Journal of Ethnopharmacology, 29 (1990) 117 - 125 Elsevier Scientific Publishers Ireland Ltd. 117 EFFECT OF ALLIUM SAZ’IVUM ON EPIDIDYMAL SPERMATOZOA, ESTRADIOL-TREATED MICE AND GENERAL TOXICITY ABDULLAH M. AL-BEKAIRI, ARIF H. SHAH* and SHOEB QURESHI Experimental Animal Care Centre, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh-11451 lSaudi Arabial (Accepted October 24, 1989) Summary A water extract of A&urn sativum (garlic) was given to male and female mice in drinking water (100 mglkgldayl for three months. There was a signifi- cant increase in the weight of seminal vesicles and epididymides of male animals as compared to controls and the sperm count was significantly elevated. There was no increase in the body weight of the test animals, and weights of heart, liver and spleen were reduced as compared to controls. Haematological studies revealed an increase in WBC and a decrease in RBC levels of the test animals. Three-day intraperitonial treatment (500 mg/kg) with the A. sativum extract failed to exhibit any estrogenic or antiestrogenic activity. Introduction Allium sativum L. (Lilliaceae) is a very widely accepted plant serving both as a part of diet and as a medicine (Flares, 1951; Binding, 1975; Kirtikar and Basu, 1984). In the various traditional systems of medicine, it is commonly used as a tonic, aphrodisiac, emmenogogue, digestive, anthelmentic, heart tonic and treatment for spleen and liver diseases, wounds, inflammations, leucoderma, asthma, tuberculosis and tumors. Garlic juice is also believed to restore virility in men, maintain strength and stamina and counteract the symptoms of ageing (Binding, 1975; Kirtikar and Basu, 1984; Ageel et al., 19871. The antimicrobial, hypolipidemic, antihypertensive, antihepatotoxic and anthelmentic activities of A. sativum and its effect on platelet aggrega- Correspondence to: Dr. Arif H. Shah. *Present address: Central Drug and Food Analysis Labs., P.O. Box 59082, Riyadh-11525 (Saudi Arabia). 0378-8741/$03.50 01990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
Transcript
Journal of Ethnopharmacology, 29 (1990) 117 - 125
Elsevier Scientific Publishers Ireland Ltd. 117
EFFECT OF ALLIUM SAZ’IVUM ON EPIDIDYMAL SPERMATOZOA, ESTRADIOL-TREATED MICE AND GENERAL TOXICITY
ABDULLAH M. AL-BEKAIRI, ARIF H. SHAH* and SHOEB QURESHI
Experimental Animal Care Centre, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh-11451 lSaudi Arabial
(Accepted October 24, 1989)
Summary
A water extract of A&urn sativum (garlic) was given to male and female mice in drinking water (100 mglkgldayl for three months. There was a signifi- cant increase in the weight of seminal vesicles and epididymides of male animals as compared to controls and the sperm count was significantly elevated. There was no increase in the body weight of the test animals, and weights of heart, liver and spleen were reduced as compared to controls. Haematological studies revealed an increase in WBC and a decrease in RBC levels of the test animals. Three-day intraperitonial treatment (500 mg/kg) with the A. sativum extract failed to exhibit any estrogenic or antiestrogenic activity.
Introduction
Allium sativum L. (Lilliaceae) is a very widely accepted plant serving both as a part of diet and as a medicine (Flares, 1951; Binding, 1975; Kirtikar and Basu, 1984). In the various traditional systems of medicine, it is commonly used as a tonic, aphrodisiac, emmenogogue, digestive, anthelmentic, heart tonic and treatment for spleen and liver diseases, wounds, inflammations, leucoderma, asthma, tuberculosis and tumors. Garlic juice is also believed to restore virility in men, maintain strength and stamina and counteract the symptoms of ageing (Binding, 1975; Kirtikar and Basu, 1984; Ageel et al., 19871. The antimicrobial, hypolipidemic, antihypertensive, antihepatotoxic and anthelmentic activities of A. sativum and its effect on platelet aggrega-
Correspondence to: Dr. Arif H. Shah. *Present address: Central Drug and Food Analysis Labs., P.O. Box 59082, Riyadh-11525 (Saudi
Arabia).
0378-8741/$03.50 01990 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
118
tion have been extensively studied (Patricic et al., 1977; Bordia, 1981; Bordia et al., 1981; Chi, 1982; Chi et al., 1982; Din et al., 1982; Foushee et al., 1982; Qureshi et al., 1983a,b; Hikino et al., 19861. Some workers have also reported on the possible antitumor, mutagenic and hormone activation effects of garlic (Criss et al., 1982; Dixit and Joshi, 1982; Lutomski, 1983; Abraham and Kesa- van, 1984; Lau et al., 1986; Lutomaki, 1986).
In view of the widespread use of A. sativum and existing information on its medicinal properties including the sex-related claims and genotoxicity, the present study was designed to investigate its effects on epididymal sper- matozoa and on uterine weight in female mice. In the light of W.H.O. recom- mendations, general toxicity studies were also conducted (W.H.O. Scientific Group, 1967).
Materials and methods
Plant material Fresh A. sativum bulbs obtained from the local market in Riyadh (Saudi
Arabia1 were properly identified by the plant Taxonomy Unit of our College and cut into small pieces. About 25 ml of distilled water per 100 g of garlic was added and crushed in a mixing machine. The resultant slurry was squeezed and filtered througth a fine cloth and the filtrate quickly frozen (w/w yield = 28.7%). This product was stored in a freezer until time of use. As specified, the drug was administered in drinking water or by intraperitoneal injection. All doses in this paper are expressed in terms of the weight of this product.
Chronic treatment Home bred Swiss albino mice aged 6-7 weeks, weighing 20-25 g and fed
on Purina chow diet and water ad libitum were used for this study and main- tained under standard conditions of light, humidity and temperature. A total of 40 mice were randomly allotted to treated and control groups. To the 20 mice in the test group (10 male, 10 female), 100 mg/kg body weight of the frozen extract of A. sativum was added to the drinking water. This dose is l/5 of the pharmacologically active dose (Tanira et al., 1988) and was used for a period of 3 months (W.H.O. Scientific Group, 1967). The animals were observed for general symptoms of toxicity, effect on body weight, vital organ weight, external symptoms, mortality, visceral condition, reproductive organ weight and sperm count. The five male animals in the control and test groups were killed 24 h after the last exposure and their blood was analysed for WBC, RBC and haemoglobin using Contravas Digicell 3100 h (Zurich). Furthermore, the chronically treated animals were also analysed for sperma- togenic dysfunction using the sperm abnormality test, a reliable procedure to assess the germ cell mutagenicity and carcinogenicity (Wyrobek et al., 19831.
The caudae epididymides and vas deferens from the same animals were
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dissected out and put into centrifuge tubes containing 3 ml of Krebs Ringer bicarbonate buffer. The sperm solution was filtered through a 80-pm mesh to remove tissue fragments and 0.5 ml of 1% eosin Y was added. The solution was thoroughly mixed and slides made by placing one drop of the solution on a slide and spreading by three passes of another slide. Coded slides were examined for the following abnormalities of the sperm head: amorphous, flat head, rotated head, microcephali, megacephali and swollen achrosome (Wyro- bek and Bruce, 1975). The different parameters studied were subjected to statistical analysis by either the x2 test or Student’s t-test.
Evaluation of estrogenic activity To evaluate the estrogenic potential of A. sativum, the method described
by Wirth et al. (1981) was used. A total of 40 pre-pubertal female mice weighing 12-14 g were randomly assigned to the different control and test groups. The test group received a daily dose of 500 mg/kg of the extract (i.p.1 for three consecutive days. The animals in the positive control group were treated (i.p.1 with estradiol solubilized in corn oil at a daily dose of 50 rg/kg, and those in the negative control received only the corn oil vehicle (15 ml/ kg). Twenty-four hours following the test dose, the animals were killed by cervical dislocation and their uteri were weighed. The individual values of the uteri were converted to weight per 100 g body weight.
Results and discussion
Chronic effects The results of the present study are presented in Tables l-6. The ani-
mals were observed weekly throughout the chronic treatment and were found to be grossly normal. During this period there was a significant increase P < 0.05) in the average body weight of control animals, but there was no significant increase in body weight of the A. sativum-treated animals. This constancy in body weight of the test group animals may be due to the
TABLE 1
QUANTITATIVE DATA ON THE MORTALITY IN MICE INDUCED BY 3-MONTH TREATMENT WITH ALLIUM SATIVUM EXTRACT (100 mg/kg/day) IN DRINKING WATER
Ten male and ten female mice were used in each group.
Treatment Mortality
0 - 30 days
Male Female
31-60 days
Male Female
61- 90 days
Male Female
Lethality (‘70)
Male Female
Control 1 0 0 1 0 0 10 10
A. sativum 1 1 1 2 0 0 20 30
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TABLE 2
QUANTITATIVE DATA ON MEAN BODY WEIGHT (2 S.E.M.1 OF MICE AFTER 3-MONTH TREATMENT WITH A. SATIVUM EXTRACT (100 mg/kg/day) IN DRINKING WATER
Average weight was calculated on the number of surviving animals (see Table 11.
Treatment
Control
A. sat&m
Pre-treatment Post-treatment --.__
Male Female Male Female
30.4 f 1.50 22.9 + 1.80 35.0 2 l&o* 28.5 r l.lO*
32.4 & 1.60 25.6 + 0.96 32.5 i 0.96 26.7 + I.00
Significant relative to pretreatment values: *P < 0.05.
TABLE 3
QUANTITATIVE DATA ON ORGAN WEIGHTS (per 100 g body weight) OF MALE AND FEMALE MICE AFTER 3-MONTH TREATMENT WITH A. SATZVUM EXTRACT (100 mg/ kg/day) IN DRINKING WATER
Tabular values represent the mean ‘-e S.E.M. of 5 randomly selected surviving animals.
Treatment Mean weight f S.E.M. (gl
Heart Lungs Liver Kidney Spleen
Control 0.56 -c 0.01 0.85 r 0.09 7.48 2 0.15 1.63 & 0.05 0.81 -+ 0.10
A. sativum 0.50 + O.Ol** 1.30 ZJZ 0.20 5.99 2 0.60* 1.50 f 0.03 0.50 r?l 0.06*
Significance relative to the control group: *P < 0.05, **P < 0.01.
TABLE 4
HAEMATOLOGICAL CHANGES OBSERVED IN MALE AND FEMALE MICE AFTER 3- MONTH TREATMENT WITH A. SATIVUM EXTRACT (100 mgikgldayl IN DRINKING WATER
Tabular values represent the mean -t S.E.M. of 5 randomly selected surviving animals.
Treatment WBC x lo3 RBC x 10” Haemoglobin W/ml) w/ml) (O/o)
Control 4.22 & 0.20 8.12 ? 0.20 12.04 + 0.17
A. sativum 5.20 i 0.30” 7.35 ir 0.20* 11.76 ir 0.31
Significant relative to the control group: *P < 0.05.
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122
TABLE 6
ESTROGENIC AND ANTI-ESTROGENI~ ACTIVITY OF A. SATIVUM EXTRACT (500 mgkg i.p. for 3 days) AND/OR ESTRADIOL (50 pg/kg i.p. for 3 days) IN FEMALE MICE
Treatment N Mean 2 S.E.M. weight
Vehicle control 10 Estradiol 10 A. sativum 10 A. sat&urn + estradiol 9
Significance relative to vehicle control: *P < 0.001.
cholesterol and lipid biosynthesis inhibiting properties of A. ~a~~~~~ (Chi, 1982; Chi et al., 1982; Din et al., 1982; Adetumbi et al., 1986).
During the chronic treatment, one male animal initially developed leg inflammation in the test group but recovered during the next week. The mortality rate as compared to the control was insignificant and the animals appeared healthy in both control and test groups. However, the haematologi- cal studies revealed a significant decrease in RBC level and rise in WBC level of the chronically treated animals as compared to the controls. A. sativum is known to contain alliin, allicin, ajoen, etherial oils, enzymes, thio- glyeosides, nucleosides, &sitosterin, aesin, garlicin, amino acids, fatty acids, eaffeic acid, flavonoids, sterols, carbohydrates, vitamin C, vitamin B-complex, choline, sinistrin and different trace elements including Se, Mg, Fe and I (Flares, 1951; Plant et al., 1962; Binding, 1975; Starke and Herrman, 19’76; Kamanna and Chandrasekhara, 1980; Stoianova-Ivonova et al., 1980; Lutom- ski, 1986) which are held responsible for antibiotic and other effects of garlic. The haematological changes observed during our present study may be attributed to the glucoseS-phosphate dehydrogenase decreasing properties of A. sativum (Chanarin et al., 1980; Bolton et al., 1982; Chi, 1982).
The general visceral condition of the animals in the test group was com- parable to the control group. However, there was a significant decrease in heart, liver and spleen weights of rl. sativum-treated animals. Garlic is reputed to inhibit cholesterol and lipid synthesis (Din et al., 1982; Qureshi et al., 1983a,b; Adetumbi et al., 19863, affect platelet aggregation, reduce plasma and liver cholesterol, sugar and triglyeeroids levels and decrease hepatic glucose-(i-phosphate dehyrogenase and malic enzyme activities in addition to reducing liver weight and adipose tissues (Bordia et al., 1981; Rao et al., 1981; Chi, 1982; Chi et al., 1982; Foushee et al., 1982; Zaman, 1982; Qureshi et al., 198313; Serivastava, 1984; Jamaluddin et al., 1988). The reduction in the weights of some vital organs observed during our present study are in agreement with the previous findings in other experimental models.
The average weight of seminal vesicles and epididymides was found to be significantly increased in the A. sat&urn-treated males as compared to the
123
controls. The weight increase in testes was, however, statistically not signifi- cant. In the garlic-treated group, a highly significant increase (P < 0.01) in the sperm count was observed. These effects tend to support the androgenic activity of the extract (Qureshi et al., 19891 and the claimed aphrodisiac activity of garlic. In earlier reports, A. sativum has been reported to effect hormonal levels and increase the excretion of neutral and acidic steroids (Chi et al., 1982; Lutomski, 19861 which may play an important role in the reputed aphrodisiac effect.
A. sativum has been reported to be mutagenic in bacteria (Kada et al., 1978; Takemura and Shimizu, 1978) and Drosophila (Abraham and Kesavan, 19851. It is interesting to note that in our present study, sperm abnormalities after chronic treatment with garlic extract were lower thanin control ani- mals. However, this effect was statistically not significant. The presence of caffeic acid, selenium and vitamin C may contribute to the observed antioxi- dant property of A. sativum as these compounds are known to possess this property (Poirier and Milner, 1983; Raj et al., 1983; McCarty, 1984; Lutomski, 1986; McCarty, 19861. Our results confirm the report of Abraham and Kesa- van (19841 where orally administered garlic was reported not to enhance the frequency of micronucleated polychromatic erthyrocytes indicating it to be devoid of genotoxicity.
Es trogenic studies To assess the estrogenic potential of A. sativum in female mice, our study
was conducted on pre-pubertal female mice. The results (Table 61 indicate a very highly significant increase (P < 0.001) in the mean uterine weight of the estradiol-treated mice as compared to the controls confirming the strong estrogenic activity of estradiol. In A. sativum-treated animals, no change in the mean uterine weight was observed and body weights did not differ from the controls. This suggests that garlic possesses no estrogenic effect in mice at the doses used.
To investigate the possible anti-estrogenic potential of A. sativum, a group of animals was treated with estradiol and extract (i.p.1 simultaneously. No decrease in the mean uterine weight was noticed, and the effect was the same as observed in the estradiol-treated group indicating no anti-estrogenic effect for garlic (P > 0.051.
The results of our present study suggest that there may be some basis for the claimed aphrodisiac potentials of A. sativum and establish that this her- bal drug possesses no estrogenic or anti-estrogenic activity at the doses administered.
Acknowledgement
We are thankful to Professor Dr. J. Lutomski, Institute of Medicinal Plants, Poznan, Poland for his kind remarks and references.
124
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