INTRODUCTION:

Ficus bengalensis Linn. (Moraceac) is a large genus of trees. Nearly 800 species are known to occur in nature and are widely distributed throughout the tropics of both hemispheres, but particularly abundant in South East Asia. Vernacularly it is known as a Banyan tree. The plant was authenticated by Department of Botany, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur and specimen voucher (Acc. No. 6423/U) were deposited for the same.

Three new methyl ethers of leucoanthocyanins - delphinidin - 3-0-2-L- rahmnoside (I) Pelargonidin - 3-0-2-L-rahmonoside (II) and leucocyanidin 3-0-B-D- galactosylcellsbioside (III) - along with a methyl ether of leucoauthocyamidin isolated from bark. β - sitosterol - α - D - glucoside and meso - inositol also isolated from the bark extraction¹.The antifertility activity has not been validated by researchers. Data on the efficiency and biologic activity of this plant for fertility control are not available. In this study, the dried alcoholic extract of Ficus bengalensis bark was tested on rats to verify its properties as an abortificient agent and to determine possible mechanisms of action.

MATERIALS AND METHODS:

1) Animals:

Swiss albino rats used in the present investigation (the colony was maintained at the Dept. of Biochemistry P.G.T.D. Nagpur) weighing 250-300 mg/ body weight.

2) Animal husbandary:

Rats were housed in polypropylene cages, balanced pelleted rats feed and community tap water was provided, ad libitum. Standard laboratory environment was maintained (temperature 20-25°C, humidity 50-70%)

3) Extraction of Bark:

Air-dried, coarsely powdered bark of the plant collected from the Local areas of Nagpur and charged into Soxhlet apparatus for extraction with petroleum ether followed by alcohol. Each time before extracting with the next solvent, the powdered materials were air dried to remove the traces of solvent. Extract was filtered and concentrated under reduced pressure below 50°C to a viscous brown mass, and dried under high vacuum for 2 hr to remove the last traces of the solvent. The alcoholic extracts thus obtained were used for detailed biological investigation. This extract was evaluated for antifertility and estrogenic activities were assessed according to the procedure following WHO protocol for Natural products Research on Antifertility Agents.^2,3

DETERMINATION OF LD - 50 and ED – 50:

Rats were fed with the 250 / 500 / 750 / 1000 mg/kg body weight for 21 days of the experiment to determine the LD - 50 and ED - 50.

EXPERIMENTAL PROTOCOLS:

Adult female rats showing a regular pattern of estrous cycle were mated with males 3:1 sex ratio of know reproductive vigor. The mating performance was determined from a vaginal plug or sperm positive smear.

The mated females were treated orally with effective dose of 250 mg/kg body weight with the extract suspended in 500 ml of tween-20 and 1 ml saline. Control rats received only vehicle control changes in the Ostrous cycle was evaluated for 3 consecutive cycles. On 14th day of gestation the females were laprotomised and the incidence of pregnancy as well as the size and number of implants were recorded. The abdomen was closed surgically and pregnancy was allowed to continue.

On the 21^th day of gestation the females were dissect, opened and fetuses were delivered by caesarean sections. The fetal loss and sex ratio were recorded. The ovaries and uteri of non-pregnancy females were fixed in 10% formalin for histology.

HISTOLOGICAL STUDIES:

Tissue fixed in 10% formalin were washed, trimmed, dehydrated in a graded series of ethanol and cleared in xylene. The tissue was embedded in Paraffin wax and blocks were prepared. Sections of 6-7 μm thickness were stained with hematoxylin and eosin and maintained in DPX. The histological changes were observed under the light microscope.

STATISTICAL ANALYSIS:

Results are expressed as the mean ± SD and significance was determined by student's t test. A probability level of less than 5% was confirmed significant.

RESULTS:

The LD-50 and ED-50 observed was 500 mg/kg body weight and 250 mg/kg body weight respectively. Methanolic extract of Ficus bengalensis bark showed 0.3% w/w yield of starting crude material with the presence of saponin.

Rats treated with the extract exhibited decrease in body weight and the alteration of vaginal smear along with the reduction in the pituitary, uterine and ovarian weight at effective dose, suggests a diminuation in the gonadal steroidogenesis after treatment with methanolic extract.

In the present study, the total absence of implants was observed in either of the uterine horns of the treated female rats. This resulted in a decrease in the overall reproductive capacity of the treated group due to inhibition of implantation. However, only one female rat that received the extract showed dead foetus with anencephaly, exophthalmia and a protruding tongue was found in the group that received the extract.

In uterus, endometrial glands were more degenerated and non functional (Photo 1). Degeneration in all types of follicle and corpus luteum. Oocytes of the follicle were completely destroyed (Photo 2).

Fallopian tube showed complete degeneration in the epithelial cells. Villi, folds of fallopian tube reduced considerably due to degeneration of the cells. Muscular coat of the fallopian tube also reduced considerably (Photo 3).

In the vaginal mucosa cavities were formed due to degeneration of the cells. Marked changes were observed in epithelial layer of the vagina. Lamia propria on which the epithelium rests was completely disappeared. The basement membrane disappeared and polymorphonuclear leukocytes invaded the epithelium and passed through it and confirmed mostly to the superficial layers of the epithelium. In some areas epithelial layer was completely disappeared as marked by arrow. These observations were matched with the disease stage of the rat. Vaginal lumen reduced significantly (Photo 4).

Photo 1. Part of endometrium showing regressive changes in uterine epithelium and endometrial glands after the treatment (X110).

EG = Endometrial gland

END = Endometrium

EP = Epithelium

Photo 2. Methanolic extract of Ficus bengalensis bark in the developing, mature follicles and corpus luteum (X100)

Photo 3. T.S. of fallopian tube showing epithelial cells degeneration and accumulation in the lumen.

Photo 4. Section of vagina showing thin vaginal epithelium layer (X100)

DISCUSSION:

It assumes that inhibition of cyclic changes of vaginal smear in methanolic extract treated rats were due to demedation in the ovarion steroidogenesis. The stages of estrus cycle and their interconnections are governed by the synthesis of ovarian harmones, which are controlled by the secuation of pituitary and gonadetropine and hypothelmic releasing factor. In investigations on antifertility activity of seeds of Nelumbo nucifera, mice have shown that the seeds affect the estrus cycle by blocking the biogenesis of ovarian steroids⁴. T.T. treated rat uterus shows degeneration in endometrial gland.

Similar histological results were observed in the CRE treated rats showed rupture and desquamation of endometrial epithelium, degeneration of endometrial glands and infiltration of polymorphonuclear leucocytes in the stroma.⁵

Ficus bengalensis alcoholic extract showed degeneration in all types of follicles and corpus leuteum. Similarly, there was an increasing small atretic and hemorrhagic dilated follicles in RAG treated rabbits⁶

In the endometrium of the control rats of the present work, microvilli on cell surface appeared more numerous and well developed during the proestrous and estrous, the periods which are under estrogenic stimulation while they are least abundant and poorly developed during the period of relative estrogen deprivation, i.e. metaestrous and diestrous⁷. The metaestrous and diestrous which are the luteal phase of rat estrous cycle showed the presence of sea-anomonelike⁸ or morel-like⁹ structures characteristic of progesterone stimulation on endometrium. With the absence of these structures, which are also called pinopoids¹⁰ owing to their pinocytic activity, the endometrium in methanolic extract treated rat lost the progesterone conditioned intranterine environment in which blastocysts reside. Clumping of the microvilli forming microridgenes, desquamation of surface epithelium and appearance of polymorphs may lead to functional aberration of the uterine tissue interfering with the process of nidation; similar to action of certain progestational agents¹¹. As well, this abnormal morphological feature of treated rat uterus may retard the movement of ova and spermatozoa, since the abundance of cilia in uterine luminal epithelium serves as a useful tool for sperms transport¹². The fluid current created by the ciliary action of microvilli provides a transport vehicle for ova and spermatozoa¹².

In the present experiment it could possible that oral administration of methanolic extract attend the endometrial structure and function by loss and reducing of microvilli and its distribution, resulting in appearance of polymorphs at the surface epithelium. This structural disparity has been thought to contribute to antifertility in methanolic extract treated rats suppressing implantation during the estrous cycle.

CONCLUSION:

In conclusion, the present study shows that Post-mating oral administration of drug interferes with crucial reproductive events such as estrus cycle, mating behavior, fertilization and implantation. Studies are in progress to elucidate the possible mechanism, hormonal profile and also chemical constituents present in the extract of Ficus bengalensis Linn.

REFERENCES:

1. Compendium of Indian Medical Plants: Vol I, (Glossary Indian Med. Plants, Chopra, Nayar and Chopra, PID, New Delhi, 1956, 118.)

2. World Health Organization Protocol of MB-30; A method for examining the effect of plant extracts administered orally during first ten days of pregnancy in rats. Geneva; WHO, 1982: 7732.

3. World Health Organization Protocol of MB-70; A method for detecting estrogenicity in plant extracts administered orally in rats. Geneva; WHO, 1983: 9856.

4. Muzumdar VK, Malayagupt G, Parmanik RK, Mukhopadhyay S and Sarkar S, Antifertility activity of seeds of Nelumbo nucifera in mice. India J. Expt. Biol. 1992, 30:533-534.

5. Sharma HN, Mahanta HC, Modulation of morphological changes of endometrial surface epithelium by administration of composite root extract in albino rats contraception.: 62 and 51-54.

6. Salhab AS, Shomat MS, Gharaibeli MN and Amer NA, Effects of Castor Bean Extract and Ricin achain on Ovulation and implantation in Rabbits. 1999, 59:395-399.

7. Hammer RE, Samarion RA, Mitchell JA, Alterration in surface morphology of the antimesometrial uterine epithelium of the rat; effects of ovarian steroid hormones. Scan Elec. Micros Vol. II. 1978:701 -706.

8. Greep RO, Astwood EB, Handbook of physiology, Vol. 3, Part II. Baltimore: University Park press 1973:198-215.

9. Neilson O., Ultrastructure of the process of secretion in the uterine epithelium during delayed implantation. Jour. of Ultraster Res. 1972; 40:572 - 580.

10. Enclers AC, Nelson DN Pinocytic activity of the uterus of the rat. American Jour. of Anatomy 1973:138, 277-300.

11. Moghissi KS, Reame NE, Effect of progestational agents on the female reproductive tract, Proceedings of the workshop on the SEM in Reproductive Biology IIT Research Institute Chicago, Scamming Electron Microscopy Part VI 1976:373 -378.

12. Fadel H E, Dennis Bern B S, Lourens J D, The human uteratubal functionl; a scamming electron microscope study during different phases of the menstrual cycle. Fertil steril 1976; 27:1176-1186.

Received on 21.11.2009 Modified on 25.03.2010

Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1285-1287