B. Vijaykumar, I. Sangamma, A. Sharanabasappa, Saraswati B. Patil

Plants have served as a natural source of antifertility substances. Henshaw [1] has listed many plants used by primitive people in different countries to control fertility. Though many indigenous plants have been shown to prevent birth, only a few have so far been investigated for antispermatogenic activity [2-5]. Crotalaria juncea Linn. (Papilionaceae), commonly known as Sunn hemp, is cultivated throughout India and Indian Ayurvedic medicine indicated that various parts of C. juncea have analgesic, astringent, emmenagogue and abortifacient activities and is also used for the treatment of skin diseases. The seeds of this plant have been reported to possess contraceptive activity [6], however, no detailed study is available so far on its possible male antifertility activity. The present study was designed to clarify this point in albino mice.

The fresh seeds of C. juncea were obtained from a local source during October and November 2000 and were authenticated by Dr. Y. N. Sitharam, Department of Botany, Gulbarga University, Gulbarga (HGUG No. 71), India, where voucher specimens were deposited.

The seeds were shade-dried, powdered and subjected to Soxhlet extraction successively and separately with petroleum ether (B. P. 60-80 ), benzene and ethanol (95 %). The decoctions produced were evaporated under reduced pressure below 45 . The residual extracts thus obtained (500 g of C. juncea seed produces: 3.8 g of petroleum ether extract, 1.2 g of benzene extract and 2.73 g of ethanol extract) were screened for their antifertility activity in mice. Before use, the extract was suspended in Tween-80 (1 %) normal saline with a final concentration of 25 mg/mL.

A significant (P<0.01) reduction in the weight of testis was observed in the ethanol, petroleum ether and benzene extracts groups. Histometric data were similar to those of weight changes: a significant (P<0.01) reduction in the diameters of the testis and seminiferous tubules with all the 3 extracts (Table 1).

The number of spermatogonia, spermatocytes and spermatids are significantly reduced (P<0.01) with the ethanol extract, while the petroleum ether and benzene extracts significantly reduced the number of spermatogonia and spermatocytes (P<0.01). In all treated groups, there were pyknosis in the primary and secondary spermatocytes and degeneration of Leydig cells, spermatozoa were completely absent from the seminiferous tubular lumen and the sperm density was significantly reduced in the cauda epididymis (P<0.01) (Table 1).

The cholesterol content of the testis was significantly (P<0.01) increased in the ethanol and benzene extract groups, while the protein content was significantly reduced in the ethanol extract group (P<0.01) and in the petroleum ether and benzene extract groups (P<0.05). The glycogen content was significantly reduced with the benzene and ethanol extracts (P<0.01), but not with the petroleum ether extract (Table 1).

The weight of epididymis was significantly reduced in the ethanol and benzene groups (P<0.01), but not in the petroleum ether group. The cholesterol content of the epididymis was significantly increased (P<0.01) and the protein and glycogen contents of epididymis were significantly reduced (P<0.01) with all the three extracts (Tables 2 & 3).

A significant reduction in the weight of vas deferens (P<0.01) was observed in the ethanol extract group, but not in the other 2 groups. The protein content of vas deferens was significantly reduced (P<0.01) with the ethanol extract, whereas insignificant with petroleum ether and benzene extracts. Though all the 3 extracts reduced the glycogen content of vas deferens, it was significant (P<0.01) only with the ethanol extract (Tables 2 & 3).

The weight of seminal vesicle was significantly (P<0.01) reduced with all the three extracts. The weight of prostate was significantly reduced (P<0.01) with the ethanol extract, but not with the petroleum ether and benzene extracts (Table 2).

The weights of the bulbourethral gland and levator ani showed a significant (P<0.01) reduction in the benzene, ethanolic and petroleum ether groups (Table 2).

Testosterone administration caused a highly significant (P<0.01) increase in the wet weights of epididymis, vas deferens, seminal vesicles, prostate, bulbourethral gland and levator ani. On the contrary, the ethanol extract significantly decreased the wet weights of these organs (P<0.01) compared to the controls, indicating an antiandrogenicity of the ethanol extract (Table 4).

Administration of the petroleum ether, benzene and ethanol extracts of C. juncea reduces the testicular weight, which may be due to a decreased production of seminiferous tubular fluid that contributes to the weight of testis [9]. The reduced protein content may be another reason as the growth rate of any organ is proportional to its protein content. The observed reduction in the number of spermatogonia, spermatocytes and spermatids may indicate lowered availability of FSH and LH/ICSH, which are essential for initiation and maintenance of spermatogenesis. For this reason, the seminiferous tubular lumen is devoid of spermatozoa.

The glycogen content in the cell represents the energy storage. The Sertoli cells and spermatogonia contain glycogen and provide nourishments to the seminiferous tubular cells and the glycogen content is found to be directly proportional to the steroid hormone levels [10]. A decrease in the glycogen content of the testis reduces the energy source for spermatogenic activity.

The increased cholesterol content of testis may reflect a reduced conversion of cholesterol to androgens, which is dependent on the availability of LH/ ICSH. The reduction or non-availability of androgens is further supported by the reduction in the weight of accessory organs. All these organs play important roles in sperm maturation and mobility and the formation of semen. The ethanol extract when tested in immature mice also showed antiandrogenic effect. In conclusion, various extracts of C. juncea seeds arrest spermatogenesis and are likely to have an antiandrogenic activity.