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African Journal of
Pharmacy and
Pharmacology
Volume 6 Number 26 15 July, 2012
ISSN 1996-0816

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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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Editorial Board
Prof. Fen Jicai
School of life science, Xinjiang University,
China.
Dr. Ana Laura Nicoletti Carvalho
Av. Dr. Arnaldo, 455, São Paulo, SP.
Brazil.
Dr. Ming-hui Zhao
Professor of Medicine
Director of Renal Division, Department of Medicine
Peking University First Hospital
Beijing 100034
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Guangdong Cardiovascular Institute, Guangdong General
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China.
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Faculty of Engineering and Applied Science,
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Medical Intensive Care Unit
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Department of Gynaecology and Obstetrics, the 1st
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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,
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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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African Journal of Pharmacy and Pharmacology
International Journal of Medicine and Medical Sciences
Table of Contents: Volume 6 Number 26 15 July, 2012
ences
ARTICLES
Research Articles
Antibacterial and synergistic effects from aerial part of Kalimeris
yomena Kitamura extract against methicillin-resistant Staphylococcus
aureus 1883
Young-Hwa Kim, Dae-Ki Joung, Dong-Young Shin, Kyung-Hee Lee,
Dong-Yeul Kwon, Jang-Gi Choi, Dong-Soo-Ha, and Dong-Won Shin
New solid phase extraction reversed phase high performance liquid
chromatography ultraviolet (RP-HPLC-UV) method for simultaneous
determination of tenofovir and emtricitabine in Chinese population 1890
Rui Tan, Bo Yan, Jingchuan Shang, Junqing Yang, Wenxiang Huang,
Xiaoni Zhong, Ailong Huang and Mingfeng Wang
Cardio-tonic effect of the aqueous extract of whole plant of Crataegus
aroniasyn: azarolus (L) on isolated Rabbit’s heart 1901
Abdullah S. Shatoor
Efficacy and safety profile of moxifloxacin in treatment of urogenital
system infections: A meta-analysis of randomized controlled trials 1910
Yanping Mu, Jinyu Huang, Ruilin Liu, Xun Deng, Liqing Wang, Ping Fang,
Meidan Wei, and Yong Wang
The expression of tumor metastasis suppressor gene KAI1 and matrix
metalloproteinase 2 in breast cancer tissues 1923
Tong Zhang, Guoping Ren, Zhenhai Zhang, Rui Zhang and Yuanhang Li
Comparative excretion of vitexin-2"-O-rhamnoside in mice after oral and
intravenous administration 1927
Ye An, Chaoshen Zhang, Yang Du, Lin Zhao, Zhongzhe Cheng, Wenjie Zhang,
Xixiang Ying and Tingguo Kang

Table of Contents: Volume 6 Number 26 15 July, 2012
ences
ARTICLES
Comparison of side effects and marital satisfaction between the women
taking cyclofem and depo-medroxyprogesterone contraceptive ampoules 1933
Gholamitabar Tabari M, Moslemi L., Esmaelzadeh S. and Bijani A.
Effect of salidroside on Bcl-2/Bax protein levels in rats with hemisection-
induced spinal cord injury 1938
Ming Wang, Ning Zhang, Rili Ji and Jing Zhao
Evaluation of the phytochemical and hemostatic potential of Jatropha
multifida sap 1943
Tamègnon Victorien Dougnon, Jean Robert Klotoé, Julien Sègbo,
Jean-Marc Atègbo, Aléodjrodo Patrick Edorh, Fernand Gbaguidi, Armelle
Sabine Hounkpatin, Carlos Dandjesso, Lauris Fah, Brice Fanou, Karim
Dramane and Frédéric Loko
Effects of penehyclidine hydrochloride and anisodamine on acute kidney
injury induced by two-hit Rats 1949
Dong-Ting Chen, Chun-Shui Lin, Miao-Ning Gu, Zhen-Long Zhao, Rang-Hui Yu,
Jin-Dong Xu and Man-Ling Tan
Protective effect of green tea on CCl4 induced hepatoxicity in
experimental rats 1958
Syed M. Shahid, Sana Shamim and Tabassum Mahboob
Preparation and biological activity of saponin from Ophiopogon japonicus 1964
Shuang-Li Xiong, Da-Bin Hou, Ni Huang and Anlin Li
Statistical analysis of medical experiment data for discovering groups of
correlated symptoms 1971
Chenghe Shi, Qingqiong Deng, Peng Lu, Minquan Zhou and Gang Xiong

Table of Contents: Volume 6 Number 26 13 July, 2012
ences
ARTICLES
Comparative analysis of distinct diuretics through urine analysis 1977
Zain Ullah, Musa Kaleem Baloch, Jameel Ahmed Khader, Naser Mohamed
AbdElsalam, Riaz Ullah, Arif-ullah Khan, Muhammad Talha, Naeem Khan
and Iqbal Hussain
Antimicrobial and anticoagulant activities of Citrullus colocynthis Schrad.
leaves from Tunisia (Medenine) 1982
Belsem Marzouk, Ehsen Haloui, Najoua Akremi, Mahjoub Aouni, Zohra
Marzouk and Nadia Fenina

African Journal of Pharmacy and Pharmacology Vol. 6(26), pp. 1883-1889, 13 July, 2012
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP11.791
ISSN 1996-0816 ©2012 Academic Journals
Full Length Research Paper
Antibacterial and synergistic effects from aerial part of
Kalimeris yomena Kitamura extract against methicillinresistant
Staphylococcus aureus
Young-Hwa Kim 1 , Dae-Ki Joung 1 , Dong-Young Shin 1 , Kyung-Hee Lee 1 , Dong-Yeul Kwon 2 ,
Jang-Gi Choi 2 , Dong-Soo-Ha 3 and Dong-Won Shin 1 *
1 Department of Oriental Medicine Resources.College of bio Industry Science, Sunchon National University,
Sunchon Jeonnam 540-742, Republic of Korea.
2 Department of oriental pharmacy, college of Pharmacy, Wonkwang Oriental Medicines Research Institute,
Wonkwang University, Jeonbuk 570-749 Republic of Korea.
3 Department of Chemistry Education Sunchon National University, Sunchon National University,
Sunchon Jeonnam 540-742, Republic of Korea.
Accepted 7 May, 2012
Methicillin-resistant Staphylococcus aureus (MRSA) is recognized as a serious clinical problem
worldwide. Consequently, a recent investigation was conducted on the antibacterial activity and aerial
part of Kalimeris yomena Kitamura. The antibacterial activities of ethanol (EtOH) extract of K. yomena
and its n-hexane, EtOAc, n-BuOH and H2O fractions were evaluated against 15 strains of MRSA and 1
standard methicillin-susceptible S. aureus (MSSA) strain. The n-hexane fraction showed a positive
antibacterial activity against all the tested strains. The minimum inhibitory concentrations (MICs) of nhexane
fraction were in the range of 31.3 to 250 μg/ml. The disc diffusion test diameter of the inhibition
zone was in the range of 12 to 17 mm. Thereafter, the combination of n-hexane fraction of K. yomena
(HFK) + ampicillin (AM) or oxacillin (OX), fractional inhibitory concentrations index (FICI) values for HFK
+ AM ranged between 0.25 and 0.5, and HFK + Ox ranged between 0.5 and 0.56, which show increased
synergistic effects. Determining time-kill curves also confirmed the improved synergism of HFK and AM
combination against 4, 8, 12 and 24 h cultured MRSA. This result encouraged us to think that n-hexane
fraction of aerial part of K. yomena can be used as a natural antibacterial agent.
Key words: Kalimeris yomena Kitamura, aster yomena, antibacterial, ethanol (EtOH) extract, combination
methicillin-resistant Staphylococcus aureus (MRSA).
INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA) has
been a problem since the 1960s as its infection is
associated with higher mortality and increase cost in the
hospitals (Klevens et al., 2007). It becomes more and
more evident that bacteria, when faced with a new
developed drug, respond with clever mechanisms of
resistance (Tenover, 2006). Today, with this emergence
*Corresponding author. E-mail: sdw@sunchon.ac.kr. Tel: +82
61 750 3665.
of antibiotic resistant pathogens like MRSA, a new
approach to natural products must be taken. These
natural products are increasingly in demand due to their
non-side effect benefit (Ghosh et al., 2008). Therefore,
our ongoing efforts to find bioactive natural products have
led us to study the antibacterial activity of Kalimeris
yomena. K. yomena (Asteraceae) is a perennial herb that is
found in Korea, Japan, China and Siberia. As a kind of
folk remedy, it has been used for the treatment of cough,
asthma, insect bite (Jung et al., 2005; Lee, 1996). In
addition, though not scientifically tested until now, its
cancer prevention qualities (Jung et al., 2005),

1884 Afr. J. Pharm. Pharmacol.
Table 1. Staphylococcus aureus strains used in the experiments.
S. aureus strain Class mecA gene β-Lactamase activity Antibiotic resistance pattern
ATCC 33591 MRSA + + AM, OX
ATCC 25923 MSSA - - -
Clinical isolates MRSA
DPS-1 a
MRSA + - AM, OX
DPS-2 MRSA + + AM, OX
DPS-3 MRSA + - AM, OX
DPS-4 MRSA + - AM, OX
DPS-5 MRSA + - AM, OX
DPS-6 MRSA + - AM, OX
DPS-7 MRSA + + AM, OX
DPS-8 MRSA + + AM, OX
DPS-9 MRSA + + AM, OX
DPS-10 MRSA + + AM, OX
DPS-11 MRSA + - AM, OX
DPS-12 MRSA + + AM, OX
DPS-13 MRSA + - AM, OX
DPS-14 MRSA + - AM, OX
(+), positive; (-), negative; AM, ampicillin; OX, oxacillin.
antioxidant qualities (Bae and Kim, 2009) has been
proven. The essential oils were obtained from K. yomena
and were analyzed for its chemical composition by gas
chromatography-mass spectrometry; there were 68
constituents in the essential oil: 48 hydrocarbons, 8
alcohols, 3 ketones, 2 oxides, 2 acetates, 1 aldehyde, 1
acid, and 1 anhydride. The Major constituents were
germacrene D (11.56%), camphor (5.23%),
caryophyllene oxide (3.38%), caryophyllene (3.18%) and
germarcrene B (3.09%). Through solid-phase microextraction
(SPME), 13 constituents were identified: 12
hydrocarbons and 1 ketone. Major constituents of the
SPME extracted sample were germarcrene D (45.75%),
γ-gurjunene (9.39%), γ-selinene (8.33%), camphor
(4.81%) and β-caryophyllene (3.05%). (Yeon et al., 2011)
However, the antibacterial activity of K. yomena against
MRSA has not been studied. The present study
investigated antibacterial activity of k. yomena and the
synergistic effects of the mixture of standard antibiotics
[Ampicillin (AM), Oxacillin (OX)], performed minimum
inhibitory concentrations (MICs), the disc diffusion
method, and a time-kill assay to evaluate the
susceptibility of K. yomena against 16 MRSA strains.
MATERIALS AND METHODS
Plant material and sample preparation
Aerial parts of K. yomena were collected from suncheon, southern
republic of Korea, in September, 2010. Samples were identified by
Prof. Dong-young Shin of the Department of Development in Plant
Resources; a voucher specimen was deposited in the Laboratory of
Oriental Pharmacology (N.7718). Thereafter, Aerial part of K.
yomena was air-dried to 200 g, which were then boiled in 2 L of
ethanol (EtOH) for 3 h. The EtOH extract aerial part of K. yomena
(10.68% w/w) was partitioned with organic solvents of different
polarities to yield n-hexane, EtOAc, n-BuOH and H2O fractions, in
sequence. The samples were stored at 4°C.
Equipment
An incubator (vision co, seoul, Korea).
Test microorganisms
Among the 16 S. aureus strains used in this study (Table 1),
Fourteen (14) clinical isolates (MRSA) were obtained from 14
different patients at Wonkwang University Hospital (Iksan, South
Korea). The Other 2 strains were S. aureus ATCC 33591
(Methicillin-resistant strain) and S. aureus ATCC 25923 (Methicillinsusceptible
strain).
ATCC 25923 (American Type Culture Collection, Manassas, VA,
USA) and ATCC 33591 were commercially purchased. Before use,
all bacteria were stored in 30% glycerol and frozen at -70°C. The
bacteria were cultured in Mueller-Hinton broth (MHB) and Mueller-
Hinton agar (MHA) (Difco Laboratories, Baltimore, MD, USA).
Bacteria were suspended in MHB and then incubated at 37°C for
24 h.
Antibiotics
AM and OX (Sigma Chemical Co. St.Louis, M0, USA).

Minimum inhibitory concentration (MIC)
The MIC was determined using the broth microdilution method
according to the clinical and Laboratory Standards Institute
guideline (CLSI, 2000). Briefly, a preparation of the microorganisms
inoculated was done on a 24 h broth cultures, and the suspensions
were adjusted to a 0.5 McFarland standard turbidity (approximately
1.5�10 8 CFU/ml). Final inoculums were adjusted to the 1.5�10 5
CFU/ml. These serially diluted cultures were then incubated at 37°C
for 18 h. MIC was defined at the lowest concentration of AM, OX,
aerial part K. yomena extract, Fractions (n-hexane, EtOAc, n-BuOH,
H2O).
Disc diffusion method
The disc diffusion method was as prescribed by the Clinical and
Laboratory standards Institute standards, using a modified agarwell
diffusion method (CLSI, 2001). Bacterial strains grown on MHA
at 37°C for 18 h were suspended in MHB and adjusted to a turbidity
of 0.5 McFarland standard scale (approximately 1.5�10 8 CFU/ml).
The MHA was poured into petri dishes and inoculated with 100 μl of
the suspension sterile paper discs (diameter 6mm: Tokyo Roshi
Kaihsa, Japan) were punched in the agar and filled with 500 and
250 μg. The dissolution of the organic extracts was facilitated with
the addition of 50% (v/v) dimethyl sulfoxide (DMSO) (50% DMSO
was not active against all strains). (DMSO, Sigma, USA) AM and
OX was used as positive controls, and the disks treated with 50%
DMSO were used as the negative control. The plates were placed
in an incubator (Vision Co, Seoul, Korea) at 37°C for 24 h, after
which the diameter of the zone of inhibition around each of the
discs was measured and recorded. Each experiment was
performed in triplicate.
Checkerboard dilution test
The synergistic combinations were investigated in the preliminary
checkerboard method performed using the MRSA, methicillinsusceptible
S. aureus (MSSA) and one clinical isolate strains via
MIC determination, according to the CLSI guidelines (CLSI, 2006).
The MIC was defined as the lowest concentration of drug alone or
in a combination that inhibited the visible growth. The in vitro
interaction was quantified by determining the fractional inhibitory
concentration (FIC). The FIC index (FICI) was calculated as follows:
FIC = (MIC of drug A in combination/MIC of drug A alone) + (MIC of
drug B in combination/MIC of drug B alone). FIC indices (FICI) were
interpreted as follows: 1.0 to 4.0, indifference; and >4.0,
antagonism. Finally, the varying rates of synergy between the two
agents were determined (Mazumdar et al., 2005; Guadalupe et al.,
2006). All experiments were independently repeated three times.
The time-kill curve assay
The time-kill curve assay was performed according to the method
described (Nascimento et al., 2007) in order to study the combined
effects of time and antimicrobial agent concentration on the
bacterial growth. For this assays, a standard inoculums of
approximately 10 6 CFU/ml of an overnight culture was used. nhexane
fraction of K. yomena (HFK) (0.5 MIC) and AM (0.5 MIC)
were used. Combinations of HFK + AM were also evaluated. A test
plate containing only MHB was inoculated and served as control.
Counts of viable strains were carried out at different intervals up to
24 h at 37°C. The rate and extent of killing was determined by
plotting viable colony counts (CFU/ml) against time in MHA.
According to the method described (Nascimento et al., 2007), all
experiments were independently repeated three times.
Kim et al. 1885
Colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl
tetrazolium bromide (MTT) test
A colorimetric assay based on MTT for rapid detection of the
presence of bacteria was performed as previously described
(Scheuber et al., 1983; Abate et al., 1998; Shi et al., 2008). Briefly,
a stock solution of 5 mg/ml MTT (Sigma) was prepared in
phosphate-buffered saline and kept at – 70°C. A final concentration
of 1 mg/ml of MTT was used in the assay. After 24 h of incubation a
37°C, 20 μl of the yellow MTT was added to the 96-well microtiter
plate (0.3 ml volume) and incubated for an additional 20 min. The
presence of a blue color indicates the presence of bacteria.
RESULTS
Antimicrobial activity of K. yomena EtOH extract and nhexane,
EtOAc, n-BuOH and H2O fractions for exhibited
antimicrobial activities against 15 MRSA and 1 MSSA
strains.
Among them, the n-hexane fraction was remarkable
which was even higher than that of the EtOH extract
shown on Table 2. Other fractions (except EtOAc) did not
show antimicrobial activity against MRSA.
The n-hexane fraction showed a significant
antibacterial activity against all the tested strains. The
MICs of n-hexane against MRSA strains ranged from
31.3 to 250 μg/ml and excellent antibacterial effect than
AM and OX against some clinical isolates (DPS-2, 8, 10)
MRSA (Table 3).
Antibacterial activity n-hexane fraction used as the
control group showed no significant difference AM, and
seven other strains (DPS-7, 8, 9, 10, 11, 12, 14) rather
showed better antibacterial activity. OX did not indicate
any antibacterial activity within clinical isolates strains
used in the control group, however, n-haxane fraction
showed more antibacterial activity (Table 4).
For the determination of in vitro combinations test we
selected a clinical isolate MRSA, a standard MRSA
strain, and a standard MSSA strain. This test was
performed to determine the action of HFK alone as well
as its synergistic action with AM or OX against the 3
strains. HFK lowered the MICs of AM and OX against the
MRSA strains. For the standard MSSA strain, the HFK
lowered the MICs of both AM and OX. The combined
activity of HFK and two antibacterial agents (AM, OX)
against 2 MRSA strains resulted in FICI ranged from 0.25
to 0.5 and from 0.5 to 0.56, respectively (Tables 5 and 6).
So the combination effect of HFK with AM and OX was
found to be synergistic or partially synergistic. We found
that HFK reduced MICs of AM and OX.
Time-kill tests were performed to study the
synergisticeffects of HFK and AM with time. Figures 1
and 2 show the test results of the time-kill assay against
a standard MRSA strain, and the DPS-3 strain. On the
tested MHA plates, remarkably lower numbers of
colonies were detected as compared to the MHA plates

1886 Afr. J. Pharm. Pharmacol.
Table 2. Antimicrobial activity aerial part of K. yomena EtOH extract and fractions against S. aureus strain ATCC33591.
Minimal inhibitory concentration (MIC)(μg/ml)
S. aureus strain Extract Fractions Antibiotic resistance pattern
EtOH n-hexane EtOAc n-BuOH H2O Ampicillin Oxacillin
ATCC 33591 500 250 2000 ND ND 125 125
ATCC 25923 250 62.5 1000 ND ND 0.24 0.49
DPS-1 a 500 125 2000 ND ND 62.5 500
DPS-2 250 31.3 1000 ND ND 250 125
DPS-3 500 125 2000 ND ND 62.5 500
DPS-4 500 125 2000 ND ND 62.5 500
DPS-5 250 250 2000 ND ND 15.63 500
a , DPS indicates S. aureus strains from the Department of Plastic Surgery, Wonkwang University Hospital. ND, no detected activity at this
concentration.
Table 3. Antimicrobial activity aerial part of K. yomena n-hexane fraction against S. aureus strain ATCC33591, ATCC25923,
and 14 MRSA.
S. aureus strain
Minimal inhibitory concentration (MIC) (μg/ml)
n-hexane fraction Ampicillin Oxacillin
ATCC 33591 (MRSA) 250 125 125
ATCC 25923 (MSSA) 62.5 0.24 0.49
Clinical isolates MRSA
DPS-1 a
125 62.5 500
DPS-2 31.3 250 125
DPS-3 125 62.5 500
DPS-4 125 62.5 500
DPS-5 250 15.6 500
DPS-6 62.5 31.3 500
DPS-7 125 125 500
DPS-8 62.5 125 500
DPS-9 125 125 500
DPS-10 62.5 125 500
DPS-11 125 125 500
DPS-12 125 125 500
DPS-13 250 62.5 500
DPS-14 125 125 500
a , DPS indicates S. aureus strains from the Department of Plastic Surgery, Wonkwang University Hospital.
treated with each HFK and AM. The effect of HFK with
AM was synergistic in FICIs. Each of these combination
regimens were then tested together in a time kill at 4
times the respective isolate MIC. In this same time kill,
HFK significantly enhanced AM activity. (Figures 1 and 2)
DISCUSSION
Due to the recent appearance of MRSA and the "Super
Bacteria" showing the resistance to multiple antibiotics,
the development of new antibiotics is urgently required,
which is even tendered as a social issue. The most
effective method is to develop antibiotics from the natural
products without having any toxic or side effects and
develop substances showing synergistic effect by the
combined application with conventional antibiotics.
Therefore, there is a need to develop alternative
antimicrobial drugs for the treatment of infectious
diseases. (Ahmad et al., 1998; Berahou et al., 2007).
Conclusion
We conclude that the aerial part of K. yomena extract has
an antibacterial effect on MRSA and MSSA. Especially
within n-hexane fraction for the case of MRSA was found

Kim et al. 1887
Table 4. Antimicrobial activity aerial part of K. yomena n-hexane fraction against S. aureus strain ATCC33591, ATCC25923,
and 14 MRSA.
Zone of Inhibition (mm) (μg/ml)
S. aureus strain n-hexane fraction Ampicillin Oxacillin
500 μg 250 μg 500 μg 250 μg 500 μg 250 μg
ATCC 33591 16 15 17 14 18 14
ATCC 25923 16 13 46 44 41 39
Clinical isolates
DPS-1 a
15 13 18 14 ND ND
DPS-2 17 15 19 18 20 19
DPS-3 15 14 19 18 ND ND
DPS-4 16 13 19 14 ND ND
DPS-5 16 14 19 14 ND ND
DPS-6 15 13 17 14 ND ND
DPS-7 15 14 13 11 ND ND
DPS-8 15 14 13 12 ND ND
DPS-9 14 13 14 12 ND ND
DPS-10 15 14 12 9 ND ND
DPS-11 14 12 13 11 ND ND
DPS-12 14 12 14 11 ND ND
DPS-13 15 12 18 13 ND ND
DPS-14 15 14 14 11 ND ND
a DPS indicates S. aureus strains from the Department of Plastic Surgery, Wonkwang University Hospital. ND, no detected activity at this
concentration.
Table 5. The interpreted FICI response for HFK + AM combinations against a standard MRSA strain, a standard MSSA strain
and an MRSA isolate.
MIC of HFK + AM (μg/mL)
Strian HFK Ampicillin
S. aureus strain Alone with AM Alone with HX FICI b Outcome
ATCC 33591 250 31.3 125 15.6 0.25 Synerge
ATCC 25923 62.5 0.06 0.24 0.015 0.13 Synerge
Clinical isolates
DPS-3 a
125 31.3 62.5 15.6 0.5 Partial synergy
a , DPS indicates S. aureus strains from the Department of Plastic Surgery, Wonkwang University Hospital. b FICI, fractional inhibitory
concentration index; HFK, n-hexane fraction aerial part of K. yomena Kitamura.
Table 6. The interpreted FICI response for HFK + OX combinations against a standard MRSA strain, a standard MSSA strain
and an MRSA isolate.
MIC of HFK + OX (μg/ml)
Strain HFK Oxacillin
S. aureus strain Alone with OX Alone with HX FICI b Outcome
ATCC 33591 250 62.5 125 15.6 0.5 Partial synergy
ATCC 25923 62.5 3.9 0.48 0.03 0.06 Synerge
Clinical isolates
DPS-3 a
125 125 500 31.7 0.56 Partial synergy
a , DPS indicates S. aureus strains from the Department of Plastic Surgery, Wonkwang University Hospital.

1888 Afr. J. Pharm. Pharmacol.
Log10 (CFU/ml)
Time (h)
Figure 1. The time-kill curves of HFK and AM against the standard MRSA ( ATCC 33591) strain.
Log10 (CFU/ml)
Time (h)
Figure 2. The time-kill curves of HFK and AM against the Clinical Isolate MRSA ( DPS-3) strain.
to have good antimicrobial activity. Additional research in
the future with detailed analysis to determine exact
composition and development of a new antibiotic by
deriving natural products such as K. yomena should be
positively considered.
ACKNOWLEDGEMENT
This study was supported by Sunchon National University
Research Fund in 2012.
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African Journal of Pharmacy and Pharmacology Vol. 6(26), pp. 1890-1900, 15 July, 2012
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP12.128
ISSN 1996-0816 ©2012 Academic Journals
Full Length Research Paper
New solid phase extraction reversed phase high
performance liquid chromatography ultraviolet
(RP-HPLC-UV) method for simultaneous determination
of tenofovir and emtricitabine in Chinese population
Rui Tan 1 , Bo Yan 2 , Jingchuan Shang 2 , Junqing Yang 1 *, Wenxiang Huang 3 , Xiaoni Zhong 3 ,
Ailong Huang 3 and Mingfeng Wang 1
1 Department of Pharmacology, Chongqing Medical University, Chongqing, China.
2 Department of Pharmaceutical Analysis, Chongqing Medical University, Chongqing, China.
3 Institute for Viral Hepatitis, Key Laboratory of Molecular Biology on Infectious Diseases, Ministry of Education,
Chongqing Medical University, Chongqing, China.
Accepted 23 May, 2012
The objective of this study is to develop and validate a method of reversed phase high performance
liquid chromatography (RP-HPLC) assay with ultraviolet (UV) detector after solid phase extraction (SPE)
for simultaneous measure tenofovir and emtricitabine in Chinese population. Detection of analytes was
performed with a Phecda C18 (250 � 4.6 mm, 5 μm), reversed-phase analytical column and a security
guard column C18 (4 � 3.0 mm, 5 μm). An obviously extrusive separation was successively followed with
the SPE method using BOND ELUT-C18 Varian columns and a 30 min gradient elution consisting of
potassium phosphate monobasic buffer with 0.08% of triethanolamine (pH 3.52) and methanol with UV
detector at 270 nm. The results of this study showed that the method used in this study was simple,
accurate and sensitive with a wide linear range from 10 to 5000 ng/ml for both tenofovir and
emtricitabine when 300 μl aliquots was analyzed. The intra- and inter-day precision and accuracy for
both analytes were lower than ±20% at the limit of quantification (LOQ) and ±15% at the other quality
control (QC) levels. The absolute recoveries for tenofovir and emtricitabine were 80.6 and 86.5%,
respectively. This method is suitable for routinely monitor plasma concentrations of tenofovir and
emtricitabine in Chinese population.
Key words: High performance liquid chromatography ultraviolet (HPLC-UV), solid phase extraction (SPE),
pre-exposure chemoprophylaxis (PrEP), tenofovir, emtricitabine.
INTRODUCTION
With a global estimate of 2.7 million, new human
immunodeficiency virus (HIV) infections occurring
worldwide each year and even more undiagnosed cases
waiting to be discovered, discovering novel methods to
help stem the spread of this virus is critical. While
behavior change programs have contributed to dramatic
reductions in the number of annual infections in the U.S.
*Corresponding author. E-mail: cqjqyang2004@yahoo.com.cn.
Tel: +86 23 6848 5071. Fax: +86 23 6848 5161.
and many other nations, far too many individuals remain
at high risk. With an effective vaccine years away, there
is a growing body of evidence that antiretroviral drugs
may be able to play an important role in reducing the risk
of HIV infection (http:// www. cdc. gov, 2008). Preexposure
chemoprophylaxis (PrEP) is being explored as
a novel strategy to interrupt the spread of HIV in at-risk
patient populations such as men who have sex with men
(MSM), injecting drug users (IDU), and female sex
workers (Clauson et al., 2009). The Centers for Disease
Control and Prevention (CDC) is sponsoring three clinical
trials of PrEP, and is participating in a University of

Figure 1. Chemical structure of tenofovir disoproxil fumarate (TDF).
Figure 2. Chemical structure of emtricitabine.
Washington-sponsored trial in Kenya and Uganda.
Similar PrEP trials are also being conducted by Family
Health International (FHI), the National Institutes of
Health (NIH), FHI and the Microbicide Trials Network
(MTN) (http:// www. cdc. gov, 2008). Recently, a project
named feasibility study of PrEP to decrease the infection
of HIV for High Risk Group in Western China has been
initiated by Chongqing Medical University to study the
safety, compliance and resistance of tenofovir and
emtricitabine for PrEP.
Some researchers believe that an antiretroviral drug
taken as a daily oral preventative is one of the most
important new prevention approaches of HIV infection
and several sources of data suggest that the use of
antiretroviral drugs in this manner may be effective.
Animal studies have demonstrated that pre-exposure
administration of tenofovir disoproxil fumarate (TDF) plus
emtricitabine provided significant protection to monkeys
exposed repeatedly to an HIV-like virus. These data,
combined with the drug’s favorable resistance, safety
profiles and long intracellular half times make TDF and
TDF plus emtricitabine ideal candidates for HIV
prevention trials (http:// www. cdc. gov, 2008).
Tenofovir and emtricitabine are nucleoside/tide reverse
transcriptase inhibitors (NRTI), commonly used to treat
persons infected with HIV-1. VIREAD ® is a trademark for
TDF of Gilead Sciences, Inc., which is a fumaric acid salt
Tan et al. 1891
of the bis-isopropoxycarbonyloxymethyl ester derivative
of tenofovir. It has a molecular formula of
C19H30N5O10P•C4H4O4 and a molecular weight of 635.52.
It’s structural formula is shown in Figure 1
(http://www.accessdata.fda.gov, 2008). It is an oral
prodrug of tenofovir, an acyclic nucleoside phosphonate
(nucleoside) analog of adenosine 5 , -monophosphate,
which has potent activity against retroviruses and
hepadnaviruses. Upon oral administration, it is converted
in vivo to tenofovir by esterase hydrolysis, which is taken
up by cells. And then tenofovir is activated to tenofovir
disphosphate through phosphorylation reaction, the
active form of tenofovir. Tenofovir disphosphate
competes with the natural substate deoxyadenosine 5’triphosphate
for incorporation into DNA during HIV
transcription, which blocks the action of HIV reverse
transcription, prevents further DNA chain elongation, and
cause termination of viral DNA replication (Kwarney et
al., 2004).
Emtricitabine differs from other cytidine analogs in that
it has a fluorine in the 5-position. It has a molecular
formula of C8H10FN3O3S and a molecular weight of
247.24. Its structural formula is shown in Figure 2. It is
phosphorylated by cellular enzymes to form emtricitabine
5’-triphosphate, which inhibits the activity of the HIV-1
reverse transcriptase (RT) by being competing with the
natural substrate deoxycytidine 5’- triphosphate and by

1892 Afr. J. Pharm. Pharmacol.
Table 1. The gradient elution table of mobile A and B over 30 min of run time.
Time (min) Mobile phase A (%) Mobile phase B (%)
0.00 90 10
2.00 90 10
18.00 80 20
19.00 10 90
21.00 10 90
22.00 90 10
30.00 90 10
being incorporated into nascent viral DNA which results
in chain termination (http://www. accessdata.fda. gov,
2008).
In the project of feasibility study of PrEP to decrease
the infection of HIV for High Risk Group in Western
China, measurement of the two drug levels in plasma of
Chinese people was required to define pharmacokinetic
properties, monitor the drug administration compliance
,design rational PrEP modalities, and understand the
correlation between drug levels and protection. Several
methods for determination of tenofovir or tenofovir
combined with emtricitabine were described. Avolio et al.
(2008) set up an assay based on solid phase extraction
(SPE) procedure with liquid chromatography-mass
spectrometry (LC-MS) to measure plasmatic concentrations
of tenofovir and emtricitabine in HIV infected
patients. Gomes developed a LC-tandem MS (LC-
MS/MS) method for simultaneous determination of
tenofovir and emtricitabine (Gomes et al., 2008). Also,
Delahunty et al. (2006) developed and validated an
LC/MS/MS assay for the determination of tenofovir.
Theses assay methods were costly because of expensive
performance apparatus and it was not really convenient
for the developing countries, in which the infection
percentage of HIV was in a large population. Barkil used
LC method coupled to UV and single MS detection to
determine tenofovir in human plasma, and concluded that
both detections were allowed to obtain a reliable
quantification of tenofovir (Barkil et al. 2007).
The aim of this study was to set up a simple and
reliable method for simultaneous determination of
tenofovir and emtricitabine in plasma of Chinese people
with high performance liquid chromatography-ultraviolet
(HPLC-UV) detector.
MATERIALS AND METHODS
Chemicals and regents
Chemical standards of tenofovir and lamivudine (internal standard,
IS.) were kindly provided by Yingpu Science and Technology
Development Co., Ltd. (Hangzhou, China). Emtricitabine was
supplied by Sichuan Baili Pharmaceuticals.
Methanol of HPLC grade was obtained from Hanbon Science
and Technology Co., Ltd (Jiangsu, China). High purity water used in
the mobile phase and solution preparation was freshly prepared
from Milli-Q Academic (Millipore, Bedford, USA). Potassium
phosphate monobasic used for the preparation of mobile phase
was provided by Bo Yi, Chongqing Chemical Regant Co., Ltd
(Chongqing, China). Triethanolamine used for mobile phase A was
provided by Zhengzhou Zhongtian Test Instruments (Zhengzhou,
China). Phosphoric acid for pH regulation of mobile phase was
obtained from Chengdu Kelong Chemical Regant Factory
(Chengdu, China). Ammonium acetate of analytical grade used in
SPE was purchased from Chuandong Chemical Industry Group
Co., Ltd. (Chongqing, China), while sodium hydroxide was from
Zhengzhou Chemical Factory.
Drug free (blank) heparinised human plasma was obtained from
healthy volunteers and was stored at -20°C prior to use. SPE
columns (3.0 ml, 200 mg, BOND ELUT-C18) purchased from Varian
(Lake Forest, CA, USA) was employed.
VIREAD ® for volunteers was provided by Gilead Sciences, Inc.
and emtricitabine was purchased from Hebei Pharmaceutical
company, Hebei Medical University.
Chromatographic conditions and instrumentation
The chromatographic separation was performed at 35°C, on a
Phecda C18 analytical column (4.6 � 250 mm, 5 μm, Hanbon
Science and Technology, China), with a Phenomenex ® C18 security
guard column (4.0 � 3.0 mm). The total run time for each sample
was 30 min, delivered with a flow rate of 1.0 ml/min. Run time was
achieved with a gradient elution (Table 1) consisting of potassium
phosphate monobasic buffer with 0.08% of triethanolamine (pH
3.52) as mobile phase A, and methanol as mobile phase B. The
buffer solution was filtered through a 0.45 um membrane filter. For
UV detection, wavelength was monitored at 270 nm in order to
detect tenofovir, emtricitabine and lamivudine, simultaneously.
A HPLC system of LC-2010A HT LIQUID CHROMATOGRAPH
from SHIMADZU CROPORATION run on a Dell computer
(operated with Windows 2000 Professional), was used for this
method.
Preparation of stock standard solutions, working standard
solutions, plasma calibration curve samples and quality
controls
Standards were accurately weighed and then dissolved with HPLCgrade
water to produce the corresponding stock standard solutions
(40 μg/ml for lamivudine, and 1 mg/ml for each for tenofovir and
emtricitabine), refrigerated at 4°C until use.
A 9-point working standard solutions (both tenofovir and
emtictabine) for calibration curve were prepared by diluting the
stock solutions with HPLC-grade water from 1 to 500 μg/ml; and
plasma calibration curve standards were obtained from 10 to 5000

ng/ml by 4.9 ml blank plasma with 50 ul stock standard solution of
both tenofovir and emtricitabine at each point.
Quality control (QC) samples were prepared at three
concentration levels of 20, 200 and 2000 ng/ml for both standards
in the same manner with the preparation of plasma calibration
curve samples.
Sample preparation
A 300 μl aliquot of human plasma sample was mixed with 20 μl of
IS working solution (30 μl/ml of lamivudine) and 180 ul of 10%
trichloroacetic acid solution. Then the solution was vortexed for 30 s
and centrifuged at 13,000 rpm for 10 min, at room temperature. The
supernatant fluid was neutralized by 240 μl of 1% sodium hydroxide
solution and then loaded in the SPE cartridge, which was
conditioned by 3 ml methanol and 3 ml of 150 mM ammonium
acetate (pH 5.0). After washed with 900 μl of 100 mM ammonium
acetate (pH 7.0) in order to obtain less interference at the retention
time of each analyte, the column was eluted by 500 μl of methanol
to receive the drugs. Finally, the elute was evaporated to dryness
by using a concentrator (eppendorf ® , Germany) in vacuum at room
temperature. The extracted sample was reconstituted with 120 μl of
water and methanol solution (90:10,v/v) and 100 µl was injected
into the chromatographic system.
Method validation
Selectivity
Selectivity of the method was investigated by analyzing standards
solutions, blank plasma samples of four different blank plasma
samples and the spiked samples, in order to determine if any
interference of endogenous compounds existed at the retention
time of tenofovir, emtricitabine, and lamivudine.
Linearity
Both calibration curves of two drugs were established by plotting
the peak area ratio (tenofovir/lamivudine or emtricitabine
/lamivudine) as a function of plasma concentration of the aim
analytes. Each calibration curve was obtained using 9 calibration
points (n = 5) of plasma calibration curve samples prepared as 2.4,
which were 10, 20, 50 ,100, 200, 500, 1000, 2000 and 5000 ng/ml,
respectively.
The limit of determination and quantification
The limit of quantification (LOQ) was the smallest analytical
concentration which can be detected with a signal noise ratio more
than 3. The lower LOQ (LLOQ) was the smallest analytical
concentration which could be measured with accuracy and
precision still less than 20%.
Accuracy and precision
Accuracy, intra- and inter-day precision were evaluated by replicate
analysis (n = 5) of the three QC levels at the concentrations of 20,
200 and 2000 ng/ml.
Five replicates at each level of quality controls were assayed at
the first day for the accuracy and intra-day precision experiment.
Another five replicates at each level of quality controls were
assayed within five different days for the inter-day precision
experiment.
Tan et al. 1893
Accuracy is defined as the percent relative error (%RE) and was
calculated by using the formula:
%RE = (E-T)(100/T)
Where E is the experimentally determined concentration and T is
theoretical concentration.
Intra- and inter-day precision were expressed as relative
standard deviation:
(%R.S.D.) = (S.D./M)(100),
Where M is the mean of the experimentally determined
concentration and S.D. is the standard deviation of M at each
concentration.
Recovery
Recovery of the tenofovir and emtricitabine after extraction
procedures was assessed by comparing the peak area of the
extracted plasma samples (n=5) with those of non-extracted
standard solutions at concentrations corresponding to the three QC
values (20, 200 and 2000 ng/ml).
Stability
Stability of tenofovir and emtricitabine in spiked plasma control
samples was determined in triplicate at three concentrations (20,
200 and 2000 ng/ml) including freeze-thaw, auto sampler and longterm
stabilities. The acceptance criterion was ±15% difference
between the peak area of observed and theoretical concentrations.
Freeze-thaw stability of analytes was determined by assaying the
three levels of samples in triplicate over three freeze-thawing
cycles. The long-term stability of spiked plasma at -20°C was also
evaluated by assaying weekly in triplicate, three concentration
levels of samples stored at −20°C for a 2-month period.
Additionally, stability of extracted samples after the final
reconstitution during storage in the auto sampler for 24 h at room
temperature was also verified.
RESULTS
The mean retention times around 7.7 ± 0.2 min for the IS
(lamivudine), around 6.5 ± 0.2 min for tenofovir and 12.5
± 0.1 min for emtricitabine. No endogenous substances
interfered with any of the analytes in blank plasma.
Selectivity
Chromatogram of standard for tenofovir, emtricitabine
and lamivudine is shown in Figures 11, 12, and 13.
Representative chromatogram of the blank human
plasma is shown in Figure 3. Figure 4 shows
chromatogram of the extracted spiked plasma with the
concentration of 200 ng/ml for tenofovir, emtricitabine and
IS.
Linearity
The peak area ratios of analyte to IS for calibration

1894 Afr. J. Pharm. Pharmacol.
Figure 3. Chromatogram of drug-free human plasma of volunteers.
Figure 4. Chromatogram of plasma spiked with tenofovir and emtricitabine at 200 ng/ml.
standards were proportional to the concentration of each
drug in plasma over the range tested. The calibration
curves recorded have satisfied the requirement of
regression coefficient (R 2 ) higher than 0.999, with 0.9998
for tenofovir and 0.9997 for emtricitabine. Across the 9
points taken as calibration standards, the R.S.D. and
%RE obtained were within ±20% at the LLOQ and ±15%
for the rest of the tested concentration.

Figure 5. Chromatogram of plasma spiked with tenofovir and emtricitabine at the highest level of QC
concentration (2000 ng/ml).
Figure 6. Chromatogram of plasma spiked with tenofovir and emtricitabine at the lowest level of QC
concentration (20 ng/ml).
The limit of determination and quantification
The LOQ was 8.0 ng/ml for tenofovir and 3.0 ng/ml for
emtricitabine with the signal noise ratio more than 3. The
LLOQ for both analytes was determined as 10 ng/ml,
which was the lowest concentration that could be
measured with a R.S.D. within 20% and accuracy
between 80 and 120%. The upper LOQ was 5,000 ng/ml.
Tan et al. 1895
Chromatogram for the QC samples 20, 200 and 2000
ng/ml are shown in Figures 4, 5 and 6, respectively.
Accuracy and precision
Results of the validation of the method are shown in
Table 2. Intra- and inter-day period expressed as percent

1896 Afr. J. Pharm. Pharmacol.
Table 2. Summary of accuracy and precision (%) for method validation at low .medium and high concentrations.
Analyte
Tenofovir
Emtricitabine
Theoretical concentration
(ng/ml)
Observed concentration
(ng/ml)
Accuracy
(%RE)
Intra-day precision
(R.S.D%)
Inter-day precision
(R.S.D%)
20 22.1 110.5 9.6 9.3
200 210.8 105.4 3.8 6.3
2000 2122.0 106.1 2.4 5.4
20 22.1 110.5 7.1 13.4
200 219.2 109.6 5.6 7.0
2000 2152.0 107.6 2.6 5.6
R.S.D. were ≤6.3% for tenofovir and ≤7.0% for
emtricitabine, whereas at the LOQ levels was ≤9.6 for
tenofovir and ≤13.4% for emtricitabine, respectively. The
accuracy in terms of %RE was within the range of 105.4
to 110.5% and 107.6 to 110.5% for tenofovir and
emtricitabine, respectively.
Recovery
The mean recovery obtained by analyzing two pairs of
samples has values of 80.6% for tenofovir
(%R.S.D.:4.8%), 86.5% for emtricitabine (%R.S.D.:3.3%)
and 81.7% for IS-lamivudine (%R.S.D.:3.5%),
respectively.
Stability
The analytes stability was demonstrated in various
conditions by the deviation of observed concentration
from the nominal one in the range within ±15%, as the
requirements stipulated. The result revealed that
tenofovir and emtricitabine were stable for at least 24 h in
the auto sampler at room temperature. It was confirmed
that repeated freeze and thawing (three cycles) of spiked
plasma samples did not affect the stability. And in the
long-term, stability results also indicated that both
analytes, stored in a freezer at -20°C in matrix, remained
stable for at least 2 months.
Analysis of samples from volunteers
Described method of this study was applied by analyzing
plasma samples collected from 8 healthy volunteers
(males). Volunteers were administrated with a single
dose of tenofovir Viread ® (300 mg) plus emtricitabine
(200mg). The study was approved by the Ethics
Committee on Human Research of Chongqing Medical
University. Figure 7 was the chromatogram of the healthy
volunteer sample.
The pharmacokinetics of tenofovir and emtricitabine in
the healthy population of western China in vivo was in
accordance with two-compartment model. The main
parameters of single administration of tenofovir were as
follows: t1/2 was 12.72 ± 2.83 h, tmax was 1.28 ± 0.29 h,
Cmax was 420.84 ± 96.71 ng/ml, AUC0-t and AUC0-∞ were
3869.42 ± 962.85 and 4107.09 ± 974.82 μg·h·L -1 ,
respectively, and CL/F was 1.28 ± 0.28 L·h -1 ·kg -1 . And the
main parameters of single administration of emtricitabine
were as follows: t1/2 was 4.20 ± 0.71 h, tmax was 1.40 ±
0.21 h, Cmax was 2143.97 ± 315.43 ng/ml, AUC0-t and
AUC0-∞ were 9972.97 ± 1571.03 and 10413.41 ± 1611.73
μg·h·L -1 , respectively, and CL/F was 0.39 ± 0.07 L·h -1 ·kg -
1 .
DISCUSSION
The combination of tenofovir and emtricitabine was
supposed to be used as a daily oral preventative of HIV
infection. In order to prove the hypothesis, some projects
around the world are being implemented. It is necessary
to establish a simple and reliable method for
simultaneous determination of tenofovir and emtricitabine
in human plasma.
In this study, we established a method for simultaneous
determination of tenofovir and emtricitabine in plasma
with HPLC-UV. The present assay was not only accurate
and sensitive, but also reproducible and with lower cost
for monitoring the plasma concentration of both tenofovir
and emtricitabine in clinic. Compared with the other costly
methods such as HPLC-MS and HPLC-MS/MS, the
method of HPLC-UV cost a little longer time. However,
the method of HPLC-UV was more economic and
convenient for simultaneous determination of tenofovir
and emtricitabine.
For extraction procedure in this study, SPE was chosen
considering the high polarity of tenofovir. To get a high
level of extraction efficiency, several columns were tried,
such as OASIS MCX 1 CC/30 mg from waters, SampliQ
C18 3 ml/200 mg from agilent, Strata-X 1 ml/30 mg from
phenomenex and BOND ELUT-C18 3 ml/200 mg from
VARIAN. Finally, BOND ELUT-C18 was more suitable for
our aim analytes.

Figure 7. Chromatogram of a healthy volunteer sample taken 3 h after administration of 300 mg dose of
VIREAD ® and 200 mg dose of emtricitabine. The sample contained 1311.9 ng/ml of FTC and 195.5 ng/ml of
TNF.
Figure 8. Result of tenofovir for scanned absorbance values at different wavelengths.
For the UV detection wavelength of the all the three
drugs, we had scanned absorbance values at different
wavelengths to ensure the best wavelength for
Tan et al. 1897
simultaneous detection of three drugs, and the result for
scanning is shown in Figures 8, 9 and 10.
Compared with the SPE method reported by Rezk et al.

1898 Afr. J. Pharm. Pharmacol.
Figure 9. Result of emtricitabine for scanned absorbance values at different wavelengths.
Figure 10. Result of lamivudine for scanned absorbance values at different wavelengths.

Figure 11. Chromatograms of standard tenofovir (2000 ng/ml).
Figure 12. Chromatograms of standard emtricitabine (2000 ng/ml).
(2005), our SPE method showed relatively low recovery.
However, in the method established by us, after once
extracted, the column could be washed and activated by
Tan et al. 1899
methanol and water for three times. So, each column for
SPE can be repeatedly used at least 3 times by a
repeated validation experiment (% R.S.D < 15%) in our

1900 Afr. J. Pharm. Pharmacol.
Figure 13. Chromatograms of standard lamivudine (2000 ng/ml).
study, in which spiked plasma replicates were extracted
by one column 3 times successively at each three
concentration levels (20, 200 and 200 ng/ml),
respectively.
Studies showed that the pH of the mobile phase was
found to be an extremely considerable factor for the
separation of tenofovir with the endogenous substance
and for the optimized shape of the peaks (Kuklenyik et
al., 2009). For chromatographic conditions, potassium
phosphate monobasic buffer with 0.08% of
triethanolamine (pH 3.52) and methanol was chosen as
the mobile phase A and B in our study. Under this pH of
the buffer, the chromatographic peak tailing was
minimized with an obviously extrusive separation for the
aim analytes and the IS. The gradient condition in our
paper makes it feasible for satisfactory separation of
emticitabine and tenofovir and their endogenous
interference. Due to the HPLC instrument, wavelength for
UV detection was chosen at 270 nm in order to detect
tenofovir, emtricitabine and lamivudine, simultaneously.
Conclusion
The method of HPLC-UV established by us in this study
is simple, reliable and economic for routinely monitor
plasma concentrations of tenofovir and emtricitabine in
Chinese population and it can be considered as a
technical support for plasma concentration measure of
tenofovir and emtricitabine of patients and volunteers
especially in developing countries.
ACKNOWLEDGEMENT
This study was supported by National Science and
Technology Major Project, China (No 2008ZX10105).
REFERENCES
Avolio AD, Sciandra M, Baietto L, Siccardi M, Requena DG, Bonora S,
Perri GD (2008). A New Assay Based on Solid-Phase Wxtraction
Procedure with LC-MS to Measure Plasmatic Concentrations of
Tenofovir and Emtricitabine in HIV Infected Patients. J. Chromatogr.
Sci., 46: 524-528.
Barkil M, Gagnieu MC, Guitton J (2007). Relevance of a combined UV
and single mass spectrometry detection for the determination of
tenofovir in human plasma by HPLC in therapeutic drug monitoring. J
Chromatogr. B., 854: 192-197.
CDC trials of pre-exposure prophylaxis for HIV prevention, http:// www.
cdc. gov/ hiv/ resources/Factsheets/PDF/prep.pdf
Clauson KA, Polen HH, Joseph SA, Zapantis A (2009). Role of the
pharmacist in pre-exposure chemoprophylaxis (PrEP) therapy for HIV
prevention. J. Pharm. Pract., 7: 11-18.
Delahunty T, Bushman L, Fletcher CV (2006). Sensitive assay for
determining plasma-tenofovir concentration by LC/MS/MS. J.
Chromatogr. B., 830: 6-12.
Gomes NA, Vaidya VV, Pudage A, Loshi SS, Parekh SA (2008). Liquid
chromatography-tandem mass spectrometry(LC-MS/MS) method for
simultaneous determination of tenofovir and emtricitabine in human
plasma and its application to a bioequivalence study. J. Pharm.
Biomed. Anal., 48: 918-926.
Http://www.accessdata.fda.gov/drugsatfda_docs/label/2008/021752s01
7lbl.pdf
Kuklenyik Z, Martin A, Pau CP, Garcia-Lerma G, Heneine W, Pirkle JL,
Barr JR (2009). Effect of Mobile Phase pH and Organic Content on
LC–MS Analysis of Nucleoside and Nucleotide HIV Reverse
Transcriptase Inhibitors. J. Chromatogr. Sci., 47: 365-372.
Kwarney BP, Flaherty JF, Shah J (2004). Tenofovir Disproxil Fumarate
Clinical Pharmacology and Pharmacokinetics. J. Clin.
Pharmacokinet., 43: 595-612.
Rezk NL, Crutchley RD, Kashuba ADM (2005). Simultaneous
quantification of emtricitabine and tenofovir in human plasma using
high-performance liquid chromatography after solid phases
extraction. J. Chromatogr. B., 822: 201-208.

African Journal of Pharmacy and Pharmacology Vol. 6(26), pp. 1901-1909, 15 July, 2012
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP12.135
ISSN 1996-0816 ©2012 Academic Journals
Full Length Research Paper
Cardio-tonic effect of the aqueous extract of whole
plant of Crataegus aronia syn: azarolus (L) on isolated
Rabbit’s heart
Abdullah S. Shatoor
Department of Internal Medicine, Cardiology Section, College of Medicine, King Khalid University (KKU), Abha 24121,
Saudi Arabia. E-mail: asshalghamdi@yahoo.com.
Accepted 19 June, 2012
The aim of this work is to study the effect of aqueous extract of whole plant of Crataegus aronia on the
force of contraction and heart rate of isolated rabbit’s heart. Six Isolated rabbit’s hearts were perfused
through aorta in a Langendorff mode. Heart rate and contractility were determined for 5 min in the
presence of 8 concentrations of C. aronia aqueous extract (1, 2, 5, 10, 20, 40, 100 and 200 mg/ml) or
adrenaline (0.05 mM) as control drug. The changes after each treatment were compared with their
baseline values. Data were collected with the help of PowerLab data acquisition and analyzed by
Labchart Pro 7 software. At all time intervals recorded, there were no significant changes in the force of
contraction nor the heart rate (HR) after infusion of low concentrations of the extract (1, 2, 5 and 10
mg/ml). The maximum increase in force of contraction occurred at a dose of 40 mg/ml, while the
maximum decrease in HR occurred at a dose of 20 mg/ml (P< 0.01). The highest doses of the extract
(100 and 200 mg/ml) caused hardening of the heart, stopping of the perfusion fluid from entering and
stopped the beating of the heart. Therefore, the aqueous extract of whole plant of C. aronia syn:
azarolus (L) showed a positive inotropic and negative chronotropic effects on isolated rabbit’s heart.
Key words: Crataegus aronia, cardiovascular, rabbits.
INTRODUCTION
Cardiac disorders are of serious medical concern, and
are increasing throughout the world (Brauwald et al.,
2001). Several drugs with therapeutic value in congestive
heart failure and those with positive inotropic effect such
as cardiac glycosides and phosphodiesterase inhibitors
(PDI), have several side effects that limit their use
(Packer et al., 1991; Uretsky et al., 1990). Medicinal
plants have over the years constituted indispensable
tools for research and development of new drugs (Bonati,
1980). Coupled with the fact that there are still many
plants whose medicinal values have not been exploited, it
is reasonable to describe the plant kingdom as a sleeping
giant for potential drug development (Harvey, 2000).
Hawthorn (Crataegus) is a plant native to
Mediterranean region, North Africa, Europe and Central
Asia. The medicinal use of its extracts date back to
ancient times and now listed officially as herbal drugs in
pharmacopoeias of countries, such as Germany, France,
China and England (Chang et al., 2002b). There are
more than 200 species of hawthorn worldwide, but only
very few were tested and used medicinally to treat
cardiovascular diseases, such as C. oxycantha, C.
laevigata, C. monogyna, C. orientalis and C. pinnatifida
(Bahorun et al., 2003). These studies showed that some
of these species increase contraction of the heart, dilates
coronary and peripheral blood vessels, and improves
blood supply to the heart, thereby help in treating heart
disease and mitigating symptoms in early stage of heart
failure (Rewerski, 1967; Petkov et al., 1981; Wagner and
Grevel, 1982; Leuchtgens, 1993; Reutxer, 1994; Rigelsky
and Sweet, 2002). Crataegus aronia syn: azarolus (L),
the predominant species which populates the mountains
of the Mediterranean basin, has not been subjected to
adequate scientific research. Several ethnobotanical and
ethnopharmacological surveys on the therapeutic use of
indigenous plants in Jordan and the Palestinian area

1902 Afr. J. Pharm. Pharmacol.
revealed the use of C. aronia in the Arab traditional
medicine to treat cardiovascular diseases, as well as
cancer, diabetes and sexual weakness (Ali-Shtayeh et
al., 2000; Said et al., 2002).
Despite the extensive researches of the different
species of hawthorns on cardiovascular system, most of
these studies were carried out in vivo. At a practical level,
the isolated heart, especially from small mammals,
provides a highly reproducible preparation which can be
studied quickly and in large numbers at relatively low cost
(Hearse and Sutherland, 2000). It allows broad spectrum
of biochemical, physiological, morphological and
pharmacological indices to be measured. These
measurements can be made in the absence of the
confounding effects of other organs, such as systemic
circulation and circulating neuro-hormonal factors. Thus,
it reflects the intrinsic responsiveness of the heart
independent of the systemic hemodynamic and neurohormonal
alterations. In addition, there is a deficient
supporting evidences from previous researches on the
effect of hawthorn on isolated heart, which is consider a
limitation for previous researches. Only very few studies
using the most popular hawthorn species (C. oxycantha)
or standardized commercial extracts, were studies on
isolated heart (Popping et al., 1995; Almak et al., 2009).
To the best of our knowledge, no single evaluation
study of C. aronia on cardiovascular system has been
reported prior to this report either in an in vivo or in vitro
study. Therefore, the aim of our current study is to
examine the effect of C. aronia aqueous extract on
isolated rabbit’s heart by investigating its effect on the
force of contraction (FC) and heart rate (HR).
MATERIALS AND METHODS
Preparation of the extract
This study was performed during the month of August 2011, in the
Research laboratory of Physiology Department at the Medical
School of King Khalid University. Fresh C. aronia syn: azarolus (L)
whole plant (stems, leaves and flowers) was purchased from a local
market in Jordan (Middle-east). The plant was identified, dried and
extracted in the Department of Pharmacognosy at the College of
Pharmacy of King Khalid University, Abha, Saudi Arabia. The dried
plant material was ground to a powder and extracted by maceration
using distilled water (1 kg/1 L, w/v) for 3 days at 37°C (Abdul et al.,
2009). The extract was filtered and evaporated under reduced
pressure in a rotary evaporator. The resulting residue (28 g; the
aqueous extract) was stored at 4°C. The residue was re-constituted
in Ringer-Locke solution to obtain the various concentrations (1, 2,
5, 10, 20, 40, 100 and 200 mg/ml) used in this study.
Experimental animals
Six adult white albino male rabbits weighing between 2 and 3 kg
were used for the experiments, with the approval of Ethical
Committee (REC-2011-05-01) of the medical School, King Khalid
University, Abha, Saudi Arabia. The animals were obtained from
the animal house of the College of Medicine of King Khalid
University where they were fed with standard rabbit pellets and
allowed free access to water. They were housed at a controlled
ambient temperature of 25 ± 2°C and 50 ± 10% relative humidity,
with 12-h light/12-h dark cycles. All studies were conducted in
accordance with the National Institute of Health's Guide for the
Care and Use of Laboratory Animals (National Institute of Health,
1996).
Experimental procedure
This experiment was carried out in accordance with the Langendorff
(1985) procedure. Each rabbit was injected with 1000 IU of heparin
intravenously through the marginal ear vein. Five minutes later, a
blow on the neck of the rabbit made them unconscious. The chest
was opened and the heart was dissected out with about 1 cm of
aorta attached, and washed quickly as possible with oxygenated
Ringer-Locke solution (NaCl; 45.0 g, NaHCO3; 1.0 g, D-glucose; 5.0
g, KCl; 2.1 g, CaCl2.2H2O; 1.6 g, in 5 L of distilled water). The
isolated heart was gently squeezed several times to remove as
much residual blood as possible. The heart was then transferred to
the perfusion apparatus (Radnoti isolated heart system, AD
Instruments, Australia) and tied to a stainless steel cannula through
the aorta. The perfusion fluid was Ringer-Locke solution, which was
continuously bubbled with a mixture of 95% oxygen and 5% carbon
dioxide and was applied at a constant perfusion pressure of 70 mm
Hg (Chlopicki et al., 2003). Temperature was continuously
monitored by a thermo-probe inserted into the perfusion fluid tank
and maintained between 36.5 and 37.5°C. The hearts were allowed
to stabilize for 30 min before any drug interventions. Briefly, 1 ml of
adrenaline (0.05 mM) was given before the beginning of the
experiment procedure to record the sensitivity of the heart. Then, 1
ml of Ringer-Locke solution containing different concentrations of
the extract (1, 2, 5, 10, 20, 40, 100 and 200 mg/ml) was injected
over 30 s with the aid of 1 ml syringe through the perfusion line
above the aortic line, and the changes in the cardiac parameters
were recorded (Figures 1 and 2). After each treatment, the hearts
were washed by the perfusion fluid for 10 min until the baseline
recording is achieved and the second dose was then given. The
recording before the direct perfusion of adrenaline or each extract
dose was considered as baseline reading for each dose.
Parameters measured were: 1) mean force of contractions (mean
cycle height in g); 2) heart rate (beats/min) and 3) an
electrocardiogram (ECG) for rhythm monitoring.
The mechanical responses of spontaneously contracting, isolated
hearts were recorded by attaching one end of a thread to the apex
of the heart using a Palmer clip and the other end of the thread to a
force transducer ((MLT 844; AD Instruments, Australia). ECG and
HR were recorded by attaching three spring clip electrodes directly
to the heart surface (MLA1210, AD Instruments, Australia). The
signal from the force transducer and the ECG electrodes were
filtered and amplified and sent to an analog-to-digital converter
(PowerLab data acquisition and analysis system: AD Instruments,
Australia) attached to a computer. The signals recorded were saved
for later analysis. FC, HR and ECG were recorded and analyzed
with the help of Labchart Pro7 software (AD Instruments, Australia).
Determination of dose response curve
Dose response curve of the effect of C. aronia extract on force of
contraction (g) was plotted by plotting the log of each dose against
its resulted increase in the force of contraction (difference between
the maximum increase in force of contraction after dose perfusion
and its baseline force of contraction). EC50 which resulted in half
maximum effect was calculated using dose response curve module
installed in LabChartpro7 software (AD Instruments, Australia)The
percent of change in FC or HR for each dose from its baseline

Shatoor 1903
Figure 1. The effect of Crataegus aronia syn: azarolus (L) aqueous extract (20 mg/ml) on heart rate; ECG and force of
contraction on rabbit’s isolated heart. The signal from the force transducer and the ECG electrodes were filtered and amplified
and sent to an analog-to-digital converter (PowerLab data acquisition and analysis system: AD Instruments, Australia). Data were
collected and analyzed by Labchart Pro7 software (AD Instruments, Australia).
Cycle height (g) Pulse (BPM) ECG (mV) Force (g)
Figure 2. The effect of Crataegus aronia syn: azarolus (L) aqueous extract (40 mg/ml) on heart rate; ECG and force of
contraction on rabbit’s isolated heart. The signal from the force transducer and the ECG electrodes were filtered and
amplified and sent to an analog-to-digital converter (PowerLab data acquisition and analysis system: AD Instruments,
Australia). Data were collected and analyzed by Labchart Pro7 software (AD Instruments, Australia).

1904 Afr. J. Pharm. Pharmacol.
Mean force of contraction (g)
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
*
*
*
*
*
*
Baseline 1 2 3 4 5
*
*
*
Time (min)
*
*
*
*
C. aronia (1 (1 mg/ml)
C. aronia (2 (2 mg/ml)
C. aronia (5 (5 mg/ml)
C. aronia (10 mg/ml)
C. aronia (20 mg/ml)
C. aronia (40 mg/ml)
Adrenaline
Figure 3. Effect of Crataegus aronia syn: azarolus (L) aqueous extract (1, 5, 10, 20 and 40 mg/ml) and adrenaline on the force of myocardial
contraction of isolated rabbit's heart. Values are given as Mean ± SD for groups of 6 rabbit’s hearts each. Analysis by one w ay ANOVA; *:
significantly different from baseline at P< 0.05. #: significantly different when compared to 20 mg/ml reading at same time interval at P< 0.05.
and : significantly different when compared to Adrenaline reading at same time interval at P< 0.05.
reading at different time intervals (5 min. intervals) was calculated
as follows:
Mean FC (g) or HR (b/min.) at a given min - mean baseline FC or HR
% of change = × 100
Mean baseline FC or HR
Statistical analysis
Results were expressed as the mean value ± SD. Statistical
differences between groups were assessed using the SPSS
software version 16 by One way ANOVA test. Values of P

Heart rate (Beats/min.)
345
330
315
300
285
270
255
240
225
210
195
180
165
150
135
120
105
90
*
*
*
*
Baseline 1 2 3 4 5
*
Time (min.)
*
* *
* *
*
*
*
Shatoor 1905
C. C. aronia (1 (1 mg/ml)
C. C. aronia (2 (2 mg/ml)
C. C. aronia (5 (5 mg/ml)
C. C. aronia (10 (10 mg/ml)
C. C. aronia (20 (20 mg/ml)
C. C. aronia (40 (40 mg/ml) mg/ml)
Adrenaline
Figure 4. Effect of Crataegus aronia syn: azarolus (L) aqueous extract (1, 5, 10, 20 and 40 mg/ml) and adrenaline on the heart
rate of isolated rabbit's heart. Values are given as Mean ± SD for groups of 6 rabbit’s hearts each. Analysis by one way ANOV A; *:
significantly different from its baseline at P< 0.05. #: significantly different when compared to 40 mg/ml reading at same time
interval at P< 0.05. &: Significantly different when compared to 20 and 40 mg/ml reading at same time interval P< 0.05.
of increases in force of contraction due to C. aronia at
this dose were 43.47, 91.3, 94.6, 145.5 and 47.8% at the
1 st , 2 nd , 3 rd , 4 th and 5 th minutes, respectively, when
compared to baseline FC (Table 2). At the fifth minute of
40 mg/ml extract dose perfusion, the FC started to
declined to its baseline reading. The ANOVA test
revealed that the maximum increase in FC at the dose of
20 mg/ml occurred at the 3 rd minute (62%), while the
maximum inotropic effect of the dose 40 mg/ml occurred
at the 4 th minute (145%).
Moreover, adrenaline produced a significant prolonged
increase in force of contraction at all time intervals and
produced 34.7, 69.5, 104.3, 139.3 and 140% increases in
the force of contraction at 1 st , 2 nd , 3 rd , 4 th and 5 th minute.
Comparing the positive inotropic effect produced by
adrenaline and the extract at the concentration of 40
mg/ml, the ANOVA test revealed that: a) increases in FC
at 1 st , and 2 nd minutes were significantly higher after
perfusion of extract; b) the effects produced by
adrenaline and the extract at 3 rd and 4 th minutes were not
significantly different; C) at the 5 th minute, the force of
contraction was significantly higher after adrenalin
perfusion. The dose response curve analysis of the effect
of the extract on force of contraction showed that the
concentration of the extract that produced 50% increase
in FC (EC50) is 27.0 ± 1.23 mg/ml (Figure 5).
There were no significant change in heart rate doses of
extract at 1-10 mg/ml while, the perfusion of 20 mg/ml of
C. aronia aqueous extract produced an initial significant
increase in HR at 1min (percent of change +10.27%),
followed by a progressive significant decrease (p <
0.0001) at 2 nd , 3 rd , 4 th and 5 th minutes (Figure 4) with
percents of inhibition of 38.76, 41.6, 43.87 and 47.37%,
respectively (Table 2). On the other hand, the perfusion
of C. aronia aqueous extract at a dose of 40 mg/ml
produced a significant decrease (p < 0.0001) in heart rate
after 3 rd , 4 th and 5 th minutes, with the percents of inhibition
of 11.0, 18.96 and 33.4%, respectively (Figure 4). The
ANOVA test revealed that the negative chronotropic
effect produced by the dose of concentration of 20 mg/ml
on heart rate during the first 5 min intervals after extract
perfusion was significantly higher than the effect
produced by the dose of concentration of 40 mg/ml.
Adrenaline produced a significant positive chronotropic
effect with percents of increases of 17.0, 33.75, 42.0,
64.0 52.3% at 1 st , 2 nd , 3 rd , 4 th and 5 th minutes, respectively
(Table 2). No abnormal rhythm was recorded during the
period of study.

1906 Afr. J. Pharm. Pharmacol.
DISCUSSION
Table 1. Percent of changes in force of contraction of isolated rabbit’s heart
during the first 5 min after perfusion of different Crataegus aronia syn: azarolus
(L) aqueous extract doses and adrenaline.
Dose (mg/ml)
Percent of changes (%)
1 st min 2 nd min 3 rd min 4 th min 5 th min
1 -4.76 0.0 -4.76 -4.7 -2.72
2 +4.2 0.0 0.0 0.0 +4.76
5 0.0 +2.27 +1.36 +1.81 0.0
10 +7.4 +8.26 +3.9 -4.3 0.0
20 +25 +45 +62.5 -4.16 -4.16
40 +43.47 +91.3 94.6 +143.5 +47.8
Adrenaline (0.05 mM) +34.7 +69.5 +104.3 +139.3 +140
Average EC 50 = 27.0 ± 1.23 mg/ml
Figure 5. Dose response curve for the effect of Crataegus aronia syn: azarolus (L) aqueous extract on force of
contraction of isolated rabbit’s heart. EC50 was given as mean ± SD for group of 6 rabbit’s hearts each. EC50=
27.0 ± 1.23 mg/ml, Hill slop= 1.67. Dose response (g) for each dose was calculated as the average increase in
force of contraction during the first 5 min of extract perfusion, Pre-dose baseline correction was applied during
the calculation. Data were collected and analyzed by Labchart Pro7 software (Dose response module, AD
Instruments, Australia).
In this study, higher extract concentrations (100 and 200
mg/ml) caused ballooning of the heart, stopping of
perfusion solution circulation and stopping of heart
beating. One explanation for such effect at these high
doses could be due to the high viscosity of the extracts,
which caused a blockage in the small coronary vessels
(capillaries) with subsequent prevention of the perfusion
solution from circulating through its normal pathway in
Langendorff preparation and instead flowing back
through aortic valve into the left ventricle and thus
resulting in ballooning of the left ventricle. Langendorff
preparation involves the cannulation of the aorta which is
then attached to a reservoir containing oxygenated
perfusion fluid. This fluid is then delivered in a retrograde
direction down the aorta either at a constant flow rate
(delivered by an infusion or roller pump) or a constant
hydrostatic pressure (usually in the range of 60 to 100
mmHg). In both instances, the aortic valves are forced
shut and the perfusion fluid is directed into the coronary
ostia, thereby perfusing the entire ventricular mass and
then drained into the right atrium via the coronary sinus
(Langendorff, 1985).
The positive effect of C. aronia aqueous extract on the
force of contraction of isolated rabbit’s heart reported in
this study is in agreement to most previously published in
vivo and in vitro studies that showed a similar effect of
other extract prepared from other species of hawthorn
(Reutxer, 1994; Popping et al., 1995). However, varying
results have been observed regarding the effect of
hawthorn and its constituents on HR. In majority of in

Table 2. Percent of changes in heart rate of isolated rabbit’s heart during the
first 5 min after perfusion of different Crataegus aronia syn: azarolus (L)
aqueous extract doses and adrenaline.
Dose (mg/ml)
Percent of changes (%)
1 st min 2 nd min 3 rd min 4 th min 5 th min
1 -0.8 -0.8 -2.58 -0.8 0.18
2 -3.25 -3.39 -7.34 -5.07 -5.0
5 +0.56 +3.04 +3.04 +0.56 +0.53
10 -0.02 -2.68 -3.09 -4.07 -2.60
20 +10.27 -38.76 -41.6 -43.87 -47.37
40 +4.8 +0.56 -11.0 -18.96 -33.4
Adrenaline (0.05 mM) +17.9 +33.75 +42.0 +64.2 +52.3
vitro studies, an increase in HR has been observed, while
most of the in vivo studies reported a decrease in HR
which is similar to our finding (Ammon and Kaul, 1994).
In fact, there was an increase in heart rate at 1 min with
both doses of extract (20 and 40 mg/ml) but maximum
seen with 20 mg/ml followed by quick fall at the 2 nd
minute, which then continued to fall slowly thereafter.
This difference in response of HR could be due to the
effect of extract on vagal tone with the in vivo studies and
possibly the species difference used in our study, as no
effect has been previously reported on C. aronia syn:
azarolus (L) extract on isolated heart (Petkov et al.,
1981). However, we did not investigate the mechanism of
action by which C. aronia aqueous extract exerts its
positive inotropic and negative chronotropic effects. Other
in vivo and in vitro studies are running now in our
laboratory, aiming to demonstrate the mechanism of the
inotropic effect of C. aronia extract. However, at this
stage, we may postulate some of the possible
mechanisms based on previously published works.
The mechanism underlying the enhanced FC is an
enhanced Ca 2+ membrane influx (Chang et al., 2002a). In
this study, the positive increase in FC after extract
perfusion strongly suggests that the C. aronia syn:
azarolus (L) could act on rabbit’s heart by opening the
membrane L-type Ca 2+ channels. Inhibition of myocardial
Na + /K + ATPase, which is an integral membrane enzyme
that maintains cardiac resting potential and inhibition of
the enzyme phosphodiesterase (PDE) that ultimately
results in an increase in intracellular cyclic nucleotides,
have been reported to occur in different studies with
different species of hawthorn (Holzl et al., 1988; Reutxer,
1994; Popping et al., 1995). Both ways eventually
enhance the opening of L-type Ca 2+ with subsequent
increase in FC.
Flavonoids, tannins, saponins, terpenes and sterols are
the main constituents of C. aronia syn: azarolus (L)
aqueous extract (Shatoor, 2011). Most of the
pharmacological actions of hawthorn are attributed to the
flavonoids contents (Yao et al., 2008). The reported
flavonoid contents of C. orientalis and C. oxycantha are:
hyperoside, along with apigenin, apigenin 7-glucoside,
Shatoor 1907
ursolic acid, vitexin and vitexin 4′-rhamnoside (Melikoglu
et al., 1999). Ursolic acid interacts with the digitaloid
binding site for Na + /K + ATPase, while catechin, the
flavonoid vitexin and flavonol kaempferol were found to
be structurally similar to papaverine and theophylline, the
two chemical agents known to inhibit PDE. On the other
hand, saponins are mainly plant-derived glycosides,
occurring as triterpenoid or steroid saponins. Steroid
saponins have been found to have multiple interesting
biological and pharmacologic effects including negative
chronotropic, positive inotropic, diuretic, antibacterial,
anti-inflammatory, hypocholesteremic (Francis et al.,
2002; Lacaille-Dubois and Wagner, 1996). Furthermore,
administration of oral standardized C. oxycantha extract
to an ischemic/reperfusion rat model effectively protected
animals from reperfusion induced arrhythmias and
hypotensive crisis (Krzeminski and Chatterjee, 1993).
The mechanisms which may account for the slow
diastolic depolarization, seen between two successive
action potentials of myocardial pacemaker cells, may
include one of the following: 1) a slow inward Na + current,
If, the so-called ‘funny current’ that is induced by cell
hyperpolarization; 2) a temporal decrease of the outward
K + current due to a time-dependent decay of the
membrane K + conductance; 3) a low background K +
outward current; 4) an inward Na + /Ca ++ exchange current,
and 5) an inward T-type and L-type Ca 2+ current (Lipsius
et al., 1996). The individual contributions of these
currents to pacemaker function are controversial. To
decrease the HR, one or more of the aforementioned
mechanisms, could be altered. HR is tightly coupled to
myocardial oxygen consumption (Laurent et al., 1956;
Braunwald, 1971). Hence, myocardial oxygen demand is
reduced through bradycardia in patients with severe heart
failure (HF) treated with digitalis (Erdmann, 1998).
Furthermore, slowing of HR, prolong diastolic period and
improve diastolic flow through coronary arteries.
Therefore, HR reduction became well established
strategy for the treatment of various ischemic heart
diseases (IHD), and HF since the introduction of βadrenoceptor
blockers (Gillam, 1965; Goethals et al.,
1993; Lechat, 1998). β-Adrenoceptor blockers have in

1908 Afr. J. Pharm. Pharmacol.
common, a negative inotropic effect which may worsen
HF symptoms and have to be used carefully in patients
with impaired left ventricular function. Therefore, the
newer agents like Ivabradine, and Zatebradine which
specifically inhibit sinus node pacemaker current without
direct effect on contraction is gaining more attraction for
treating patients with IHD and/or HF (Gillam, 1965;
Goethals et al., 1993; Lechat, 1998; Borer et al., 2003).
The currently available inotropic agents such as
Dobutamine, milrinone, enoximone and Vesnarinone are
limited by the proarrhythmic effects, increased mortality
and are not recommended for routine use in patients with
HF and/or IHD (Focaccio et al., 1996; Cuffe et al., 2002;
Abraham et al., 2005; Dec, 2005). Almost all inotropic
agents exhibit their positive inotropic effects by increasing
the influx of calcium into the cytosol (Lorell et al., 1988).
This process leads to intracellular calcium overload and
may trigger serious arrhythmias, while slowing of HR
prolongs the diastolic period and contribute to
intracellular calcium release (Brutsaert et al., 1993;
Martin et al., 2011). It is not yet known how hawthorn
handles the intracellular calcium. However, combining
inotropic agents such as enoximone which act through
inhibition of PDE with β-adrenoceptor blockers improve
survival in advanced cases with HF (Shakar et al., 1998).
It seems that the presence of multiple active ingredients
in the aqueous extract of the whole plant of C. aronia syn:
azarolus (L) may exert the inotropic and chronotropic
effects through a multiple mechanisms of action.
Conclusion
The results of this study showed that the aqueous extract
of C. aronia syn: azarolus (L) produced a positive
inotropic and negative chronotropic effects on isolated
rabbit’s heart. Further investigation is therefore needed to
define the mechanism of action underlying the enhanced
force of contraction and the negative chronotropic effects
of the aqueous extract of C. aronia syn: azarolus (L).
ACKNOWLEDGEMENT
I would like to express my sincere gratitude and
appreciation to the Research Deanship, King Khalid
University, for their financial support.
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African Journal of Pharmacy and Pharmacology Vol. 6(26), pp. 1910-1922, 15 July, 2012
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP12.183
ISSN 1996-0816 © 2012 Academic Journals
Full Length Research Paper
Efficacy and safety profile of moxifloxacin in treatment
of urogenital system infections: A meta-analysis of
randomized controlled trials
Yanping Mu 1 , Jinyu Huang 2 , Ruilin Liu 3 , Xun Deng 1 , Liqing Wang 1 , Ping Fang 4 , Meidan Wei 1 *,
and Yong Wang 1
1 Department of Clinical Pharmacology, Zhujiang Hospital, Southern Medical University, Guangzhou 520282, China.
2 Department of Stomatology,Zhujiang Hospital, Southern Medical University, Guangzhou 520282, China.
3 hujiang Hospital, Southern Medical University, Guangzhou 520282, China.
4 Department of Urology, Zhujiang Hospital, Southern Medical University, Guangzhou 520282, China.
Accepted 26 March, 2012
This meta-analysis was performed to evaluate the efficacy and safety profile of moxifloxacin in
treatment of urogenital system infections. PubMed, EMBASE, Science Direct, ClinicalTrials.gov,
Cochrane Central Register of Controlled Trials, CBM (Chinese Biomedical Literature Database), CNKI
(Chinese National Knowledge Infrastructure), Wan Fang Data and VIP INFORMATION were searched
from January 1999 to May 2011 to comprehensively collect randomized controlled trials (RCTs) that
compared moxifloxacin with conventional antibiotics therapy in patients with urogenital system
infections. Clinical cure rates, clinical effective rates, pathogens eradication rates and incidence of
adverse drug reactions were pooled using meta-analysis performed by Review Manager 5.1 software.
Relative risk (RR) and 95% confidence interval (95%CI) were calculated in a random-effects model or in
a fixed-effects model. Twenty-two trials including a total of 3940 patients were included for metaanalysis.
The results of meta analysis showed that clinical cure rates, clinical effective rates and
pathogens eradication rates of moxifloxacin were higher than conventional therapy [RR = 1.08, 95%CI
(1.02, 1.14), P = 0.008; RR = 1.09, 95%CI (1.04, 1.14), P = 0.0005; RR = 1.04, 95%CI (0.99, 1.09), P = 0.08];
the incidences of adverse drug reactions between moxifloxacin and control group were not
statistically significant [RR = 0.88, 95%CI (0.72, 1.06), P = 0.17]. In three large studies of pelvic
inflammatory disease (PID) patients, no statistically significant difference was found between
moxifloxacin monotherapy group and control group about clinical cure rates, microbiological success
rates and the incidences of adverse drug reactions [RR = 0.98, 95%CI (0.95, 1.02), P = 0.33; RR = 1.06,
95%CI (0.96, 1.16), P = 0.25; RR = 0.85, 95%CI (0.68, 1.05), P = 0.13]. Moxifloxacin can be suggested as
the regimen of choice for treatment of urogenital system infections.
Key words: Meta-analysis, moxifloxacin, efficacy, urogenital system infections, pelvic inflammatory disease.
INTRODUCTION
Urogenital system infections are among the most
frequently seen and encountered infectious diseases of
humans in the world. They are one of the leading causes
of acute diseases and chronic health impairment (Skerk
et al., 2010; Hannan et al., 1993). Urinary tract infections
mainly include pyelonephritis, cystitis, urethritis, while
*Corresponding author: E-mail: ywang43555@sina.com.
genital system infections cover pelvic inflammation
disease (PID), vaginitis, and cervicitis in women and
prostatitis in men. Anatomically, the urinary and genital
systems are close, and susceptible to cross-infection.
Sexually transmitted infections are important public
health problem, numerous complications leaving
permanent consequences on the human health as well
as large expenses that health-care system and
individuals have to pay for their detection, prevention and
treatment (Skerk et al., 2010; Minichiello et al., 2011).

Urinary tract infections are the most common bacterial
infections in humans and the most common reason for
justified antibiotic prescriptions (Foxman ., 2002).
Urogenital system infections are mostly caused by
Escherichia coli, Enterococcus, Staphylococcus,
Klebsiella pneumoniae, Pseudomonas aeruginosa and
Proteus mirabilis (Bebear et al., 2008). For the past few
years, some atypical pathogens, such as Mycoplasma
and Chlamydia, are accounting for more and more
infections (Wagenlehner and Naber, 2006).
Fluoroquinolones which are among the alternatives for
empirical antibiotic treatment of some urogenital system
infections have become widely accepted for treatment of
urogenital system infections because of their favorable
pharmacokinetic and pharmacodynamic properties
(Pathania and Sharma., 2010; Wagenlehner and Naber.,
2006; Naber, 2001). Clinical practice has proved that
fluoroquinolones are a good choice for urogenital system
infections (Wagenlehner and Naber, 2006). However, the
most important drawback in the treatment of urogenital
system infections is that bacterial resistance quickly
appears. A four-year prospective study reported that
there is high intrinsic resistance to the quinolones among
strains of Pseudomonas aeruginosa (43.4%), E. coli
(26.3%) and Proteus spp. (17.1%), and rising rates of
resistance were observed in P. aeruginosa (14.6%
increase), Staphylococcus aureus (9.8%), and E. coli
(9.7%) after four years (Omigie et al., 2009).
Moxifloxacin, a new fluoroquinolone antibiotic that acts
by inhibiting bacterial topoisomerases II and IV, not only
possesses increased activity against typical, atypical and
anaerobic bacteria, but also has enhanced potential to
minimize the emergence of bacterial resistance (Bebear
et al., 2008; Lode and Schmidt., 2008a; Boswell et al.,
2002; Brueggemann et al., 1997). There is no known
cross-resistance between moxifloxacin and other kinds of
antimicrobials, such as beta-lactams, macrolides,
aminoglycosides and tetracyclines (Keating and Scott,
2004). Moxifloxacin achieves good tissue penetration and
high concentrations in clinically relevant tissues and fluids
(Lode and Schmidt, 2008b). Based on the safety profile
and the pharmacokinetic behavior of moxifloxacin, a
dosage regimen of 400 mg given once daily, are effective
and well tolerated for the treatment of various infections
(Stass et al., 2001). Moxifloxacin, taken as respiratory
fluoroquinolone, is mainly used for the treatment of acute
bacterial exacerbation of chronic bronchitis, communityacquired
pneumonia (Miravitlles, 2005), acute bacterial
sinusitis (Lode et al., 2008b), complicated skin and skinstructure
infections (Muijsers and Jarvis , 2010) and
complicated intra-abdominal infections (Cheadle et al.,
2010). Because of the desirable pharmacokinetic and
concentration in tissues and fluids, we want to find out
whether moxifloxacin can be considered as an alternative
for the treatment of urogenital infections. Although Naber
et al. (Bebear et al., 2008) discovered that the urinary
excretion of moxifloxacin (20%) was lower than
Mu et al. 1911
ciprofloxacin (43%), ofloxacin (81%) and levofloxacin
(84%) and the indications of moxifloxacin are not
included urogential infections (Foxman et al., 2011), in
some countries, such as China, France, Russia, and
England, moxifloxacin is used for treatment of urogenital
system infections (Zheng, 2010; Su et al., 2010; Judin et
al., 2010; Meng and Ding, 2009; Tang et al., 2009; Wang
and Pei, 2009a, b; Jin et al., 2009; Wang et al., 2009;
Zhang et al., 2009; Heystek and Ross, 2009; Li, 2008;
Per et al., 2009; Zhang et al., 2007; Sun, 2007; Ross et
al., 2006; Luo et al., 2006; Luo et al., 2006; Gao et al.,
2005; Tian et al., 2005; Zhang et al., 2004; Cai et al.,
2003).
Research findings from those clinical studies of the
efficacy and safety profile of moxifloxacin in treatment of
urogenital system infections have been inconsistent.
Therefore, aiming to compare the efficacy and safety profile
of moxifloxacin monotherapy with conventional antibiotic
treatment for treatment of urogenital infections, we conduct a
meta-analysis of randomized controlled trials (RCTs).
MATERIALS AND METHODS
Data sources
Studies were identified by extensively searching the PubMed,
EMBASE, Science Direct, ClinicalTrials.gov, Cochrane Central
Register of Controlled Trials, CBM (Chinese Biomedical Literature
Database), CNKI (Chinese National Knowledge Infrastructure),
Wan Fang Data and VIP INFORMATION were searched from
January 1999 to May 2011. The search terms were moxifloxacin,
Avelox, urinary tract infections, urogenital infections, pelvic
inflammatory disease and nongonococcal urethritis. The language
of the literatures was not restricted to English. In addition,
references of the relevant articles were reviewed in order to identify
additional studies not detected by the initial search.
Study selection
Two reviewers (Yanping Mu and Xun Deng) independently
searched literatures and examined relevant randomized controlled
trials (RCTs). Any disagreement about study selection or data
extraction was resolved by consensus with the third reviewer (Yong
Wang, MD). For meta-analysis, all studies had to meet the following
inclusion criteria: (1) A study described as RCT; (2) patients with
urogenital infections had no statistically significant differences in
baseline characteristics; (3) comparison of the efficacy and safety of
moxifloxacin and other conventional antibiotics; (4) The outcome
measures were clinical treatment success (defined as “clinical cure”
and “clinical effective”. “Clinical cure” was the normalization of acute
signs, symptoms related to infection and laboratory test results with
no requirement for further antibiotic therapy, and “clinical effective”
was the aggregation of cure and improvement), microbiological
treatment success (defined as the eradication of baseline
pathogens) and adverse drug reactions.
Non-randomized studies were excluded, as well as case reports,
reviews with insufficient details to meet the inclusion criteria,
abstracts in the proceedings of scientific conferences, experimental
trials and trials focusing on pharmacokinetics or
pharmacodynamics, and children and pregnant female.
Data extraction
Two of the authors independently extracted data from the trials that

1912 Afr. J. Pharm. Pharmacol.
112 potentially relevant articles identified from database
47 articles from initial screening
23 potentially RCT trials identified
22 trials included in the meta-analysis
Excluded 65:
11 reviews
54 experimental trials
Excluded 24:
7 non-randomized trials
14 no comparative trials
3 Russian trials not meet inclusion criteria
Excluded 1:
Experimental group used moxifloxacin and
terazosin
Figure 1. Flow diagram of the process for selecting articles for a meta-analysis of
moxifloxacin compared with conventional medical treatment in the treatment of
urogenital infections.
met the inclusion criteria using. Authors would be contacted for
missing data when necessary. For each trial, the following data
were extracted: disease; number of patients in each group; mean
age and sex distribution of each group; drug regimen, including
doses and treatment duration; clinical cure rates; clinical effective
rates; pathogens eradication rates and the incidences of adverse
drug reaction.
Assessment of study quality
Quality assessment of the RCTs included in the meta-analysis was
independently performed by the same reviewers according to
Cochrane Handbook 5.0.1 and Juni et al. (Juni et al., 2001; Jadad
et al., 1996), which assesses the descriptions of randomization
procedures, allocation concealment, double blinding and
dropouts\withdrawals of the included trials. Each author rated the
quality of the trials using Jadad grade (maximum grade=A;
minimum grade = C; grade ≥ B = good quality).
Statistical analysis
Data were analyzed using Review Manager 5.1. Included articles
were pooled and weighed (Juni et al., 2009). Relative risk (RR) and
95% confidence interval (95%CI) were calculated in a randomeffects
model or in a fixed-effects model. Heterogeneity was
assessed by calculating a � 2 test and the quantity of heterogeneity
was measured with I 2 statistic. If heterogeneity (P50%)
was found among the trials, random-effects model would be
chosen, otherwise fixed-effects model chosen. If heterogeneity was
evident (I 2 >70%), the inferior quality study should be eliminated to
analyze.
RESULTS
Study selection process
The flow diagram (Figure 1) shows the process of
selecting articles for meta-analysis of moxifloxacin
compared with conventional therapy for urogenital
infections. Twenty-two (Zheng, 2010; Su et al., 2010;
Judin et al., 2010; Meng et al., 2009; Tang et al., 2009;
Wang and Pei, 2009a; Wang and Pei, 2009b; Jin et al.,
2009; Wang et al., 2009; Zhang et al., 2009; Heystek and
Ross, 2009; Li, 2008; Per et al., 2009; Zhang et al., 2007;
Sun, 2007; Ross et al., 2006; Luo et al., 2006; Luo et al.,
2006; Gao et al., 2005; Tian et al., 2005; Zhang et al.,
2004; Cai et al., 2003) were finally selected from the 112
articles.
Study characteristics
The main characteristics of twenty-two included RCTs
are presented in Table 1. Twenty-two RCTs involving
3940 patients were ultimately confirmed that met the
criteria for inclusion in the meta-analysis. Among the
patients included in the trials, the mean age was 36.87
years, and 82.61% (3255/3940) of the patients were
women. All of the trials enrolled patients with urogential
system infections, nine of which were researched

Table 1. Study characteristics of included RCTs.
Study Country
Zheng
HZ 23
2010
Su XD 24
2010
Judlin 25
2010
Meng
W 26
2009
Tang
JD 27
2009
Wang
G 28
2009(a)
Wang
G 29
2009(b)
China UTI
Type of
disease
China NGU
France PID
China Acute PID
China UTI
China NGU
China UTI
Treatment
No. of
patients
(male/female)
Mean age,
y
Drug regimen
MFX 32 (20/12) 40.8 ± 4.1 400 mg, po, qd × 10d
LVFX 31 (19/12) 39.8 ± 3.7 200 mg, po, bid × 10d
MFX 60 (60/0) 32 400 mg, po, qd × 14d
AZI 60 (60/0) 34 500 mg, po,qd × 14d
MFX 228 (0/228) 35.2 ± 8.4 400 mg, po, qd × 14d
LVFX/MTZ 232 (0/232) 35.4 ± 8.7
500 mg LVFX,po,qd + 500
mg MTZ, po, bid × 14d
MFX 25 (0/25) 400 mg po, qd ×14d
CTRX/AZI 25 (0/25) 19~61
CTRX250 mg, im, qd +
AZI500 mg,po,qd ×14d
MFX 50 (8/42) 400 mg, iv, qd × 3d or 14d
OFLX 50 (7/43) 43.1 ± 25.6 200 mg, iv, bid × 3d or 14d
MFX 80 (0/80) 18 ~ 58 400 mg, po, qd ×14d
AZI 80 (0/80) 500 mg, po, qd × 14d
MINO 80 (0/80) 100 mg, po, bid × 14d
MFX 150 (0/150) 400 mg, po, qd × 7d
GAT 149 (0/149) 31 ± 0.5 400 mg, po, qd × 7 d
Clinical
cure rate
75.0
(24/32)
64.5
(20/31)
40.0
(24/60)
33.3
(20/60)
78.4
(152/194)
81.6
(155/190)
92.0
(23/25)
80.0
(20/25)
86.0
(43/50)
84.0
(42/50)
56.3
(45/80)
55.0
(44/80)
57.5
(46/80)
60.0
(90/150)
56.4
(84/149)
Clinical
effective rate
Pathogen
eradication rate
Mu et al. 1913
Incidence of
ADR
90.6 (29/32) 87.5 9.4 (3/32)
87.1 (27/31) 74.2 6.6 (2/31)
90.0 (54/60) - 6.7 (4/60)
71.7 (43/60) - 10.0 (6/60)
- 90.0 (27/30)
- 84.6 (22/26)
56.6
(129/228)
56.9
(132/232)
100.0 (25/25) - 16.0 (4/25)
96.0 (24/25) - 32.0 (8/25)
92.0 (46/50) 91.5 (43/47) 6.0 (3/50)
90.0 (45/50) 91.3 (42/46) 6.0 (3/50)
91.3 (73/80) 90.0 (72/80) 10.0 (8/80)
91.3 (73/80) 91.3 (73/80) 6.3 (5/80)
93.8 (75/80) 92.5 (74/80) 11.3 (9/80)
96.0
(144/150)
95.3
(142/149)
89.9 (169/188) 8.0 (12/150)
87.1 (162/186) 8.7 (13/149)

1914 Afr. J. Pharm. Pharmacol.
Table 1 cont
Jin X 30
2009
Wang X 31
2009
Zhang
YH 32
2009
Heystek 33
2009
Li SQ 34
2008
Pei YH 35
2008
Zhang
GH 36
2007
China UTI
China NGU
China UTI
England PID
China UTI
China Female RTI
China
Urogential
infections
MFX 121 - 400 mg, po,qd × 10d
SPFX 121 300 mg ,po,qd × 10d
MFX 60 (0/60) - 400 mg, po, qd × 12d
CLA 60 (0/60) 500 mg, po, qd × 12d
MFX 40 (23/17) 39 ± 8 400 mg, iv, qd × 7d
GAT 40 (24/16) 37 ± 9 400 mg, iv, qd × 7d
MFX 232 (0/232)
DOX/MTZ/CPFX 202 (0/202)
29.1 ±
7.2
28.5 ±
7.0
400mg,po, qd ×14d
DOX 100 mg, bid × 14d +
MTZ400 mg, tid × 14d +
CPFX500 mg
MFX 90 400 mg, po, qd × 10d
LVFX 86 200mg,po,bid × 10d
MFX 62 (0/62) 30.5 400mg,po,qd ×14d
CPFX 50 (0/50) 200 mg, po, bid × 14d
MFX 38 (26/12) 45 ± 13 400 mg, po, qd × 7d
GAT 38 (25/13) 45 ± 12 200 mg, ivgtt, bid × 7d
93.4
(113/121)
92.6
(112/121)
63.3
(38/60)
48.3
(29/60)
87.5
(35/40)
85.0
(34/40)
96.5
(224/232)
98.0
(198/202)
94.4
(85/90)
86.0
(74/86)
66.1
(41/62)
40.0
(20/50)
60.5
(23/38)
63.2
(24/38)
95.1
(115/121)
95.9
(116/121)
95.7
(116/121)
96.7
(117/121)
9.1 (11/121)
9.1 (11/121)
81.7 (49/60) - 5.0 (3/60)
65.0 (39/60) - 0 (0/60)
95.0 (38/40) 77.5 (31/40) 5.0 (2/40)
92.5 (37/40) 76.9(30/39) 7.5 (3/40)
- 93.5 (43/46)
- 89.7 (35/39)
44.0
(151/343)
49.7
(162/326)
97.8 (88/90) 96.7 (87/90) 7.8 (7/90)
91.9 (79/86) 94.2 (81/86) 9.3 (8/86)
91.9 (57/62) 89.5 (51/57) 8.1 (5/62)
62.0 (31/50) 72.7 (32/44) -
86.8 (33/38) 91.3 (42/46) 2.6 (1/38)
89.5 (34/38) 92.0 (46/50) 5.3 (2/38)

Table 1. contd.
Sun GQ 37
007
Ross 38
2006
Luo XM 39
2006
Luo JL 40
2006
Gao HY 41
2005
Tian YP 42
2005
Zhang
WF 43
2004
China NGU
England PID
China UTI and RTI
China
China UTI
China
China
Urogential
infections
Urogential
infections
Urogential
mycoplasma
infection
Mu et al. 1915
MFX 45 (26/19) 28.3 ± 11 400 mg, po, qd × 12 ~ 14d 75.0 (33/44) 93.2 (41/44) 91.8 (45/49) 18.2 (8/44)
AZI 41 (26/15) 27.36 ± 12 500 mg, po, qd × 12~14d 29.3 (12/41) 75.6 (31/41) 75.6 (31/41) 14.6 (6/41)
MFX 384 (0/384) 30.1 ± 8.4 400 mg, po, qd × 14d 90.2(248/275) - 87.5 (49/85) 22.5 (85/378)
OFLX/MT
Z
365 (0/365) 30.5 ± 8.5
OFLX400 mg +
MTZ500mg,po,bid ×14d
90.7(262/289) - 82.1 (46/56)
30.9(112/363
)
MFX 56 (0/56) 27.6 ± 3.1 400mg,po,qd×12d 78.6 (44/56) 91.1(51/56) - 12.5 (7/56)
DOX 56 (0/56) 27.5 ± 2.0 1 pill, po, bid × 14d 51.8 (29/56) 76.8(43/56) - 73.2 (41/56)
MFX 52 18 ~ 60 400 mg, po, qd × 14d 65.4 (34/52) 92.3 (48/51) 93.3 (56/60) 9.6 (5/52)
GAT 50 200 mg, po, bid × 14d 64.0 (32/50) 90.0 (45/50) 88.5 (54/61) 10.0 (5/50)
CPFX 48 200 mg, po, bid × 14d 41.7 (20/48) 64.6 (31/48) 57.9 (33/57) 10.4 (5/48)
MFX 22 42.6 ± 10
Morning 400 mg + one
placebo, evening two
placebo, po × 7-14d
52.6 (10/19) 84.2 (16/19) 100.0 (14/14) 65.0 (13/20)
LVFX 22 (1/43) 43.5 ± 17.7 200 mg, po, bid × 7-14d 47.1 (8/17) 82.4 (14/17) 72.7 (8/11) 40.9 (9/22)
MFX 29 (41/17) 19 ~ 45 400 mg, po, qd × 7d 79.3 (23/29) 93.1 (27/29) 89.7 (26/29) 55.2 (16/29)
THI 29 500 mg, po, tid × 7d 58.6 (17/29) 65.5 (19/29) 58.6 (17/29) 62.2 (18/29)
MFX 43 400 mg, po, qd × 14d 74.4 (32/43) 95.3 (41/43) 90.7 (39/43) 9.3 (4/43)
SPFX 43 (33/96) 29.6 200 mg, po, qd × 14d 65.1 (28/43) 90.7 (39/43) 88.4 (38/43) 11.6 (5/43)
LVFX 43 200 mg, po, bid × 14d 53.5 (23/43) 69.8 (30/43) 67.4 (29/43) 16.3 (7/43)
MFX 20 (1/19) 42 ± 13
Morning 400 mg + one
placebo, evening two
placebo, po × 7 - 14d
90.0 (8/20) 95.0 (19/20) 94.7 (18/19) 27.2 (6/22)
LVFX 20 (1/19) 42 ± 14 200 mg, po, bid × 7 - 14d 85.0 (17/20) 90.0 (18/20) 100.0 (19/19) 27.2 (6/22)
MFX: Moxifloxacin; LVFX: levofloxacin; AZI: azithromycin; OFLX: ofloxacin; MTZ: metronidazole; SPFX: sparfloxacin; GAT: gatifloxacin; CTRX: ceftriaxone sodium; CLA: clarithromycin; CPFX:
ciprofloxacin; TRZ: terazosin; CAZ: ceftazidime; DOX: doxycycline; MINO: Minocycline; THI: thiamphenicol; UTI: urinary tract infections; NGU: non gonococcal urethritis; PID: pelvic inflammation
disease; RTI: reproductive tract infection.
patients with urinary tract infections (UTI), four
pelvic inflammation disease (PID), five nongonococcal
urethritis (NGU), four urogenital
infections and one reproductive tract infection
(RTI).
Patients in the moxifloxacin group received
moxifloxacin at dosage of 400 mg once a day
(orally or intravenously), while the control group
received the recommended dose of other
antibiotics (according to different countries), such
as levofloxacin, azithromycin, ofloxacin,
metronidazole, sparfloxacin, gatifloxacin

1916 Afr. J. Pharm. Pharmacol.
Table 2. Quality assessment of included RCTs.
Study ID Randomization Allocation concealment Double blinding Dropouts\withdraws Jadad Grade
Zheng HZ 2010 Yes Unclear Unclear No B
Su XD 2010 Yes Unclear Unclear No B
Judlin 2010 Adequate Yes Yes Yes A
Meng W 2009 Yes Unclear Unclear No B
Dang JD 2009 Yes Unclear Unclear No B
Wang G
2009(a)
Yes Unclear Unclear No B
Wang G
2009(b)
Yes Unclear Unclear No B
Jin X 2009 Yes Unclear Unclear No B
Wang X 2009 Yes Unclear Unclear No B
Zhang YH 2009 Yes Unclear Unclear No B
Heystek 2009 Yes Unclear Yes Yes B
Li SQ 2008 Yes Unclear Unclear No B
Pei YH 2008 Yes Unclear Unclear No B
Zhang GH 2007 Yes Unclear Unclear No B
Sun GQ 2007 Yes Unclear Unclear Yes B
Ross 2006 Yes Unclear Yes Yes B
Luo XM 2006 Yes Unclear Unclear No B
Luo JL 2006 Yes Unclear Unclear No B
Gao HY 2005 Adequate Unclear Yes Yes B
Tian YP 2005 Yes Unclear Unclear No B
Zhang WF 2004 Yes Unclear Unclear No B
Cai SF 2003 Adequate Unclear Yes No B
(sparfloxacin and gatifloxacin have been withdrawn
from the American market, respectively, in 2005 due to
QT prolongation and in 2006 due to dysglycemia). All
patients included in the meta-analysis received
antimicrobial treatment for a period of no less than seven
days and a maximum of fourteen days.
The quality assessment of included RCTs is presented
in Table 2. There studies clarified adequate
randomization procedures, one reported allocation
concealment, five used double blinding and five reported
numbers of dropouts\withdrawals. One were eventually
assessed to be good in terms of methodology with a
Jadad score A (Juni et al., 2001), the other twenty-one
were B.
Comparisons of effectiveness
Clinical success
All the twenty-two studies provided specific data for
analysis of clinical cure. Clinical cure rate in the
moxifloxacin group [1513 (77.7%) of 1947 patients] was
higher than control group [1370 (72.91%) of 1879
patients], whilst statistically significant difference was
found [RR = 1.08, 95%CI (1.02, 1.14), P = 0.008] (Figure
2).
19 studies (Zheng, 2010; Su et al., 2010; Meng et al.,
2009; Tang et al., 2009; Wang et al., 2009a; Wang et al.,
2009b; Jin et al., 2009; Wang et al., 2009; Zhang et al.,
2009; Li, 2008; Per et al., 2009; Zhang et al., 2007; Sun,
2007; Luo et al., 2006; Luo et al., 2006; Gao et al., 2005;
Tian et al., 2005; Zhang et al., 2004; Cai et al., 2003)
provided relevant data for analysis of clinical
effectiveness which consisted of clinical cure and
improvement. Clinical effective rate in the moxifloxacin
group [1156 (92.9%) of 1244 patients] was higher than
the control group [1035 (85.0%) of 1217 patients], while
statistically significant difference was found [RR = 1.09,
95%CI (1.04, 1.14), P = 0.0005] (Figure 3).
Microbiological treatment success
17 (Judin et al., 2010; Tang et al., 2009; Wang et al.,
2009a; Wang et al., 2009b; Jin et al., 2009; Zhang et al.,
2009; Heystek and Ross., 2009; Li, 2008; Per et al.,
2009; Zhang et al., 2007; Sun, 2007; Ross et al., 2006;
Luo JL et al., 2006; Gao et al., 2005; Tian et al., 2005;
Zhang et al., 2004; Cai et al., 2003) of the 22 RCTs
provided microbiological eradication data. 1088 (90.8%)
of the 1198 patients in the moxifloxacin group and 1001
(86.5%) of the 1157 patients in the control group
achieved eradication of the baseline pathogens. The
overall pathogens eradication rate in the moxifloxacin
group (90.8%, 1088/1198) was higher than control group

Study or Subgroup
Cai SF 2003
Gao HY 2005
Heystek2009
Jin X 2009
Judlin 2010
Li SQ 2008
Luo JL 2006(1)
Luo JL 2006(2)
Luo XM 2006
Meng W 2009
Pei YH 2008
Ross2006
Su XD 2010
Sun GQ 2007
Tang JD 2009
Tian YP2005
Wang G2009 (a1)
Wang G2009 (a2)
Wang G2009 (b)
Wang X2009
Zhang GH 2007
Zhang WF 2004(1)
Zhang WF 2004(2)
Zhang YH 2009
Zheng HZ 2010
Total (95% CI)
Total events
Heterogeneity: Tau² = 0.01; Chi² = 67.49, df = 24 (P < 0.00001); I² = 64%
Test for overall effect: Z = 2.67 (P = 0.008)
moxifloxacin conventional therapy Risk Ratio Risk Ratio
Events
18
10
224
113
152
85
34
34
44
23
41
248
24
33
43
23
45
45
90
38
23
32
32
35
24
1513
Total
20
19
232
121
194
90
52
52
56
25
62
275
60
44
50
29
80
80
150
60
38
43
43
40
32
1947
Events
17
8
198
112
155
74
32
20
29
20
20
262
20
12
42
17
44
46
84
29
24
28
23
34
20
1370
Total
20
17
202
121
190
86
50
48
56
25
31
289
60
41
50
29
80
80
149
60
38
43
43
40
31
1879
Weight
3.7%
0.7%
10.1%
9.0%
7.9%
7.9%
2.8%
1.7%
2.8%
3.8%
2.4%
9.6%
1.2%
1.1%
5.5%
2.0%
3.0%
3.1%
4.7%
2.3%
2.0%
2.9%
2.3%
5.2%
2.3%
100.0%
M-H, Random, 95% CI
1.06 [0.84, 1.34]
1.12 [0.58, 2.16]
0.99 [0.95, 1.02]
1.01 [0.94, 1.08]
0.96 [0.87, 1.06]
1.10 [0.99, 1.21]
1.02 [0.77, 1.36]
1.57 [1.06, 2.31]
1.52 [1.14, 2.02]
1.15 [0.92, 1.44]
1.02 [0.75, 1.41]
0.99 [0.94, 1.05]
1.20 [0.75, 1.93]
2.56 [1.55, 4.25]
1.02 [0.87, 1.21]
1.35 [0.95, 1.94]
1.02 [0.78, 1.35]
0.98 [0.75, 1.28]
1.06 [0.88, 1.29]
1.31 [0.95, 1.81]
0.96 [0.67, 1.36]
1.14 [0.86, 1.51]
1.39 [1.00, 1.93]
1.03 [0.86, 1.23]
1.16 [0.84, 1.62]
1.08 [1.02, 1.14]
Figure 2. Meta-analysis of cure rates of moxifloxacin and conventional therapy.
Study or Subgroup
Cai SF 2003
Gao HY 2005
Jin X 2009
Li SQ 2008
Luo JL 2006(1)
Luo JL 2006(2)
Luo XM 2006
Meng W 2009
Pei YH 2008
Su XD 2010
Sun GQ 2007
Tang JD 2009
Tian YP2005
Wang G2009 (a1)
Wang G2009 (a2)
Wang G2009 (b)
Wang X2009
Zhang GH 2007
Zhang WF 2004(1)
Zhang WF 2004(2)
Zhang YH 2009
Zheng HZ 2010
Total (95% CI)
Total events
Heterogeneity: Tau² = 0.01; Chi² = 68.35, df = 21 (P < 0.00001); I² = 69%
Test for overall effect: Z = 3.46 (P = 0.0005)
M-H, Random, 95% CI
0.5 0.7 1 1.5 2
Favours conventional Favours moxifloxacin
moxifloxacin conventional therapy Risk Ratio Risk Ratio
Events
19
16
115
88
48
48
51
25
57
54
41
46
27
73
73
144
49
33
41
41
38
29
1156
Total
20
19
121
90
51
51
56
25
62
60
44
50
29
80
80
150
60
38
43
43
40
32
1244
Events
18
14
116
79
45
31
43
24
31
43
31
45
19
73
75
142
39
34
39
30
37
27
1035
Total
20
17
121
86
50
48
56
25
50
60
41
50
29
80
80
149
60
38
43
43
40
31
1217
Weight
3.8%
2.0%
7.2%
6.8%
5.5%
3.0%
4.1%
5.6%
2.8%
3.7%
3.5%
5.2%
2.1%
6.0%
6.3%
7.4%
2.9%
4.1%
5.4%
3.2%
5.5%
3.8%
100.0%
M-H, Random, 95% CI
1.06 [0.88, 1.26]
1.02 [0.76, 1.37]
0.99 [0.94, 1.05]
1.06 [0.99, 1.14]
1.05 [0.93, 1.17]
1.46 [1.17, 1.82]
1.19 [1.00, 1.40]
1.04 [0.93, 1.16]
1.48 [1.18, 1.86]
1.26 [1.05, 1.50]
1.23 [1.02, 1.49]
1.02 [0.90, 1.16]
1.42 [1.07, 1.88]
1.00 [0.91, 1.10]
0.97 [0.89, 1.06]
1.01 [0.96, 1.06]
1.26 [1.01, 1.57]
0.97 [0.82, 1.14]
1.05 [0.94, 1.18]
1.37 [1.11, 1.68]
1.03 [0.92, 1.15]
1.04 [0.87, 1.24]
1.09 [1.04, 1.14]
Figure 3. Meta-analysis of clinical effective rates of moxifloxacin and conventional therapy.
M-H, Random, 95% CI
0.5 0.7 1 1.5 2
Favours conventional Favours moxifloxacin
Mu et al. 1917

1918 Afr. J. Pharm. Pharmacol.
(86.52%, 1001/1157), while statistically significant
difference was found [RR = 1.04, 95% (0.99, 1.09),
P=0.08] (Figure 4).
Comparisons of safety
All of the twenty-two RCTs provided adverse drug
reaction data. In the total evaluable population, the
incidence of adverse drug reaction between moxifloxacin
group [509 (23.8%) of the 2136 patients] and control
group [580 (27.9%) of the 2077 patients] had no
statistically significant difference [RR = 0.88, 95%CI
(0.72, 1.06), P = 0.17] (Figure 5).
Comparisons of PID patients
Judin (Judin et al., 2010), Heystek (Heystek et al., 2009)
and Ross (Ross et al., 2006) altogether researched 1643
pelvic inflammatory disease (PID) patients. As the their
studies recommended moxifloxacin as first-line therapy
for pelvic inflammatory disease, we exclusively performed
a meta-analysis for them. For the primary measure of
efficacy [clinical cure rate at test-of-cure (TOC)],
moxifloxacin was non-inferior to comparator
(moxifloxacin: 624/701, 89.0%; comparator: 615/681,
90.3%) with no statistically significant difference between
moxifloxacin and control group [RR = 0.98, 95%CI (0.95,
1.02), P = 0.33] (Figure 6). Microbiological success rates
were 90.2% (119/132) for moxifloxacin and 85.1%
(103/121) for comparator, whilst no statistically significant
difference was found [RR = 1.06, 95%CI (0.96, 1.16), P =
0.25] (Figure 7). No statistically significant difference was
found [RR = 0.85, 95%CI (0.68, 1.05), P = 0.13] (Figure
8) between moxifloxacin [38.5% (315/949)] versus the
comparator [45.9% (406/884)] about adverse drug
reaction rates.
DISCUSSION
We conducted this meta-analysis to compare the efficacy
and safety of moxifloxacin monotherapy with
conventional antimicrobial therapy for urogenital system
infections. The overall RR of clinical rate, clinical effective
rate and pathogens eradication rate of moxifloxacin were
higher than control group. The results of meta-analysis
indicated that efficacy of moxifloxacin was superior to
conventional antibiotics therapy with statistically
significant difference [Clinical cure rate: RR = 1.08,
95%CI (1.02, 1.14), P = 0.008; clinical effective rate: RR
= 1.09, 95%CI (1.04, 1.14), P = 0.0005; pathogens
eradication rate: RR = 1.04, 95% (0.99, 1.09), P = 0.08].
The safety profile analysis regarding the incidence of
adverse drug reactions had no significant difference
between moxifloxacin and control
group [RR = 0.88, 95%CI (0.72, 1.06), P = 0.17].
Moreover, the efficacy and safety profile for PID patients
(Judin, 2010; Heystek, 2009; Ross, 2006) were
separately analyzed. These three large randomized
controlled trials supported the efficacy of moxifloxacin for
the treatment of uncomplicated PID. Clinical cure rates
observed in the MONALISA (Judin et al., 2010) study
were 78.4% for moxifloxacin versus 81.6% for
levofloxacin plus metronidazole. Heysteck (2009)
reported a clinical cure rate of 81.5% in women treated
with moxifloxacin versus 83.2% in those treated with the
doxycycline plus metronidazole plus one dose of
ciprofloxacin. In the MAIDEN (Ross et al., 2006) study,
clinical cure rate was achieved in 90.2% for moxifloxacin
versus 90.7% for ofloxacin plus metronidazole. Metaanalysis
indicated that the difference of efficacy between
moxifloxacin mono- therapy group and combination
therapy group was not statistically significant (Clinical
cure rates: RR = 0.98, 95%CI (0.95, 1.02), P = 0.33). In
addition, no statistically significant differences were found
in pathogens eradi- cation rates (RR = 1.06, 95%CI (0.96,
1.16), P = 0.25) and the incidences of adverse drug
reactions (RR = 0.85, 95%CI (0.68, 1.05), p = 0.13).
Moxifloxacin monotherapy, 400 mg once daily for 14
days, is an effective and well-tolerated treatment for
women with PID. Therefore, moxifloxacin can be
recommended as the first-line therapy for uncomplicated
PID.
In all the included studies, moxifloxacin had the
advantage of using single dose per day and did not need
combination because of broad coverage to pathogens.
Monotherapy is always associated with greater
compliance than combination therapy (Haggerty and
Ness, 2007), especially for outpatients. The adminis-
tration of moxifloxacin was intravenous or oral and its
medication time was free from diet (Instruction of
Moxifloxacin Hydrochloride Tablets). Moxifloxacin can be
accessible to inpatients and outpatients with greater
compliance. The most common adverse events reported
in the included studies were gastrointestinal disturbances
and central nervous system reactions, such as nausea,
diarrhea, vomiting, dizzy and headache. Serious adverse
events were not reported in the enrolled articles. Balfour
et al. (1999) demonstrated that in contrast to some other
fluoroquinolones, moxifloxacin appears to have a low
propensity for causing phototoxic and central nervous
system (CNS) excitatory effects, and the most common
adverse events are gastrointestinal disturbances.
This meta-analysis is not without limitations. Certain
limitations affecting the results of this meta-analysis
should be taken into account. Firstly, our findings may be
affected by the quality of trials included in the metaanalysis.
Only five of the included trials were double
blinding, and only three clarified adequate randomization
procedures. A sensitivity analysis was performed
including only trials that were double-blinded, the
comparisons of pathogens eradication rates and
incidence of adverse drug reaction were consistent with
the primary results. The primary clinical effective rate of
moxifloxacin was superiority to conventional antibiotics

Study or Subgroup
Cai SF 2003
Gao HY 2005
Heystek2009
Jin X 2009
Judlin 2010
Li SQ 2008
Luo JL 2006(1)
Luo JL 2006(2)
Pei YH 2008
Ross2006
Sun GQ 2007
Tang JD 2009
Tian YP2005
Wang G2009 (a1)
Wang G2009 (a2)
Wang G2009 (b)
Zhang GH 2007
Zhang WF 2004(1)
Zhang WF 2004(2)
Zhang YH 2009
Total (95% CI)
Total events
Heterogeneity: Tau² = 0.01; Chi² = 49.43, df = 19 (P = 0.0002); I² = 62%
Test for overall effect: Z = 1.74 (P = 0.08)
moxifloxacin conventional therapy Risk Ratio Risk Ratio
Events
18
14
43
116
27
87
56
56
51
49
45
43
19
72
72
169
42
39
39
31
1088
Total
19
14
46
121
30
90
60
60
57
56
49
47
29
80
80
188
46
43
43
40
1198
Events
19
8
35
117
22
81
54
33
32
46
31
42
29
73
74
162
46
38
29
30
1001
Total
19
11
39
121
26
86
61
57
44
56
41
46
29
80
80
186
50
43
43
39
1157
Weight
5.1%
1.3%
5.6%
8.9%
3.4%
8.3%
6.3%
2.9%
3.5%
4.6%
3.7%
5.8%
2.3%
6.8%
7.0%
8.0%
5.9%
5.1%
2.9%
2.7%
100.0%
M-H, Random, 95% CI
0.95 [0.82, 1.09]
1.36 [0.94, 1.99]
1.04 [0.91, 1.19]
0.99 [0.94, 1.04]
1.06 [0.87, 1.30]
1.03 [0.96, 1.10]
1.05 [0.94, 1.18]
1.61 [1.28, 2.03]
1.23 [1.01, 1.51]
1.07 [0.91, 1.25]
1.21 [1.00, 1.47]
1.00 [0.88, 1.14]
0.66 [0.51, 0.86]
0.99 [0.89, 1.09]
0.97 [0.88, 1.07]
1.03 [0.96, 1.11]
0.99 [0.88, 1.12]
1.03 [0.89, 1.19]
1.34 [1.07, 1.69]
1.01 [0.79, 1.28]
1.04 [0.99, 1.09]
M-H, Random, 95% CI
0.5 0.7 1 1.5 2
Favours conventional Favours moxifloxacin
Figure 4. Meta-analysis of pathogens eradication rates of moxifloxacin and conventional therapy.
Study or Subgroup
Cai SF 2003
Gao HY 2005
Heystek2009
Jin X 2009
Judlin 2010
Li SQ 2008
Luo JL 2006(1)
Luo JL 2006(2)
Luo XM 2006
Meng W 2009
Ross2006
Su XD 2010
Sun GQ 2007
Tang JD 2009
Tian YP2005
Wang G2009 (a1)
Wang G2009 (a2)
Wang G2009 (b)
Wang X2009
Zhang GH 2007
Zhang WF 2004(1)
Zhang WF 2004(2)
Zhang YH 2009
Zheng HZ 2010
Total (95% CI)
Total events
Heterogeneity: Tau² = 0.07; Chi² = 46.31, df = 23 (P = 0.003); I² = 50%
Test for overall effect: Z = 1.37 (P = 0.17)
moxifloxacin conventional therapy Risk Ratio Risk Ratio
Events
16
13
151
11
129
7
5
5
7
4
85
4
8
3
16
8
8
12
3
1
4
4
2
3
509
Total
22
20
343
121
228
90
52
52
56
25
378
60
44
50
29
80
80
150
60
38
43
43
40
32
2136
Events
6
9
162
11
132
8
7
5
41
8
112
6
6
3
18
5
9
13
0
2
5
7
3
2
580
Total
22
22
326
121
232
86
32
48
56
25
363
60
41
50
29
80
80
149
60
38
43
43
40
31
2077
Weight
4.6%
5.9%
12.9%
4.1%
13.0%
3.0%
2.6%
2.2%
4.8%
2.6%
11.6%
2.1%
3.0%
1.4%
8.2%
2.6%
3.4%
4.4%
0.4%
0.6%
2.0%
2.3%
1.1%
1.1%
100.0%
M-H, Random, 95% CI
2.67 [1.29, 5.53]
1.59 [0.88, 2.88]
0.89 [0.75, 1.04]
1.00 [0.45, 2.22]
0.99 [0.85, 1.17]
0.84 [0.32, 2.21]
0.44 [0.15, 1.27]
0.92 [0.28, 2.99]
0.17 [0.08, 0.35]
0.50 [0.17, 1.45]
0.73 [0.57, 0.93]
0.67 [0.20, 2.24]
1.24 [0.47, 3.27]
1.00 [0.21, 4.72]
0.89 [0.58, 1.37]
1.60 [0.55, 4.68]
0.89 [0.36, 2.19]
0.92 [0.43, 1.94]
7.00 [0.37, 132.66]
0.50 [0.05, 5.28]
0.80 [0.23, 2.78]
0.57 [0.18, 1.81]
0.67 [0.12, 3.78]
1.45 [0.26, 8.11]
0.88 [0.72, 1.06]
M-H, Random, 95% CI
0.1 0.2 0.5 1 2 5 10
Favours moxifloxacin Favours conventional
Figure 5. Meta-analysis of the incidences of adverse drug reactions of moxifloxacin and conventional therapy.
Mu et al. 1919

1920 Afr. J. Pharm. Pharmacol.
Study or Subgroup
Heystek2009
Judlin 2010
Ross2006
Total (95% CI)
Total events
Events
224
152
248
624
Total
232
194
275
701
Heterogeneity: Chi² = 0.42, df = 2 (P = 0.81); I² = 0%
Test for overall effect: Z = 0.97 (P = 0.33)
Experimental Control Risk Ratio Risk Ratio
Events
198
155
262
615
Total
202
190
289
681
Weight
33.9%
25.1%
41.0%
100.0%
M-H, Fixed, 95% CI
0.99 [0.95, 1.02]
0.96 [0.87, 1.06]
0.99 [0.94, 1.05]
0.98 [0.95, 1.02]
M-H, Fixed, 95% CI
0.5 0.7 1 1.5 2
Favours conventional Favours moxifloxacin
Figure 6. Meta-analysis of clinical cure rates of moxifloxacin and conventional therapy in the three PID studies.
Study or Subgroup
Heystek2009
Judlin 2010
Ross2006
Total (95% CI)
Total events
Events
Heterogeneity: Chi² = 0.06, df = 2 (P = 0.97); I² = 0%
43
27
49
119
Total
46
30
56
132
Test for overall effect: Z = 1.16 (P = 0.25)
Experimental Control Risk Ratio Risk Ratio
Events
35
22
46
103
Total
39
26
56
121
Weight
35.3%
21.9%
42.8%
100.0%
M-H, Fixed, 95% CI
1.04 [0.91, 1.19]
1.06 [0.87, 1.30]
1.07 [0.91, 1.25]
1.06 [0.96, 1.16]
M-H, Fixed, 95% CI
0.5 0.7 1 1.5 2
Favours conventional Favours moxifloxacin
Figure 7. Meta-analysis of microbiological success rates of moxifloxacin and conventional therapy in the three PID studies.
Study or Subgroup
Heystek2009
Judlin 2010
Ross2006
Total (95% CI)
Total events
Events
151
129
85
365
Total
343
228
378
949
Heterogeneity: Tau² = 0.03; Chi² = 8.44, df = 2 (P = 0.01); I² = 76%
Test for overall effect: Z = 1.50 (P = 0.13)
Experimental Control Risk Ratio Risk Ratio
Events
162
132
112
406
Total
326
232
326
884
Weight
35.5%
35.7%
28.8%
100.0%
M-H, Random, 95% CI
0.89 [0.75, 1.04]
0.99 [0.85, 1.17]
0.65 [0.51, 0.83]
0.85 [0.68, 1.05]
M-H, Random, 95% CI
0.5 0.7 1 1.5 2
Favours moxifloxacin Favours conventional
Figure 8. Meta-analysis of the incidence of adverse reaction of moxifloxacin and conventional therapy in the three PID studies.
therapy with statistically significant difference, but not
finding statistically significant difference at sensitivity
analysis. Secondly, heterogeneity was found among the
included trials, we cannot make symmetrical funnel plots.
Publication bias can lead to overestimation in metaanalysis.
The funnel plot is frequently used to detect
publication bias which can lead to overestimation in
meta-analysis (Souza et al., 2007). However,
heterogeneity does not preclude pooling of the results
because individual patients are directly compared only
with other patients within the same trial, and not across
trials (Lau et al., 1998; Thompson, 1994) Although some
limitations exist in this meta-analysis, we believe that
moxifloxacin can be considered using for the treatment of
urogenital infections for the following reasons. First, the
results of this meta-analysis revealed that the clinical

cure rate and clinical effective rate achieved with
moxifloxacin tended to be higher than obtained in the
groups that received conventional antibiotic treatment
and the incidence of adverse drug reactions of
moxifloxacin was not significantly different from the
control group. Second, the compliance of patients is
influenced by the complexity of the dosage regimen. The
simple usage of moxifloxacin (400 mg, once a day,
PO/IV) offer benefits compared with regimens that
require combination therapy or multiple dosing.
Moreover, it does not need to adjust the dosage of
moxifloxacin for elderly patients and those patients with
renal or mild hepatic impairment (Ball et al., 2004). Third,
in a review article, Bambeke et al. (Van and Tulkens,
2009) concluded that moxifloxacin did not reveal
significantly higher incidences of drug-related adverse
effects than for comparators. It was coincident with result
of this meta-analysis. Certainly, further high quality RCTs
are required.
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African Journal of Pharmacy and Pharmacology Vol. 6(26), pp. 1923-1926, 15 July, 2012
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP12.197
ISSN 1996-0816 ©2012 Academic Journals
Full Length Research Paper
The expression of tumor metastasis suppressor gene
KAI1 and matrix metalloproteinase 2 in
breast cancer tissues
Tong Zhang 1 *, Guoping Ren 1 , Zhenhai Zhang 1 , Rui Zhang 1 and Yuanhang Li 2
1 Department of Oncology, Xinxiang Central Hospital, He Nan, China.
2 Department of Internal Medicine, Liaoning Provincial Tumor Hospital, Liao Ning, China.
Accepted 8 June, 2012
The objective of this study is to investigate the expression of tumor metastasis suppressor gene KAI1
protein and matrix metalloproteinase 2 (MMP-2) in breast carcinoma. We retrospectively analyzed 60
cases of patients from June, 2005 to June, 2011 treated with radical mastectomy for invasive ductal
breast cancer in our department. The carcinoma tissue was stained for KAI1 and MMP2 with
immunohistochemistry. In breast cancer group, the positive expression rate of KAI1 was 41.7%, which
was significantly lower than control (83.3%) (� 2 = 17.328, P < 0.05); the positive expression rate of MMP-
2 was 80.0%, which was significantly higher than control group (16.7%) (� 2 = 41.637, P < 0.05). Within
the breast cancer patients, the group with lymph node metastasis showed higher positive rate of KAI1
and MMP2 expression than those without lymph node metastasis (P < 0.05). The patients with tumor
diameter more than 5 cm showed lower positive rate of KAI1 but higher positive rate of MMP-2
expression than patient group with tumor diameter less than 5 cm. Additionally, tumor-nodesmetastasis
III (TNM III) grade patients showed lower positive rate of KAI1 but higher positive rate of
MMP-2 expression than patient group in I and II grades. KAI1 and MMP-2 were highly expressed in
breast cancer tissues. The positive expression of KAI1 and MMP-2 were associated with tumor
metastasis, tumor size, TNM grades etc.
Key words: Breast cancer, KAI1, matrix metalloproteinase 2 (MMP-2), immunohistochemistry.
INTRODUCTION
Breast cancer is one of the most common malignant
cancers in woman, with increasing incidences in recent
decades. The causes of mortality by breast cancer mainly
include the lymph node and blood metastasis (Budach,
2011; Ruiterkamp and Ernst, 2011). Therefore, the
investigation of the underlying molecular mechanisms
controlling tumor metastasis is critical in breast cancer
control and treatment.
KAI1 is a newly identified inhibitory gene for tumor
metastasis, while its role in breast cancer metastasis has
not been fully investigated (Dong et al., 1995; Ueda et al.,
1996; Yang et al., 1997). Additionally,
matrix
*Corresponding author. E-mail: zhongtong_cancer@163.com.
Tel: +86 373 2039 091.
metalloproteinase-2 (MMP-2) has also been known for its
roles in tumor invasion and metastasis due to its
degrading function of the basal membrane and the
extracellular matrix (ECM) components (Cockett et al.,
1998; Bachmeier et al., 2005; Jezierska and Motyl,
2009). However, whether these two factors jointly
involved in tumor/breast cancer metastasis is unknown.
The present study investigated the changes of KAI1 and
MMP-2 in breast cancer patients and correlated the
results with clinical pathological characteristics.
MATERIALS AND METHODS
Clinical data
Sixty (60) cases of patients (24 to 70 years old with a mean of 43.6
± 11.5 years old, median age 45 years old) from June, 2005 to

1924 Afr. J. Pharm. Pharmacol.
Figure 1. A, In invasive ductal carcinoma tissue KAI1 is lowly expressed in cell
membrane and cytoplasm (×200); B, In benign breast tissue, KAI1 is highly
expressed in cell membrane and cytoplasm (×200); C, In invasive ductal
carcinoma tissue MMP-2 is positively expressed in cell membrane and cytoplasm
(×200); D, In benign breast tissue MMP-2 is lowly expressed in cell membrane
and cytoplasm (X×200).
June, 2011 treated with radical mastectomy for invasive ductal
breast cancer in our department was retrospectively recruited in this
study. The diagnoses were confirmed with pathological sample
dissected from the surgery, and all patients were without prechemotherapy.
The patients were divided according to tumornodes-metastasis
(TNM) grades: 14 cases of stage I, 29 cases of
stage II, and 17 cases of stage III, or by histological grades: 29
cases of stage I, 17 cases of stage II, and 14 cases of stage III.
Twenty-seven (27) cases of patients showed tumor size more than
5 cm. Twenty-one (21) cases of patients showed lymph node
metastasis. Fifteen (15) patients have post-menopausal.
Thirty (30) cases of subjects (25 to 70 years old, averaged at
44.8 ± 9.7 years old) with benign breast cancer tissue samples
were recruited as control group. There were no differences in the
age composition of the two groups (P>0.05). The study has been
approved by Local Ethic Committee of human research and has the
informed written consent of all patients.
Immunohistochemistry
The samples obtained during surgery were fixed in 10% neutral
formalin solution, and processed for 5 µm paraffin sections. The
mouse-anti human KAI1 and MMP-2 monoclonal antibody (1:200,
Jinqiao, Beijing) were incubated overnight at 4°C and finally the
reaction was visualized with 3,3'-diaminobenzidine (DAB) approach.
Then sections were examined under microscope and 5 areas per
section were randomly selected for counting of positive cells. The
positive cell rate were recorded as negative (–) for < 5%, positive
(+) for 5 to 25%, ++ for 25 to 50%, and +++ for >50%.
Statistics
All data were represented as mean ± standard deviation (SD) and
analyzed with SPSS 13.0 software (Chicago, US). The � 2 test was
used to examine the relationship between the expression of
KAI1/MMP-2 and the clinical characteristics; the correlationship was
examined with Pearson test; P

Table 1. The expression of KAI1 and MMP-2 in breast cancer and control groups.
Group n
Positive (cases)
KAI1
Negative (cases) Positive rate (%) Positive (cases)
MMP-2
Negative (cases) Positive rate (%)
Breast cancer 60 25 35 41.7* 48 12 80.0*
Control 30 25 5 83.3 5 25 16.7
x 2 5.328 12.777
P 0.000 0.000
*, P0.05
Lymph node metastasis
Tumor size
TNM stage
Histological stages
Menstruation
The expression of KAI1 and MMP-2 in breast
cancer patients with different clinical features
Within the breast cancer patients, the group with
lymph node metastasis showed higher positive
rate of KAI1 and MMP2 expression than those
without lymph node metastasis (P

1926 Afr. J. Pharm. Pharmacol.
KAI1 could mediate the interactions between cells or cells
and the ECM to change the ability of cell adhesion and
migration (Yoshida et al., 1998). It has been shown that
low differentiation of the cancer cells was associated with
lower expression of KAI1 (Chen et al., 2011). This is in
consistent with the present results: the KAI1 expression
was down-regulated in breast cancer tissues, and the
patients with lymph node metastasis showed lower KAI1
expression than those without. These data suggested
that decreased KAI1 expression is closely related to the
tumor progression and metastasis.
MMPs were considered as one of the most important
proteolytic enzymes involving in tumor growth and
invasion (Cockett et al., 1998). MMP-2 has been shown
to be involved in breast cancer metastasis, by
degradation of the ECM and induction of angiogenesis
(Jezierska and Motyl, 2009). The present study showed
that in breast cancer group, the MMP-2 was highly
expressed; within the cancer group, MMP-2 expression is
positively correlated with tumor metastasis, but not with
age and menopause factors.
Taken together, our studies proved that changes of
KAI1 and MMP-2 were associated with the development
and progression of breast cancer in our population.
These provided rational bases for molecule-based
diagnosis of breast cancer and prognosis. Future studies
are still required to recruit more patients to verify these
results.
REFERENCES
Bachmeier BE, Iancu CM, Jochum M, Nerlich AG (2005). Matrix
metalloproteinases in cancer: comparison of known and novel
aspects of their inhibition as a therapeutic approach. Expert Rev.
Anticancer Ther., 5:149-163 doi: 10.1586/14737140.5.1.149
Budach W (2011). Radiotherapy in patients with metastatic breast
cancer. Eur. J. Cancer, 47 Suppl., 3:S23-27 doi: 10.1016/S0959-
8049(11): 70143-70145.
Chen X, Xu Z, Wang Y (2011) Recent advances in breast cancer
metastasis suppressor 1. Int. J. Boimarkers, 26:1-8.
Cockett MI, Murphy G, Birch ML, O'Connell JP, Crabbe T, Millican AT,
Hart IR, Docherty AJ (1998). Matrix metalloproteinases and
metastatic cancer. Biochemical Society symposium 63:295-313.
Dong JT, Isaacs WB, Isaacs JT (1997). Molecular advances in prostate
cancer. Curr. Opin. Oncol., 9:101-107.
Dong JT, Lamb PW, Rinker-Schaeffer CW, Vukanovic J, Ichikawa T,
Isaacs JT, Barrett JC (1995). KAI1, a metastasis suppressor gene for
prostate cancer on human chromosome 11p11.2. Science, 268:884-
886.
Jezierska A, Motyl T (2009). Matrix metalloproteinase-2 involvement in
breast cancer progression: a mini-review. Medical science monitor :
international medical J. Exper. Clin. Res., 15:RA32-40.
Ruiterkamp J, Ernst MF (2011). The role of surgery in metastatic breast
cancer. Eur. J. Cancer, 47 Suppl., 3:S6-22 doi: 10.1016/S0959-
8049(11): 70142-70143.
Ueda T, Ichikawa T, Tamaru J, Mikata A, Akakura K, Akimoto S, Imai T,
Yoshie O, Shiraishi T, Yatani R, Ito H, Shimazaki J (1996).
Expression of the KAI1 protein in benign prostatic hyperplasia and
prostate cancer. Am. J. Pathol., 149:1435-1440.
Yang X, Welch DR, Phillips KK, Weissman BE, Wei L (1997). KAI1, a
putative marker for metastatic potential in human breast cancer.
Cancer Lett., 119:149-155.
Yoshida BA, Chekmareva MA, Wharam JF et al., (Provide Complete
Name) (1998). Prostate cancer metastasis-suppressor genes: A
current perspective. In vivo 12:49-58.

African Journal of Pharmacy and Pharmacology Vol. 6(26), pp. 1927-1932, 15July, 2012
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP12.276
ISSN 1996-0816 ©2012 Academic Journals
Full Length Research Paper
Comparative excretion of vitexin-2"-O-rhamnoside in
mice after oral and intravenous administration
Ye An 1 , Chaoshen Zhang 1 , Yang Du 2 , Lin Zhao 2 , Zhongzhe Cheng 2 , Wenjie Zhang 2 ,
Xixiang Ying 2 * and Tingguo Kang 2
1 Department of Pharmacy, the General Hospital of Shenyang Military Region, Shenyang, Liaoning, 110015, China.
2 School of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian, Liaoning, 116600, China.
Accepted 03 April, 2012
The aim of the present study was to characterize comparative excretion of pure vitexin-2"-O-rhamnoside
(VR) in mice following oral and intravenous administration at dose of 30 mg/kg, therefore, a sensitive
and specific high-performance liquid chromatography (HPLC) method using vitexin-4"-O-glucoside as
internal standard developed and validated for quantitative analysis of VR. The results of elimination of
VR in urinary and fecal excretion following oral and intravenous dosing indicated that VR was mainly
excreted as prototype for both routes of administration, and biliary and renal excretions are two major
ways for the elimination of VR.
Key words: Excretion, high-performance liquid chromatography (HPLC), Vitexin-2"-O-rhamnoside.
INTRODUCTION
Vitexin-2"-O-rhamnoside (VR) abundantly exists in fruits
and leaves of Crataegus pinnatifida Beg. var major
(hawthorn and hawthorn leaves), both of which are very
popular herbal materials in traditional Chinese medicine
(TCM) and are well used in treating cardiovascular
diseases (PRC, 2010). As VR is one of the main
components of flavonoid of hawthorn leaves (Ding et al.,
1990) and also a bioactive constituent on cardiovascular
system in hawthorn (Liang et al., 2007), many
pharmacological studies of VR have been reported until
now, such as protective effect on the injured cardiac
myocytes and endothelial cells (Zhu et al., 2003, 2006)
and strongly inhibiting deoxyribonucleic acid (DNA)
synthesis in MCF-7 human breast cancer cells (Ninfali et
al., 2007). In recent years, many articles focus on in vitro
study and pharmacokinetic studies (Cheng et al., 2007;
Ying et al., 2007, Du et al., 2011). However, there is little
research on the comparative excretion of pure VR
isolated from hawthorn leaves following oral and
intravenous routes of administration. In our study, a
*Corresponding author. E-mail: yingxixiang@163.com.
sensitive and specific high-performance liquid
chromatography (HPLC) method using vitexin-4"-Oglucoside
(VG) as internal standard thereby was first
established to fully evaluate the urinary and fecal
excretion content of VR following oral and intravenous
route of administration. In addition, the differences of
excretion after two forms of administration can be
identified.
MATERIALS AND METHODS
Reagents and chemicals
The water used in all experiments was purified by a Milli-Q ® Biocel
Ultrapure Water System (Millipore, Bedford, MA, USA). Methanol,
acetonitrile and tetrahydrofuran were all of HPLC grade and
purchased from Xinxing Chemical Reagent Plant (Shanghai,
China). All other chemicals of analytical grade were obtained from
Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).
Plant
Leaves of C. pinnatifida Bge.var major were collected in Shenyang,
Liaoning Province, China and identified by Prof. Ting- Guo Kang. A

1928 Afr. J. Pharm. Pharmacol.
(A)
HO
H3C O OH
O
OH
HO
O
OH
O
OH
OH OH
O
OH
HO
OH
OH
OH
O
HO
O
HO
O
OH
O
OH
OH
OH
Figure 1. (A) Chemical structure of vitexin-2"-O-rhamnoside. (B) Chemical structure of vitexin-4"-
O-glucoside (I.S.).
voucher specimen (20110921) was deposited in Liaoning University
of Traditional Chinese Medicine.
Extraction and isolation
A sample of the leaves (2 kg) of C. pinnatifida Bge. var. major was
cut in small pieces and refluxed with 60% aqueous ethanol for two
times, each for 2 h. The crude extract after concentrated under
reduced pressure was then adsorbed on a porous-polymer resin
(AB-8, Tianjin, China) column, removed impurity with water and
eluted with a gradient of 30, 50 and 70% ethanol. The fraction
eluted with 30% ethanol was evaporated under reduced pressure to
obtain extract, which was then chromatographed on silica gel
column and eluted with ethyl acetate/butanone/formic acid/water
3:3:1:1. Fractions of similar composition were pooled on the basis
of TLC analysis (UV monitoring at 365 nm) were repeatedly
subjected to silica gel column chromatography and eluted with ethyl
acetate/butanone/formic acid/water 5:3:1:1 to directly obtain crystal
of VR (1 g) and VG (0.2 g) internal standard (I.S.), and purities of
them were both over 99% by HPLC analysis. The chemical
structures of VR and VG, confirmed by 1 HNMR, 13 CNMR and MS
data, are shown in Figure 1.
Chromatographic system
The analysis was carried out on an Agilent 1100 series HPLC
system (Agilent technology, Palo Alto, CA, USA) which consisted of
a quaternary Pump (G1310A), a vacuum degasser (G1322A), a
UV-VIS spectrophotometric detector (G1314A) and Chemstation
software (Agilent). The analytical column was a Diamonsil C18
column (150 mm × 4.6 mm i.d., 5 μm, Diamonsil, USA) protected by
a KR C18 guard column (35 × 8.0 mm, i.d., 5 μm, Dalian Create
Science and Technology Co., Ltd., China). The optimal mobile
phase used for separation is a mixture consisting of methanolacetonitrile-tetrahydrofuran-1%
glacial acetic acid (6:2:18:74,
v/v/v/v). All the chromatographic measurements were performed at
room temperature and a flow rate of 1 ml/min with the detection
(B)
O
wavelength of 330 nm.
Preparation of standards and quality control samples
Standard stock solutions of VR and I.S. were both prepared in
methanol to yield the concentrations of 4000 and 276 μg/ml,
respectively. Stock solution of VR was serially diluted with methanol
to desired concentrations over the range of 0.16 to 3200 μg/ml. All
the solutions were stored at 4°C before use. The calibration
samples were prepared by spiking 200 μl blank mouse urine or 500
μl blank feces homogenates successively with appropriate amount
of working solutions of VR (50 μl), acetic acid (20 μl) and I.S.. The
volume of I.S. is 40 μl for urine samples and 80 μl for feces
samples. The quality control samples of VR in the method
validation were similarly prepared to the standard calibration
samples.
Animals and dosing
SPF male Kunming strain mice (20 ± 2 g) were obtained from the
Experimental Animal Center of Liaoning University of Traditional
Chinese Medicine (Shenyang, China) and housed in an aircontrolled
breeding room for a week. Before starting the
experiment, all the mice formerly free access to standard laboratory
food as well as water ad libitum were fasted for more than 12 h. All
experiments involving animals were approved by the animal ethics
committee of Liaoning University of traditional Chinese medicine
and performed according to the Guidelines for Animal
Experimentation of this institution. For excretion studies, ten mice
were randomly divided into two groups to be administrated VR at
dose of 30 mg/kg. Among the two groups, one was orally
administrated and the other intravenously administrated. All the
mice were respectively housed in stainless-steel metabolism cages
to collect urine and feces at different time. Two hours after
administration, water and standard laboratory food were offered.
Urine and feces samples were collected at 0 to 2, 2 to 4, 4 to 6, 6 to
8, 8 to 12 and 12 to 24 h post-dosing. The volume of each collected
urine sample and the weight of each collected feces sample were

Figure 2. Typical chromatograms of urine excretion study (A-D) respectively obtained from blank
urine sample, blank urine sample spiked with standard analyte and I.S., and urine samples collected
from 4 to 6 h following oral and intravenous routes of administration of VR at dose of 30 mg/kg.
separately recorded. All the samples were stored at -20°C until
analysis.
Sample processing
To 200 μl urine samples, 20 μl of acetic acid, 40 μl of I.S. and 1 ml
of methanol were successively pipetted, followed by vortex mixing
for 1 min. After samples being centrifuged at 890 g for 15 min, the
supernatant was separated and evaporated to dryness under a
gentle stream of nitrogen at 50ºC. The residue was diluted in 200 μl
of mobile phase and again centrifuged at 15,092 g for 10 min. Then
an aliquot (20 μl) of clean supernatant was injected into HPLC
column for analysis. Feces (0.2 g) were homogenized in 0.5 ml of
An et al. 1929
saline solution. The homogenate was successively added 20 μl of
acetic acid, 80 μl of I.S. and 2 ml of methanol and then treated as
urine samples.
RESULTS AND DISCUSSION
HPLC chromatograms
Typical chromatograms of urinary and fecal excretion
studies are shown in Figures 2 and 3, respectively. The
total run time was no more than 20 min and the retention

1930 Afr. J. Pharm. Pharmacol.
Figure 3. Typical chromatograms of fecal excretion study (A-D) respectively obtained from blank feces
sample, blank feces sample spiked with standard analyte and I.S., and feces samples collected from 6
to 8 h following oral and intravenous routes of administration of VR at dose of 30 mg/kg.
times of I.S. and VR were approximately 6.6 and 8.7 min,
with no interfering peaks detected at the retention times
of VR or I.S.
Method validation
Method validation involves linearity, precision, limit of
detection (LOD), limit of quantification (LOQ), recovery
and stability. The linear range for urine and feces were
within 0.4 to 400 μg/ml with R 2 > 0.99. The LOD (S/N > 3)
and the LOQ (S/N > 10) were respectively 0.121 and
0.363 μg/ml in urine and 0.121 and 0.363 μg/g in feces.
Both of the precision (RSD%) and accuracy (RE%) were
below 15%, conforming to the criteria for the analysis of
biological sample according to guidance of USFDA. The
extraction recoveries of VR in urine and feces ranged
from 82.36 ± 5.82% to 108.9 ± 8.47%. The results of
short-term stability, long-term stability and freeze-thaw
stability indicated that no remarkable degradation
occurred during chromatography, extraction and sample
storage processes for excreta samples.

Excretion studies
Figure 4 shows the urinary and fecal excretion time
profiles of VR following oral and intravenous
administration at dose 30 mg/kg. The urinary and fecal
cumulative ratios of unchanged VR amounted to 4.13 ±
(A)
Urinary cumulative ratio of VR (%)
Fecal cumulative ratio of VR (%)
25
20
15
10
5
0
(B)
0 5 10 15 20 25
60
50
40
30
20
10
0
Time (h)
Time (h)
Intravenous administration
Oral administration
Intravenous administration
Oral administration
0 5 10 15 20 25
Figure 4. (A) Urinary cumulative ratio of vitexin-2"-O-rhamnoside in
mice (mean ± S.D., n = 5) following oral and intravenous routes of
administration at dose of 30 mg/kg. (B) Fecal cumulative ratio of
vitexin-2"-O-rhamnoside in mice (mean ± S.D., n = 5) following oral
and intravenous routes of administration at dose of 30 mg/kg.
An et al. 1931
0.01% and 38.89 ± 3.04% after oral administration, and
amounted to 12.83 ± 0.03% and 24.65 ± 1.75% after
intravenous administration. VR as prototype was
detected in excreta by comparing HPLC chromatogram of
blank biological samples with the tested one. The total
VR recoveries of excreta were 37.48 ± 1.78% (12.83 ±

1932 Afr. J. Pharm. Pharmacol.
0.03% in urine; 24.65 ± 1.75% in feces) following
intravenous dosing and 43.02 ± 3.05% (4.13 ± 0.01% in
urine; 38.89 ± 3.04% in feces) following oral dosing,
demonstrating that biliary excretion and renal excretion
are two major ways of elimination of VR and VR
underwent extensive first-pass effect after oral
administration and that not being absorbed was mainly
excreted as feces. Recently, Ma et al. (2010) studied the
excretions of VR after orally administrated hawthorn
leaves extract and found that the total recovery of the
dose in 24 h was 89.01% (0.72% in urine; 88.29% in
feces) for VR (Ma et al., 2010), which is different from the
results of monomer administration of VR were 43.02 ±
3.05% (4.13 ± 0.01% in urinary excretion; 38.89 ± 3.04%
in fecal excretion) following oral dosing, suggesting that
various kinds of components in hawthorn leaves extract
affect the concentration and duration of VR in vivo.
ACKNOWLEDGEMENT
This study was supported by the Natural Science
Foundation of Liaoning Province (20072073), China.
REFERENCES
Cheng S, Qiu F, Huang J, He J (2007). Simultaneous determination of
vitexin-2"-O-glucoside, vitexin-2"-O-rhamnoside, rutin, and
hyperoside in the extract of hawthorn (Crataegus pinnatifida Bge.)
leaves by RP-HPLC with ultraviolet photodiode array detection. J.
Sep. Sci. 30:717-721.
Ding XB, Jiang YQ, Zhong Y, Zuo CX (1990). Studies on the chemical
constituents of the leaves of the Crataegus pinnatifida Beg. var.
major N. E. Br. China J. Chin. Mater. Med. 15:39.
Du Y, Wang F, Wang D, Li HB, Zhang WJ, Cheng ZZ, Ying XX, Kang
TG (2011). Tissue distribution and pharmacokinetics of vitexin-2"-Orhamnoside
in mice after oral and intravenous administration. Lat.
Am. J. Pharm. 30:1519-1524.
Liang MJ, Xu W, Zhang WD, Zhang C, Liu RH, Shen YH, Li HL, Wang
XL, Wang XW, Pan QP, Chen CL (2007). Quantitative LC/MS/MS
method and in vivo pharmacokinetic studies of vitexin rhamnoside, a
bioactive constituent on cardiovascular system from hawthorn.
Biomed. Chromatogr. 21:422-429.
Ma LY, Liu RH, Xu XD, Yu MQ, Zhang Q, Liu HL (2010). The
pharmacokinetics of C-glycosyl flavones of Hawthorn leaf flavonoids
in rat after single dose oral administration. Phytomedicine 17:640-
645.
Ninfali P, Bacchiocca M, Antonelli A, Biagiotti E, Di Gioacchino AM,
Piccoli G, Stocchi V, Brandi G (2007). Characterization and biological
activity of the main flavonoids from Swiss Chard (Beta vulgaris
subspecies cycla). Phytomedicine 14: 216-21.
The Pharmacopoeia Commission of PRC (2010). The Pharmacopoeia
of the People's Republic of China. Part 1, In: Li Y et al (eds), Chin.
Med. Sci. Technol. Press. Beijing, pp. 30.
Ying XX, Gao S, Zhu WL, Bi YJ, Qin F, Li XQ, Li FM (2007). Highperformance
liquid chromatographic determination and
pharmacokinetic study of vitexin-2''-O-rhamnoside in rat plasma after
intravenous administration. J. Pharm. Biomed. Anal. 44:802-806.
Zhu XX, Li LD, Liu JX, Liu ZY, Ma XY (2006). Effect of vitexiarhamnoside
(V-R) on vasomotor factors expression of endothelial
cell. China J. Chin. Mater. Med. 31:566-569.
Zhu XX, Li LD, Liu ZX, Liu ZY, Ma XY (2003). The Protective effects of
vitexia-rhamnoside (VR) on the cultured cardiacmyocytes damaged
by hypoxia and reoxygenation. Chin. J. Nat. Med. 1:44-49.

African Journal of Pharmacy and Pharmacology Vol. 6(26), pp. 1933-1937, 15 July, 2012
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP12.308
ISSN 1996-0816 ©2012 Academic Journals
Full Length Research Paper
Comparison of side effects and marital satisfaction
between the women taking cyclofem and depomedroxyprogesterone
contraceptive ampoules
Gholamitabar Tabari M. 1 , Moslemi L. 1 *, Esmaelzadeh S. 2 and Bijani A. 3
1 Department of Midwifery, Islamic Azad University Of Sari, Sari, Iran.
2 Fatemehzahra Infertility and Reproductive Health Research Center, Babol University of Medical Science, Babol, Iran.
3 Non-communicable Pediatric Disease Research Center, Amircola Hospital, Babol University of Medical Science, Babol,
Iran.
Accepted 21 May, 2012
None of the contraceptive methods is fault-free and all come with some complications. In this study we
investigated the comparison of complications and marital satisfaction between the women taking
cyclofem contraceptive ampoule and depo-medroxyprogesterone acetate (DMPA). This study was
performed on 300 married women using cyclofem and depot medroxyprogesterone for family planning,
with 150 people in each group. Data collection tools included two types of questionnaires; one for
studying the complications and the other for examining the marital satisfaction (Enrich Couple
Questionnaire). The results obtained indicated that increased days of menstrual bleeding in depomedroxy
consumers were more than those of cyclofem consumers, and there was a significant
difference (P = 0.004). Furthermore, both groups are similar in terms of weight changes, mood
disorders, libido changes and all other complications and there was no significant difference. It was
also found that cyclofem ampoule was used by 62% for one year, while depo-medroxy ampoule was
used by 60% for one year; the continuation rate of using depo-medroxyprogesterone was more than
cyclofem (P = 0.02). There was no significant relationship between marital satisfaction and age,
education, number of children and job. Average marital satisfaction in women using depomedroxyprogesterone
contraceptive method was 116.79± 8.62, which was significantly higher than the
average marital satisfaction in women using cyclofem contraceptive method as 114.53± 7.16 (P = 0.01).
Based on the results achieved in the present and similar studies, it can be concluded that the
complications caused by cyclofem and depo-medroxyprogesterone ampoules are similar and they have
no significant difference; and these complications are not fatal and irreversible.
Key words: Side effects, family planning, cyclofem, depo-medroxy.
INTRODUCTION
Although in recent years, world population (Nowels and
Veillette, 2010) as well as Iran’s population growth has
been declining (Mohammad, 2009; UNFPA, 2008), there
is still a need for family planning to reduce poverty and to
enjoy more health for mother and child as well as for
better training (Cleland et al., 2006). On the other hand,
*Corresponding author. E-mail: moslemi.iausari@gmail.com.
Tel: +98-911-1168064.
recent reports indicate that the rate of unwanted
pregnancies (Faghihzadeh et al., 2003) and induced
abortion is high in Iran (Majlessi et al., 2008; Nojomi et
al., 2006). Based on the past studies, one-third of
pregnancies in large cities (Tehran) are unwanted,
resulting in increased rate of illegal abortions and thus
endangering maternal health (Nojomi et al., 2006).
Nowadays, there are a variety of contraceptive
methods, including hormonal contraceptives such as
cyclofem and depo-medroxyprogesterone. In Some
countries, the use of injectable contraceptive is more than

1934 Afr. J. Pharm. Pharmacol.
other family planning methods (Gizaw and Regassa,
2011). In its report in 2000, prepared based on the
researches conducted in relation to reproductive issues,
the World Health Organization (WHO) wrote that though
depo-medroxyprogesterone acetate (DMPA) ampoules
are highly effective contraceptives, although the rate of
discontinuation is still remarkable due to side effects. For
this reason, researchers are seeking a better alternative
to this ampoule (Biennale Report, 2000). Considerable
efforts have been made to reduce hormonal compounds
doses in order to minimize the complications risk. Such
efforts made for reducing hormonal content were based
on “low as much as possible and high as much as
necessary” (Endricat et al., 2001). Injection hormonal
method as one of the contraception methods with high
efficacy and assumed suitable because it is easy to use
and has no interference with sexual intercourse (Rominjo
et al., 2005).
Cyclofem ampoule is a very effective method of
contraception with 1-month intervals; it contains 25 mg of
medroxyprogesterone acetate and 5 mg of estradiol
cypionate. DMPA is also an effective hormonal
contraception; it is injected intramuscular once every 12
weeks (Stubblefield et al., 2007). They do not need to be
repeated every day and their acceptability are more than
pills (Adebara and Ijaiya, 2010). However, none of the
contraceptive methods is fault-free and all come with
some complications. Among these complications of
hormonal methods are: menstrual disorders,
amenorrhea, irregular uterine bleeding, weight gain,
headache, decreased libido, nervousness, fatigue, mood
changes, etc. (Polaneczky et al., 1996; Hagh et al., 1996;
Fraser and Dennerstein, 1994; Nelson, 1996). Studies
have shown that such complications occur with different
rates in different communities (Nelson, 1996; Kaunitz,
1998; Davidson et al., 1997) Thus, the complaints of side
effects occurrence for the clients could be reduced by
careful consideration of side effects, and their satisfaction
would be promoted.
Consumers of such hormonal methods also face
physical, psychological and mental disorders due to
unwanted complications, which sometimes affect marital
satisfaction. Marital satisfaction is a condition in which
married couples are happy and satisfied with each other
(Mirahmadizadeh et al., 2003). Many variables are
effective on how couples communicate with each other
during their common life, including income, employment,
children, illness and sexual satisfaction (The reasons of
marital satisfaction, Culture and research.
Available at www.fpm.ir/archive/no-169/farsi:2006)
Changes in physical and psychological status of these
individuals may lead to unfavorable reactions and
behaviors by avoiding their housekeeping obligations as
well as routine duties and functions. Although marital
satisfaction and factors affecting it has been the focus of
attention by Iranian researchers, there are no studies on
this particular issue on these people. Therefore,
considering the shortages and the importance of this
topic, we decided to study the comparison of
complications and marital satisfaction between the
women taking cyclofem contraceptive ampoule and
DMPA.
MATERIALS AND METHODS
This study is comparative and was conducted by cluster sampling.
The population under study was women using contraceptive
methods of cyclofem and DMPA provided from health centers in
Babol, Iran. Sampling was done in 15 health centers allocated
among 42 urban and rural Health center with cluster. The duration
of sampling was 6 month. Precondition for entering the study was at
least two 3-month periods; that is 6-month use of depomedroxyprogesterone
ampoule and at least 3 one-month periods;
that is 3 months use of cyclofem ampoule due to adjustment of side
effects of the hormonal methods after first few months of use.
Furthermore, the samples should not use any other hormonal
method while using this method. The women should have at least 1
child, do not have any illness that interfered with complication and
do not have any psycho logic disorder and no recent death of a
close relative. With the use of pilot study, sample size was
determined as 150 people in each group.
Data collection tools included two types of questionnaires; one
for studying the complications and the other for examining the
marital satisfaction. Questionnaires related to complications were
prepared considering the previous studies and the existing scientific
resources. And for validity, 10 expert persons and gynecologist
review the questions that consist in two parts: (1) profile consisting
of 6 demographic questions involving age, occupation, education,
number of children, method of contraception, duration of
consumption of the method and (2) a part related to the
complications of ampoules including 15 questions about menstrual
disorder, mood disorder, weight, libido, hirsutism, vomiting,
headache, breast sensitivity, backache, early exhaustion, hair
shedding, foot muscle contraction, abdominal bloat, face rash,
vertigo and decrease power. These variables are defined as
follows:
Demographics variables: Age: The women age between 15-45
years, those use family planning method;
Number of children: The number of childbearing that they have;
Occupation: The women divided in 3 groups.1-household 2-work
out of house 3-work in the house; Education: The women divided in
5 groups .1-no education 2-guidance school 3-high school 4-
Diploma 5- university education.
Menstrual disorder: Amenorrhea: stop menstruation during
consumption.
Decreasing days of bleeding: Reduce the days of bleeding lower
than 3 days or lower than use previous method, Increase days of
bleeding: Increase days of bleeding more than 7 days or more than
use previous method,
Increase volume of bleeding: Use 6 or more than 6 pads at 3 first
days.
Regular menstruation: menstruation occurs in ordinary duration
between 28-30 days.
Irregular menstruation: menstruation occur in untidy duration.
Change in libido: Decrease or Increase sexual tendency in
comparison previous method.
Change in weight: Decrease or Increase in weight in comparison
previous method.
We used binary questions and nominal variable for all complication
variables. Enrich Couple Questionnaire (Fowers and Olson, 1989)

Table 1. Statistical results of the variables analysed in this study.
Gholamitabar et al. 1935
Side effect
Depo-medroxy
N (%)
Cyclofem
N (%)
P value Side effect
Cyclofem
N (%)
Depo-medroxy
N (%)
P value
Amenorrhea 5 / 3)
47(
48 (72) P = 0.77 Painful coit 5 / 3)
8(
6 ) 9(
P=0.80
Decreasing days of bleeding 1 / 3)
2(
1 / 3)
2(
P=0.99 Leukorrhea 8 / 4)
13(
7 ) 6(
P=0.09
Breakthrough bleeding 19 / 3)
29(
22 / 4)
31(
P=0.77 Decrease libido 11 / 3)
14(
17 ) 21(
P=0.48
Increase days of bleeding 5 / 3)
8(
2 P=0.004 Increase libido 8 ) 12(
5 / 3)
8(
P=0.35
Increase bleeding 1 / 3)
2(
2 P=0.15 Weight Without change 79 / 3)
47(
51 / 3)
44(
P=0.72
Regular menstruation 2.3 ) 1(
2 P=0.31 Increase weight 37 ) 51(
33 / 3)
52(
P=0.90
Irregular menstruation 6 / 4)
12(
6 ) 9(
P=0.81 Decrease weight 17 ) 21(
13 / 3)
22(
P=0.86
Mood Without change 74 / 3)
41(
79 / 3)
47(
P=0.72 Hirsutism 13 / 3)
22(
11 / 3)
14(
P=0.59
Sensitivity 15 / 3)
23(
16 / 4)
25(
P=0.75 Vomiting 4 / 3)
11(
4 / 3)
11(
P=1
Easy for crying 1 / 3)
2(
2 ) 3(
P=0.65 Headache 19 / 3)
29(
22 / 4)
31(
P=0.77
Nervousness level 16 / 4)
25(
18 / 4)
28(
P=0.65 Breast tendency 17 ) 21(
13 / 3)
22(
P=0.86
Early exhaustion 16 ) 27(
12 / 4)
16(
P=0.17 Backache 23 / 3)
35(
28 / 4)
73(
P=0.29
Decrease power 3 / 3)
5(
2 / 4)
7(
P=0.73 Vertigo 12 / 4)
16(
12 ) 18(
P=0.71
Foot muscle contraction 36 / 4)
55(
37 ) 51(
P=0.62 Face rush 8 ) 12(
9 / 3)
17(
P=0.68
Hair shedding 17 / 4)
22(
19 / 3)
29(
P=0.28 Abdominal bloat 27 ) 36(
23 / 3)
35(
P=0.89
with 92% validity and consisting of 35 questions translated
by Asoodeh et al. (1389), including 4 subscales and 35
questions about satisfaction, communication and conflict
resolution, was used to examine the marital satisfaction.
This questionnaire as a valid research instrument has been
used in many researches and clinical works. And Likert
scale was used for Evaluation; the score lower than 30
presented intensive dissatisfaction, scores between 30-40
presented dissatisfaction, scores between 60-70 presented
very satisfaction, while scores more than 70 presented
intensive marital satisfaction between couple. Finally, the
data so collected were analyzed by statistical software
SPSS/v19 applying descriptive statistics, Chi-square test, ttest
and Pearson’s Correlation.
RESULTS
All respondents were in the age range of 17-50
years old. Average total age of the respondents
was 31.57 ± 8.03 and there was no significant
difference between two groups using cyclofem
and depo-medroxyprogesterone in respect of age,
number of children, occupation and education.
According to Table 1, two groups using depomedroxyprogesterone
and cyclofem contraceptive
methods were similar for menstrual disorders,
including amenorrhea, decreased days of
bleeding, breakthrough bleeding, increased bleeding,
more regular menstrual cycles and irregular
menstrual cycles, and they have no significant
statistical differences. However, increased days of
menstrual bleeding in depo-medroxy consumers
were more than those of cyclofem consumers,
and there is a significant difference (P = 0.004).
Furthermore, both groups are similar in terms of
weight changes, mood disorders, libido changes
and all other complications and there was no
significant difference.
Cyclofem ampoule has been used by 62% for
one year, 22.7% for two years and 15.3% for
three years and more. On the other hand, depomedroxy
ampoule has been used by 60% for one
year, 16.7% for two years and23.3% for three
years and more. A comparison between the
consumption periods of two depomedroxyprogesterone
and cyclofem ampoules showed
that there is a significant difference between
consumption period and type of contraceptive
method (P = 0.02); it means that continuation rate
of using depo-medroxyprogesterone ampoule for
consumers was more than cyclofem ampoule.
Pearson’s correlation showed no significant
relationship between the continuation in the use of
the related method and the age of the consumers
in two groups of women using cyclofem injection
and depo-medroxyprogesterone ampoule and
continuous use of contraceptive method in women
who have used Cyclofem ampoule and their

1936 Afr. J. Pharm. Pharmacol.
age (P = 0.07, Rho = 0.14). However, a significant
relationship has been observed between continuous use
of contraceptive method in women who have used depomedroxyprogesterone
ampoule and their age. (P = 0.01,
Rho = 0.20).
Furthermore, descriptive analysis showed that there
was no significant relationship between marital satisfaction
and job. Pearson’s correlation test showed that
there was no significant relationship between marital
satisfaction and age, education and number of children.
Score of marital satisfaction in women using cyclofem
ampoule was minimum 100 and maximum 136. Score of
marital satisfaction in women using depomedroxyprogesterone
ampoule was minimum 102 and maximum
142. T-test analysis showed that the average marital
satisfaction in women using depo-medroxyprogesterone
contraceptive method was 116.79 ± 8.62, which was
significantly higher than the average marital satisfaction
in women using cyclofem contraceptive method (14.53 ±
7.16; P = 0.01).
DISCUSSION
In this study, there was no significant relationship
between amenorrhea, reduced days of bleeding,
breakthrough bleeding, increased bleeding, regular and
irregular menstrual cycles with cyclofem and depomedroxyprogesterone
contraceptive methods. However,
the increased incidence of bleeding days in depomedroxyprogesterone
method (5.3%) was significantly
more than that in cyclofem method (0%). Moreover, the
most common menstrual disorder in the consumers of
cyclofem and depo-medroxyprogesterone was
amenorrhea at 48 and 50.3%, respectively. In a study
conducted by Afkari et al. (2003) in Kermanshah
province, the most common menstrual disorder in
consumers of depo-medroxyprogesterone was
amenorrhea as 53.2%, which is consistent with present
study. However, according to Yazdanpanah et al. (2010),
the most common disorder in the consumers of cyclofem
was the lasted bleeding and then amenorrhea, which was
not inconsistent with this study; incidence of amenorrhea
in depo-medroxyprogesterone consumers is 50/3% in this
study. Similar studies report this amount higher and
others have reported it less. Moradan et al. (2009) stated
the incidence of amenorrhea as 39.2% in Semnan
province, and Kamalifard et al. (2011) reported it as 50%
in East Azerbaijan province. In the present study,
incidence of amenorrhea was 48% in Cyclofem
consumers, while Kamalifard et al. (2011) reported it as
22% in East Azerbaijan province and Yazdanpanah et al.
(2010) announced it as 14.7% in Kerman in his studies.
In his study on Iranian women conducting an immunechemistry
survey, Symber showed that there is no
difference for endometrial and endometrial atrophic
vessels density in depo-medroxyprogesterone and
cyclofem consumers after 3 - 6 months consumption
(Simbar et al., 2007). In this study, increased
nervousness level was the most common type of mood
changes in cyclofem and depo-medroxyprogesterone
consumers as 16.7 and 18.7%, respectively. In his study
in Kermanshah, Afkari et al. (2003) reported this level as
25.4% in depo-medroxyprogesterone consumers that
was higher than that in the present study; perhaps due to
difference in culture context of the races under study.
However, no study was found on mood changes in
cyclofem consumers. In the present investigation, oneyear
continuation rates of Cyclofem and Depo-
medroxyprogesterone in consumers were 62% and 60%
respectively, while in his study Kamalifard et al. (2011)
reported that one-year continuation rate of cyclofem and
depo-medroxyprogesterone in consumers of these
methods are 27 and 42%, respectively. Yazdanpanah et
al. (2010) also stated that one-year continuation rate of
Cyclofem is 21.2%. In some studies conducted in Kenya
(Rominjo et al., 2005), the one-year continuation of
cyclofem and depo-medroxyprogesterone consumption
were reported as 56 and 75%, respectively. In a similar
study conducted on Muslim countries like Indonesia and
Tunisia (Garza-Flores, 1998), one-year continuation rate
of cyclofem were reported as 66.5 and 28.2%,
respectively. Difference in continuation rates in different
studies may be due to cultural, economic and social
differences of the population under study as well as the
quality of advices before starting to use injection
methods.
Generally, based on the results achieved in the present
and similar studies, the complications caused by
cyclofem and depo-medroxyprogesterone ampoules are
similar and they have no significant difference. Moreover,
these complications are not fatal and irreversible. Health
staff may help the women using such methods through
their advices and adequate training especially about the
possible side effects in higher application and continuity
of these methods.
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African Journal of Pharmacy and Pharmacology Vol. 6(26), pp. 1938-1942, 15 July, 2012
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP12.329
ISSN 1996-0816 ©2012 Academic Journals
Full Length Research Paper
Effect of salidroside on Bcl-2/Bax protein levels in rats
with hemisection-induced spinal cord injury
Ming Wang 1 , Ning Zhang 2 , Rili Ji 3 and Jing Zhao 4 *
1 Department of Urology, The second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310000,
Zhejiang Province, P. R. China.
2 Department of Orthopedics, The second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310000,
Zhejiang Province, P. R. China.
3 TCM pharmacy, Zhaohui Community Health Service Center, Hangzhou 310000, Zhejiang Province, P. R. China.
4 Pharmaceutical Institute, Zhejiang Chinese Medical University, Hangzhou 310000, Zhejiang Province, P. R. China.
Accepted 18 June, 2012
The effect of salidroside on Bcl-2/Bax protein levels in rats with hemisection-induced spinal cord injury
(SCI) was studied. The Sprague Dawley (SD) rats were randomly divided into six groups: Sham
operation group, SCI model group, methylprednisolone sodium succinate (MPSS) treatment group,
salidroside-low dosage treatment group, salidroside-moderate dosage treatment group, and
salidroside-high dosage treatment group. The SD rats were hemisected at the spinal cord, at the T8
vertebra to establish SCI model. 24 h after operation, different dosage of salidroside increased and
reduced the protein levels. Noticeably, salidroside at the 100 mg/kg dosage exhibited similar effects as
MPSS, which has been frequently used for clinical acute SCI. These results suggested that salidroside
can significantly inhibit apoptosis in acute SCI.
Key words: Salidroside, spinal cord injury (SCI), Bcl-2/Bax, apoptosis.
INTRODUCTION
Spinal cord injury (SCI) is a leading cause of permanent
disability, resulting in partial or complete loss of motor
and sensory function below the lesion site (Wells et al.,
2003). Potentially toxic substances are activated and
released in injured spinal cords, including free radicals,
inflammatory cytokines, phospholipases and lipid
peroxidases, which lead to oxidative stress damage,
thereby resulting in neuronal necrosis or apoptosis and
progressive secondary nerve tissue destruction (Bao et
al., 2006). There are also numerous agents for the
treatment of the SCI, such as anti-inflammatory,
antioxidants, antipoptosis agents and myelin-associated
growth inhibitors (Wong et al., 2011; Mallei et al., 2005;
Yang and Piao, 2002; Festoff et al., 2006; Tian et al.,
2007; Xie et al., 2012). Methylprednisolone sodium
succinate (MPSS) is the only Food and Drug
*Corresponding author. E-mail: zhaoj31@126.com.
Administration (FDA) approved and clinically used agent
for the treatment of acute SCI (Bracken et al., 1998; Ding
et al., 2012). Nevertheless, high-dose of MPSS
applications in acute SCI have high risks of serious side
effects (Qian et al., 2005). Obviously, some novel and
safe agents for the treatment of the SCI are urgently
needed.
Salidroside (p-hydroxyphenethyl-b-D-glucoside,
C14H20O7, structure is shown in Figure 1) is one of the
major active constituents in Rhodiola Crenulata. It has
been reported to possess various pharmacological
properties including resisting anti-inflammation,
antioxidative, anti-apoptotic, anti-fatigue, neuroprotective,
hepatoprotective and cardioprotective effects (Diaz et al.,
2001; Ma et al., 2009; Nan et al., 2003; Wang et al.,
2004, 2009; Zhang et al., 2007). Previous studies have
shown that salidroside plays important roles in apoptosis
related diseases. Therefore, we proposed that salidroside
might have some effects on hemisection-induced SCI in
rats.

HO
CH 2OH
O
OH OH
OCH 2CH 2 OH
Figure 1. The chemical structure of salidroside.
In our present work, we established rat models of SCI
by hemisecting the spinal cord at the T8 vertebra and
attempted to study the effects of salidroside on Bcl-2/Bax
protein levels in rats with hemisection-induced SCI. The
Bcl-2/Bax protein levels were evaluated by
immunohistochemistry methods.
MATERIALS AND METHODS
Salidroside was purchased from China pharmaceutical and
biological products inspection (Lot: 110818-201005). MPSS was
obtained from Pharmacia and Upjohn Company (Belgium).
Animals
A total of 54 healthy, female, Sprague Dawley rats, aged 2 months,
weighing 180 to 220 g, were purchased from the Laboratory Animal
Centre of Zhejiang University, China. Animal care and experiments
were performed in accordance with the Guidelines for the Care and
Use of Laboratory Animals of Zhejiang University.
Establishment of SCI models
The SD rats were anesthetized with chloral hydrate, and placed in a
prone position on a heating pad to maintain a constant body
temperature. A longitudinal incision was made at the midline of the
back and the paravertebral muscles were exposed. These muscles
were dissected and thoracic level 7 to 11 (T7 to 11) vertebrae were
exposed. A laminectomy at T7 to 11 was performed. Acute SCI was
induced by hemisection at the T8 vertebra to a depth of 0.5 cm. The
wound was sutured layer-by-layer (Kim et al., 2009). Rats in the
Sham operation group experienced the same procedures, while
without the hemisection was at the T8 vertebra. Following spinal
cord hemisection, the rat tails swung spastically, and the affected
hind limbs exhibited flaccid paralysis after several spastic seizures.
Rats were sacrificed 24 h after administration of salidroside or 0.9%
normal saline (NS).
Drug treatment and sample preparation
The sham operation group underwent laminectomy to expose the
spinal cord without hemisection, and received 0.9% NS (2 ml/kg).
The SCI model group underwent laminectomy followed by SCI, and
received 0.9% NS (2 ml/kg). The MPSS treatment group, positive
control group underwent laminectomy followed by SCI, and was
administered 100 mg/kg single dose of MPSS (2 ml/kg, i.p.) 5 min
after hemisection. The 25, 50, and 100 mg/kg salidroside groups
Wang et al. 1939
underwent laminectomy followed by SCI, and were given a single
dose of 25, 50 or 100 mg/kg of salidroside (dissolved in 0.9% NS, 2
ml/kg, i.p.) 5 min after hemisection. 24 h after administration, the
rats were anesthetized with chloral hydrate (0.3 g/kg) transcardially
perfused with 150 ml of 0.9% NS and 200 ml of 4%
paraformaldehyde in 0.1 M PBS. Approximately 2 cm of spinal cord
segments between the T7 and T11 levels were obtained and
cryopreserved at -70°C for measurements of Bcl-2/Bax protein
levels.
Immunohistochemistry (ICH)
The spinal cord segments were cut on a freezing microtome into six
adjacent series of 4 μm-thick coronal sections. The sections were
dehydrated through an alcohol series. Prior to immunohistochemical
processing, sections were rinsed in 2% PBS-Triton X-100
and mounted onto gelatine-coated slides. Immunohistochemistry
was performed on slide-mounted sections utilizing the following
antibodies: Bax or Bcl-2 (dilution 1:100). The sections were
incubated overnight at room temperature with the primary antibody
diluted in PBS-bovine serum albumin (BSA). After rinsing, sections
were incubated for 1 h at room temperature in biotinylated goat
antimouse serum (1:500), sections were incubated for 1 h in avidin–
biotin–horseradish peroxidase complex (1:200). Following rinses,
sections were placed for 30 min in chromagen solution consisting of
0.05% diaminobenzidine and 0.01% H2O2. The reaction was
monitored visually and stopped by rinses of 0.1 M PBS. In order to
minimize variability, sections from all animals were stained
simultaneously. Cell counts were performed blindly in all sections
using a Nikon Eclipse E800 microscope. Counts were made in six
randomly selected optical fields under 400× magnification by
individuals who were blinded to diagnosis. Bcl-2 or Bax
immunoreactivity were assessed semi-quantitatively using Image
Pro Plus software Version 4.5.129 (Media Cybernetics). The
percentage area covered by immunoreactivity was measured and
the mean value taken.
Statistical analysis
Data were expressed as Mean ± SD, and analyzed by one-way
analysis of variance followed by Least Significance Difference
multiple comparison or Dunnett’s multiple comparison tests using
SPSS 16.0 software. Multiple comparison tests were used when
appropriate. A P-value of 0.05 was considered statistically
significant.
RESULTS
Quantitative analysis of experimental animals
Of the 72 selected adult SD rats, 60 were used to
establish the model of spinal cord hemisection and were
assigned to 5 groups (n=12), which were intraperitoneally
injected, respectively, with 25, 50 and 100 mg/kg of
salidroside, 100 mg/kg of MPSS, and 2 ml/kg of NS. The
remaining 12 rats served as the Sham operation group. A
total of 60 rats were finally included in the final analysis.
Bcl-2 expression following SCI
As shown in Figure 2 and Table 1, the protein levels of

1940 Afr. J. Pharm. Pharmacol.
Figure 2. Bcl-2 positive cells of spinal cord in different treatment group (SP×200). A: Bcl-2
positive cells of spinal cord in SCI model group; B: Bcl-2 positive cells of spinal cord in
salidroside group.
Table 1. Blc-2 protein level of rats among different groups.
Group Blc-2 protein level (n = 12)
Sham operation 37.20 ± 6.41
SCI model 11.27 ± 4.27
MPSS 29.40 ± 6.21
25 mg/kg of salidroside 19.30 ± 4.82 df
50 mg/kg of salidroside 22.54 ± 5.21 de
100 mg/kg of salidroside 29.12 ± 6.36 c
(VS Sham: a , P

A
B
Figure 3. Bax positive cells of spinal cord in different treatment group (SP×200). A:
Bax positive cells of spinal cord in SCI model group; B: Bax positive cells of spinal
cord in salidroside group.
Bcl-2 decreased in the spinal cord tissues at 24 h
following SCI compared with Sham operation group
(P0.05).
Bax expression following SCI
As shown in Figure 3 and Table 2, the protein levels of
Bax increased in the spinal cord tissues at 24 h following
Wang et al. 1941
SCI compared with Sham operation group (P0.05).
DISCUSSION AND CONCLUSION
The “commitment” step of apoptosis is regulated by the
expression of genes of the Bcl-2 family, including proapoptotic
(Bax, Bcl-xS, and Bak), and anti-apoptotic (Bcl-

1942 Afr. J. Pharm. Pharmacol.
Table 2. Bax protein level of rats among different groups.
Group Bax protein level (n = 12)
Sham operation 14.56 ± 3.24
SCI model 53.82 ± 6.49
MPSS 36.93 ± 4.33
25 mg/kg of salidroside 46.73 ± 5.68 df
50 mg/kg of salidroside 44.89 ± 4.36 de
100 mg/kg of salidroside 37.89 ± 4.68 c
(VS Sham: a , P

African Journal of Pharmacy and Pharmacology Vol. 6(26), pp. 1943-1948, 15 July, 2012
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP12.351
ISSN 1996-0816 ©2012 Academic Journals
Full Length Research Paper
Evaluation of the phytochemical and hemostatic
potential of Jatropha multifida sap
Tamègnon Victorien Dougnon 1,2 *, Jean Robert Klotoé 1,3 , Julien Sègbo 1 , Jean-Marc Atègbo 3 ,
Aléodjrodo Patrick Edorh 2,4 , Fernand Gbaguidi 5 , Armelle Sabine Hounkpatin 2 ,
Carlos Dandjesso 1 , Lauris Fah 1 , Brice Fanou 1 , Karim Dramane 3 and Frédéric Loko 1
1 Polytechnic School of Abomey-Calavi, Research Laboratory in Applied Biology, University of Abomey-Calavi,
01 BP 2009 Cotonou, Benin.
2 Interfaculty Center of Formation and Research in Environment for the Sustainable Development,
Laboratory of Toxicology and Environmental Health, University of Abomey-Calavi (UAC), 01 BP 1463 Cotonou, Benin.
3 Department of Animal Physiology, Faculty of Science and Technology, University of Abomey-Calavi (UAC),
01 BP 526 Cotonou, Benin.
4 Department of Biochemistry and Cellular Biology, Faculty of Science and Technology, University of Abomey-Calavi
(UAC), 01 BP 526 Cotonou, Benin.
5 Laboratory of Pharmacognosy and Essential Oils (Porto-Novo), Benin.
Accepted 14 June, 2012
Jatropha multifida is a plant traditionally recognized for its medicinal properties. This study was
undertaken not only to assess the hemostatic potential of the sap of J. multifida, but also to elucidate
its chemical profile. Wistar rats were injured at various locations and time of bleeding was evaluated
based on the addition of J. multifida’s sap or distilled water. Phytochemical study was performed on
sap. J. multifida’s sap significantly reduced bleeding time. Indeed, more blood flow was noticed on the
side that received physiological water than the one which received the sap. This is even truer when the
cut is deep with times of bleeding reduction of 39.06, 47.89, and 46.68%, respectively for superficial
wounds, saphenous vein, and the femoral vein clippings. The phytochemical screening of J. multifida
showed the presence of tannins, flavonoids, saponins, leucoanthocyanes, mucilage and reducing
compounds. The power of the hemostatic sap is explained by the strong presence of tannins and
flavonoids. All this proves that using the sap of J. multifida in traditional medicine is justified.
Key words: Hemostatic, Wistar rats, tannins, flavonoids, bleeding, Jatropha multifida.
INTRODUCTION
The World Health Organization has encouraged research
on medicinal plants since 1978 in order to improve,
secure and reduce the cost of medical products (OMS,
2002).
No less than 170.000 bioactive molecules have been,
to date, identified from plants (OMS, 2002). Despite these
results, it should be noted that few plants have been
studied for their therapeutic properties, and some
pharmacological activities such as hemostatic effects
*Corresponding author. E-mail: victorien88@hotmail.com or
dougnonv@yahoo.fr. Tel: 00 (229) 97 73 64 46/64 67 51 19.
remain unexplored. In Benin, for example, only about 500
plants were studied among the 3000 species of plants
(Adjanohoun et al., 1989; Akoègninou et al., 2006). No
studies have looked specifically at the inventory of
hemostatic plants. The few existing data are from
ethnobotanical surveys on the flora as a whole (de
Souza, 1988; Adjanohoun et al., 1989; Akoègninou et al.,
2006). However, there are some hemostatic plants
including Jatropha multifida which is used in Vodou ritual
practices to stop external bleeding (Adjanohoun et al.,
1989).
Interest for hemostatic plants is justified by the fact that
hemorrhage is the first cause of early death in surgery
bleedings (Sauaia et al., 1995). It is now a leading cause

1944 Afr. J. Pharm. Pharmacol.
of maternal mortality worldwide (Goodburn and
Campbell, 2001; Brücker et al., 2006; Fourn et al.,
2007). Hemorrhages are unpredictable in 84% of cases
(Cinat et al., 1999). Although, it often stops on its own
(minor injuries), in many situations, the use of mechanical
barriers, thermal and hemostatic drugs is essential
(Kozen et al., 2008; Wedmore et al., 2006). Thus, the
variability in the severity of bleeding today justifies the
existence of a variety of hemostatic. Some hemostatics
are human, animal, vegetable or synthetic. These
substances are administered topically, orally or by
injection (Abaut et al., 2008). It is important for African
researchers to investigate African plants known for their
therapeutic properties by the legends and traditions. The
identification of efficient hemostatic could, for this
purpose, improving the management of bleeding in all
medical disciplines.
J. multifida, in traditional medicine, is used as a
hemostatic, usually to treat wounds. It is also used as
herbal tea to treat microbial infections (Adjanohoun et al.,
1989). In Benin, no scientific study has, to date, proved
hemostatic potential of this plant. However, preliminary
studies and ongoing work has shown good results of this
sap on in vitro tests (Dougnon, 2012); hence, the need to
assess its potential phytochemical and its effects in vivo.
MATERIALS AND METHODS
Phytochemical tests were conducted at the Laboratory of
Pharmacognosy and Essential Oils of Benin Center for Scientific
and Technical Research. Hemostatic and biochemical tests were
conducted at the Laboratory for Research in Applied Biology at the
University of Abomey-Calavi (UAC).
Plant
The plant material is made from the sap of J. multifida. It was
collected directly into Eppendorf tubes after leaf cutting. Samples
were kept refrigerated at 4°C.
Animals
Seven male Wistar rats of 16 to 20 weeks and weighing between
240 and 270 g were kept in the Research Laboratory in Applied
Biology at constant temperature of 22 ± 1°C with a 12 h light and 12
h in the dark. They were fed with pellets and water ad libitum.
Phytochemical tests
Phytochemical analysis is a qualitative test based on staining
reactions and/or differential precipitation of the major groups of
chemical compounds of plants. This analysis was performed on the
sap using the methodology described by Houghton and Rama
(1998). The different reactions of active compounds are
summarized in Table 1.
Hemostatic potential of J. multifida’s sap
Minor cuts, deep cuts (type 1), and deep cuts (type 2) were
performed in both legs simultaneously on seven Wistar rats. Wistar
rats were intramuscularly anesthetized with ketamine (80
mg/kg). The anesthesia was supplemented by a local administration
of 2% Xylocaine (10 mg/kg) on the chosen site for a total
stunning of the animal.
Superficial cuts included carrying out the incisions of a millimeter
in length and 3 mm deep at the plantar surface of rats localized at 1
cm below the knee. A sterile blade was used. The cuts were made
simultaneously at the two legs. Deep cuts of type 1 consisted of
sectioning saphenous vein (Figure 1) to two inches above the
knee. The two veins of the animal were sectioned simultaneously
after partial dressing of the rat. Deep cuts of type 2 consisted of
severing femoral vein (Figure 1) which is an extension of the
saphenous vein at the groin. The two veins of the animal were
sectioned simultaneously after partial dressing of the rat.
After each cut, sap was applied topically to the wound located on
one of the legs and the physiological water (0.9% NaCl) on the
other leg. The volumes applied were 50, 150, and 200 µl for
superficial cuts, deep cuts of type 1, and type 2, respectively.
Bleeding time was determined in all three cases with a stopwatch. It
was defined as bleeding time, elapsed time between the onset of
bleeding (from the cut) and stopping blood flow.
Statistical analyses
Comparisons of comparing two by two the average using the
Student t test, p (T>t) = 0.05 were made. The softwares used are
Microsoft Excel 2010 and 2011 XL Stat.
RESULTS
Chemical composition of the sap of J. multifida
The phytochemical tests carried out on the sap of J.
multifida have revealed the presence of tannins (gallic
and cathechic), flavonoids, saponins, leucoanthocyanes,
mucilage, and reducing compounds (Table 2).
Assessment of bleeding time
Averages are recorded as shown in Figure 2. The effect
of applying the sap of J. multifida on bleeding time is
reflected by Figure 3. The cavity marked by blue arrow
received physiological water, while the one marked by
the green arrow has received sap after section of the
femoral vein. More blood flow toward the blue arrow has
been noticed than the other. The blood clotted faster on
the side receiving the sap.
To better explain the relationship between the depth of
the wound and the bleeding time, the percentage
reductions are summarized in Table 3.
DISCUSSION
The power of hemostatic sap is really explained by the
strong presence of tannins and flavonoids. Indeed, the
tannins can stop the bleeding and fight against
infections. The tannins-rich plants are used to make

Table 1. Summary of specific reactions of the activeplant.
Classes of active substance Specific reagent and reaction
Alkaloids Mayer → yellowish precipitate
Quinone derivatives Born-Träger → purplish red color
Cathetic and gallic tannins
-Reagent of Stiasny → pink precipitate
-Saturation by acetate of Na+ FeCl3 → blue-dark green or black
Flavonoids Shinoda → orange color, red or purple
Cyanogenic derived Guignard (picric acide) → brown coloration
Steroids and triterpenoids
-Libermann-Burchard → violet-blue or green
-Kedde → reddish purple or red wine
Dougnon et al. 1945
Saponins Determination of the foam index (MI): positive test if MI >100
Anthocyanins Red coloration of the filtrate increased in acid medium and blue-violet in alkaline medium
Leuco-anthocyanins Chloridric alcohol → cherry red color
Mucilages Absolute alcohol → flocculent precipitate
Reducing compounds Fehling's hot → brick-red precipitate
Coumarins Ammonia 25% → intense fluorescence
Anthracene derivatives Ammonia 50% → intense red color
Saphenous
vein
Femoral
vein
Figure 1. Sections of saphenous and femoral veins.
tissues soft, as in the case of varicose veins, to drain the
excess secretions and to repair tissue damaged by burns
or eczema (Djabou, 2006). Rira (2006) has also shown
that the biological properties of tannins are mainly related

1946 Afr. J. Pharm. Pharmacol.
Table 2. Result of reactions to characterize the sap of Jatropha multifida.
Classes of active ingredients sought Result
Alkaloids -
Quinone derivatives -
Cathetic and gallic tannins +++
Flavonoids +++
Cyanogenic derived +++
Steroids and triterpenoids +++
Saponins -
Anthocyanins -
Leuco-anthocyanins +++
Mucilages -
Reducing compounds +++
Coumarins +++
Anthracene derivatives +++
Figure 2. Bleeding time variation by type of cuts.
to their ability to form complexes with macromolecules,
especially proteins. Tannins have astringent properties.
They are also anti-inflammatory in burns (Paris
and Moyse, 1965). Nacoulma (1996) also showed that
flavonoids are able to reduce bleeding. They are
particularly active antioxidant substances in maintaining
blood circulation. They contribute to the increased
production of nitric oxide in blood platelets, which limits
the formation of clots by preventing platelets from sticking
together (Diallo, 2005).
The study of the hemostatic properties of the sap of J.
multifida reinforces the potential of plants used in
traditional hemostasis. According to Kerharo and Adam
(1974), the decoction of the bark of Entada africana’s
trunk is regarded as hemostatic, and tannins and
flavonoids were found there. The decoction of its root or
stem is also used to clean wounds and the powder of the
same parts of the plant is applied on wounds (Sangaré,
2005). Flavonoids were also found in Calandula
officinalis, a medicinal plant (Hussain et al., 2012).
The results of this study showed that the sap of J.
multifida significantly reduced the bleeding time. This is
even more true when the cut is deep, probably because
the blood volume charged at this time is important. The
effect of the sap of J. multifida on bleeding time is
comparable to Ankaferd Blood Stopper, a mixture of
traditional plants (Thymus vulgaris, Glycyrrhiza glabra,
Vitis vinifera, Alpinia officinarum, and Urtica dioica) acting
on hemostasis reducing bleeding time (Duz et al., 2010).
Traditional knowledge deserves to be appreciated. This
enhancement involves the production of improved
traditional medicines. After the study, it was proved that

Figure 3. Effects of the sap of Jatropha multifida on bleeding time.
Table 3. Percentage reductions in bleeding time according to the type of injury.
Tube
Dougnon et al. 1947
Superficial cutting Saphenous cutting Femoral cutting
Control Test Control Test Control Test
Means ± Standard deviations 91.42 ± 33.07 a 55.71 ± 23.22 b 95 ± 37.26 a 49.5 ± 28.76 b 103.16 ± 25.82 a 55 ± 38.35 b
Percentage of reduction 39.06 47.89 46.68
Means with the same letters are not significantly different at significance level α = 0.05.
the traditional use of the sap of J. multifida is justified as
a hemostatic. Its effects on bleeding time were
investigated. The different constituents of this substance
have been revealed and its hemostatic potential has
been well explained. This offers interesting perspectives
in therapy, because the sap of J. multifida could be used
as local hemostatic in both normal subjects as those with
coagulation disorders such as hemophilia.
ACKNOWLEDGEMENTS
Authors give thanks to Dr. Paulin YOVO and Dr. Jacques
DOUGNON for their scholarly contributions.
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d’Almeida J, Akpovo F, Bouke K, Chadare M, Cusset G, Dramane K,
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SE (1999). Improved survival following massive transfusion in
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Diallo S (2005). Etude des propriétés antioxydantes et antiplasmodium
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Djabou N (2006). Sambucus Nigra L., une plante de la pharmacopée
traditionnelle Nord-africaine. Mémoire de Magistère, Université Abou
Bekr Belkaid, Algérie.
Dougnon V (2012). Evaluation des propriétés hémostatiques de la sève
de Jatropha multifida L. (Euphorbiaceae), Mémoire de Master,
Université d’Abomey-Calavi, Bénin.
Duz E, Logman A, Ismail A, Irfan B, Abdullah K, Harun A, Eda O (2010).
The investigation on the effect of the vegetal origin Ankaferd Blood
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J.A.V.A.A. 9 (10): 1491- 1494.
Fourn L, Fayomi EB, Yacoubou M (2007). Mortalité maternelle évitable
en milieu hospitalier dans un département au Bénin. M.A.N. 47(1) :
22-26.
Goodburn E, Campbell O (2001). What can a health system deliver if it
can’t deliver a baby. B.M.J., 7(2): 12-17.
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(2012). Study on the medicinal plant Calandula officinalis. Afr. J.
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Kerharo J, Adam JG (1974). La pharmacopée sénégalaise
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Kozen BG, Kircher SJ, Henao J, Godinez FS, Johnson AS (2008). An
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African Journal of Pharmacy and Pharmacology Vol. 6(26). pp. 1949-1957, 15 July, 2012
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP12.399
ISSN 1996-0816 © 2012 Academic Journals
Full Length Research Paper
Effects of penehyclidine hydrochloride and
anisodamine on acute kidney injury induced by
two-hit Rats
Dong-Ting Chen 1 , Chun-Shui Lin *1 , Miao-Ning Gu 1 , Zhen-Long Zhao 1 , Rang-Hui Yu 2 , Jin-Dong
Xu 1 and Man-Ling Tan 3
1 Department of Anesthesiology, Nan Fang hospital, Southern Medical University, Guangzhou 510515, China.
2 Department of ICU, Hospital of Guangdong Province Hydropower Group Company Limited, Guangzhou 511340, China.
3 Department of Otorhinolaryngology, Nan Fang hospital, Southern Medical University, Guangzhou 510515, China.
Accepted 25 May, 2012
Anticholinergics have effects on organs with hemorrhage-reperfusion injury. The present study
investigated the benefit effects of penehyclidine hydrochloride (PHC) and anisodamine (ANI) on
hemorrhage-reperfusion and lipopolysaccharide (LPS) (two hits) induced acute kidney injury (AKI).
Administration of PHC and ANI do not only remarkedly reduced the plasma concentrations of tumor
necrosis factor alpha (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6), but also reduced
malondialdehyde (MDA) content, myeloperoxidase (MPO) activity, and enhanced superoxide dismutase
(SOD) activity. Histopathological observation showed that PHC and ANI treatment markedly relieved
renal histopathological damages, and inflammatory cells infiltration. Furthermore,
immunohistochemistrical study presented that intercellular adhesion molecule-1 (ICAM-1) was
increased after two hits management, which could be attenuated by PHC and ANI treatment. These data
indicated that PHC and ANI had the benefit effects on AKI resulting from two-hits in rats. The effects of
PHC treatment were better than ANI.
Key words: Anisodamine, penehyclidine hydrochloride, two hits, acute kidney injury.
INTRODUCTION
It is generally accepted that severe infection, trauma, and
hemorrhagic shock can activate inflammatory action
leading to systemic inflammatory response syndrome
(SIRS). In recent years, it has been increasingly recognized
that SIRS could contribute to the induction of acute
kidney injury (AKI) (Zager et al., 2005). In the surgical
intensive care unit, critically sick patients suffered from
various insults that resulted in their organ function
deterioration, while inflammation after renal ischemiareperfusion
injury (IRI) was a leading contributor to renal
cell death, and SIRS was an important factor and the
potential mechanism to AKI ( Lee et al., 2007; Aregger et
al., 2009).
*Corresponding author. E-mail: lifeholder@126.com. Fax: +86
020 61641881.
The two-hit theory has become increasingly accepted.
It is believed that a less severe traumatic or hemorrhagic
insult as “the first hit” to activate an inflammatory
environment; and the subsequent insults, infectious or
non-infectious, as “the second hit” may amplify the preexisting
inflammatory state into SIRS (Saadia and
Schein, 1999). In order to investigate the clinical pathophysiology
changes of AKI which resulted from various
insults, the two-hit model was duplicated by two-hits of
hemorrhage-reperfusion and lipopolysaccharide (LPS),
which can reflect the pathology, pathophysiology and
complexity of clinically severe hemorrhage and sepsis
shock.
Anisodamine (ANI) is extracted from a Chinese herb
Anisodus tanguticus. Because of its capability of
improving the microcirculatory flow and splanchnic
perfusion, ANI has been reported to work as an antishock
medicine to treat burn shock, septic shock, and

1950 Afr. J. Pharm. Pharmacol.
ischemia shock (Sheng et al., 1997; Ruan et al., 2001;
Hu and Sheng, 2002). However, the side effects of ANI
such as dry mouth and accelerating heart rate (HR) have
limited its wide clinical application. Therefore,
penehyclidine hydrochloride (PHC), a new anticholinergic
drug, was synthesized (Han et al., 2005). Compared to
other anticholinergics, PHC is noticeable benefits for its
selectively blocked M1, M3, N receptors, and few M2
receptor-associated cardiovascular side effects (Zhan et
al., 2007). Recent experimental and clinical studies
showed that PHC do not only improve microcirculation,
reduced permeability of capillaries, decreased release of
lysosomal enzymes (Han et al.,2005), but also was
widely used in preoperative medication (Yan et al., 2006),
and the treatment of organophosphate poisoning (Liang,
2007) .
AKI is a hazardous clinical issue correlated with high
morbidity and mortality, and pharmacological therapy has
been proven to be beneficial in some preclinical studies;
however, the prevention and treatment of AKI is still
highly unsuccessful (Ferenbach et al., 2010; Bajwa et al.,
2009). There was no research concerning whether
anticholinergic drug was effective to antagonise two-hits
induced AKI. Regarding the AKI, characteristics of
pathophysiology and histopathology are free radicals
accumulation, inflammatory mediator activation, tubular
necrosis, and glomerular damage, etc (Shah et al., 2010).
The purposes of the present study were to determine
whether anticholinergic drug: 1) decreased oxygen free
radicals accumulation, 2) reduced inflammatory
mediators and cytokine activation, 3) lessened kidney
histopathological damages, and 4) ANI or PHC was more
effective.
MATERIALS AND METHODS
Experimental animals
Forty-eight (48) Wistar rats (250 to 280 g) were randomly divided
into four groups: sham group, two-hit (TH) group, TH treated with 1
mg/kg PHC (TH + PHC), and TH treated with 5 mg/kg ANI (TH +
ANI). The doses of PHC and ANI were determined by the results of
our preliminary experiment. All the rats were purchased from the
Laboratory Animal Center of Southern Medical University. All
experiments were conducted in accordance with protocols
approved by the Institutional Animal Care and Use Committee of
Southern Medical University.
Duplication of two-hits model
The two-hits model was duplicated by modified Zhou’s method
(Zhou et al., 2003). The rats were anesthetized with 20% urethane
(5 ml/kg) by intramuscular injection. Under sterile conditions, the
femoral artery was cannulated with 24-gauge tubing for blood
exanguinating, mean arterial pressure (MAP) monitoring and blood
sample collecting. The femoral vein was cannulated with 24-gauge
tubing for liquid resuscitation and medicine administration. The first
hit was initiated by blood withdrawal and reduction of the MAP to 35
± 5 mmHg within 15 min. This blood pressure was maintained by
further blood withdrawal if the MAP was >45 mmHg, and by infusion
of 0.5 ml Ringer’s lactate (RL) if the MAP was

Figure 1. A schematic diagram showing the experimental protocol in the present studies. At
the end of the resuscitation period, 2 mg/kg LPS was injected via the femoral vein. Sham
animals underwent the same surgical procedures, but neither the first hit nor second hit was
performed. 1 h later, sham group and group TH received NS 5 ml/kg, while groups TH + PHC
and TH + ANI received 1 mg/kg PHC and 5 mg/kg ANI, respectively.
two fluorescent dye. These particles are irradiated by two separate
lasers in the Luminex 100 analyzer (Qiagen, Germany). The
machine distinguished the coded particles and detected the fluorescence
intensity from the reporter molecules (Ren et al., 2011).
TNF-α, IL-1, and IL-6 levels of plasma were determined in a 96well
microtiter plate by using a Procarta Cytokine Assay Kit
(Affymetrix, Inc, USA) according to the manufacturer’s guidelines.
Put the plate in Luminex 100 analyzer, the data acquisition was
done with the integrated system software of the Liquidchip
workstation, and data analysis with Microsoft Excel.
Histological examination of kidney
The renal specimens were fixed in 10% paraformaldehyde for 24 h
and then embedded in paraffin, a series of microsections (5 mm)
were stained with hematoxylin and eosin staining for light
microscopy observation. The experimental conditions were
examined with an inverted biological microscop (Eclipse Ti-S,
Nikon, Japan). The severity of tissue damage was graded on a
scale from 1 to 5. The different grades were defined as follows
(Kong et al., 2010): grade 0 = no damage, grade 1 = minimal
damage 0.05). Cr levels in group TH + PHC (83.25 ± 14.9 pg/ml,
P < 0.05) and group TH + ANI (102.1 ± 18.5, P>0.05)
were lower than group TH (122 ± 19.9 pg/ml). Cr levels in
group TH + ANI were significantly higher than group TH +
PHC (P < 0.05).
The SOD activities, MDA contents and MPO activities
of renal tissue
SOD activities in renal tissues were investigated, and
these data are shown in Figure 3A. SOD activities in all
the rats experienced two-hits were significantly lower
than sham group (220.7 ± 45.5 U/mg, P < 0.01 or P <
0.05). Compared with group TH (99.8 ± 21.2 U/mg),
treatment with 1 mg/kg PHC (159.1 ± 23.7 U/mg, P <
0.01) and 5 mg/kg ANI (127.7 ± 18.1 U/mg, P < 0.05)
significantly prevented reduction in SOD activities. The
data of MDA contents and MPO activities are shown in
Figures 3B and 3C. Significantly increased MDA contents

1952 Afr. J. Pharm. Pharmacol.
Figure 2. The levels of BUN and Cr of plasma in four groups. Data presented means ±SD. **P

Chen et al. 1953
Figure 4. Concentration of TNF-α, IL-1 and IL-6 of plasma in four groups. (A) IL-1; (B); IL-6; (C) TNF-α. Data presented means ±SD. **P < 0.01 versus sham group; #P < 0.05, ## P 0.05) were lower than group TH (48.7 ± 7.9
U/g). MDA content in group TH + PHC was lower
than group TH + ANI, but no significant difference
was found in these two groups (P > 0.05). On the
other hand, MPO activity in group TH + PHC was
lower than group TH + ANI (P < 0.05).
The concentrations of,IL-1, IL-6, TNF-α in
plasma
Figure 4 shows the data of IL-1 (Figure 4A), IL-6
(Figure 4B) and TNF-α (Figure 4C) concentration
in plasma. Compared with sham group (IL-1: 9.8 ±
3.1 pg/ml; IL-6: 16.8 ± 7.4 pg/ml; TNF-α: 6.4 ± 3.7
pg/ml), the concentrations of IL-1, IL-6, and TNF-α
(P < 0.01) in groups suffered two-hits were significantly
elevated. Group TH + PHC and group TH +
ANI notably lessened the concentrations of IL-1,
IL-6, and TNF-α in plasma when compared with
group TH (IL-1:781.3 ± 145.1 pg/ml; IL-6:723.9 ±
163.9 pg/ml; THF-α: 736.2 ± 177.1 pg/ml; P <
0.05). The concentrations of IL-1 (453.1 ± 193.7
pg/ml), IL-6 (481.2 ± 176.9 pg/ml) and TNF-α
(494.2 ± 166.8pg/ml) in plasma in group TH + ANI
were notably higher than group TH + PHC (IL-1:
211.3 ± 67.8 pg/ml; IL-6: 273.7 ± 90.1 pg/ml; THFα:
262.4 ± 162.1 pg/ml, P < 0.05).
Histological examination
The structure of the renal glomeruli and tubuli,
proximal and distal convoluted tubes were seen
clearly in the sham group (Figure 5A). Severe
tubular dilatation and obstruction, glomerulus
sclerosis and necrosis, and the development of
protein casts were presented in the kidney of the
rats in group TH (Figure 5B). Patchy glomeruli
and interstitial hemorrhage, tubular epithelium
swelling, were found in group TH + PHC (Figure
5C) and group TH + ANI (Figure 5D), the damage
severity of renal tissues in these two groups (TH +
PHC: 9.8 ± 1.2; TH + ANI:18.1 ± 1.2) were
significantly lower than group TH (27.7 ± 1.2, P <
0.05). The mean grade scored of the rats treated
with PHC was lower than the rats treated with ANI
(P < 0.05).
Immunohistochemical detection of ICAM-1
Figure 6 indicates the representative
immunohisto-chemical detections of ICAM-1

1954 Afr. J. Pharm. Pharmacol.
Figure 5. Representative photomicrographs of kidneys. (A) Regular morphology with glomeruli and tubuli were
seen in the sham group; (B) glomeruli and tubuli were seriously damaged in the group TH; (C) damages relieved
in animals treated with PHC; (D) damages relieved in animals treated with ANI; Original magnification: �200; (E)
Mean grading scale of histological appearance of sham group, group TH, and groups treated with PHC or ANI
after TH. **P < 0.01 versus sham group; # P < 0.05 versus group TH; △P < 0.05 versus group TH + PHC. Data
are presented as means ±SD.

Figure 6. Representative photomicrographs of immunohistochemical detection of ICAM-1 expression in
(A), sham group; (B), TH group; (C), TH + PHC group; (D), TH+ANI group. Immunohistochemical staining
mean density = integrated option density /positive area. (E), Mean density of ICAM-1 expression of sham
group, TH group, groups treated with PHC or ANI after TH. **P < 0.01 versus sham group; # P < 0.05
versus group TH; △P < 0.05 versus group TH + PHC. Data are presented as means ±SD.
expression in renal tissues. Sham rats did not exhibit
detectable levels of ICAM-1 expression (Figure 6A). Rats
subjected to TH showed a notably increased ICAM-1
expression (Figure 6B, 0.46 ± 0.11, P < 0.01). Compared
Chen et al. 1955
with group TH, ICAM-1 expression in groups TH + PHC
(0.31 ± 0.12, P < 0.05) and TH + ANI (0.39 ± 0.07, P <
0.05) were notably decreased. ICAM-1 expression in
group TH + PHC (Figure 6C) were lower than group TH

1956 Afr. J. Pharm. Pharmacol.
+ ANI (Figure 6D, P < 0.05).
DISCUSSION
Inflammation after renal IRI is not only a major contributor
of renal cell death, but also a potential mechanism to
initiate and maintain renal cell necrosis and apoptosis
(Saadia and Schein, 1999). Aregger et al. (2009) did not
consider blood loss which was the main cause of AKI in
multivariate analysis; however, SIRS after transfusion
was a high risk factor and the possible mechanism of
AKI. Since AKI has a high morbidity and mortality, the
study of prevention and therapy required an animal
model truly replicated the complex pathogenesis of
human AKI. In the present study, two-hit model was
adopted to present a similar pathogenesis, pathology,
and complexity of the patients suffering AKI that was
secondary to trauma, hypotension shock and severe
infection.
As we know, inflammation process involves multiple
inflammatory mediators, neutrophils recruitment, and
macrophages infiltration. Neutrophils rapidly respond to
injury and release MPO and proteolytic enzymes, which
generate reactive oxygen species. Macrophages produce
pro-inflammatory cytokines that can stimulate the activity
of other leukocytes. Analysis of kidney infiltrating
macrophages demonstrated that these leukocytes were
the major producer of the cytokines IL-1, IL-6, and TNF-α
(Bajwa et al., 2009). TNF-α has been proven to play a
“master-regulator” role in orchestrating the cytokine cascade
in many inflammatory diseases (Parameswaran and
Patial, 2010). IL-1 and IL-6 have been reported as good
indicators of activation of cytokine cascade in various
conditions (Oda et al., 2005). These pro-inflammatory
cytokines were essential during the early phase of IRI,
inflammation, and endotoxemia (Ishii et al., 2010; Lloyd
et al., 2003). Therefore, it is important to inhibit the
release of IL-1, IL-6, and TNF-α for lessening inflammatory
responses. The present study found the plasma
concentrations of IL-1, IL-6, and TNF-α in group TH was
significantly higher than that in the groups treated with
anticholinergics, the data demonstrated that PHC and
ANI acted positively to inflammatory response. The
protection against two-hits induced AKI by PHC and ANI
can probably be ascribed to the reduced generation of IL-
1, IL-6, and TNF-α generated by macrophages.
MPO is an enzyme mainly located in the primary
granules of neutrophils; the tissue MPO levels may
indicate neutrophils infiltration (Tuğtepe et al., 2007).
MDA, a lipid peroxidation end product, is widely worked
as a marker of oxidative stress (Gaweł et al., 2004).
According to our findings, PHC and ANI treatments
significantly suppressed the activity of MPO and the MDA
contents in renal tissue, which illustrated that
anticholinergics treatment could inhibit the infiltration of
neutrophils into renal parenchyma or medulla nephrica
and attenuate oxidative stress. SOD is an enzyme that
exists in cells removing oxyradicals, whose activity
variation may represent the degree of tissue injury
(Macarthur et al., 2000). The present study also revealed
that SOD activities in group TH were remarkably lower
than the groups treated with PHC and ANI. These
findings implied that the feature of anticholinergics are to
reduce MDA contents and enhance SOD abilities, which
prevented renal tissue from cellular membrane destroy
and chondriosome dysfunction attacked by oxygen free
radicals.
ICAM-1 is found abundantly in endothelial, epithelial,
and mesangial cells and fibroblasts. It is up-regulated in
vitro and in vivo by cytokines such as TNF-α and IL-1
(Burne et al., 2001). Park et al. (1008) reported that IL-1
and TNF-α could regulate the expression of leukocytebinding
adhesion molecules in endothelial cells derived
from human glomerulus. Our study showed that ICAM-1
expression decreased significantly in the groups treated
with anticholinergics while it was compared with the
group TH. These data suggested that ICAM-1 played a
significant role during the neutrophil dependent injury
phase after renal ischemia and reperfusion. As a result,
suppressing adhesion molecule may have potential to
against AKI induced by two hits.
In our study, we also found that the plasma BUN and
Cr levels in group TH were significantly higher than that
in groups treated with anticholinergics. A histopathological
observation indicated that the cellular structures of
the kidney in the sham group were normal, while
congestion, degeneration and necrosis were found in the
group TH, and mild lesions were found in the groups
treated with PHC and ANI. The pathologic changes of
group TH were increased infiltration of neutrophils,
glomerular sclerosis, tubular obstruction and necrosis.
The glomerulus and tubular damage degree of PHC and
ANI treatment groups were alleviated when compared
with group TH. It suggested that anticholinergics had a
beneficial effect on the kidney in two-hit rats.
ANI, a classical anticholinergic, has been prescribed for
the treatment of certain diseases such as COPD,
Alzheimer disease, and urinary incontinence for its capability
to relieve small blood vessel spasm and improve
microcirculation (Han et al., 2005). However, the side
effects such as accelerating HR and short effective drug
duration have restricted its clinical application. M2
receptors are distributed in the atrial myocardium. Liang
et al. (2008) reported a potential value of M2 receptor
antagonists in the treatment of certain types of arrhythmia
and atrial fibrillation. The new traits of PHC are selective
blocking M1 and M3 receptors, faster reaction time and
long effective drug duration, making PHC having less
cardiovascular side effects such as sychnosphygmia and
arrhythmia than ANI. Therefore, PHC could reduce
myocardial consumption of oxygen and heart burden.
The improvement of cardiac preload and cardiac function
is profited to antagonize hemorrhagic shock and sepsis

shock. The features of PHC can explain the information
we found in the present study. Cr and BUN levels,
oxidative stress products, and inflammatory cytokines in
TH + PHC group were lower than group TH + ANI. PHC
treatment in two-hits induced rats AKI can result in a
better therapy than ANI by attenuating cytokines
activation, lessening renal histopathological damage, and
decreasing oxygen free radicals accumulation.
Conclusion
The investigation found that both PHC and ANI had
beneficial effects on two-hits induced rats AKI, whose
underlying mechanism was probably to inhibit the production
of inflammatory cytokines IL-1, IL-6, and TNF-α,
stabilize the cell membrane and resist oxygen free
radicals, attenuate neutrophils recruitment and
macrophages infiltration, and suppress the expression of
ICAM-1 in renal tissue. It is the first time these findings
are reported with regard to AKI. This study may provide a
novel strategy for the clinical physician against two-hit
induced AKI.
ACKNOWLEDGMENT
We thank Jian-xin Diao for excellent technical assistance.
This work was supported by the Nature Science Fund of
Guangdong Province (7005155).
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African Journal of Pharmacy and Pharmacology Vol. 6(26), pp. 1958-1963, 15 July, 2012
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP12.474
ISSN 1996-0816 ©2012 Academic Journals
Full Length Research Paper
Protective effect of green tea on CCl4 induced
hepatoxicity in experimental rats
Syed M. Shahid 1 *, Sana Shamim 2 and Tabassum Mahboob 2
1 The Karachi Institute of Biotechnology and Genetic Engineering (KIBGE), University of Karachi,
Karachi-75270, Pakistan.
2 Department of Biochemistry, University of Karachi, Karachi-75270, Pakistan.
Accepted 31 May, 2012
Liver diseases are among the major health problems worldwide. Despite their increasing frequency,
high morbidity and mortality, medical management is supposed to be insufficient; no therapy has
successfully prevented the progression of hepatic diseases. Green tea is one of the commonly used
beverages and considered as powerful antioxidant. This study was designed to investigate the
protective effects of green tea administration on CCl4 induced liver cirrhosis in experimental rats. A
total of 24 male Wistar rats were selected and divided into 4 experimental groups (6 each) as: Group-1:
normal healthy untreated rats; Group-2: CCl4 (0.8 mg/kg) induced cirrhotic rats; Group-3: treated with
5% green tea orally; Group-4: treated with CCl4 (0.8 mg/kg) intraperitoneally once a week for 8
weeks+5% oral administration of green tea. The preventive effects of green tea were measured by
means of plasma alanine aminotransferase (ALT), alkaline phosphatase (ALP), total and direct bilirubin,
tissue malondialdehyde (MDA), tissue superoxide dismutase (SOD) and tissue catalase. Induction of
cirrhosis by CCl4 was indicated by high levels of plasma ALT, direct bilirubin, tissue MDA and low levels
of tissue SOD. Results showed that administration of green tea reduced these changes significantly in
cirrhotic rats by putting beneficial effects on antioxidant and liver enzymes as well as total and direct
bilirubin. This study convinced the possible protective effect of green tea in relation to antioxidant and
liver enzymes. It is also conclusive that the chronic and sub chronic administration of green tea
extracts has counter effects on hepatotoxicity caused by CCl4 administration.
Key words: Green tea, total and direct bilirubin, cirrhosis, alanine aminotransferase (ALT), alkaline
phosphatase (ALP).
INTRODUCTION
Liver diseases are among the major health problems
worldwide. Despite their increasing frequency, high
morbidity and mortality, medical management is
supposed to be insufficient, no therapy has successfully
prevented the progression of hepatic diseases, even
*Corresponding author. E-mail: smshahid@uok.edu.pk. Tel:
009221-99261181 or 0092300-2759170. Fax: 009221-
34823886.
though newly developed drugs have been used to treat
chronic liver disorders, these drugs have often side
effects (Wolf et al., 2008). Liver cirrhosis is a result of late
stage scarring in chronic liver disorder. It is progressive
damage to the liver tissue starting with subendothelial or
hepatic fibrosis and develops with nodule formation,
which is actually called cirrhosis (Richard et al., 2008).
This inflammation is produced by free radicals generated
by viruses, toxins, unhealthy fats, alcohol and some
drugs or antibodies that are attacking liver cells
(Tsukamoto et al., 1997).

Administration of single or repeated dose of CCl4 is one
of the common methods to investigate the possible
mechanisms of hepatic injury in rats. This model has
been implemented in various studies for the deposition of
extracellular matrix in the cases of liver cirrhosis and
fibrosis (Luckey and Petersen, 2001; Nakade et al.,
2002). The biotransformation process of CCl4 causes the
formation of haloalkane free radicals which can damage
the hepatocytes making liver an important target for CCl4
(Weber et al., 2003; Ozardali et al., 2004). Herbal
medicine derived from plant extracts is increasingly used
to treat various medical problems. Plant extracts have
been used by traditional medical practitioners for the
treatment of liver disorders for centuries. It is being
acknowledged that plants contain non-nutritional
constituents with beneficial health effects, such as antiinflammatory
and anti-carcinogenic properties (Amuta et
al., 2010). A number of previous studies suggested that
oxidative stress and DNA damage is responsible to
initiate the tumor formation and the normal process of
oxidation produces highly reactive free radicals (Kyung et
al., 2007).
Green tea, one of the popular beverages of the world,
contains polyphenolic antioxidants, which are thought to
contribute to cancer prevention (Jin et al., 2008).
Although, an association between these beverages and
liver cancer has been speculated, epidemiologic
evidence is insufficient and varies by beverage (WCRF,
2007). Camellia sinensis (Tea) exhibits a wide range of
effects on human and animal health. It is antiinflammatory
(Dona et al., 2003) and has beneficial
effects in collagen-induced arthritis, inflammatory bowel
disease and carrageenan-induced paw edema (Das et
al., 2002). An increased consumption of green tea may
reduce the risk of liver disease (Jin et al., 2008). An
imbalance between antioxidants and reactive oxygen
species results in oxidative stress, leading to cellular
damage. These free radicals readily react and damage
biomolecules and DNA. Green tea polyphenol prevents
oxygen free radical-induced hepatocyte lethality, prevent
lipopolysaccharide-induced liver injury through inhibition
of inducible nitric oxide synthase and tumor necrosis
factor-α expression and inhibits carcinogen or toxininduced
liver oxidative DNA damage (Cai et al., 2002).
Catechin isolated from green tea, has antioxidant
properties and is thought to act as an antioxidant in
biological systems. The protective effects of tea extracts
or tea polyphenol against liver fibrosis and liver cirrhosis
in rats have been reported. The polyphenols contained in
the tea are antimutagenic and anticarcinogenic by
inhibiting cancer cell proliferation and induction of
apoptosis (Bun et al., 2006).
By keeping in mind the beneficial effects of green tea
administration on liver damage, this study was conducted
to evaluate the hepatoprotective effects of green tea
against CCl4 induced cirrhosis in experimental rats.
MATERIALS AND METHODS
Study animals
Shahid et al. 1959
A total of 24 male albino Wistar rats weighing 200 to 250 g body
weight (g.b.w), purchased from the animal house of International
Center for Chemical and Biological Sciences (ICCBS), University of
Karachi were selected for the study. Rats were acclimatized
according to the laboratory environment more than one week
before the commencement of experiment. All the rats were caged
with saw dust covered floor, in a quiet and temperature controlled
room (23 ± 4°C). The rats were given free access to standard rats’
diet and water. All the protocols regarding this study were approved
by institutional ethical committee and conducted according to the
ethical guidelines for the use of animals in laboratory experiments.
Study design
The age and sex-matched rats were randomly divided into 4
experimental groups consisting of 6 rats per group: Group-1
contained controls, fed on standard diet and water; Group-2
contained rats treated with CCl4 (0.8 mg/kg b.w., i.p), the dose was
given intraperitoneally at 11:45 a.m., once a week for 8 weeks;
Group-3 contained rats that received 5% green tea extract prepared
in distilled water orally on daily basis, the volume of green tea
extract consumed by each rat was measured on 11:30 a.m. every
morning, and the mean intake of green tea extract was 48.7 ± 12.56
ml on the day-1, which was increased to 120.3 ± 8.86 ml on day-45
of treatment; Group-4 contained rats that received CCl4 (0.8 mg/kg
b.w., i.p) weekly for 8 weeks as well as green tea extract (5%) orally
on daily basis, the volume of green tea consumed by each rat was
measured on 11:30 a.m. every morning, and the mean intake of
green tea extract in these rats was 40.5 ± 12.56 ml on day-1, which
was increased to 110.5 ± 15.45 ml on day-45 of treatment.
Collection of sample
After 8 weeks treatment, animals were decapitated and blood
samples were collected from head wound in the lithium heparin
coated tubes. A portion of blood was separated to collect the
plasma. Liver were excised, trimmed of connective tissues, rinsed
with saline to eliminate blood contamination, dried by blotting with
filter paper and weighed. The tissues were then kept in freezer at -
80°C until analysis.
Preparation of liver homogenate
A portion of liver was weighed, perfused with saline and
homogenized in chilled potassium chloride (1.17%) using a
homogenizer. The homogenates were 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
post mitochondrial supernatant which was used to assay
superoxide dismutase (SOD), catalase (CAT) and malondialdehyde
(MDA) activities.
Estimation of liver enzymes
Plasma alanine aminotransferase (ALT), alkaline phosphatase
(ALP), total and direct bilirubin were analyzed using commercially
available reagent kits from Randox ® Laboratories Ltd. UK (Reitman
and Frankel, 1957).

1960 Afr. J. Pharm. Pharmacol.
Estimation of CAT activity
CAT activity was assayed by the method of Sinha et al. (1972).
Briefly, the assay mixture consisted of 1.96 ml phosphate buffer
(0.01 M, pH 7.0), 1.0 ml hydrogen peroxide (0.2 M) and 0.04 ml
phenazine methosulfate (PMS) (10%) in a final volume of 3.0 ml.
About 2 ml dichromate acetic acid reagent was added in 1 ml of
reaction mixture, boiled for 10 min, and was cooled. Changes in
absorbance were recorded at 570 nm.
Estimation of SOD
Levels of SOD in the cell free supernatant were measured by the
method of Kono (1978). Briefly, 1.3 ml of solution A (0.1 mM
ethylenediaminetetraacetic acid (EDTA) containing 50 mM Na2CO3,
pH 10.5), 0.5 ml of solution B (90 mm nitro blue tetrazolium (NBT)
dye), 0.1 ml of solution C (0.6% TritonX-100 in solution A) and 0.1
ml of solution D (20 mM hydroxylamine hydrochloride, pH 6.0) was
mixed and the rate of NBT reduction was recorded for 1 min at 560
nm. 0.1 ml of the supernatant was added to the test cuvette as well
as reference cuvette, which do not contain solution D. Finally, the
percentage inhibition in the rate of reduction of NBT was recorded
as described earlier. One enzyme unit was expressed as inverse of
the amount of protein (mg) required inhibiting the reduction rate by
50% in 1 min.
Assessment of tissue lipid peroxidation
Butylated hydroxytoluene (BHT) of 10 µl (0.5 M in acetonitrile) was
added to prevent homogenate from oxidation and the homogenate
was stored at -70°C until analysis for MDA.
Estimation of MDA
The MDA content, a measure of lipid peroxidation, was assayed in
the form of thiobarbituric acid reacting substances (TBARS)
(Ohkawa et al., 1979). Briefly, the reaction mixture consisted of 0.2
ml of 8.1% sodium dodecyl sulphate, 1.5 ml of 20% acetic acid
solution adjusted to pH 3.5 with sodium hydroxide and 1.5 ml of
0.8% aqueous solution of thiobarbituric acid was added to 0.2 ml of
10% (w/v) of PMS. The mixture was brought up to 4.0 ml with
distilled water and heated at 95°C for 60 min. After cooling with tap
water, 1.0 ml distilled water and 5.0 ml of the mixture of n-butanol
and pyridine (15:1 v/v) was added and centrifuged. The organic
layer was taken out and its activity was measured at 532 nm and
was compared with those obtained from MDA standards. The
concentration values were calculated from absorption measurements
as standard absorption.
Estimation of total and direct bilirubin
The total and direct bilirubin was estimated by previously described
method (Jendrassik and Grof, 1938).
Statistical analyses
The results are presented as mean ± standard error of the mean
(SEM). Statistical significance and differences from control and test
values were evaluated by Student’s t-test. Statistical probability of
P

Table 1. Effects of green tea on SOD and CAT activity in CCl4 induced liver cirrhosis in experimental rats.
Shahid et al. 1961
Parameter
Group-1
(Control)
Group-2
(CCl4 treated rat)
Group-3
(Green tea treated rat)
Group-4
(CCl4 + Green tea treated rat)
SOD (U/gm of tissue) 26.08 ± 2.9 9.37 ± 0.9* 18.57 ± 1.1* 21.4 ± 3.8
CAT (mmol/g of tissue) 3.97 ± 0.3 1.505 ± 0.3** 3.24 ± 0.5 2.739 ± 1.6
Table 2. Effects of green tea on liver enzymes in CCl4 induced liver cirrhosis in experimental rats.
Parameter
Group-1
(Control)
Group-2
(CCl4 treated rat)
Group-3
(Green tea treated rat)
Group-4
(CCl4 + Green tea treated rat)
ALT (IU/L) 52.55 ± 3.4 896.49 ± 39.1* 69.91 ± 9.5** 487.73 ± 18.7*
ALP (IU/L) 484.16 ± 19.1 947.16 ± 27.1** 362.15 ± 21.5** 528.83 ± 69.6
Table 3. Effects of green tea on total and direct bilirubin in CCl4 induced liver cirrhosis in experimental rats.
Parameter
Group-1
(Control)
Group-2
(CCl4 treated rat)
Group-3
(Green tea treated rat)
Group-4
(CCl4 + Green tea treated rat)
Total bilirubin (µmol/L) 13.45 ± 2.7 24.82 ± 0.9* 11.90 ± 1.3** 8.87 ± 0.3*
Direct bilirubin (µmol/L) 4.32 ± 1.1 16.77 ± 3.5* 3.45 ± 0.4** 6.58 ± 0.7*
Table 4. Effects of green tea on tissue lipid peroxidation in CCl4 induced liver cirrhosis in experimental rats.
Parameter
Group-1
(Control)
Group-2
(CCl4 treated rat)
Group-3
(Green tea treated rat)
Group-4
(CCl4 + Green tea treated rat)
MDA (nmol/g of tissue) 1.09 ± 0.3 2.17 ± 0.6* 0.84 ± 0.2 1.83 ± 0.5*
Values are mean ± SEM. Significant difference between controls and test groups by Students’ t-test. *p < 0.01, as compared to controls.
cancer cells was a negative feedback of the multiplication
of cancer cells and loss of lipid peroxidation explains the
malignancy of hepatocarcinoma, and enhanced lipid
peroxidation in liver cancer cells may cause the necrosis
(Alia et al., 2006; Bechman and Koppenol, 1996).
In the present study, the levels of SOD in green tea
treated rats were significantly low (P

1962 Afr. J. Pharm. Pharmacol.
place of origin. That may be reason why the combined
effects of green tea and CCl4 suggested the higher levels
of catalase activity in cirrhotic rats as compared to the
controls.
This study also indicates an altered liver enzyme
activity (ALT and ALP) (Table 2) and total and direct
bilirubin levels (Table 3) which strongly indicates liver
tissue injury. The increased serum levels of ALT and ALP
have been attributed to the damaged structural integrity
of the liver, because these are cytoplasmic in location
and are released into circulation after cellular damage.
The ALT level was significantly increased in CCl4 treated
rats where the cells of liver have been inflamed and ALT
leaked into blood stream, while ALP is significantly
decreased in CCl4 treated rats. ALP is synthesized in the
bile canalicular cells and appears in the blood stream
only whenever biliary duct is inflamed or blocked. It might
be possible that CCl4 produced only hepatic damage not
biliary. Low ALP level is associated with magnesium
deficiency as ALP activity is almost inhibited due to
chelation of zinc and magnesium, the enzyme cofactors
(Zaidi et al., 2005). Direct bilirubin concentrations was
elevated in CCl4 treated rats, indicating that it may be
due to an increased production, decreased uptake by the
liver, decreased conjugation, decreased secretion from
the liver or blockage of bile ducts (Bun et al., 2006),
decreasing amount of reducing equivalents, that is,
NADPH reductase, reduced glutathione (GSH). GSH
maintains the integrity of red blood cells (RBCs)
membrane; its reduced level increases the hemolysis and
increases the bilirubin level. Green tea increases the
biliary flow and bile helps to eliminate the bile salts, fats,
and toxins from the body. Herbal polyphenolic
compounds in the cell can function as an antioxidant and
prooxidant by scavenging reactive oxygen species via
enzymatic and non-enzymatic reactions (Pyo et al.,
2004).
The level of MDA in cirrhotic rats was found to be high
as compared to the controls. Green tea treated group
showed low levels of MDA (Table 4) as polyphenol rich
green tea extracts inhibit lipid peroxidation in
experimental rats as well as in human (Zaidi et al., 2005).
During this study, the antioxidant system of cirrhotic rats
was severely impaired, causing a high level of MDA in
cancer tissues as compared to controls. The oxidative
tissue damage in cirrhosis causes a significantly low level
of catalase. During the process of inflammation oxidative
stress occurs which leads to a significant decrease in
antioxidant enzyme system. The main target of oxidative
stress is the poly unsaturated fatty acids in cell
membranes causing lipid peroxidation and excessive
formation of MDA which may lead to damage of the cell
structure and function (Kuper et al., 2000).
The results of this study are convincing that the
administration of green tea has beneficial effects in CCl4-
incuded liver cirrhosis in experimental rats. The protective
benefits of this herbal extract may be due in part to their
potential antioxidant properties and ability to reduce
oxidative stress. These should be supplemented with diet
over a significant period of time to reduce the risk of
hepatic damage caused by free redicals. In conclusion,
these herbal products may be used for protective
purpose which expose to hepatotoxic agents. However,
more detailed studies are sought to refine the understanding
of relationship between the hepatotoxicity and
protective effects of herbal antioxidants.
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African Journal of Pharmacy and Pharmacology Vol. 6(26), pp. 1964-1970, 15 July, 2012
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP12.484
ISSN 1996-0816 © 2012 Academic Journals
Full Length Research Paper
Preparation and biological activity of saponin from
Ophiopogon japonicus
Shuang-Li Xiong*, Da-Bin Hou, Ni Huang and Anlin Li
College of Biological Science and Engineering, Southwest University of Science and Technology,
Mianyang 621010, Sichuan, China.
Accepted 11 June, 2012
Saponin from Ophiopogon japonicus was isolated and preliminary identified by physical and chemical
methods. The antioxidant and macrophages-modulating activities of the saponin were also investigated
employing various established systems in vitro. The results of physical-chemical identification
indicated that saponin was steroidal saponin. It was observed that the scavenging activity of saponin
on 2, 2-diphenyl-1-picry-hydrazyl (DPPH) significantly increased in a concentration-dependent manner.
The scavenging rate of it on DPPH radical was 99.64% when the concentration of the saponin was up to
5 mg/ml. For scavenging hydroxyl radical, it first increased and then decreased gradually with the
increase of concentration, and reached the strongest scavenging rate at the concentration of 2 mg/ml.
Saponin also exhibited prominent macrophages-modulating activity by the promotion of phagocytic
capacity, macrophage viability, nitric oxide (NO) production and interleukin-1 release. It follows that
saponin from O. japonicus can be explored as a novel and potential natural antioxidant and
immunostimulants.
Key words: Ophiopogon japonicas, saponin, antioxidant activity, macrophages.
INTRODUCTION
Saponins are a diverse group of natural compounds
occurring in a wide variety of plants, food, and a few
marine animals. They are classified into two categories
(steroidal saponins and triterpenoid saponins) according
to the nature of their aglycone skeleton (Sparg et al.,
2004). Although, saponins are extremely toxic to coldblooded
animals, their oral toxicity to mammals is low
(Dini et al., 2001). Many plant drugs and folk medicines
contain saponins that are found to have several kinds of
bioactivities, such as antiviral, anti-inflammatory, antiparasitic,
immune-enhancing, anti-cancer, and antimicrobial
activities (Navarroa et al., 2001; Estrada et al., 2000).
Thus, plants containing saponins are becoming research
focus all over the world.
The tuber of Ophiopogon japonicus (Thunb.) Ker-Gawl,
*Corresponding author. E-mail: lxberry225@yahoo.com.cn. Tel:
86 816 6089531.
the liliaceous plant, is an important traditional Chinese
herbal medicine and has many health benefits in treating
a wide range of disorders, mainly thrombosis, myocardial
ischemia, arrhythmias, respiratory disease and
hyperglycemia (Li et al., 2010) based on a wide variety of
bioactive components, such as homoisoflavonoid,
saponins, and polysaccharide (Anh et al., 2003; Chen et
al., 2011). It has always been the food material which can
be used as medicine and food in China. In recent years,
some studies have shown that saponins from tuber of O.
japonicus have the pharmacological activity of antimyocardial
ischemia (He and Dai, 2005). However, few
studies have so far been carried out regarding the
antioxidant and immunoregulatory activity. In the context
of the present work, we have isolated and evaluated
antioxidant (2, 2-diphenyl-1-picry-hydrazyl (DPPH) and
hydroxyl radical scavenging assay) and macrophagesmodulating
activities (phagocytic activity, macrophage
viability, NO release and interleukin-1 production) of
saponins from tuber of O. japonicus.

EXPERIMENTAL PROCEDURE
Materials and reagents
Fresh tuber of O. japonicus was collected in Mianyang known as
Sichuan O. japonicus. Butylated hydroxyanisole (BHA), DPPH
radical, concanavalin A (ConA) and lipopolysaccharide (LPS) were
purchased from Sigma Chemical Co. (St. Louis, MO, USA). Roswell
Park Memorial Institute (RPMI) Medium 1640 and fetal bovine
serum (FBS) were obtained from GIBCO (Grand Island, NY, USA).
3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl tetrazolium bromide
(MTT) were purchased from Amresco. All the reagents were of
analytical grade.
Extraction and isolation of saponin
Crushed tuber of O. japonicus was firstly extracted with 70%
alcohol at 60°C in ultrasonic washing machine (200 W) for 1.5 h.
The extracts were evaporated of alcohol at 45°C in vacuum on
rotary evaporation instrument (SHANG HAI HU XI ANAYSIS
INSTRUMENT FACTORY CO., LTD). The resulting solution was
extracted with diethyl ether, and the treatment was repeated twice.
The obtained water layer was again extracted with normal butyl
alcohol saturated with water, and the treatment was repeated four
times. The obtained normal butanol layer was re-extracted with 0.4
M NaOH (twice). The normal butanol layer was evaporated just to
dryness with a vacuum evaporator at 45°C to obtain saponin which
was again purified by non-polar macroporous adsorption resin D
101 (particle size of 0.3 to 1.25 mm, pore size of 6.0 to 10.0 nm,
TIANJIN HAIGUANG CHEMICAL CO., Ltd., China). A portion of it
was dissolved in deionized water and loaded onto a
chromatographic column (2.0 × 20 cm). It was firstly eluted with
0.1% of NaOH to colourless eluent, then eluted by deionized water
for removing carbohydrate. It was again eluted by 70% of ethanol at
a flow rate of 2 BV/h for collecting saponin after eluent exhibited
Mollish negative reaction. The purified saponin was evaporated just
to dryness with a vacuum evaporator at 45°C and was stored at
20°C for further evaluation of antioxidant and macrophagesmodulating
activities.
Physico-chemical properties of saponin
Saponin was preliminary identified by chemical methods including
foam experiment and Liebermann reaction, and thin layer
chromatography (Chen, 2004). Briefly, sample containing methanol
solution was spotted on silica gel G thin-layer plates, with
chloroform-ether (1:9) as developing solvent. Vanillin-sulfuric acid
reaction system was used for identification of saponin.
DPPH radical scavenging activity of saponin
The DPPH radical scavenging activity of saponin was measured
using the method reported by Song et al. (2010) with slight
modification. Briefly, 1 ml sample with different concentrations (1 to
5 mg/ml) was added to screw-capped tube containing 1.5 ml of
DPPH (40 mg/L, m/v) anhydrous alcohol solution. The mixture was
vortexed and set for 30 min, then the absorbance was measured at
517 nm against a blank (water instead of test sample solution)
using UNICO 2102 spectrophotometer (UNICO Instruments Co.,
Ltd., Shanghai, China). Ascorbic acid and BHA were used for
positive comparison. DPPH radical scavenging rate (SC) is
calculated as follows:
DPPH radical scavenging rate (%) = 100 × (A0 - A1)/A0.
where A0 is absorbance of blank control and A1 is absorbance value
Shuang-Li et al. 1965
of the sample. The IC50 value was calculated by nonlinear
regression algorithm SC via sample concentration. This is defined
as the amount of antioxidant necessary to reduce the concentration
of DPPH radical by 50%.
Hydroxyl radical scavenging activity of saponin
Hydroxyl radical (·OH) scavenging activity of saponin was assessed
by the method of Zhu et al. (2010). Briefly, 2 ml deionized water or
tested sample was added to screw-capped tubes which contained
0.35 ml of 8.8 mM H2O2 and 0.35 ml of 6 mM FeSO4. The mixture
was thoroughly shaken and set for 10 min before salicylic acid was
added, and shaken vigorously, which was set for 10 min and
absorbance at 510 nm was recorded. The ·OH scavenging activity
was calculated according to the following equation:
·OH scavenging rate (%) = 100 × (A0 - A1)/A0
where A0 is absorbance value of blank and A1 is absorbance value
of the sample. IC50 value was the same as that of DPPH radical.
Isolation and culture of peritoneal macrophage
Male Kunming strain mouse (7 to 8 weeks old, 19 ± 0.5 g body
weight) was purchased from the Experimental Animal Institute of
Sichuan Academy of Medical Science. Main members in research
teams have obtained the permission to work with experimental
animals. Mouse was intraperitoneally injected with 5% soluble
starch three days in advance before experiments. Macrophages
were prepared from mouse as described earlier with some
modifications (Li et al., 2011; Yang et al., 2008). Briefly, peritoneal
cell suspensions were harvested by peritoneal cavity lavage with 5
ml ice-cold sterile phosphate buffer solution (PBS). The recovered
peritoneal fluid was centrifuged at 2380 g for 10 min, the cell pellets
were suspended with RPMI-1640 cell culture media supplemented
with 10% (v/v) heat-inactivated FBS, and was cultivated for 3 h at
37°C in a humidified 5% CO2 incubator. Non-adherent cells were
removed by gentle washing twice with sterile PBS. 1 ml fresh RPMI-
1640 was added to the adherent macrophages and it was adjusted
to the concentration of 5 × 10 5 cells/ml.
Assay of phagocytic activity
The effects of saponin on the phagocytic activity of macrophage
were determined using neutral red assay as reported earlier with
some modifications (Yi et al., 2010; Xiong et al., 2011). In brief, 100
μl of macrophages (5 × 10 5 cells/ml) were pre-incubated for 3 h in a
96-well plate before removal of non-adherent with RPMI- 1640.
Various tested samples and RPMI-1640 supplemented with 10%
FBS were added to each well, and were incubated for 48 h. 100 μl
of 1% neutral red physiologic saline solution was then added. After
an additional incubation for 3 h, the mixture was removed of
supernatant and was washed 3 times with PBS prior to the addition
of cytolysate (glacial acetic acid:alcohol, 1:1, 100 μl). The
absorbance at 540 nm was recorded on Multiskan Spectrum
Microplate Spectrophotometer (Thermo Scientific, America) after
cytolysis. Higher absorbance indicates stronger phagocytic activity.
Assessment of macrophage viability
The effect of saponin on the macrophage viability was detected by
the MTT assay as previously described with some modifications
(Davicino et al., 2006). Briefly, 20 μl of MTT was added to
macrophage (100 μl, 5 × 10 5 cells/ml) which was pre-incubated for

1966 Afr. J. Pharm. Pharmacol.
44 h. After additional cultivation for 4 h, supernatant was removed
and 100 μl of dimethylsulfoxide was added in each well. The purple
MTT-formazan products formed by the action of mitochondrial
enzymes in living cells were determined by measuring the
absorbance at 540 nm on microplate spectrophotometer. The
absorbance correlated positively with macrophage viability.
Measurement of nitric oxide (NO) production
Various samples were added to macrophages (5 × 10 5 cells/ml)
suspensions. The mixtures were incubated for 48 h before they
were centrifuged with 4850 × g for 10 min. 100 μl of the supernatant
and isochoric Griess (1% sulfanilamide and 0.1% N-[1-naphthyl]-
ethylenediamine dihydrochloride in 5% phosphoric acid) were
combined and shaken slightly for 5 min at room temperature. The
nitrite concentration was recorded at 570 nm on microplate
spectrophotometer using NaNO2 as standard (Lee et al., 2010; Liu
et al., 2005).
Determination of interleukin 1
LPS or saponin could stimulate macrophage for producing IL-1 in
vitro which again coordinated ConA to stimulate mitochysis reaction
of thymocyte. The mitochysis intensity of thymocyte was used for
determining the activity of IL-1 (Lin, 1999). Firstly, test sample was
added to each cell culture plate containing macrophage suspension
at a density of 5 × 10 5 cell/ml, respectively, and was cultivated for
48 h for induction of IL-1. The supernatants were collected and
freezed for determining IL-1 activity. Secondly, thymocyte
suspension that was obtained from thymus isolated aseptically by
grinding in RPMI-1640 contained 10% FBS. It was filtered through
8-layer pre-moistened sterile absorbent gauze. The filtrate was
centrifuged at 1650 ×g for 10 min. The purified thymocyte was
harvested by discarding supernatant and washing repeatedly
sediments with PBS. It was then adjusted to 1 × 10 7 cell/ml with
RPMI-1640 and was stimulated 3 days by 2 μg/ml of ConA.
Obtained thymocyte was again washed 3 times with PBS for
eliminating remnant Con A. Then, 100 μl of thymocyte (5 × 10 5 ml -1 )
and 100 μl of supernatants (IL-1 inducer) were added to cell culture
plate and were cultivated for 67 h at 25°C before MTT (50 μg/well)
was added. Cultures were centrifuged at 1650 × g for 10 min.
DMSO was added in precipitum and the absorbance of it was
measured at 492 nm on microplate spectrophotometer.
Statistics
All experiments were performed in triplicate. Results are given as
averages ± standard error (SE). Statistical significance among
various samples was evaluated by one-way analysis of variance
(ANOVA). Significant differences between two means were
determined by least significant difference (LSD) multiple-range
tests. P-values of less than 0.05 were considered to be statistically
significant.
RESULTS AND DISCUSSION
Primary identification of saponin
The aqueous solution containing saponin would produce
persistent foam, because it can decrease the surface
activity of aqueous solution, which has been used as the
identification method of saponin. Under anhydrous
conditions, strong acid reacts with saponin, which leads
to the dewatering of saponin, structural changes, such as
double bond shift, formation of carbonium ion salt, and
color change. Different saponins present different change
of color. The results of foam experiment indicated that
extracts of butanol contain saponin. The color change
from yellow to red showed that it was steroidal saponin.
Claret-color spot on thin layer chromatography (TLC)
plate confirmed it as saponin.
Scavenging activity of saponin on DPPH radical
DPPH radical is usually used as a stable substrate to
evaluate antioxidative activity of antioxidants. The method
is based on the reduction of absorbance at 517 nm of
DPPH solution in the presence of proton-donating
substance, owing to the formation of the non-radical
forming DPPH-H by the reaction. As shown in Figure 1,
the scavenging ability of saponin on DPPH radicals
exhibited a concentration-dependent tendency within the
tested concentrations. The activity of ascorbic acid and
BHA had higher free radical scavenging activity than
those of saponin when the concentration was lower than
3 mg/ml. It was worth noting that the activity of saponin
was significantly stronger than that of BHA and close to
ascorbic acid (94.62% for saponin and 99.64% for
ascorbic acid, respectively, 4 mg/ml) when the
concentration was more than 4 mg/ml, although, IC50
(1.46 mg/ml) of saponin was significantly lower than
ascorbic acid (1.1 µg/ml) and BHA (3.08 µg/ml) (data not
shown). The scavenging rate of saponin on DPPH radical
was 99.64% when the concentration of it was up to 5
mg/ml, which was significantly higher than that of BHA.
This implies that saponin can be an effective free radical
scavenger. The possible mechanism of the saponin
acting as an antioxidant may be attributed to its electron
or hydrogen donation power to the DPPH radicals,
thereby terminating the radical chain reaction (Lai et al.,
2010).
Scavenging activity of saponin on hydroxyl radical
Among the reactive oxygen species, the hydroxyl radical
is the most reactive and could induce severe damage to
all classes of biological macromolecules in the form of
abstracting hydrogen atoms, addition reaction and
electron transportation (Lai et al., 2010). It is also
believed to be an active initiator for peroxidation of lipids.
Therefore, it was used extensively as the free radical to
evaluate antioxidants. As shown in Figure 2, the
scavenging ability of saponin on hydroxyl radical showed
significant increase in a concentration-dependent pattern
with the concentration ranging from 0.5 to 2 mg/ml. The
scavenging ability of saponin was significantly stronger
than that of BHA and close to ascorbic acid when the
concentration was more than 1.5 mg/ml, and being the

Scavenging rate (%)
Figure 1 Scavenging activity of
Figure 1. Scavenging activity of saponin on DPPH radical.
saponin on DPPH radical
highest scavenging rate at the concentration of 2 mg/ml.
It is important to note that the scavenging ability of
saponin decreased gradually as the concentration of it
was over 2 mg/ml. For hydroxyl radical, there are two
type of antioxidation mechanism: one suppresses the
generation of the hydroxyl radical and the other
scavenges the hydroxyl radicals generated (Hu et al.,
2010). Steroidal saponin is composed of a steroidal
aglycone and one or more sugar residues. The
scavenging activity of saponin on hydroxyl radical may be
due to the inhibition of radical generation by ion (such as,
Fe 2+ and Cu 2+ Figure 1 Scavenging activity of
saponin on DPPH radical
) chelation of hydroxyl group and the
reaction of reductones in it with precursors of peroxide
which thus prevented peroxide formation. The exact
mechanism underlying the DPPH radical and hydroxyl
radical scavenging activity exerted by saponin needs to
be further investigated.
Macrophage activation by saponin
Macrophages are versatile cells that play many roles.
Macrophages execute immunomodulatory functions in
host defense directly and indirectly, such as phagocytosis
of pathogens and cell debris, secretion of cytokines, and
proteolytic processing and presentation of foreign
antigens (Xie et al., 2008). In addition, they carry receptors
for lymphokines that allow them to be "activated" into
single-minded pursuit of microbes and tumour cells, and
also play key roles in novel immunotherapeutic
approaches to the treatment of cancer (Klimp et al.,
2002). The precondition of the macrophage work is
macrophage activation. An increase in phagocytic activity
is one of the most distinguished features of macrophage
activation. Phagocytic activity of saponin was examined
by the uptake of neutral red (0.1%) in this experiment.
LPS, the major structural component of the outer wall of
Gram-negative bacteria, is a potent activator of
macrophages and is often used in screening for immunity
Shuang-Li et al. 1967
Figure 2 Scavenging activity of
saponin Figure on 2 hydroxyl Scavenging radical activity of
saponin on hydroxyl radical
Figure 2. Scavenging activity of saponin on hydroxyl radical.
regulator (Fujihara et al., 2003). As shown in Figure 3
(the left), the phagocytic activities increased significantly
in all saponin-administered groups in comparison to the
control group receiving PBS (P < 0.01), and exhibited
remarkable concentration-dependent characteristics with
the saponin concentration in the range of 100 to 400
μg/ml.
In order to further explore the macrophage-activating
potential of saponin, the effects of saponin on
macrophage viability was also evaluated using MTT
methods. As shown in Figure 3 (the right), there was also
significant increase in macrophage viability rate (A540)
when peritoneal macrophages were incubated with LPS
and different concentrations of saponin in comparison
with blank group (P < 0.01). A dose-dependent
enhancement of macrophage viability, treated with 100 to
400 μg/ml was also discovered, which is similar to
phagocytic activity. Especially, macrophage viability was
significantly stronger than that of LPS when the
concentration of saponin exceeded 400 μg/ml. Based on
the activation expression of saponin on macrophage, it
was concluded that it was potent activators of
macrophage.
Effects of saponin on NO release and IL-1 production
In the innate and adaptive immune responses, activated
macrophages play an important role by producing
cytokines, interleukin-1 beta, NO, and other inflammatory
mediators. More efforts have been made to develop
therapeutic agents that regulate NO production and
secretion for pivotal role of it in regulation of the immune
response. To assess the role of saponin in the NO
production, we cultivated macrophage with saponin or
LPS. Then, NO produced from the cells was determined
in terms of nitrite by using the Griess method, because
NO is known to be converted to nitrite soon after its

1968 Afr. J. Pharm. Pharmacol.
No (µmol/L)
Absorbance at 540 nm
Figure 3 Effects of saponin on phagocytic activity (the left ) and macrophage viability
Figure 3. Effects of saponin on phagocytic activity (the left) and macrophage viability (the right). All values represent
triplicate
(the
mean
right)
values
All
±
values
SE. Different
represent
uppercase
triplicate
letters on
mean
different
values
columns
± SE.
are significantly
Different uppercase
different between
letters
groups
on
by
LSD (P different < 0.01). The columns same letters are suggested significantly that differences different between groups groups were not by statistically LSD (P same 0.05).
letters suggested differences between groups were not statistically significant (P>0.05).
A
D
B
Various samples
Figure 4 Effects of saponin on NO production All values represent triplicate mean values ± SE. Different uppercase
Figure letters on 4. different Effects columns of saponin are significantly on NO different production. between groups All by values LSD (P0.05). letters on different
columns are significantly different between groups by LSD (P < 0.01).
The same letters suggested that differences between groups were not
statistically significant P>0.05).
production (Granger et al., 1996). The effects of saponin
on NO production in peritoneal macrophage are shown in
Figure 4. Macrophages treated by LPS and saponins with
various concentrations produced larger amounts of NO
than that treated with PBS (P

A
D
Figure 5 Figure Effects 5. of Effects saponin of saponin on IL-1 on production IL-1 production. All All values values represent represent triplicate mean
values ± triplicate SE. Different mean values uppercase ± SE. letters Different on uppercase different columns letters on are different significantly different
between columns groups are by LSD significantly (P (P>0.05). 0.05).
proved that steroidal saponin from tuber of O. japonicus
would regulate immune response such as anti-infection,
anti-tumor, and promote proliferation and differentiation of
lymphocyte, etc.
Conclusion
Based on the aforementioned results, it was concluded
that saponin from O. japonicus belonged to steroidal
saponin. It exhibited strong free radical-scavenging
activity on DPPH radical and hydroxyl radical. The
scavenging rate of it on DPPH radical was 99.64% when
the concentration of it was up to 5 mg/ml. For hydroxyl
radical, it ascended first and then descended gradually
with the increase of concentration, and being the highest
scavenging rate at the concentration of 2 mg/ml, which
was significantly stronger than that of BHA and close to
ascorbic acid. Saponin also demonstrated remarkable
macrophages-modulating activities by the promotion of
phagocytic capacity, macrophage viability rate, NO and
interleukin-1 production, all of which appeared in an
evident dose-dependent manner. To further address the
correlation between the structure, and the antioxidant and
immunostimulating activities of this saponin, purification
and chemical characterization of dominant saponin
compounds from Ophiopogonis japonicas is in progress.
ACKNOWLEDGEMENT
This work was supported financially by Doctor
Foundation of Southwest University of Science and
Technology (06zx7122).
B
C
E
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African Journal of Pharmacy and Pharmacology Vol. 6(26), pp. 1971-1976, 15 July, 2012
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP12.493
ISSN 1996-0816 © 2012 Academic Journals
Full Length Research Paper
Statistical analysis of medical experiment data for
discovering groups of correlated symptoms
Chenghe Shi 1# , Qingqiong Deng 2# , Peng Lu 3# , Minquan Zhou 2 and Gang Xiong 4 *
1 Department of Traditional Chinese Medicine, Peking University Third Hospital, Beijing 100191, P. R. China.
2 College of Information Science and Technology, Beijing Normal University, No. 19 Xin-Jie-Kou-Wai Street, Beijing
100875, P.R China.
3 Institute of Automation, Chinese Academy of Sciences, Beijing 100190, P. R. China.
4 Department of Physics, Beijing Normal University, No. 19 Xin-Jie-Kou-Wai Street, Beijing 100875, P. R. China.
Accepted 3 May, 2012
Three ways of statistical analysis, that is, Pearson correlation (PC), Spearman correlation (SC) and
mutual information (MI), are applied on medical experiment data to obtain correlation matrix. When the
results of this study’s analysis were combined with those of professional analysis, it was found that the
method based on MI may be the best way for discovering groups of correlated symptoms.
Key words: Association, symptom combination, coronary heart disease.
INTRODUCTION
Coronary heart disease (CHD) remains the single leading
cause of death for adults worldwide (Lloyd-Jones et al.,
1999). Effective prevention and therapy for CHD poses a
major challenge to the entire medical community.
Traditional Chinese medicine (TCM) has fought against
CHD and its related diseases for more than 1000 years,
and has accumulated thousands of prescriptions as well
as clinical literatures (Chen and Jia, 2010; Guo et al.,
2009; Liu et al., 2010). Therefore, more and more
patients all over the world take TCM as a complementary
and alternative avenue to treat CHD.
TCM is a medical system with at least 3000 years’s
uninterrupted clinical practice and it has the advantage of
collecting macroscopic information (including symptoms,
tongue and pulse recognition) of a patient for diagnosis,
while syndrome is the core of diagnosis and target of
herbal remedy in TCM. Nowadays, syndrome in TCM has
been always studied in the context of a specific disease
or biomedical condition and several literatures have
demonstrated that syndromes are significantly associated
with diseases (Chen and Jia, 2010, Guo et.al., 2009).
TCM is taken by most people in China as a
*Corresponding author. E-mail: phgxiong@bnu.edu.cn.
# These authors contributed equally to this work.
complementary therapeutic alternative since herbal
remedies have the advantage over Western medicine in
that it has less side effects and are less costly. TCM has
always been regarded as a key component in 5000 years
of Chinese civilization history. In ancient times before
modern medicine was born, people all over the world
mainly benefit from three traditional medicines, among
which only TCM is still alive today; while Chaldaic and
ancient Hindu medicines only have extremely rare
documents as evidence that they ever existed in history.
TCM, whose core is syndrome, is on the way to
modernization. It is aiming to be accepted as a science,
like Western medicine (Bohigas et al., 1984; Seba, 2003;
Zhong et al., 1998; Zhong and Geisel, 1999).
Unstable angina (UA) is a type of CHD. It describes a
biomedical condition that is intermediate between
myocardial infarction (MI) and the more chronic state of
stable angina. UA is now a heavy burden on the society
and families in both industrialized and developing
countries. So UA presents a better example and context
for investigating diagnosis method and biological basis of
syndromes in TCM.
The syndrome is the basic pathological unit and the key
concept in TCM theory since herbal remedy is prescribed
according to syndrome or syndromes a patient catches Li
et.al., 2007).
Therefore, identification and determination of syndrome

1972 Afr. J. Pharm. Pharmacol.
Table 1. Clustering and validation results.
No. Patterns
1
2
3
4
5
Chest pain, chest tightness, short breath,
palpitation, hypodynamia, spontaneous perspiration
Xerostomia, dizziness, amnesia;
vértigo, tinnitus
Sighing, depression, short and yellow urine, low
speaking voice
Inappetency, abdominal distension, stomach
discomfort, eructation
Dry and hot face, swelling in the costal regions,
light color in lips and nails
Syndrome
diagnosed by
experts
Qi deficiency and
blood stasis
Deficiency of Yin
Qi stagnation
Deficiency of
spleen
Corresponding
treatment
Tong Xin Luo
capsule
Jisheng Shenqi
pills
Composite Salvia
Dropping Pill
Ren sen Jian Pi
pill
Number of
cases of
patterns
The maximum
Number of
syndrome
Sensitivity
of each
pattern (%)
560 500 89.29
240 225 93.75
150 135 90
108 96 88.89
Yu re syndrome Sijunzi decoction 54 48 88.89
6 Night sweat, feverishness in palms and soles; Deficiency of Yin Pingwei powder 124 116 93.55
7 Fear of cold and cold limbs, insomnia; lumbago
(s) in CHD patient become significantly important
for TCM physicians. Nevertheless, there are few
docu- ments dedicated to this issue.
In this paper, we carried out a clinical
epidemiology survey and we proposed a novel
unsupervised data mining model, in which we
treat mutual information (MI) as an association
measure of two variables. In our effort, we tried to
discover syndromes in CHD data and clinically
verify these syndromes to test the performance of
our model without supervision. Based on revised
MI, we proposed an unsupervised pattern
discovery algorithm to self-organize allocate
significantly associated symptoms to patterns. By
using diagnostic data, each pattern is verified to
Deficiency of Yang
(Subtype)
have clinical meaning.
MATERIALS AND METHODS
Study population
Syndrome is diagnosed according to symptom com-
binations. As shown in Table 1, we choose 72 symptoms
that are closely related to CHD. The pulse information of
every patient was not included for its bad consistency
during the process of survey. In the survey, the data set
was recruited from six clinical centers located in six
provinces from the same demographic area and at the
same time from October, 2005 to March, 2006, where a
total of 1069 patients who suffer from CHD were surveyed.
Eligible patients in this paper were defined as with UA
Sijunzi decoction 206 192 93.20
based on diagnosis criteria of UA established in 2002 by
ACC (American College of Cardiology) and AHA (American
Heart Association), that is, chest pain at rest and transient
S-T segment changes, without significant increases in
creatine kinase and creatine kinase MB fraction (Zhong et
al., 1998).
The criterion for enrollment was admission within 48 h
after the onset of chest pain. Moreover, the exclusion
criteria were composed of four conditions: (1) Besides UA,
a patient also suffers from other cordis diseases such as
acute myocardial infarction, myocarditis and cardiac nerve
functional disease; (2) A patient with angina caused by
other diseases, for example, rheumatic fever, syphilis,
congenital coronary anomalies, hypertrophic cardio-
myopathy and cardiac mitral stenosis; (3) Besides UA, a
patient also suffers from stroke, diabetes, nephritis, renal
failure, pulmonary infection, urinary tract infection,

Figure 1. Level-spacing distribution function P(s) of MI matrix with
cut-off value rcut =0.02 and rcut =0.1.
rheumatism, osteoarthritis and serious diseases caused by liver,
renal, haematogenous system and incretion system; (4) A woman
patient in gestation or lactation.
Every case is with 72 symptoms, together with the basic
information of each subject. The frequencies of 72 symptoms are
shown in Table 1; each variable (symptom) has four categories, that
is, none, light, middle and severe, represented by 0, 1, 2, 3,
respectively. The latter three categories of each variable means
that the symptom has appeared and then separated into light,
middle and severe by clinical doctors, who are strictly and uniformly
trained to reach a high consistency.
All subjects gave informed consent and were approved by the
Medical Ethics and Human Clinical Trial Committee at
Guanganmen Hospital.
Data mining methods
It is essential to develop powerful computational methods to extract
as much information as possible from medical experiment data.
In this paper, the data we dealt with are 70 symptoms of 1,070
patients. What we did was to find a way based on correlation
analysis to category these symptoms into groups. We used three
widely used statistical methods: Pearson correlation (PC),
Spearman correlation (SC) and MI. The standard PC coefficient
between two symptoms xi and xj is defined as:
The standard SC coefficient between two symptoms xi and xj is
defined as:
with R(xi,k) the rank of xi,k. The MI coefficient between two
symptoms xi and xj is defined as:
Where H(xi) and H(xj) are information entropies of distributions of
each symptom, and H(xi,xj) is information entropy of joint
distribution of the two symptoms. Using PC, SC and MI coefficients,
(1)
(2)
(3)
Shi et al. 1973
we can construct three matrices of rank 70 from experiment data,
respectively. The non-diagonal elements of these matrices give
information of correlations among the 70 symptoms.
We shall analyze these matrices in the following way similar with
Zhong et al. (1998). We set a cut-off value rcut that non-diagonal
elements with its absolute value smaller than rcut are neglected
and set to be zero. At the starting point rcut = 0, we expect strong
correlations among the 70 symptoms. By tuning rcut from zero to
the maximum value, the correlation induced by non-diagonal
elements is expected to decrease and the 70 symptoms should be
separated into uncorrelated groups. If we consider the symptoms
as sites forming a random network and the non-diagonal elements
as existence probability of bonds connecting those sites of that
random network, the procedure of decreasing non-diagonal
elements is similar with the bond-percolation transition of a random
network. At the beginning rcut = 0 the network forms a wholly
percolated cluster. With increasing rcut, the network begins to split
into separate parts and each part corresponds to a group of
correlated symptoms.
The stated percolation transition can be studied by considering
eigenvalue spacing distribution of the real symmetrical matrices
obtained from the network of symptoms. According to the random
matrix theory (RMT) (Wigner, 1967), eigenvalue spacing distri-
butions of real symmetrical random matrices fall into two universal
types depending on the strength of eigenvalue correlation induced
by non-diagonal elements (Hofstetter and Schreiber, 1993). Strong
correlation between eigenvalues leads to the so-called Gaussian
orthogonal ensemble (GOE) while weak correlation between
eigenvalues leads to Poisson ensemble (PE) ( Plerou et.al., 1999).
The nearest neighbor spacing distribution (NNSD) of eigenvalues in
the case of GOE is close to the Wigner-Dyson distribution (Wigner,
1967).
While the distribution in the case of PE is the Poisson distribution
It should be noted that the obtained result of RMT holds only for
statistical results of large number of eigenvalue spacings, while in
the case, we study the rank of obtained matrix and it is 70 and not
large. Thus statistical fluctuation is expected strong in NNSD.
However, we still decide to have a try. We obtain the eigenvalues Ei
of the matrices and line them in order. Then we unfold Ei by
dividing them with their average value
and obtain
the normalized eigenvalues ei = Ei/ME. Thus, the eigenvalue
spacings are obtained as si = ei+1-ei.
RESULTS AND DISCUSSION
Figure 1 shows numerical NNSD P (s) obtained from the
MI correlation matrix with cut-off value rcut = 0, rcut =0.02
and rcut =0.1, respectively. We can see that P (s) with
rcut =0 and rcut =0.02 show a peak around s =0.1 which
is similar with the behavior of PGOE.
However, P (s) with rcut =0.1 is monotonically
decreasing with increasing s similar to PPE. Figures 2
and 3 are NNSD obtained from correlation matrices of PE
and SP. One can see that with increasing rcut, they also
(5)
(4)

1974 Afr. J. Pharm. Pharmacol.
Figure 2. Level-spacing distribution function P(s) of SC matrix with
cut-off value rcut =0.2 and rcut =0.4.
Figure 3. Level-spacing distribution function P(s) of SC matrix
with cut-off value rcut =0.2 and rcut =0.4.
Figure 4. Number of group Ng versus cut-off
value rcut by MI analysis. Black and red dots
correspond to strong and weak criterion,
respectively.
show similar behavior as that of MI. Therefore, although
the number of eigenvalues in our case is not large
enough to reach the thermodynamic limit, one is still able
to see a transition from a typical GOE behavior to a
typical PE with increasing rcut. This means that with
increasing rcut the finite network of correlations among
Figure 5. Number of group Ng versus cut-off
value rcut by SC analysis. Black and red dots
correspond to strong and weak criterion,
respectively.
Figure 6. Number of group Ng versus cut-off value
rcut by PC analysis. Black and red dots correspond
to strong and weak criterion, respectively.
the 70 symptoms also encounters a percolation
transition, turning from a connected network into separate
groups, which is just what we expect to see.
Considering the structure of the correlation network of
the symptoms, since the number of symptoms is not too
large, we can directly consider the number of correlated
groups the symptoms forms with increasing rcut. We
used two different standards to determine whether a
symptom shall be added into a group. One is a weak
criterion that the symptom is added into a group if at least
one symptom in that group has non-zero correlation
coefficient with it. The other is a strong criterion that the
symptom is added into a group only if every symptom in
that group has non-zero correlation coefficient with it.
Figures 4, 5 and 6 show the curve of group number Ng
versus rcup.

Number of cases
Pattern
Shi et al. 1975
Figure 7. X-axis is pattern; Y-axis is corresponding number of cases in the data. For purple circle point, the data is symptoms data, while
for green diamond point, the data is syndrome data. The percentage around each point indicates the sensitivity of the corresponding
pattern. The average of them is sensitivity of the algorithm -96.48%.
One can see that the curves of the two criterions by MI
are quite similar, while the curves of the weak criterion by
PE and SP show that the network is not split into groups
at smaller value of rcut. Therefore, we may conclude that
MI analysis is easier to obtain the form of uncorrelated
groups.
Finally we evaluate clinical meaning of the clusters by
two avenues.
The first avenue is using clinical theory of TCM and
expert domain knowledge to estimate pattern results. As
shown in Figure 7, the pattern results recognized by MI
analysis (green diamond points) are highly accordant with
the results diagnosed by TCM physicians (purple circle
point).
The second avenue is to objectively estimate each
pattern discovered by above algorithm using a
supervised validation method of the following three steps.
Step 1. Each pattern S is returned to the unsupervised
data, if all variables of the pattern simultaneously appear
(their values are non-zero) on a patient, then serial
number of the patient is recorded as shown in Table 1. All
serial numbers are stored in a vector with Ls dimensions
denoted as Vs.
Step 2. We track the vectors back to the syndrome data
by adding up the vectors one by one to generate a new
vector. The maximal number in the new vector and the
corresponding syndrome are stored as shown in Table 1.
Step 3. The accuracy of a pattern is defined as the ratio
of maximal number and total number, and the accuracy
of the algorithm is determined by averaging the accuracy
of all patterns. We found that the pattern results of MI
analysis reached a high accuracy as depicted in Figure 8.
The optimal rcut is 0.04. and the corresponding accuracy
is 91.04%.

1976 Afr. J. Pharm. Pharmacol.
Conclusion
Accuracy (%)
Figure 8. The optimal selection step (fourth step).
In summary, we present a kind of correlation based
cluster algorithm to cluster symptoms in medical data.
We also propose a supervised validation algorithm to
objectively evaluate the clustering results. We find that MI
is better fit to describe correlation between symptoms in
the data. The corresponding clustering results reached
an accuracy of 91.04%. The algorithm presented here
paves a significant basis for discover new pattern in
medical data.
ACKNOWLEDGEMENT
Authors acknowledge the support of CNSF under Grant
No. 10604006 and SCC of Beijing Normal University.
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African Journal of Pharmacy and Pharmacology Vol. 6(26), pp. 1977-1981, 15 July, 2012
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP12.606
ISSN 1996-0816 ©2012 Academic Journals
Full Length Research Paper
Comparative analysis of distinct diuretics through urine
analysis
Zain Ullah 1 , 2 , Musa Kaleem Baloch 1* , Jameel Ahmed Khader 3 , Naser Mohamed AbdElsalam 3 ,
Riaz Ullah 4 , 5 , Arif-ullah Khan 6 , Muhammad Talha 7 , Naeem Khan 8 and Iqbal Hussain 8
1 Department of Chemistry, Gomal University, Dera Ismail Khan, Pakistan.
2 Diabetis and Nutritional Sciences Division, King’s College London, UK.
3 Riyadh Community College, King Saud University, Saudi Arabia.
4 Department of Chemical Engineering, College of Engineering, King Saud University Riyadh, Saudi Arabia.
5 Department of Chemistry, Govt Degree College Ara Khel, Jawaki, FR Kohat, Pakistan.
6 Institute of Pharmaceutical Sciences, Kohat University of Science and Technology, Kohat-26000, Pakistan.
7 College of Science Research Centre, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia.
8 Department of Chemistry, Kohat University of Science and Technology, Kohat-26000, Khyber Pakhtunkhwa, Pakistan.
Accepted 25 June, 2012
Diuretics are used for different diseases and are taken without knowing their adverse/side effects. To
create awareness, four popular diuretics, Lasix, Spiromide, Aldactone and Conium were investigated
for their effect on body. These were experimented over four volunteers with single and double doses.
The urine of these volunteers was collected for analysis of sodium and potassium. The obtained results
showed that the effect of medicine remained up to 6 to 12 h. In this duration, the sodium excreted value
went up for a certain period and then became almost normal and same was true for potassium. The
increase in dosage increased the rate of excretion of the cations and also prolonged the time period of
their effects. It is concluded that although Lasix, a potential diuretic but its effects over body were also
severe in terms of excretion of ions etc. Similarly, Spiromide was also noted to be a reasonably
effective diuretic and its effects are totally different from Lasix in terms of excretion of ions and body
weight loss. The other two medicines (Aldactone and Conium) did not show much effect upon excretion
etc.
Key words: Urine, Lasix, Spiromide, Aldactone, Conium.
INTRODUCTION
Any substance that tends to increase the flow of urine,
which causes the body to get rid of excess water, is
known as diuretic drugs. Substances that augment
"diuresis," or the removal of fluids from the body through
urination, are considered diuretics. Some people use
diuretics as a weight loss aid, usually when a large
amount of weight needs to be lost in a short amount of
time (Capps et al., 1952). The fact is that diuretics are not
proven to promote the loss of fat; they simply remove
retained fluid. While the scale may show the loss of a few
*Corresponding author. E-mail: zain_kust@yahoo.com. Tel: 020
7848 4247, 075 5063 3668. Fax: 020 7848 4500.
pounds, it is a temporary loss. This is not a healthy way
to lose weight. Diuretics are widely used and generally
safe, but like any therapeutic agents, they may causeside
effects. They enjoy a very high clinical reputation for
safety and efficacy. However, more than 3 decades of
clinical investigation have disclosed a number of
abnormalities in fluid electrolyte handling, metabolism
and other adverse effects that can complicate therapy
with diuretic drugs. These include extracellular fluid
volume depletion, associated orthostatic hypotension,
and prerenal azotemia (Brown et al., 2003). Others are
not a direct action of the diuretic, but can be explained as
intranephronal compensation to the diuretic action.
Diuretics are used for many reasons. They may be

1978 Afr. J. Pharm. Pharmacol.
indicated for people who suffer from edema, high blood
pressure and heart related diseases (Staessen and
Wang, 2001). Over-diuretics can lead to dehydration and
sometimes severe potassium deficiencies, which can be
dangerous (Briggs et al., 1998).
There are four major classes of diuretics and all of
them inhibit sodium (and water) re-absorption in different
segments of the nephron, therefore, increasing urinary
excretion of sodium and urine volume:
1) Carbonic anhydrase inhibitors
2) Loop diuretics
3) Thiazides
4) Potassium-sparing diuretics
The first one is a mild alkaline diuresis in the proximal
tubule. Acetazolamide are used to reduce the intraocular
pressure in glaucoma or to alkalinize urine. The second
one is the most potent diuretics and can induce
significant volume depletion. These diuretics are organic
anions that are secreted in the lumen of the proximal
tubule. They bind to the chloride site of the Na-K-2Cl cotransporter
in the luminal membrane located in the thick
ascending limb of Henle. The Lasix used here is
furosemide and is loop diuretic. Loop diuretics may cause
a change in the levels of potassium and other electrolytes
in the blood. In some cases, loop diuretics may cause a
decrease in the amount of magnesium in the blood. The
third one is the organic anions secreted by the proximal
tubule that bind to the chloride site of the Na-Cl cotransporter
in the luminal membrane of the distal
convoluted tubule. Thiazides can induce hyponatremia
because they inhibit urine dilution and the forth one is
weak because not much sodium is reabsorbed in their
site of action. Spironolactone is an aldosterone antagonist
useful in the treatment of congestive heart failure
and hepatic cirrhosis (Pickkers et al., 1998).
Diuretics side effects are a result of the change in the
amount of levels of minerals due to the action of these
water pills. Although the side effects of diuretics may be
mild and they may be felt during the initial days of
treatment, a physician should be consulted in case the
side effects persist for a long period of time (Buchet and
Lauwerys, 1973).
Lasix, Spiromide, Aldactone and Conium are used as
diuretics for many reasons. They may be indicated for
people who suffer from edema, an intense accumulation
of fluids in the body's tissues, and those who suffer from
high blood pressure or other heart related diseases.
Increasing the production of urine not only releases fluid,
but also helps rid the body of excess salts and may
reduce blood volume (Birtwhistle et al., 2004). Although it
can be administered as single dosage but as there are
four volunteers to be chosen, therefore double dosage is
much more valuable and productive (Pickkers et al.,
1998). We have tried to use and follow all the drugs
according to ABCD rules of British Hypertension
Society’s (BHS) July 29, 2005 (Williams et al., 2004;
Birtwhistle et al., 2004). The present work is unique and
novel type because the literature survey reveals that no
symmetric study has been carried out about the efficiency
of the diuretics and the adverse effects of them
(Martinez-Maldonado et al., 1990). The objective of the
work is to take different diuretics and investigate them
with reference to their action and side effects
MATERIALS AND METHODS
Selection of drugs and subjects
For comparison, the drugs are selected in such a way that the
origin, mechanism of action be different from each other and these
must be available in the market harmful for the person to take.
Keeping in view, these facts the drug selected for this purpose were
purely diuretic including, Lasix, Spiromide, Aldactone and Conium
(Staessen et al., 2003). Further, the following considerations were
kept in mind:
1) These drugs are easily available for laboratory study.
2) The action be rapid and quick and be at least in 24 h.
3) These should increase the volume of urine excreted.
4) The medicine must not have dangerous adverse effects so that
the model is ready to take these and their lives might not be in
danger (Braunwald et al., 2004).
To conduct experiments, four volunteers were chosen as models in
hostel dispensary. The four volunteers were divided into two
groups. The volunteers were having the same characteristics.
Further, the following considerations were made during the
selection:
1) Physically and mentally normal up to the possible extent.
2) Provided with the same dietary intake up to large extent.
3) Same age within permissible range and is young.
4) Not taking any medicine in those days except the ones provided
by us.
5) Available throughout the study period that is, the whole year.
Dosing and sample collection
The four volunteers selected were divided into two groups as stated
earlier. One group was given a single dosage of the respective
diuretic drug and the other provided with a double dosage. In both
cases, the sample of urine were taken regularly and analyzed for
sodium and potassium (Buchet and Lauwerys, 1973). Before giving
the medicine, the urine was collected in early morning on same day
and considered to be at zero time. The medicine was given at an
appropriate time (normally 8:00 am) on Sunday and the urine
samples were collected after each hour at regular intervals of time.
These samples were collected in washed, cleaned and tight
fattened containers (Carlberg et al., 2004). The samples were
collected up to twelve hours (8:00 pm) after giving the medicine.
The blank sample was used as a reference for the variation in
concentration of various ions after the administration of drugs
(Psaty et al., 2004). Quantitative analysis was carried out to
determine the variations in concentration in different ions excreted
through urine by the intake of the drugs.
Determination of sodium and potassium electrolytes
0.2543 g of NaCl provided by EMerck Germany and analytical
grade was taken after drying at 11°C in oven. This amount of

sodium was then dissolved in 1000 ml of de ionized water to form
100 ppm stock solution of NaCl. For this purpose, 1 L volumetric
flask was used. This solution was further diluted to give reference
solution of 5, 10, 20, 30, 40 and 50 ppm (Brown et al., 2000). These
reference solutions were taken into test tubes, the flame
photometer was switched on at least half an hour before the
measurements were taken. Deionized water was then taken in a
test tube and the sucker of flame photometer was inserted into it.
The instrument was adjusted to zero and the standard solution was
sucked by flame photometer gradually, and the emission for each
reference concentrations was determined and noted. The emission
was plotted versus concentration to get a standard graph which
was then used to determine the Na + concentration in a given urine
sample. In the same the cooled urine sample were sucked by
photometer and absorption was noted. From these results and
using standard graph the concentration of sodium was determined.
For potassium detection, 1.9 g of dried KCl was dissolved in 1000
ml of de ionized water in a volumetric flask. This gave 100 ppm
stock solutions. FrOm this solution, reference solution of 5, 10, 20,
30, 40, and 50 ppm was prepared. These solutions were used for
the determination of K + urine samples. The analysis were carried
out be flame photometer and the method used was the same as
that for sodium determination.
Statistical analysis
Quantitative analysis was carried out to determine the variations in
concentration in different ions excreted through urine by the intake
of the drugs
RESULTS AND DISCUSSION
The urine collected from the models after administration
of single dosage of Lasix was diluted to several times and
was analyzed for sodium and potassium. For sodium, the
dilution factor was 200. For potassium, the urine samples
were diluted to 50 times. The results obtained for sodium
Na + concentration (ppm)
Time (h)
Figure 1. Effect on sodium excretion in urine by single dose of
different diuretics.
Ullah et al. 1979
analysis after the intake of single dosage of Lasix,
Spiromide, Aldactone and Conium shows that sodium
excretion increases with time and reaches to maximum
after 6 hours and then goes down, as shown in Figure 1.
When the diuretics were administered in double dose, it
can be noted from the results the maximum exertion
reaches much earlier than that of single dosage while the
maximum excretion value is almost the same as
observed for single dosage, as shown in Figure 2.
Similarly, the execration levels go down very fast and
come to the normal value within 12 h time (Figure 2). We
can see that the maximum effect of the medicine,
whether the dosage is single or double. Furthermore, this
dosage may affect the amount of the urine excreted by
the body but it does not affect the amount of sodium
excreted along with the urine.
The urine analyses for potassium were also performed
on the same models. The results show that the contents
of potassium in urine go down after taking the medicine
Lasix and the decrease is very sharp and reaches to
minimum value in 4 h. The effect of the medicine dies
away and the potassium approaches to a normal value,
within 4 h (Figure 3). The results obtained for double
dosage intake are shown in Figure 4, showing that
potassium decreases and reaches to minimum value and
then to the normal as in the case of single dosage. The
difference between single and double dosage is that the
minimum approaches fast that is, within 3 h for double
dosage and the decrease is also a bit higher/greater as
compared to single dosage. Though the effect of the
medicine remains even up to 12 h but the effect is very
short. In this case, we also observed that the potassium
excretion decreases with time and approaches to
minimum, as observed in case of Lasix (Figure 5). The
observations made are very much similar to that of single

1980 Afr. J. Pharm. Pharmacol.
K1 concentration (ppm)
Na concentration (ppm)
Time (h)
Figure 2. Effect on sodium excretion in urine by double dose of
different diuretics.
Time (h)
Figure 3. Effect on potassium excretion in urine by single dose of
different diuretics.
dose of Spiromide (Figure 6). The third medicine,
Aldactone was also given to four persons with two single
and two double dosages. It was also observed that the
medicines have not sharp diuretics effect (Figure 7), as
compared to the medicine discussed earlier. The fourth
and last medicine used was Conium which was a
homeopathic drug. It was noted the excreted value of
sodium firstly decreases as in the case of single dosage
but the rate of decrease with bit slower down (Figure 8).
However, the extent of decrease remained the same.
From these observations, we can say that this
medicine can be composed of two compounds one may
be having a short time and other long time effect. This
action of medicine may be useful for patient or can be
K2 concentration (ppm)
Time (h)
Figure 4. Effect on potassium excretion in urine by double dose
of different diuretics.
Concentration (ppm)
Time (h)
Figure 5. Lasix single and double dose effects on sodium and
potassium excretion.
Concentration (ppm)
Time (h)
Figure 6. Spiromide single and double dose effects on
sodium and potassium excretion.

Concentration (ppm)
Concentration (ppm)
Time (h)
Time (h)
Figure 7. Aldactone single and double dose effects on sodium and
potassium excretion.
Concentration (ppm)
Time (h)
Figure 8. Effect of Conium on sodium and potassium excretion after
single and double dose of administration.
dangerous for others depending upon the disease and
the requirements of the model patient.
Conclusions
If we compare the observation made as analysis of urine
and the report made by the model, the following
statement can be made about these drugs:
The first two medicines (Lasix and Spiromide) investigated
disturb the body system very much either by
increasing or decreasing the excreted amount of sodium
Ullah et al. 1981
or potassium up to a large extent. However, they were
very good as a diuretic that is the amount of water
excreted from the body as urine also increases up to a
large extent which is considered to be the primary task of
such medicine. Aldoctone, used for study purpose
showed very little change in sodium or potassium with
time from equilibrium value. Therefore, we can say that it
can be very safe drugs but on the other hand the urine
excretion was not very high after administration of drugs
as noted by the models. Therefore, we can say that
though this medicine is a good at one hand but does not
perform its own primary function for which it is used.
ACKNOWLEDGMENTS
The authors are thankful to the Deanship of Scientific
Research, King Saud University Riyadh for funding the
work through the research Group project No RGP-VPP-
076.
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185.

African Journal of Pharmacy and Pharmacology Vol. 6(26), pp. 1982-1988, 15 July, 2012
Available online at http://www.academicjournals.org/AJPP
DOI: 10.5897/AJPP12.098
ISSN 1996-0816 ©2012 Academic Journals
Full Length Research Paper
Antimicrobial and anticoagulant activities of Citrullus
colocynthis Schrad. leaves from Tunisia (Medenine)
Belsem Marzouk 1 *, Ehsen Haloui 2 , Najoua Akremi 3 , Mahjoub Aouni 1 , Zohra Marzouk 2# and
Nadia Fenina 2#
1 Laboratoire des maladies transmissibles et substances biologiquement actives, Faculté de Pharmacie, Monastir,
Rue Avicenne 5000, Monastir-Tunisia.
2 Unité de pharmaco-économie et développement des médicaments, Laboratoire de biologie végétale et laboratoire de
pharmacologie, Faculté de Pharmacie, Monastir-Tunisia.
3 Unité URSAM, Laboratoire de Pharmacologie, Faculté de Pharmacie, Monastir-Tunisia.
Accepted 4 June, 2012
Traditional medicine is a potential source of new drugs. Citrullus colocynthis is a Tunisian plant used in
folk medicine against dermatological, gynaecological and pulmonary infections; and against
inflammations and cardiovascular and immune-related diseases. The present study was conducted to
evaluate the in vitro antimicrobial and anticoagulant properties of leaf extracts from an endemic plant,
C. colocynthis Schrad. The extracts were screened for antimicrobial activity against Gram-negative and
Gram-positive bacteria and against four Candida spp. using the microdilution method. The
anticoagulant property was evaluated using the prothrombin time (PT) and partial thromboplastin time
tests. The antimicrobial test results showed that polar extracts using ethyl acetate, acetone and
methanol from this species strongly inhibited the growth of microorganisms while petroleum ether and
chloroform extracts had moderate antibacterial and anticandidal activities. Investigation of the
coagulant activity of different extracts showed that C. colocynthis leaves reduced or prolonged the PT
and activated or inhibited partial thromboplastin time tested on plasma according to plant extracts and
dilution degrees. This indicates that this species possesses both procoagulant and anticoagulant
activities. From these results, we confirmed the traditional use of C. colocynthis.
Key words: Traditional medicine, Citrullus colocynthis Schrad., leaves, antimicrobial, anticoagulant.
INTRODUCTION
Increasingly adverse drug reactions to the synthetic
antibiotics and the increasing resistance of some
pathogens to synthetic antibiotics, has been another
argument against the use of these chemicals as
therapeutics (Agnihotri and Vaidya, 1996; Friedman et al.,
2002; Dabai et al., 2012).
The biological value of plants has been widely studied
and is demanded by consumers, especially for protection
*Corresponding author. E-mail: belsemmarzouk@yahoo.fr. Tel:
216 73 450 389.
# These authors contributed equally.
against cardiovascular disorder, cancer and other
diseases, as well as for general health benefits
(Dapkevicius et al., 1998; Campbell and Reece, 2007;
Gboko et al., 2012).
Commercial drugs/chemicals used haphazardly in the
treatment of many diseases inevitably led multiple
drug/chemical resistance in human microorganisms
peroxidaion (Service, 1995; Loper et al., 1999). To the
best of our knowledge, antimicrobial and anticoagulant
properties of Citrullus colocynthis Schrad. leaf extracts
have not been reported before. C. colocynthis Schrad.
(Cucurbitaceae) growing in arid areas is endemic in the
south of Tunisia (Pottier-Alapetite, 1981). This medicinal
plant is widely used in Tunisian folk medicine for treating
many diseases such as rheumatism, hypertension and

various contagious diseases such as dermatological
problems and gynaecological or pulmonary infections
(Boukef, 1986; Le Flock, 1983).
Some studies have demonstrated the medicinal effect
of C. colocynthis Schrad. as anti-tumour (Tannin-Spitz et
al., 2007), immunostimulant (Bendjeddou et al., 2003),
anti-microbial
(Marzouk et al., 2009, 2010a) and
antioxidant (Marzouk et al., 2010b) and against hepatic
diseases (Gebhardt, 2003), hyperglycaemia (Al-Gaithi et
al., 2004) and hair loss (Roy et al., 2007).
The objectives of this study were to investigate the
antimicrobial and anticoagulant activities of extracts from
C. colocynthis leaves. The antimicrobial activity was
determined by using the microdilution method.
Prothrombin time (PT) and activated partial
thromboplastin time (APTT) tests on plasma are used to
determinate the coagulant-anticoagulant effects.
MATERIALS AND METHODS
Plant material
C. colocynthis Schrad. leaves were collected in August, 2007 from
nearby Mednine village, El-Araidha region, Sidi Makhlouf
municipality, Tunisia. The taxonomic identification of the plant
material was confirmed by a plant taxonomist, Marzouk, Z., in the
Biological Laboratory of the Faculty of Pharmacy of Monastir-
Tunisia- according to the flora of Tunisia 8 . A voucher specimen
(C.C-01.01) has been deposited in this laboratory.
Preparation of extracts
Aqueous extract
One hundred grams (100 g) of fresh leaves were ground with a
mixer and added to 500 ml of distilled water. The mixture was
allowed to reflux for 30 min, after which the solution was allowed to
cool (4 h at 3°C). The mixture was then filtered on filter paper
(Whatman No.1) under the vacuum of a water pump. The obtained
filtrate was lyophilized, yielding the lyophilized aqueous extract.
Soxhlet extractions
Collected plant materials were dried; the leaves were separated
from the stems, and ground in a grinder with a 2 mm in diameter
mesh. Different solvents, petroleum ether, chloroform, ethyl acetate,
acetone and methanol in ascending polarity, were used for Soxhlet
extraction to fractionate the soluble compounds from the grape
pomace. The extraction was performed with dried powder placed
inside a thimble made by thick filter paper, loaded into the main
chamber of the Soxhlet extractor, which consisted of an extracting
tube, a glass balloon and a condenser. The total extracting time
was 6 h for each solvent continuously refluxing over the sample
(grape pomace). The resulting extracts were evaporated at reduced
pressure to obtain the crude extracts.
Preliminary phytochemical screening
Aqueous and organic extracts were screened for the presence of
key families of phytochemicals (Sakar and Tanker, 1991; Trease
and Evans, 1984; Trim and Hill, 1952) using the following reagents
Marzouk et al. 1983
and chemicals: alkaloids with Dragendorff’s reagent confirmed with
Bouchardat’s (I2/MgI2) and with Meyer’s reagents (KI/MgCl2),
coumarins with diluted NaOH-UV test, flavonoids with metallic
magnesium and hydrochloric acid (HCl), anthraquinones with
Borntrager’s reagent, cardiac glycosides with Kedde’s reagent (and
confirmed with Baljet’s reagent), iridoids with diluted HCl,
saponosids for their ability to produce suds, steroids with acetic
anhydride and concentrated sulphuric acid (Liebermann reaction),
tannins in general with ferric chloride (confirmed with concentrated
HCl, Bath-Smith reaction) and gallic tannins specifically with Stiasny
reagent.
Antibacterial and antifungal activities
Organisms
The aqueous and organic extracts from C. colocynthis leaves were
individually tested against a panel of microorganisms including a
total of 8 microbial cultures belonging to 4 bacteria, than 4 Candida
species. The 4 reference strains were chosen for antibacterial
investigation: cocci gram-positive represented by Enterococcus
feacalis ATCC 29212 and Staphylococcus aureus ATCC 25923 and
bacilli gram-negative represented by Escherichia coli ATCC 25922
and Pseudomonas aeruginosa ATCC 27853. In order to determine
the antifungal effect of these extracts, a range of pathogenic
reference Candida (Candida albicans ATCC 90028, Candida
glabrata ATCC 90030, Candida kreusei ATCC 6258 and Candida
parapsilosis ATCC 22019) was tested.
Minimal inhibition concentration (MIC) and minimal
bactericidal concentration/ minimal fungicidal concentration
(MBC/MFC) determinations
The MIC was defined as the lowest concentration that prevents
visible growth bacteria. All extracts were dissolved in dimetyle
sulfoxyde (DMSO) at 10%. We have applied the dilution method
described by Berche et al. (1991). A microdilution technique using
96-well microplates was used to obtain the MIC values of extracts
against the tested strains. The concentration for extracts tested was
ranged from 6.343 to 3250 µg/ml. The lowest concentration of each
extract that inhibited the bacterial growth after incubation, at 37°C
between 18 and 24 h, was taken as the MIC.
The MBC and MFC were determined as a concentration where
99.9% or more of the initial inoculum are killed. They were
evaluated by subculture in blood agar at 37°C between 18 and 24
h. The levofloxacin was used as antibacterial positive control and
Amphotericin B for the anticandidal one.
Assay for prothrombin time (PT) and activated partial
thromboplastin time (APTT)
The method described by Brown (1988) was used for the
determination of the PT. Plasma was obtained by centrifuging
citrated blood for 15 min at 1500 � g. Thromboplastin-calcium
reagent (Sigma Diagnostics, St. Louis, MO) was reconstituted with
distilled water according to the manufacturer’s instructions. It was
then prewarmed by placing it in a water bath at 37°C for at least
10 min before commencement of the test. 100 μl of plasma was
placed in a test tube and incubated in the water bath for 180 s. For
the controls, 100 μl of serum, followed by 200 μl of the prewarmed
thromboplastin-calcium reagent was rapidly pipetted into the
plasma while simultaneously starting a timer. The test tube was
then gently tilted back and forth, until a clot formed, at which time
the timer was stopped and the clotting time recorded. For the tests,
100 μl of the prewarmed sample solution was mixed with the

1984 Afr. J. Pharm. Pharmacol.
Table 1. Yields (%) of C. colocynthis Schrad. leaf extracts.
Chemical Aque Petroleum Chlorofo Ethyl Acet Metha
group
ous ether
rm acetate one nol
Yield (%) 11.047 2.046 8.874 1.342 7.376 13.304
Table 2. Preliminary phytochemical screening of C. colocynthis Schrad. leaf extracts.
Chemical group
Aque
ous
Petroleum
ether
Chlorofo
rm
Ethyl
acetate
Acet
one
Alkaloïds + - - + + +
Coumarins + - - + - -
Flavonoïds - - - - - -
Anthracenic
heterosids
- - - - - -
Cardiotonic
heterosids
- - - - - -
Iridoïds + - - - - +
Saponosids - - - - - -
Steroïds + + - - - -
Gallic tannins + - + + - -
plasma, just before adding the thromboplastin-calcium reagent. All
experiments were carried out in at least, triplicates.
The method described by Brown (1988) suitably modified, was
used for the partial thromboplastin time tests. Alexin® (Sigma
Diagnostics), which is the partial thromboplastin with activator, and
calcium chloride (0.02 M) were prewarmed to 37°C separately in a
water bath. 50 μl of plasma was placed in a test tube. After
incubating for 180 s in the water bath, 50 μl of Alexin® was added,
and the contents were mixed rapidly. The mixture was then
incubated for another 180 s, after which 50 μl of serum, then 50 μl
of the prewarmed calcium chloride solution was added while
simultaneously starting a timer. The tube was then allowed to
remain in the water bath while gently tilting the test tube every 5 s.
At the end of 20 s, the test tube was removed from the water, wiped
clean with dry gauze, and gently tilted back and forth until a clot
was seen and the time recorded. For the test samples, 50 μl of the
material was added to the contents of the test tube just prior to the
addition of calcium chloride, and readings taken as before. For the
two tests, serum and heparin (183 IU/mg) were, respectively, used
as negative and positive controls.
RESULTS
Extraction yields and phytohemical screening
Results are shown in Tables 1 and 2. Lyophilised aquous
extract and methanol extract showed the better yields.
The phytochemical screening showed a significant
difference between the tested extracts. We note that the
present or the absence of all phytochemical groups is
related with the extract polarities and the chemical group
types.
Antimicrobial activity
The in vitro antimicrobial activity of C. colocynthis
Metha
nol
extracts against the microorganisms employed and their
activity potentials were qualitatively and quantitatively
assessed by MIC and MBC/MFC values. According to the
results shown in Tables 3 and 4, aqueous extract of C.
colocynthis had great potential of antimicrobial activities
against all bacteria and Candida species tested. Apolar to
polar fractions of the Soxhlet extracts were also found to
be effective against all microorganisms examined. MIC
and MBC values for bacterial strains, which were
sensitive to the aqueous extract and organic ones of C.
colocynthis, were in the range of 6.343 to 1625 μg/ml and
12.687 to 3250 μg/ml, respectively. The maximum was
obtained with the ethyl acetate extract against all bacteria
exept S. aureus. MIC and MFC values of the Candida
species sensitive to the tested extracts were < 6.343 to
1625 μg/ml and 6.343 to 3250 μg/ml, respectively. The
best results were observed with acetone and methanol
extracts against C. albicans and also with ethyl acetate
extract against C. Kreusei (CMI < 6.347 μg/ml and CMF =
6.347 μg/ml).
Based on these results, Soxhlet extracts have a
stronger activity and broader spectrum than the aquous
one. As emphasized elsewhere, Gram-negative bacteria
are more sensitive than Gram-positive ones (Marzouk et
al., 2010a). As well, basing on the cell wall differences of
bacteria, results show that Soxhlet extracts did possess
the same selective antimicrobial activity. By inhibiting the
growth of all human Candida tested, C. colocynthis
Schrad. exerted a broad anticandidal spectrum. The
antimicrobial nature of this studied plant organ is
apparently related to their contents, particularly alkaloids
and iridoids, and this finding is in agreement with a

Table 3. Antibacterial MIC (µg/ml) and MBC (µg /ml) of C. colocynthis Schrad. leaves.
Strain
E. coli
ATCC
25922
P.
aeruginosa
ATCC
27853
S. aureus
ATCC
25923
E. faecalis
ATCC
29212
Concentrati
on
Aque
ous
Petroleum
ether
Chlorofo
rm
Ethyl
acetate
MIC 1625 12.687 12.687 6.343
MBC 3250 25.375 25.375 12.687
MIC 812 12.687 25.375 6.343
MBC 1625 25.375 50.750 12.687
MIC 1625 25.375 25.375 25.375
MBC 3250 50.750 50.750 101.500
MIC 1625 25.375 25.375 6.343
MBC 3250 50.750 50.750 12.687
Marzouk et al. 1985
Acet
one
12.68
7
25.37
5
12.68
7
25.37
5
12.68
7
25.37
5
25.37
5
50.75
0
Metha
nol
12.687
25.375
12.687
25.375
12.687
25.375
25.375
25.375
MIC positive control, Levofloxacin (E. coli 0.61 µg/ml, P. aeruginosa 0.3 µg/ml, S. aureus 0.3 µg/ml, E. faecalis 1.22 µg/ml); E. coli,
Echerichia coli; P. aeruginosa, Pseudomonas aeruginosa; S. aureus, Staphylococcus aureus; E. faecalis, Enterococcus faecalis.
Table 4. Antifungal MIC (µg/ml) and MFC (µg/ml) of C. colocynthis Schrad. leaves.
Strain
C. albicans
ATCC
90028
C. glabrata
ATCC
90030
C. kreusei
ATCC 6258
C.
parapsilosis
ATCC
22019
Concentrati
on
Aque
ous
Petroleum
ether
Chlorofo
rm
Ethyl
acetate
MIC 1625 12.687 12.687 6.347
Acet
one

1986 Afr. J. Pharm. Pharmacol.
Table 5. PT (s) and APTT(s) times of C. colocynthis Schrad. leaf extracts.
Extract Dilution PT(s) APTT(s)
0 31.0 ± 0.2 97.5 ± 10
½ 19.2 ± 0.4 54.1 ± 7.0
¼ 16.0 ± 1.0 49.2 ± 9.0
1/5 17.0 ± 1.0 41.7 ± 0.1
Aqueous
1/10 15.3 ± 0.2 46.8 ± 1.0
1/20 15.2 ± 0.3 45.5 ± 2.1
1/50 15.2 ± 0.2 37.5 ± 0.2
1/100 15.2 ± 0.2 33.8 ± 0.5
1/1000 15.0 ± 0.4 33.9 ± 1.0
Petroleum ether
Chloroform
Ethyl acetate
Acetone
Methanol
0 15.4 ± 0.4 38.5 ± 1.3
½ 15.6 ± 0.5 38.6 ± 1.1
1/4 15.0 ± 0.8 37.9 ± 0.8
1/5 14.9 ± 0.5 37.8 ± 0.9
1/10 14.5 ± 1.0 37.3 ± 0.8
1/20 13.8 ± 0.2 37.0 ± 0.5
1/50 13.5 ± 0.8 37.0 ± 0.5
1/100 13.5 ± 0.7 36.3 ± 0.7
1/1000 13.5 ± 0.8 35.9 ± 0.8
0 14.7 ± 0.2 43.6 ± 1.0
1/2 14.5 ± 0.2 43.6 ± 1.1
1/4 14.7 ± 0.3 43.3 ± 1.0
1/5 14.1 ± 0.5 39.0 ± 0.9
1/10 13.1 ± 0.8 39.2 ± 0.9
1/20 12.7 ± 0.4 38.0 ± 0.7
1/50 12.5 ± 0.5 38.0 ± 0.7
1/100 12.2 ± 0.3 34.8 ± 0.8
1/1000 12.2 ± 0.2 32.6 ± 0.5
0 15.1 ± 1.0 41.9 ± 1.9
1/2 15.0 ± 1.0 40.1 ± 1.4
1/4 14.2 ± 0.6 40.5 ± 1.4
1/5 13.1 ± 0.7 38.3 ± 0.9
1/10 12.7 ± 0.6 37.9 ± 0.9
1/20 12.2 ± 0.5 37.0 ± 0.5
1/50 12.2 ± 0.4 36.8 ± 0.6
1/100 12.0 ± 0.2 36.7 ± 0.6
1/1000 12.0 ± 0.3 35.6 ± 0.5
0 20.3 ± 1.2 47.2 ± 2.1
1/2 18.0 ± 1.0 42.1 ± 1.9
1/4 16.2 ± 1.1 40.7 ± 1.3
1/5 16.0 ± 1.1 39.7 ± 1.2
1/10 15.6 ± 0.9 39.4 ± 1.3
1/20 15.5 ± 0.7 38.7 ± 0.8
1/50 15.5± 0.6 38.4 ± 0.7
1/100 15.1 ± 0.6 37.8 ± 0.5
1/1000 14.9 ± 0.5 33.7 ± 0.5
0 15.7 ± 0.8 43.1 ± 1.5
1/2 15.0 ± 0.7 40.5 ± 1.0
1/4 14.7 ± 0.7 40.1 ± 0.9

Table 5. Contd.
1/5 14.7 ± 0.6 39.1 ± 0.9
1/10 14.0 ± 0.6 39.6 ± 0.8
1/20 13.6 ± 0.5 38.6 ± 0.7
1/50 13.2 ± 0.5 38.1 ± 0.7
1/100 13.4 ± 0.3 36.8 ± 0.6
1/1000 13.4 ± 0.3 36.2 ± 0.5
Heparin (31.25 µg/ml) 160 ± 6.0 400 ± 10.0
Serum 13.5 ± 0.3 36.6 ± 0.2
Heparin (183 IU/mg), Positive control; Serum, negative control.
dependent manner. The soxhlet fractions of C.
colocynthis leaves generally had no significant effect on
the PTs and APTTs at low concentrations. On the other
hand, further dilutions of the aqueous extract strongly
inhibited the plasma coagulation. With the petroleum,
chloroform and ethyl acetate extracts, at high
concentrations, the plasma coagulated with PTs and
APTTs that were slightly higher than negative control
values; whereas at the most diluted solutions, we noted a
pro-coagulate effect. At high concentrations, acetone and
methanol soxhlet fractions were the most active and may
be respensable on the aqueous extract anticoagulant
effect.
DISCUSSION
C. colocynthis Schrad. leaf activity could be attributed to
contents of active components present in the extracts.
Moreover, it can be noteworthy to point out that aqueous
and organic extracts components are effective on
neutralization of microorganisms tested, especially which
are responsible for pulmonary infections and on PT and
APTT. Also, polar organic extracts exhibited stronger
antimicrobial activities than no polar ones, in accordance
with PT and APTT results. As a result, organic extracts
have excellent capacity in antimicrobial assay than
aquous extract, whereas on the PT and APTT tests,
organic extracts are less active; this efficacy difference
may be attribuated to the phenomena of synergism or
antagonism between components in the crude aquous
extract tested.
With all these wide spectrum of the antibacterial and
antifungal effects, C. colocynthis Schrad. leaves can be
considered as an effective antimicrobial agent treating
infectious diseases. Since, this plant demonstrated
activity against some prevalent bacteria and fungi in
dermatology, gynaecological and pulmonary infections,
namely polar organic extracts, the use of this plant as
antimicrobial agent is validated, scientifically supported
by the results obtained in this work.
Our data suggest that polar Soxhlet extracts from C.
colocynthis Schrad. leaves have anticoagulant properties.
The results obtained on the effect of the whole Soxhlet
Marzouk et al. 1987
extracts on the PT and APTT tests further support the
conclusion that this species contains both coagulant and
anticoagulant activities. The PT and APTT tests are used
for distinguishing between the effects of test agents on
the extrinsic and intrinsic pathways (Brown, 1988).
Substances that affect the PT are thought to act on the
extrinsic pathway factors: factors V, VII, X, prothrombin
and fibrinogen, while those that affect the APTT act on
the components of the intrinsic pathway, that is, all
coagulation factors except factors VII and XIII. The
results reported here show that the anticoagulant effect
examined by the PT test is portrayed by the APTT one.
Antimicrobial and anticoagulant properties of various
extracts from many plants are of great interest in both
academia and the food industry, since their possible use
as natural additives emerged from a growing tendency to
replace synthetic products by natural ones. In this
respect, studying with the endangered species may be of
great interest, since their bioactive properties could be
lost forever without being tapped. Owing to its strong
antimicrobial effect and moderate anticoagulant property,
the extracts from the herbal parts of C. colocynthis
Schrad. could be concluded as a natural source that can
be freely used in the pharmaceutical industry as a
medicinal herb, but, firstly, immediate and necessary
measurements should be taken for the protection of this
plant species which is known by its toxicity (leaves LD50 =
3903.2 mg/kg) (Marzouk et al., 2010b).
In conclusion, our study can be considered as the first
report on the in vitro antimicrobial and anticoagulant
properties of extracts prepared from C. colocynthis
Schrad. leaves. From now the use of this plant in
traditional medecine is validated by the results obtained
in this work. We hope that our results introduce a natural
source possessesing strong antimicrobial and moderat
anticoagulant coumpounds.
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