. Ascorbic acid is used as a control which
inhibits the free radicals. Solution of ascorbic acid was prepared by
the same method of extract.
Solution of DPPH (1-1 diphenyl 1-2 picryl- hydrazyl)
1.5 mg of DPPH is weighted and dissolved in 5 ml/5 ml methanol.
Since DPPH is light sensitive so the solution is covered in aluminum
sheet. After checking its absorbance at 517 nm, it was found to be
less than one (1). 900 µl of DPPH was added to 100 µl extract and
ascorbic acid, and incubated at 25°C for 30 min and its absorbance
at 517 nm was checked with the UV Spectra Photometer.
Scavenging activity was determined based on the formula as %
scavenging.
(DPPH ab – Sample ab/DPPH ab) × 100
Antibacterial activity
Media and bacterial strains
Nutrient broth medium (MERCK) was used for the growth of
bacteria for the preparation of inoculums. Nutrient broth medium
was prepared by dissolving 0.8 g/ 100 ml nutrient broth in distilled
water while nutrient agar medium medium was prepared by
dissolving 2 g of nutrient agar in 100 ml of dH2O. Antibacterial
activity was tested against four strains of bacteria which were
Micrococcus luteus (ATCC10240), Staphylococcus aureus
(ATCC6538), Escherichia coli (ATCC15224) and Pseudomonas
aeruginosa.
Assay procedure (agar diffusion method)
Nutrient agar medium was prepared by suspending nutrient agar 2
g in 100 ml of distilled water and was autoclaved and allowed to
cool. Petri plates (14 cm) were papered by pouring 75 ml of seeded
nutrient agar and then allowed to solidify. Five wells per plate were
made with cork borer (8 mm). Using micropipette 100 l of test
solution was poured in each well. Samples was applied to each
Petri plate and incubated at 37°C for 24 h. Then clear inhibition
zone were detected around each hole. DMSO was used as
negative control. The diameter of clear zone showing no bacterial
growth around each well was measured in mm.
RESULTS AND DISCUSSION
DPPH radical scavenging activity
DPPH (1-1 diphenyl 1-2 picryl- hydrazyl) is a free radical
and has strong electron attracting ability from antioxidant.
The scavenging activity of the methanolic extract of E.
prostratra is shown in Figure 1. The graph showed that
the concentration of methanolic extract is directly
proportional to the % scavenging of E. prostrata. The
minimum scavenging activity is shown at lowest
concentration of 50 g/ml which is 34.25%. The order of

Table 1. Antimicrobial activity of E. prostrata against different strains of bacteria.
Group Concentration
EPME
Zone(mm)
Ahmad et al. 1177
Micrococcus luteus Staphylococcus aureus Escherichia coli Pseudomonas aeruginosa
1 20 13 16 21
3 24 14 18 34
5 25 24 20 26
DMSO 1.2 0.9 1 1.4
EPME, E. prostrata methanolic extrac; DMSO, negative control. Concentration expressed in microgram per ml in respective solvent- methanol.
% scavenging activity is 50 < 100 < 150 < 200 < 250
g/ml. Ascorbic acid is used as control.
Information of the present study indicated that the
methanolic extract of E. prostrate (L) possesses marked
scavenging properties and scavenged the DPPH. The
result obtained from the present work is very closely
related to that obtained by the investigations of
Hagerman et al. (1998). The antioxidant potential of the
plants is due to the presence of components of phenolic
and polyphenolic species which are significantly inhibited
by the free radicals that cause oxidative stress. The
results obtained by Duenas et al. (2006) and Kilani et al.
(2008) are also similar to the present work.
Antibacterial assay
Result of antibacterial assay showed that the methanolic
extracts of E. prostrata possessed antibacterial activity
tested against M. luteus, S. aureus, E. coli and P.
aeruginosa. In this activity, methanolic extract of E.
prostrata were used in three different concentrations, that
is, 1, 3 and 5 mg/ml. Table 1 showed that the
concentration of extract is directly proportional to the
inhibition. The maximum inhibition for each bacterium
was shown at highest concentration of 5 mg/ml, while 3
mg/ml show moderate inhibition and lowest inhibition was
shown by 1 mg/ml. Control DMSO showed negligible
inhibition.
The result showed that methanolic extract of E.
prostrata plant had significant antibacterial activity. The
result obtained from the present work is closely related to
that of Ndhlala et al. (2009) who reported antimicrobial
potential of South African plant Aloe barberae (L). This
has strongly supported our work.
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Aruoma IO, Cuppette SL (1997). Antioxidant methodology in vivo and
in vitro concept. Illinois: AOAS press.
Bagamboula CF, Uyttendaele M, Debevere J (2003). Antimicrobial
effect of spices and herbs on Shigella sonnei and Shigella flexneri. J.
food Prot, 66: 668-673.
Dawidowicza LA, Wianowoska D, Baraniak B (2006). The antioxidants
properties of alcoholic extract from Sambucus nigra (L) (Antioxidant
properties of extracts). LWT., 39: 308-315.
Duenas M, Hernandez T, Estrella I (2006). Assisment of in vitro
antioxidant capacity of the seed coat and the cotyledon of legumes in
relation to their phenolic contents. Food Chem., 98: 95-103.
Ellof JN (1998). Which extractant should be used for the screening and
isolation of antimicrobial components from plants. J. Ethnopharmacol.,
60:1-6.
Etsuo N (2010). Assessment of antioxidant capacity in vitro and in vivo.
J. Free Radic. Biol. Med., pp. 503-515.
Hagerman AE, Riedle KM, Jones GA, Sovik KN, Hartzfeld PW (1998).
High molecular weight plant poly phenolic (tannins) as Biological
antioxidants. J. Agric. Food Chem., 46: 1887-1892.
Khan MR, Haroon J, Khan RA, Bokhari J, Rashid U (2011). Prevention
of KBrO3-induced cardiotoxicity by Sonchus asper in rat. J. Med.
Plants Res., 5(12): 2514-2520.
Khan MR, Rizvi W, Khan GN, Khan RA, Sheen S (2009). Carbon
tetrachloride-induced nephrotoxicity in rats: Protective role of Digera
muricata. J. Ethnopharmacol., 122: 91-99.
Khan RA, Khan MR, Sahreen S (2010a). Evaluation of Launea
procumbens use in renal disorders: a rat model. J. Ethnopharmacol.,
128: 452-461.
Khan RA, Khan MR, Sahreen S (2011a). Protective effect of Sonchus
asper extracts against experimentally-induced lung injuries in rats: A
novel study. Exp. Toxicol. Pathol., doi:10.1016/j.etp.2011.01.007
Khan RA, Khan MR, Sahreen S, Bukhari J (2010b). Prevention of CCl4induced
nephrotoxicity with Sonchus asper in rat. Food Chem.
Toxicol. 23: 1304-1321.
Khan RA, Khan MR, Sahreen S, Bukhari J (2010c). Antimicrobial and
Phytotoxic activity of various fractions of Sonchus asper. Afric. J.
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Khan RA, Khan MR, Sahreen S, Jan S, Bokhari J, Rashid U (2011b).
Phytotoxic characterization of various fractions of Launaea
procumbens. Afric. J. Biotechnol., 10: 5377-5380.
Kilani S, Sghaier MB, Limem I, Bouhlel I, Boubaker J, Bhouri W,
Skandrani I, Neffeti A, Ammarb RB, Dijoux-Franca MG, Ghedira K,
Chekir-Ghedira L (2008).In vitro evaluation of antibacterial,
antioxidant, cytotoxic and apoptotic activities of the tubers infusion
and extract of Cyperus rutundus. Biosource Technol., 99: 0904-9008.
Marchand LL (2002). Cancer preventive affects of Flavonoids: a review
of Biomed. Pharmacotherapy, 56: 296-301.
Marino M, Bersani C, Comi G (2001). Impedance measurements to
study the antimicrobial activity of essential oils from Lamiaceae and
Compositae. Int. J. Food Microbiol., 67: 187-195.
Mitscher LA, Drake S, Gollapuda SR, Okwute SK (1987). A modern
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radical Scavenging activities of seeds and agri-wast of some varieties
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Pratt DE, Hudson FJB (1990). Natural antioxidants not exploited

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in rat. BMC Compl. Alter. Med., 11:48 doi: 10.1186/1472-6882-11-48.

African Journal of Pharmacy and Pharmacology Vol. 5(8), pp. 1182-1184, August 2011
Available online http://www.academicjournals.org/ajpp
DOI: 10.5897/AJPP11.198
ISSN 1996-0816 ©2011 Academic Journals
Short Communication
Development of new spectrophotometric determination
of titanium in homeopathic pharmacy using Ponceau S
as a reagent
G. M. Mastoi*, M. Y. Khuhawar, Kulsoom Abbasi, Moina Akhtar, Saba Naz, Humaira Khan,
Arfana Mallah and Zuhra Memon
1 DR. M. A. Kazi Institute of Chemistry, University of Sindh, Jamshoro 76080, Sindh, Pakistan.
Accepted 21 July, 2011
Spectrophotometric method has been developed for the determination of titanium(III). The linear
calibration curve was obtained with 2-10 g/ml titanium. The titanium was determined in
pharmaceutical preparation with coefficient of variation 3.0%. The concentration of titanium was
calculated from the standard calibration curve and was found 111.25 µg/ml with coefficient of variation
3.0%. The obtained results agreed with the results obtained from hydrogen peroxide (H2O2) method.
Key words: Determination, medicine, Ponceau S, spectrophotometer, titanium.
INTRODUCTION
The paint, pigment, paper and pulp industries discharge a
very high amount of titanium. Titanium is a metal that has
been used in various and varied medical applications for
about 40 years. Although many biomaterials have come
and gone during this period, titanium is one of the few
that has seen its uses and reputation enhanced over the
years.There are various methods for the determination of
titanium utilizing a number of ligands such as
pyridylazoresorcinol (Zhou, 1990a), 5bromopyridylazodiethylaminophenol
(Zhou, 1990b),
thiazolylazoresorcinol with mordant red 19 (Zhou, 1994),
solochrome violet relay services (RS) (Wang and
Mahmud, 1986), pyrocatechol violet (Vukomanovic and
van Loon, 1994), Beryllon (III) and cupferron (Xin et al.,
1995). Ponceau S has been used for the staining of
paper electrophoresis strips for the determination of
serum albumin using cellulose acetate electrophoresis
clearing and preserving cellulose acetate electropherograms
stained with Ponceau S (Meulemans, 1960;
WuEsTm, 1965). It is used for the electrophoresis of the
*Corresponding author. E-mail: smastoi@yahoo.com.
Abbreviations: H2O2, Hydrogen peroxide; RS, relay services;
HCL, hydrochloric acid; KBrO3, potassium bromate.
high-tyrosine proteins of keratins on cellulose acetate
(Blagrove et al., 1975). Kohn and Pamel (1978)
described a method of immunofixation using cellulose
acetate immunofixation technique. This is followed by
washing and staining by Ponceau S. Heick et al. (1980)
has compared the protein of urine and cerebral spinal
fluid by using Ponceau S dye. El-Bahi et al. (1992) used
Ponceau S stained nitrocellulose strips for comparison of
detected stable diagnostic antigen from bile and feces of
Fasciola hepatica infected cattle The present work
examines the potentials of the reagent Ponceau S for the
spectrophotometric determination of titanium in pharmaceutical
preparation.
MATERIALS AND METHODS
Ponceau S (0.004 M)
Ponceau S (0.306) (BDH) was dissolved in 100 ml of distilled
water. Solution was further diluted 10 times with distilled water
before spectrophotometeric studies.
Titanium (III) solution (1000 µg//ml)
The solution (III) chloride (1.8 ml) (15% Ti Cl3 solution, E. Merck)
was added to 20 l of 37% hydrochloric acid (Merck) and diluted to
100 ml with distilled water.

Apparatus
Absorbance measurements were recorded on a Hitachi 220 UV/Vis
spectrophotometer equipped with 150 W deuterium lamp and 1 cm
quartz cells. All pH measurements were made with an Orion 420 A
digital pH meter with a combined glass-calomel electrode. A
laboratory temperature of 30 ± 2°C was used through out study. All
solutions were previously brought to this temperature.
Chemicals
GR grade chemicals of sodium acetate, acetic acid, sodium
bicarbonate, sodium carbonate, boric acid, borax, ammonium
chloride, ammonia, chloroform, acetonitrile, hydrochloric acid (HCl)
(37%) (E. Merck), were used. Freshly prepared double distilled
water for all glass was used through out the work. The buffer
solutions in the pH range 1-10 at unit interval were prepared
potassium chloride (1M), HCl (0.1M); pH 1-2, sodium acetate (1M)acetic
acid (1M); pH 3-6,ammonium acetate(1M)- acetic acid (1M);
pH 7, boric acid(1M)- borax(1M); pH 8-9 and ammonium chloride
(1M)-ammonia pH 10.
All glasses were previously soaked in 10% nitric acid for 24 h and
rinsed with double distilled water before the use.
Analytical procedure for the determination of titanium (III)
To volumetric flask (10 ml) was added 1.4 ml of Ponceau S solution
(0.4m M), 0.4 to 2.0 ml of solution containing 20-100 µg of titanium
(III) and the volume was adjusted to 10 ml with 3 M HCl. The
decrease in absorbance was measured at the wavelength of 520
nm. The blank solution was prepared following the same procedure
except addition of titanium (III).
Effect of pH and acid concentration
To different volumetric flasks (10 ml) were added Ponceau S (1.4
ml) (0.4 mM), titanium (III) (1.0 ml) (50 ppm), different buffer
solutions within the pH range 1-10, at unit interval (2 ml) and
volume was adjusted to the mark with water. Ponceau S solution
(1.4 ml) (0.4 mM) and titanium (III) (1 ml) (50 ppm) were transferred
to volumetric flask (10 ml) and added HCl (5M) 0.2-2.2 ml with an
interval of 0.2 ml. The volume was adjusted to 10 ml with water.
Ponceau S solution (1.4 ml) (0.4 mM) and titanium (III) (1ml) (50
ppm) were transferred to volumetric flask (10 ml) and added HCl
(37%) 2-6 ml with an interval of 1.0 ml. The volume was adjusted to
10 ml with water. The absorbance of each of the solution was
measured against reagent blank containing same acid
concentrations and pH.
Analysis of test solutions
The accuracy of method was checked by analysis of unknown
concentrations. Test solutions (1-3 ml) were transferred to
volumetric flask (10 ml) containing 1.4 m Ponceau S solution
(0.4mM) and analytical procedure was followed. The amount of
titanium in test solution was calculated from calibration curve.
Sample analysis for titanium
Gin Seng (B. M. Homeopathic pharmacy, Lahore, Pak) (0.5 ml) was
transferred to a crucible and was gently heated on a burner,
followed by in muffle furnace (Phoenax Furnaces Ltd, Shefflied,
England), at 550°C for 6 h. The white residue was dissolved in HCl
Mastoi et al. 1183
(3M) and volume was adjusted to 50 ml with 3 M HCl solution. To
10 ml volumetric flask, a solution 1.4 ml mM Ponceau S was
transferred and 5 ml of sample solution was added and the volume
was adjusted with 3M HCl solution. The absorbance was recorded
at 520 nm as described in analytical procedure.
Spectrophotometric determination of titanium (III) using
hydrogen peroxide
A sample solution (1.0 ml) was transferred to a conical flask (25 ml)
and the analytical procedure was followed (Stewart et al., 1974)
RESULTS AND DISCUSSION
General characteristics of the reaction
Ponceau S is a red coloured dye. It absorbs at 520 nm
with molar absorpitivity of 32500 L.mol -1 .cm -1 . Initially it
was examined for possible catalytic determination of
metal ions by its decomposition with hydrogen peroxide
(H2O2) or potassium bromate (KBrO3) but when titanium
(III) in acid solution was added a decrease in intensity of
the colored dye was observed at 520 nm. After
preliminary optimization, it was found that there was no
effect of H2O2 or KBrO3 but was due to the complex
formation with Ti (III). As titanium(III) is easily hydrolysed
in aqueous solution, therefore, the final concentration of
hydrochloric acid was examined at 3 M.
Effect of variables
The final concentration of Ponceau S solution was varied
from 0.01 mM to 0.056 mM. It was observed that when
reagent in HCl was added, the 0.016 mM solution turned
yellow immediately with the addition of titanium(III), afterwards,
there was a regular increase in the absorbance
upto 0.056 mM. The sensitivity of spectrophotometer was
poor above the concentration. Thus, the final concentration
of Ponceau S (0.056 mM) was selected for further
study and 0.4 mM, 1.4 ml was added in 10 ml.
Effect of hydrochloric acid concentration
The effect of hydrogen ion concentration on the change
in absorbance of Ponceau S with titanium (III) was
investigated. It was observed that change in absorbance
occurred in acidic media only. It was therefore the
change in absorbance with HCl concentration was further
examined. The concentration of HCl was varied from 0.5
M to 4.0 M in the final volume, and it was considered that
the solution which gave maximum difference between the
blank (B) and the analyte (A) was considered as optimal.
It was observed that a similar absorbance was obtained
using 0.5-4 M concentration of HCl. As titanium(III)
solution is readily hydrolysed in aqueous, therefore, 3 M
HCl was maintained in the final volume and was selected

1184 Afr. J. Pharm. Pharmacol.
for the prevention of Ti(III) solution.
Effect of titanium (III) concentration
The change in absorbance of Ponceau S with a
concentration of titanium (III) was examined. A linear
calibration curve was obtained by plotting (B-A) against
amount of titanium (III) within 2-10 µg/ml in the final
volume with coefficient of correlation (r) = 0.982
Interference
The effect of different cations and anions on the
determination of the 5 µg/ml of titanium (III) was
investigated for Ca (II), Mg (II), K (I), Na (I), Al (III), Mn(II),
- - - -
Co (II), Cu (II), Zn (II), Cd (II), Cr (III), NO3 , NO2 , Cl , Br ,
-
SO4 , citrate, tartrate, BrO4 and ascorbic acid, it was
observed that their concentration when added 10 times
the concentration of titanium (III), did not affect the
determination of titanium (III). However, Mo (IV), Mo (VI),
Fe (II), Fe (III), V (V) and V (VI) when added at the same
concentration of titanium (III), interfered the determination
of titanium and enhanced the decrease in the
absorbances with increase in different difference between
the blank (B) and analyte (A) absorbance (B-A) and
interfered.
Sample analysis
The titanium contents in a pharmaceutical preparation
(Gin Seng Tonic) was analysed by dry ashing, followed
by acid digestion. The spectrophotometeric determination
was carried out using analytical procedure. The concentration
of titanium was calculated from the standard
calibration curve and was found 111.25 µg/ml with
coefficient of variation 3.0%. The sample was analysed
spectrophotometrically (Stewart et al., 1974) for the
contents of titanium (III) and was found 110.21 µg/ml with
coefficient of variation 2.5%.
Conclusion
The proposed spectrophotometric method has an
acceptable sensitivity and selectivity for the determination
of titanium (III) though a few metal ion affected the
determination. The method was applied successfully for
the determination of titanium in pharmaceutical
preparation. It can also be concluded that this study
constitutes the basis of a use of Ponceau S for detecting
other transition metals.
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