Antifertility Activity of Eugenia Jambolana Seed Extract in Female Albino Rat

The present study was conducted to investigate the effect of Eugenia jambolana seeds on estrous cycle and ovulation. Petroleum ether, ethyl acetate and ethanol extracts of seeds of E. jambolana administered orally at the dose level of 200 mg and 600 mg/kg body weight to adult female rat for 30 days. Out of the three extracts, ethyl acetate extracts at both the dose level seems to be more effective resulted in irregular estrous cycle with prolonged diestrus and reduced estrus and metaestrus during the experimental period. The data revealed that the ethyl acetate extract caused a significant decline in the wet weight of ovary and uterus, as well as protein and glycogen level, however, cholesterol and total ascorbic acid level increased significantly. The extract also significantly reduced the number of developing follicles, graffian follicles and corpora lutea and increase the number of atretic follicles. Micrometric measurement of the uterus showed the decrease in the diameter of the uterus and thickness of the myometrium and endometrium significantly in the ethyl acetate extract treated rats. These observations showed the anti-ovulatory activity of Eugenia jambolana seed in female albino rats. Citation: Sarita M (2018) Antifertility Activity of Eugenia Jambolana Seed Extract in Female Albino Rat. Biochem Physiol 7: 237. doi: 10.4172/21689652.1000237

All the above treatments were given orally by using intragastric catheter for 30 days to cover 6 regular estrous cycles. The treatment was started from estrous phase only, as the ovarian activities change markedly from one phase to another phase of estrous cycle. The treatment was given orally everyday between 10.00 and 11.00 h. The stages of estrous cycle were recorded daily by observing vaginal smears according to Vogel [8]. The control and treated animals were sacrificed on day 31 st by cervical dislocation 24 hrs after the last treatment.

Organ weight
Ovaries and uteri were dissected out, freed from surrounding tissues, blotted on filter paper and weighed quickly to the nearest milligrams on an electronic balance.

Biochemical analysis
The ovary and uterus from one side of each animal were processed for biochemical analysis such as Cholesterol by Peters and Vanslyke [9], Ascorbic acid by Roe and Krether [10], Protein by Lowry et al. [11] and Glycogen by Carrol et al. [12].

Ovarian follicular kinetics
Ovary from another side of each animal were fixed in Bouin's fluid, embedded in paraffin wax, sectioned at 5 µm thickness of the ovary were prepared for the study of follicular kinetics. To quantitatively evaluate ovarian follicles, the methods described by Hirshfeild [13] Sanjay and Joshi [14] were used in the present study. Ovarian follicles were classified as primary, small preantral, large preantral, small antral and Graafian follicle according to the morphological classification scheme used by Lundey et al. [15].

Histometry
The diameter of the uterus, thickness of the myometrium, endometrium and height of epithelial cells were measured by using stage and ocular micrometer.

Statistical Analysis
One way analysis of variance (ANOVA) followed by Duncan's multiple test were used to find out significant difference among mean values of each parameter of different experimental groups by fixing minimum significance level at P<0.05. Values with same superscript letters are not significantly (P<0.05) different whereas those with different superscript letters are significantly (P<0.05) different when compared to control.

Estrous cycle
The present study revealed that the ethyl acetate extract at both dose levels showed an antifertility effect. It is observed that the administration of pet.ether at both the dose levels and low dose level of ethanol has shown no significant difference in the length of any of the phase of the estrous cycle. Ethyl acetate extract of both the doses has decreased significantly (P<0.01) the estrus and metaestrus phases and also caused prolongation of diestrus phase. High dose of ethanol extract treatment has arrested the normal estrus cycle at diestrus phase at later stages of the cycle and decreased the total number of estrus and metaestrus phases significantly (P<0.01) ( Table 1).

Organs weight
There was a significant (P<0.01) decrease in the wet weight of ovary and uterus at low dose level of ethyl acetate extract with high dose of ethyl acetate and ethanol extract administration the reduction in the weight is highly significant (P<0.001) when compared to control ( Table 2).

Biochemical change
Administration of ethyl acetate extract at both dose levels has caused highly significant decrease (P<0.001) of protein and glycogen and an increase in the cholesterol and ascorbic acid level in the ovary and uterus of rats (Table 3, 4).

Follicular kinetics
The animals treated with ethyl acetate at low dose level caused a statistically less significant (P<0.05) reduction in the number of small antral and graafian follicles with concomitant significant increase in the number of atretic follicles of the same stage. At high dose caused a highly significant decrease (P<0.001) in the number of healthy small preantral, large preantral, small antral, graafian follicles and active   and fresh corpora lutea with a concomitant significant increase in the number of atretic follicles of the same stage. The result also showed a significant reduction in the total number of follicles in the ethyl acetate extract treated ovary (Table 5).

Histometry
The diameter of the uterus, thickness of endometrium and myometrium and epithelial cell height has decreased highly significantly (P<0.001) with both the dose levels of ethyl acetate extract treated rats (Table 6).

Discussion
In the present investigation, pet.ether, ethyl acetate and ethanol extracts of E. jambolana seeds were tested for estrous cycle and ovulation. Out of these the ethyl acetate extract has shown most promising antiovulatory activity in female albino rats. In this study, treatment of ethyl acetate extract of E. jambolana at both doses for 30 days showed a significant decrease in the estrus and metaestrus phases with conmitant increase in diestrus phase, resulting in the reduction     of total number of cycles. The prolongation of diestrus phase indicates that maturation of the follicle in the preovulatory phase was delayed, leading to non-maturation of graafian follicle. Non-availability of matured graafian follicle was indicated by reduction in the metaestrus phase. As a result the extracts provoked inhibition of the ovulation with consequent reduction of the cyclicity. Similar results have been obtained with Azardirachta indica [16], Jatropha curcus [17] and Mimosa pudica [18]. This may be due to the fact that the decreased estrogen availability at regular intervals which is due to administration of the crude extract of seeds of E. jambolana. Ovulation takes place under the combined and balanced influence of ovarian and extra ovarian hormones. Imbalance in these hormones leads to irregularity in the ovarian function and duration of estrous cycle [19,20].
There is a decrease in wet weight of ovary in ethyl acetate treated groups shows the antiovulatory effect via suppression of FSH. Similar results have also obtained with administration of Artobotrys odoratissimus [21,22].
In the present study, the decrease in the relative wet weight of the uterus indicates the weak estrogenic and strong antiestrogenic effects of the ethyl acetate extract. An active antiestrogen has been reported to decrease the wet weight of uterus [23,24]. These finding clearly corroborate the potent antiestrogenic nature of the extract which was tested by using immature ovariectomized rats [7].
Protein is considered to be the building material and is involved in the alteration of almost every physiological function. In the present study the low protein content of ovary and uterus indicates the retarded growth. Glycogen is involved in providing energy to various processes like, ovulation, transportation and survival of eggs and implantation. All these changes are hormone dependent [25]. The decreased glycogen content in ethyl extract of E. jambolana seed treated rats may be due to lowered steroidogenesis, which attributed to non-availability of gonadotrophins.
Ascorbic acid plays an important role in ovarian steroidogenesis [26]. Therefore, in the present data the accumulation of ascorbic acid in the ovaries and uterus gives additional support to the inhibition of steroidogenic activities. The significant elevation in cholesterol content of ovary and uterus indicates the non-availability of pituitary gonadotrophins which are necessary for conversion of cholesterol to estrogen/progesterone [27,28]. In the present investigation, the significant elevation in cholesterol content of ovary and uterus in ethyl acetate extract treated rats suggests the non-utilization of cholesterol towards biosynthesis of hormone in ovaries.
There is an increase in the number of atretic follicles and concomitant decrease in the number of primary, preantral, antral, graafian follicles in ethyl acetate extract treated rats may be due to the non-availability of a required amount of extra ovarian regulators (FSH and LH). The formation of corpus luteum is a direct continuation of preovulatory follicle development. The decrease in the number of corpora lutea indicates that the ethyl acetate extract inhibited the conversion of the preovulatory follicles into corpus luteum arresting ovulation. Similar results have been obtained in Momordica charantia [29], Malva viscus [22].
The ethyl acetate extract caused atrophic effect on the uterine tissue as revealed by the significant reduction in the epithelial cell height and the thickness of the endometrium and myometrium. The result observed in the present study on the histology of the uterus is in agreement with the studies made by Pal [30] on Sesbania sesban seeds.

Conclusion
In conclusion, the ethyl acetate extract of E. jambolana seed exerted a strong antiovulatory effect. Administration of ethyl acetate extract of E. jambolana may block ovulation by altering estrous cycle with a prolonged diestrous, decreases the uterine and ovary weight and may cause antiovulation effect. The antiestrogenic activity of the ethyl acetate extract of E. jambolana L might be the reason for antiovulatory activity.