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| ORIGINAL ARTICLE |
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| Year : 2015 | Volume
: 35
| Issue : 2 | Page : 90-95 |
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Evaluation of the estrogenic activity of Indian medicinal plants in immature rats
Sandhya K Kamat1, Prajakt J Barde2, Sanket B Raut1
1 Department of Pharmacology and Therapeutics, King Edward Memorial Hospital and Seth Gordhandas Sunderdas Medical College, Mumbai, Maharashtra, India
2 Department of Clinical Research, Rhizen Pharmaceutical SA, Switzerland
| Date of Web Publication | 14-Dec-2015 |
Correspondence Address:
Sandhya K Kamat
Department of Pharmacology and Therapeutics, King Edward Memorial Hospital and Seth Gordhandas Sunderdas Medical College, Parel, Mumbai - 400 012, Maharashtra
India
 Source of Support: None, Conflict of Interest: None  | Check |
DOI: 10.4103/0257-7941.171669
Introduction: The objective of the study was to evaluate the estrogenic activity of four Indian medicinal plants Saraca indica (Si), Symplocos racemosa (Sr), Cyperus rotundus (Cr), Terminalia arjuna (Ta), a marketed preparation of Si (Aśokārṣṭa) and a combination of Si + Sr using an experimental model of estrogenicity.
Materials and Methods: After approval of the institutional animal ethics committee, 22 day old female rats (n = 54) were randomly allocated to 9 groups – Group 1 and Group 2: Vehicle controls, Group 3: Ethinyl estradiol, Group 4: Si (270 mg/kg), Group 5: Sr (270 mg/kg), Group 6: Cr (540 mg/kg), Group 7: Ta (270 mg/kg), Group 8: Ashokarishta (4 ml/kg), Group 9: Si + Sr (135 mg/kg). Variables studied were: Body weight, uterine weight, relative uterine weight, presence of vaginal opening, histomorphology of the uterus and total uterine glycogen content. Parametric data were analyzed using one-way ANOVA and the categorical data were analyzed using Chi-square test.
Results: All animals in the ethinyl estradiol group showed a significant change in all the variables. None of the individual test drugs, neither the marketed preparation produced change in any of the variables. The plant drug combination also did not produce a change in any of the variables studied except in histomorphology wherein it caused a slight increase in the height of the luminal epithelium of the uterus (P < 0.05 vs. Group 1).
Conclusion: The plant drugs Si, Sr, Cr, Ta and Aśokāriṣṭa did not demonstrate estrogenic activity in the immature rat model. The plant drug combination Si + Sr showed questionable estrogenic activity which needs to be evaluated in further studies. Keywords: Cyperus rotundus, immature rat biassay, phytoestrogen, Saraca indica, Symplocos racemosa, Terminalia arjuna
How to cite this article:
Kamat SK, Barde PJ, Raut SB. Evaluation of the estrogenic activity of Indian medicinal plants in immature rats. Ancient Sci Life 2015;35:90-5 |
| Introduction | |  |
Ayurvedic works recommend several plants for treatment of gynecological disorders. Saraca indica (Si), Symplocos racemosa (Sr), Cyperus rotundus (Cr), Terminalia arjuna (Ta) are few noteworthy examples. Si has been used in genital conditions such as laxity of uterus, dysmenorrhea and menorrhagia.[1],[2]Sr has been traditionally usedin menorrhagia, leucorrhoea, inflammation of uterus, dysmenorrhea and as a uterine relaxant.[3],[4],[5]Ta is used in menorrhagia and leucorrhoea.[5]Cr is said to promote uterine muscle contraction. The Romans also used it as emmenagogue in uterine complaints.[6] In fact Cr is a constituent of many polyherbal formulations that are used to treat uterine disorders.[7] Many clinical studies have evaluated the efficacy of Si, Sr and Cr in subjects having uterine disorders such as non-specific leucorrhoea, oligomenorrhoea,[8],[9] dysmenorrhea and premenstrual symptoms.[10] In addition, these plants have been evaluated in subjects with menstrual irregularity [11] and dysfunctional uterine bleeding.[12] Subjects treated with these plants, showed improvement in clinical symptoms. In a study by Ahmed et al., Ta was shown to increase bone mineral density in post menopausal women with osteoporosis.[13]
The present study was designed to investigate whether the claims put forth in Ayurvedic works and effects of these plants observed in clinical trials were due to the presence of estrogenic activity. Extensive literature search revealed that only a few studies had been carried out to explore the mechanism of action of these plants and the results were conflicting. In a study conducted by Mitra et al., a polyherbal formulation containing Si and Sr was shown to possess estrogenic activity.[14] In another study Sr was found to significantly increase the serum FSH and LH levels and enhance folliculogenesis in immature female Sprague-Dawley rats.[15] In view of this contradictory evidence, the present study was undertaken to evaluate the estrogenic activity of Si, Sr, Cr, Ta and Aśokāriṣṭa (AA), a marketed preparation of Si using the immature rat bioassay.
The immature rat bioassay was chosen as it is currently the most well established, short term assay and is universally used as a primary assay for estrogenic activity.[16] It is widely used in toxicological screening and is validated by the Organization for Economical Co-operation and Development (OECD).[17]
Ayurveda usually recommends the use of multiple plants in combination for the treatment of any particular condition. A random survey of multi-ingredient Ayurvedic formulations marketed for the treatment of gynaecological disorders revealed that Si and Sr were commonly included in a majority of these formulations. Hence it was also decided to evaluate the estrogenic activity of the combination of Si and Sr in the above-mentioned model.
| Materials and Methods | | |
Study drugs
The plant drugs selected for evaluation were the barks of Si, Sr and Ta and roots of Cr. The plants parts were procured from M/s Indian Herbs Limited, Saharanpur, India, authenticated and processed to prepare standardized dry aqueous or aqueous alcoholic extracts. All the aqueous extracts were prepared using the following procedure: Individual plant parts were dried and pulverized, water was added to the powder in a ratio of 1:4 and the mixture was boiled for 2 h. The extract obtained was bought to room temperature and then gravity filtered through a #100 nylon mesh. The procedure was repeated thrice and the three filtrates were collected and spray dried for further use. The extracts were stored at room temperature in a dessicator containing sodium bicarbonate to avoid moisture. The extractive value of the plants was Si - 20%, Sr - 16.66%, Cr - 10% and Ta - 20%. The doses of the extracts were extrapolated from the highest dose advocated for humans in therapeutic practice.[4],[18],[19],[20]
Aśokāriṣṭa (marketed formulation) was purchased from an Ayurvedic pharmacy (Batch Number BV0002). The test drugs were administered as a suspension in 0.5% carboxymethyl cellulose (CMC). 17 α ethinyl estradiol (Sigma Aldrich, USA) was administered by dissolving it in a minimal amount of 95% ethanol and diluted to the final working concentration in olive oil.[17]
Animals
The study was initiated after obtaining approval of the Institutional Animal Ethics Committee. The guidelines recommended by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India were followed during the entire study. Pregnant Wistar rats were separated and housed individually under standard laboratory conditions in polypropylene cages with paddy husk as bedding. All animals had free access to UV filtered water and food pellets (Chakan Oil Mills Ltd, Maharashtra). The temperature was maintained at 22 ± 3°C with humidity of 50-70% and 12 h light/dark cycles.
The pregnant rats were observed daily for the day of delivery and this day was marked as the 1st post natal day (PND).[21] The initial sex determination was done by observing the litters on the 14th PND for the presence of areolae with or without nipple buds [22],[23] and was confirmed by measuring anogenital distance on 22nd PND.[24],[25]
Animals showing the presence of areolae and having a smaller anogenital distance were marked as females.
Study procedure
Twenty two day old immature female Wistar rats (n = 54) weighing between 25-35 g were randomly allocated to 9 groups of 6 rats each as follows: Group 1-0.5% CMC (6 ml/kg/day), Group 2 – Olive oil (OO, 0.6 ml/kg/day),
Group 3-17 α ethinyl estradiol (EE, 3 μg/kg/day), Group 4 - Si (270 mg/kg/day), Group 5 - Sr (270 mg/kg/day), Group 6 - Cr (540 mg/kg/day), Group 7 - Ta (270 mg/kg/day), Group 8 - AA (4 ml/kg/day) and Group 9 - Si + Sr (135 mg/kg + 135 mg/kg). All compounds were administered by oral gavage for a period of 3 days. Immature rat bioassay was used to test for the estrogenic activity of test drugs. On the 4th day of experiment (25th PND), the body weights of the immature rats were noted and they were examined for the presence of vaginal opening. Following this, the rats were sacrificed, their uteri removed and the uterine wet weight was recorded. The relative uterine wet weight was calculated (shown below) to ascertain that the change in uterine weight was not influenced by small changes in body weight. The relative uterine wet weight also denotes the potency of the estrogenic compound.[26],[27]
Histomorphology of the uterus was carried out to assess height of luminal epithelium and relative areas (%) covered by the luminal epithelium, glandular epithelium, lamina propria and muscularis mucosa. Uterine glycogen was also estimated.[28]
Statistical analysis
Student's paired t-test was used to compare basal and final values of body weight. One-way ANOVA was used to compare body weight, uterine and relative uterine weight, total uterine glycogen and uterine histomorphology between the different groups. Post-hoc analysis was performed using Tukey's test. Chi-square test was used to compare the vaginal openings between the different groups. A P value of <0.05 was considered as significant and all results were expressed as Mean ± SD.
| Results | | |
The mean basal body weight of the animals was comparable in all the eight groups [Table 1]. When the body weights of the different groups were compared on the 25th PND no significant difference was observed between the groups. Wet uterine weight and relative uterine weight of Group 3 (EE) showed a significant increase (P < 0.001) as compared to Group 2 (OO) [Table 1]. None of the groups receiving the test drugs i.e. Group 4 (Si), Group 5 (Sr), Group 6 (Cr), Group 7 (Ta), Group 8 (AA) and Group 9 (Si + Sr) showed a significant increase in the uterine weights and were comparable to Group 1 (0.5% CMC) [Table 1]. | Table 1: Effects of plant drugs on body weight, uterine weight and uterine glycogen
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Only animals in Group 3 (EE) showed vaginal opening on 25th PND [Table 1]. Similarly, only animals in the EE group showed a significant increase in the mean total uterine glycogen content (P < 0.001) as compared to Group 2. None of the animals in the other groups showed presence of vaginal opening and the mean total uterine glycogen content of animals in all other groups was comparable to Group 1 [Table 1]. Again, only group 3 showed a significant increase in the height of luminal epithelium; relative areas occupied by luminal epithelium and glandular epithelium as compared to Group 2 (OO) (P < 0.001) however the relative area occupied by lamina propria and muscularis mucosa was comparable to Group 2 [Table 2]. None of the animals in Groups 4, 5, 6, 7 and 8 showed a significant increase in the height of luminal epithelium and relative areas occupied by luminal epithelium, glandular epithelium, lamina propria and muscularis mucosa as compared to Group 1 [Table 2]. Only Group 9 (Si + Sr) showed a significant increase (P < 0.05) in the mean relative area occupied by luminal epithelium as compared to Group 1 but the mean relative areas occupied by glandular epithelium, lamina propria and muscularis mucosa of Group 9 were comparable to Group 1 [Table 2].
To summarize, only the positive control EE was successful in causing vaginal opening, increasing the uterine wet weight, the relative uterine weight, height of the luminal and glandular epithelium and the total uterine glycogen. None of the test drugs caused any change in any of the variables and were comparable to control. The effects of test drugs were significantly less than that of EE. The combination of Saraca indica and Symplocos racemosa alsodid not affect any of the variables except forcausing an increasein mean relative area occupied by luminal epithelium.
| Discussion | | |
In the present study, the mean uterine wet weight of group 2 receiving olive oil was comparable to Group 1 receiving 0.5% CMC indicating that olive oil is devoid of any effect on the uterus. This group was studied because olive oil contains minute quantity of weak phytoestrogens,[29] which may act as a confounding factor and vitiate results when used as vehicle for estrogenic drugs. The mean relative uterine weight (which is the ratio of uterine weight to body weight) was also calculated to show that the change in uterine weight was not influenced by small changes in body weight but by the estrogen itself. Administration of EE in doses of 3 μg/kg/day for 3 days to rats in Group 3 produced an uterotrophic response and increased the mean uterine weight and the mean relative uterine weight as compared to Group 2 receiving olive oil.
The mean relative uterine weight is also an index of the potency of estrogenic compounds. The formulations showing positive results could be compared with each other and the positive control with respect to their estrogenic activity. None of the test drugs viz Groups 4, 5, 6, 7 and 8 produced an uterotrophic response or increased the mean relative uterine weights.
Increases in wet uterine weight that was observed in animals treated with 17 α ethinyl estradiol is due to interaction of the EE with high affinity receptors in uterine tissue [30] leading to imbibing of water in the uterine tissue and the uterine lumen. Hypertrophy and hyperplasia of the uterine tissue also contribute to the increase in weight.[31],[32] As expected, treatment with EE also induced vaginal opening and advanced the event by 7-8 days.[33] Further, the uterotrophic effects of EE also correlated well with the histomorphology where EE showed an increase in the height of the luminal as well as glandular epithelium as compared to Group 2 receiving olive oil (P < 0.001). Administration of EE also caused an increase in total uterine glycogen content in immature rats as compared to the olive oil group (P < 0.001). None of the test drugs when used alone showed a change in any of the above parameters. This was in contrast to findings observed by Mitra et al., where a polyherbal formulation containing Si and Sr was shown to possess estrogenic activity.[14] Studies reported so far have shown an increase in the uterine glycogen content after estrogen administration in adult ovariectomized rats.[28] The present study seems to be the first to observe uterine glycogen content as a variable in immature rats. Our results indicate that uterine glycogen content can be incorporated in the battery of variables selected to identify the estrogenic potential of a drug using an immature rat model.
It is possible that the test drugs vizSaraca indica, Symplocos racemosa, Cyperus rotundus, Terminalia arjuna and Aśokāriṣṭa have no estrogenic activity of their own or that their estrogenic activity is too weak to be reflected in the variables chosen for this study. Another possibility is that the administration period of 3 days is not enough for the drugs to reach effective concentrations. In the clinical studies, the drugs had been given for longer duration (3 months).[8],[10],[11],[12],[34],[35] However, the study drugs were administered according to the well established immature rat bioassay and we did not modify the model.
In Ayurveda, when the drugs are used in combination, they are not combined in the doses that are recommended for the individual plants. The doses usually vary depending on the number of ingredients to be combined and the role of individual ingredients in the given formulation. However, there are no clear guidelines for deciding the dose of an individual drug. It was therefore decided to take half of the dose mentioned for each drug. It was also felt that if the half-dose was effective, it would not only be economical but would also cause less adverse effects. However the combination of Si and Sr neither induced vaginal opening nor produced an uterotrophic response nor did it increase the total uterine glycogen content in immature rats. However, histomorphology revealed that the combination caused an increase in the height of the luminal epithelium as compared to Group 1 (0.5% CMC) suggesting that the combination has a trend towards estrogenic activity, which may be too weak. This also points out that further work should be undertaken using varying doses of the individual agents.
Though the present study provided a negative answer for the question posed, the study highlights importance of using multiple variables that provide supportive information regarding the drug activity. e.g., it was possible to identify estrogenic potential of the combination of Si and Sr using histomorphology, which would have gone unnoticed if only uterine weight would have been considered. Another lesson learnt was that a longer duration of administration would be a better solution for plant drugs for which pharmacokinetic data is not available (and not only for 3 days) so as to allow the drug/s achieved adequate concentrations in the body. This would substantiate the fact that the absence of effect was not due to inadequate concentrations in the body, thereby minimizing false negative results and the premature rejection of a potential effective drug.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]
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