Iranian Journal of Pharmacology and Therapeutics Razi Institute for Drug Research (RIDR) of Iran University of Medical Sciences and Health Services (IUMS) ISSN: 1735-2657 Vol. 4, Num. 1, 2005, pp. 43-45

Iranian Journal of Pharmacology & Therapeutics, Vol. 4, No. 1, 2005, pp. 43-45

Antisteroidogenic Activity of Ethanol Extract of Ammania baccifera (L.) Whole Plant in Female Albino Mice Ovaries

RAMAIYAN DHANAPAL, SUBRAMANI KAVIMANI, VRUSHABENDRA SWAMY BHYRAPUR MATHA, MALAYA GUPTA and SANATH KUMAR BASU

Department of Pharmaceutics (R.D.); Department of Pharmacology (V.S.B.M.), Rural College of Pharmacy, Devana-halli, Bangalore, Karnataka; Mother Theresa Research Institute of Health Sciences, Pondicherry (S.K.); Division of Pharmacology (M.G.); Division of Pharmaceutics (S.K.B.), Javadpur University, Kolkata, West Bengal, India.

Address correspondence to: Dr. R. Dhanapal, Department of Pharmaceutics, Devanahalli, Bangalore, Karnataka, India.E-mail: dhanapal2k2@sify.com

Received March 22, 2005;
Revised June 9, 2005;
Accepted June 11, 2005

Code Number: pt05011

ABSTRACT

Ethanol (90%) extract of Ammania baccifera (L.) whole plant (EEAB) was evaluated for possible antister-oidogenic activity in mature female mice ovaries. The ethanol extract at the doses of 100, 200 and 400 mg/kg body weight (i.p) arrested the normal estrus cycle at dioestrus phase and significantly decreased weight of ovaries. The cholesterol and ascorbic acid content in ovaries were significantly elevated in treated mice. The extract also significantly inhibited the activity of Δ⁵-3β-hydroxy steroid dehydrogenase (Δ⁵-3β-HSD) and Glucose-6-phosphate dehydrogenase (G-6-PD), the two key enzymes involved in ovar-ian steroidogenesis. Results of this study suggested that the ethanol extract of whole plant of Ammania baccifera (L.) acts as an antisteroidogenic agent.

Keywords: Ammania baccifera, Ovarian steroidogenesis, Δ⁵-3β-HSD, G-6-PD, Medicinal plant

Ammonia baccifera Linn (Lythraceae) is a glabrous, erect branching herb, found as weed in rice-fields and marshy localities throughout India. The leaves are acrid and used in the treatment of rheumatic pain, as laxative, rubifacient and external remedy for ring worm [ 1 ]. This plant was found to possess hypothermic, hypertensive, antiurolithiasis, antibacterial and CNS depressant activi-ties [ 2 - 4 ]. Steroids triterpenes, coumarins, flavonol and tannins were previously isolated from various parts of the plant [ 5 , 6 ]. It has come to our notice that the rural people of Tamilnadu use this plant to produce sterility in animals. In the present study, we evaluated the antis-teroidogenic activity of the ethanol extract of Ammania baccifera (L.) whole plant in female mice ovaries.

MATERIALS AND METHODS

Plant Material

The whole plant of Ammonia baccifera (L.) was col-lected from Trichy, Tamilnadu, India and was identified and authenticated by Prof. Sri. Ganesh, Botanist Madura College, Madurai, Tamilnadu. A voucher specimen MG-3 has been kept in out laboratory for future refer-ence. The whole plant was dried under shade, powdered by a mechanical grinder and was passed through 40-mesh sieve and stored in airtight container for further use.

Preparation of Extract

About 1 kg of the powdered plant material was suc-cessively extracted using petroleum ether (40-60ºC), chloroform, and then ethanol (90%) in a Soxhlet extrac-tion apparatus. The various extracts were concentrated and the traces of the solvent were completely removed under reduced pressure and were stored in a vacuum desiccator for further use. The yield was found to be petroleum ether extract (0.9%); chloroform extract (1.7%) and ethanol extract (3.6%) w/w with respect to dried powder. Preliminary qualitative chemical tests indicated the presence of steroids, triterpenoids, flavon-oids and tannins. Me turther investigation was carried out using the ethanol extract.

Animals

Adult female albino mice of Swiss strain 20 ± 2 g were acclimatized to normal environmental condition in the laboratory for one week and given a standard pellet diet (Hindustan Lever) and water ad libitum.

The experiment was performed under the guidance of the Ethical Committee, Javadpur University, Kolkata-32.

Experimental Design

Treatment of Animals. The mice showing four consecutive normal oestrus cycle were then divided into five groups (n=10). In their proestrus phase the first group received normal saline (5 ml/kg/day) whereas group 2, group 3, group 4, and group 5, received vehicle (Propylene glycol 5 ml/kg body weight), ethanol extract of whole plant of Ammania baccifera (L.) (EEAB) (100, 200 and 400 mg/kg/body weight) intraperiotoneally respectively for every alternate days for 18 days. Oes-trus cycle was observed everyday by microscopic ex-amination of vaginal smear. On the 19th day the mice were killed by cervical location, 24 hours after the final does and after 18 hours fasting. Ovaries were dissected out, weighed and kept on ice for biochemical estimations.

Biochemical Estimation. Ovarian tissues about 3 mg weight, were carefully homogenized in Potter Elvehjem homogenizer using chloroform: ethanol mixture(21) and non-polar part was extracted out and total cholesterol content was estimated according to method of Kingsley and Roscoe[7].

About 5 mg of tissue was homogenized in Potter Elvehjem homogenizer using 1 ml of normal saline and 1 ml of 0.1 M phosphate buffer (pH 7.4) and centrifuged. The activity of Δ⁵-3β-HSD was estimated as described by Rabin et al [9].

About 3 mg of ovarian tissue was again homogenized in Potter Elvehjem homogenizer using 0.5M Tris-HCL (pH 8.3) and centrifuged. The activity of G-6-PD was estimaged as described lohr and Waller [10].

Protein was estimated with Folin’s phenol reagent and the activities of enzymes were expressed in unit per mg of protein as described by Lower et al [ 11].

Statistical Analysis

Statistical analysis was done by using Student’ t-test.

RESULTS

Ethanol extract of whole plant of Ammania baccif-era (L.) (EEAB) arrested normal spelling cycle at dioes-trus phase at doses of 100, 200 and 400 mg/kg body weight after 13,10 and 6 days of treatment respectively. It was found that the EEAB significantly reduced the wet weight of ovaries in a dose dependent manner (p < 0.05 by 100mg and p < 0.01 by both 200 and 400 mg). The ethanol extract of whole plant of Ammania baccif-era (L.) (EEAB) at 100, 200 and 400 mg/kg body weight significantly increased the level of total choles-terol and ascorbic acid contents of ovaries in treated mice. The activities of Δ5-3β-HSD were inhibited sig-nificantly (p < 0.05 by 100 mg and p < 0.001 by both 200 and 400 mg). Similarly, the activities of G-6-PD were inhibited significantly (p < 0.01 by 100 mg and p < 0.001 by both 200 and 400 mg) by all the doses of whole plant of Ammania baccifera (L.) (EEAB). ( Table 1 , Fig 1 and Fig 2).

DISCUSSION AND CONCLUSION

The minimum activity of steroid hormones has been reported to occur in the dioestrus stage. [ 12 - 14 ]. This was associated with an elevation in the level of choles-terol as well as ascorbic acid content which serve as precursor for the synthesis of steroid hormones in ova-ries, suggesting, that cholesterol and ascorbic acid were not utilized [ 15 -Error! Reference source not found.]. The sterodogenesis is under the physiological control of two enzymes [ 19 , 20 ].

The ethanol extract of whole plant of Ammania bac-cifera (L.) (EEAB) at treated doses arrested normal oes-trus cycle at dioestrus phase and also elevated the ascorbic acid and cholesterol content and significantly reduced the activity of Δ5-3β-hydroxy steroid dehydro-genase (Δ5-3β-HSD) and Glucose-6-phosphate dehy-drogenase (G-6-PD) in a dose dependent manner. These results revealed that EEAB produced ovarian malfunc-tion by altering substrate and enzyme activities. Pre-liminary phytochemical tests indicated the presence of flavonoids in the ethanol extract of whole plant of Am-mania baccifera (L.) Since various flavonoids have been reported [ 21 , 22 ] to possess antifertility activity, the antisteriodogenic property of the ethanol extract of whole plant of Ammania baccifera (L.) might be due to the presence of such compounds.

From the present investigation it may be concluded that the ethanol extract of whole plant of Ammania bac-cifera (L.) may be considered as an antisteriodogenic agent.

ACKNOWLEDGEMENTS

Authors are sincerely thankful to Prof. Dr. UK Ma-zumdar, Division of Pharmaceutical Chemistry, Javad-pur University, Kolkata, for providing computer facili-ties for the help in the preparation of manuscript.

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