Antifertility effect of ethanolic extract of Amalakyadi churna in male albino mice

Y. N. Seetharam¹, H. Sujeeth¹, G. Jyothishwaran¹, Arvind Barad¹, G. Sharanabasappa¹, B. Umareddy¹, M. B. Vijaykumar², Saraswati B. Patil²

Keywords: Amalakyadi churna; testis; accessory organs; reproduction; spermatogenesis

Abstract

Aim: To evaluate the antifertility activity of the ethanolic extract of Amalakyadi churna by oral administration in male albino mice. Methods: The ethanol extract of Amalakyadi churna at the dose of 250 mg/kg and 400 mg/kg body weight was administered orally for 30 days to adult male mice. On day 31, the mice were sacrificed and the testis and accessory reproductive organs were removed and weighed. The organs were processed for biochemical estimation and histological work. Results: Treatment with Amalakyadi churna resulted in decrease in the weights of testis and accessory reproductive organs. The diameters of testis, seminiferous tubules and Leydig cell nucleus were decreased. The spermatogenic elements, like spermatogonia, spermatocytes and spermatids in the testis were reduced significantly as well as the sperm count in cauda epididymis. There was a significant reduction in the protein, glycogen, DNA and RNA contents and the activity of acid phosphatase in the testis of extract treated mice compared with the control. The cholesterol content and the alkaline phophatase activity were increased significantly in treated mice. Conclusion: Amalakyadi churna extract arrests spermatogenesis in male mice without noticeable side effects.

1 Introduction

Search for male antifertility agents in natural products remains a potential area of investigation [1-9].

The churnas are the pure and powdered form of raw drugs obtained from different parts of plants. There are more than 100 churnas and some of their therapeutic effects are described in Ayurvedic Formulary of India [10]. Among them Amalakyadi churna has been reported to possess antifertility activity. Taking in view the necessity of a potential male antifertility drug from herbs, we have undertaken the systematic work on adult male albino mice.

2 Materials and methods

Amalakyadi churna was prepared by using standard formulation prepared by an ancient Indian physician, Sarangadhara (1300 AD ~ 1400 AD), and mentioned in his treatise Sarangdhara Samhita [11].

The fruits of Phyllanthus emblica L. (Euphorbiaceae), Piper longum L. (Piperaceae), Terminalia chebuta Retz. (Combretaceae) and the roots of Plumbago Zeylamica L. (Plumbaginaceae) were collected from Sandur (Karnataka, India) in October and November 2000 and authenticated at the herbarium, Department of Botany, Gulbarga University, Gulbarga. The rock salt was purchased from a local Ayurvedic shop. The plant materials were shade-dried. The properly shade-dried plant materials and the rock salt were powdered separately and sieved through muslin cloth. They were taken in equal proportions and mixed well. This Amalakyadi churna is stored in airtight container for further processing.

Amalakyadi churna was soxhleted with ethanol. The extract was concentrated to dryness in a flash evaporator (Buchi type) under reduced pressure and controlled temperature (50 ~ 60 ). Fifteen g and 24 g of the extract was then made homogenous thin paste separately in 10 mL Tween-80 (1 %), suspended in 50 mL distilled water.

Colony bred adult, virgin Swiss strain male albino mice of 60 days ~ 70 days old, weighing 30 g ~ 35 g, were used. Animals were maintained under uniform environmental conditions with free access to standard diet (Hindustan Lever Ltd., Bombay, India) and tap water ad libitum. The animals were randomly classified into three groups of six each. Group one served as the control and received the vehicle; the other 2 groups were gavaged the ethanol extract of the Amalakyadi churna for 30 days at dose of 250 mgkg^-1d^-1 and 400 mgkg^-1d^-1, respectively.

On day 31, the mice were sacrificed by cervical dislo-cation. The testes, epididymis, seminal vesicle, prostate and Cowper's glands were dissected, freed from surrounding tissue and were weighed quickly to the nearest weight on an electronic balance. Testis from one side of the animal was fixed in Bouin's fluid, embedded in paraffin wax, sectioned at 5 mm and stained in haematoxylin and eosin for histological study. The other testis was used for biochemical estimation, including protein [12], cholesterol [13], glycogen [14] acid and alkaline phosphatase [15] and DNA and RNA [16]. The micrometric measurements like diameters of testis, seminiferous tubules and Leydig cell nucleus were calculated by the method of Deb et al. [17]. Spermatogenic elements count was made on 20 randomly chosen round cross sections. The sperm count from cauda epididymis was done by using haemo-cytometer.

Data were expressed in meanSE, if applicable. The significance of difference was analysed using Student's t-test and P<0.05 was set as significant.

3 Results

The weights of testes, epididymis, seminal vesicle, prostate and Cowper's gland were significantly (P< 0.01) reduced in the 400 mg/kg treated mice compared with the controls. Seminal vesicle and prostate weights were also reduced significantly (P<0.05) in the 250 mg/kg treated group (Table 1).

The protein and DNA levels in both treated groups decreased insignificantly. There was a significant reduction in the RNA at both doses, but more at the higher dose. The glycogen content was decreased in both treated groups, but it was significant (P<0.05) only in the 400 mg/kg treated group. The acid phosphatase level was increased in both treated groups, but was significant (P<0.01) only in the 400 mg/kg treated mice. There was no significant increase in the level of alkaline phosphatase in both treated groups (Table 2).

3.3 Histology

There was no significant change in the diameter of testis, but a significant reduction in the diameter of the seminiferous tubules and Leydig cell nucleus was observed in both treated groups. The number of spermato-gonia, spermatocytes and spermatids decreased significantly in both treated groups (Table 3, Figures 1 & Figures 2).

Figure 1. Transverse section of control mice testis showing normal seminiferous tubules with all types of spermatogenic elements and spermatozoa (400). Note the healthy Leydig cells (Lc). Spermatocytes (SPC); Spermatogonia (SPG); Spermatozoa (SZ); Spermatids (SPT).

Figure 2. Transverse section of Amalakyadi churna-treated mice testis showing shrinkage of seminiferous tubules and decreased Leydig cells. Note significant decreases in the spermatogonia (SPG), spermatocytes (SPC) and spermatids and absence of spermatozoa. In spermatocytes nuclear pyknosis (PS) is seen (400).

The cauda epididymal sperm count decreased significantly in both treated groups (Table 3).

Oral administration of an ethanolic extract of Amalakyadi Churna manifested two principal impacts on the male reproductive system of albino mice, antisper-matogenic and antiandrogenic effects. The antispermato-genic effect is reflected in the cessation of spermatogenesis and disintegration of sperm. It is evident that FSH stimulates the development of spermatogonia to spermatocytes and also maintains the spermatogenic process [18] and both FSH and LH/ICSH are necessary for meiosis and development of spermatids [19]. The androgens are necessary to induce meiosis, formation and development of spermatids in response to FSH [20]. 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. It is known that sperm production cannot proceed optimally to completion without a continuous androgen supply [21]. How-ever, the incidence of low sperm count implies Amala-kyadi Chruna induced infertility might be the consequence of an array of factors in biochemical events in tissues. The increased cholesterol content of testis after the administration of Amalakyadi churna extract indicated reduced conversion of cholesterol to androgen which is dependent on the availability of LH/ICSH [22]. The glycogen content in the cell indicates energy storage. Sertoli cells and spermatogonia often contain glycogen and secrete substrates from the blood and provide source of reserve carbohydrates for seminiferous tubular cells, and the glycogen level is found to be directly proportional to the steroid hormones [22]. The decreased glycogen content of the testis after the administration of Amalakyadi churna extract may reduce the energy source for spermatogenic activity, which might have resulted in spermatogenic arrest. Acid and alkaline phosphatase is widely distributed in the testis and is important in the physiology of sperm [23]. In the present study the changes in phosphatase activity may be indicative of spermatogenic suppression and/or suppression of exchange of materials between germinal and Sertoli cells [24], it is also indicative of extensive lytic activity. The decrease in the DNA and RNA contents of the testis in the treated mice indicates a poor growth rate.

Antiandrogenic action of this extract is reflected in the regression and disintegration of Leydig cells and the degenerative changes in the epididymis, seminal vesicle, prostate and Cowper's gland. As the administration of extract has caused reduction in the spermatogenesis and steroidogenesis, it may alter the sexual behaviour and cause infertility.

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Correspondence to: Professor (Ms.) Saraswati B. Patil, Department of Zoology, Gulbarga University, Gulbarga- 585 106, India.
E-mail: Viju_yes@rediffmail.com; saraswatibp@yahoomail.com
Received 2003-02-20   Accepted 2003-07-14

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