Effect of Alstonia scholaris bark extract on testicular function of Wistar rats

R. S. Gupta1, Rakhi Sharma¹, Aruna Sharma¹, A.K. Bhatnager², M.P. Dobhal³, Y.C. Joshi4, M.C. Sharma⁴

¹ Reproduction Physiology Section, Department of Zoology, University of Rajasthan, Jaipur - 302004, India

Asian J Androl 2002 Sep; 4: 175-178          

Keywords: Alstonia scholaris; testis; sperm motility; sialic acid; Sertoli cells

Abstract

Aim: To evaluate the antifertility effect of Alstonia scholaris bark extract in male rats. Methods: In male Wistar rats Alstonia scholaris bark extract was given by oral route at a dose of 200 mg/day for 60 days. The fertility and testicular function were assessed by mating tests, sperm motility, sperm concentration, biochemical indices and testicular cell population dynamics. Results: Oral feeding with the extract at a dose of 200 mg/day for the period of 60 days did not cause body weight loss, while the weights of testes, epididymides, seminal vesicle and ventral prostate were significantly reduced. The production of step-19 spermatids was reduced by 79.6 % in treated rats. The population of preleptotene and pachytene spermatocytes were decreased by 61.9 % and 60.1 %, respectively. Spermatogonia and Sertoli cell population were also affected. The seminiferous tubule and Leydig cell nuclear area were reduced significantly (P<0.01) when compared to the controls. Reduced sperm count and motility resulted in a total suppression of fertility. A significant fall in the protein and sialic acid content of the testes, epididymides, seminal vesicle and ventral prostate as well as glycogen content of testes were also noticed. The fructose content in the seminal vesicle was lowered whereas the testicular cholesterol was elevated as compared with the controls. The following compounds were obtained from the extract with chromatographic separation over Si-gel column: a-amyrin, b-amyrin, lupiol acetate, venenative, rhazine and yohimbine. Conclusion: At the dose level employed, Alstonia scholaris bark extract has a significant antifertility effect in male rats; the primary site of action may be post meiotic germ cells (Step 19 spermatids).

1 Introduction

Alstonia scholaris (Apocynaceae) commonly known as saptparni in sanskrit, is a plant distributed throughout the tropical and subtropical regions of the world. The plant is available in Jaipur, Rajasthan (India). The species of Alstonia are used in traditional system of medicine [1]. The extract of its root bark exhibited cytotoxic activity against two human lung cancers, adenocarcinoma and large cell carcinoma. Some of the Alstonia species have also been used for the cure of malaria [2]. The Alstonia species are indicated to be rich in alkaloids [3].

However, no attention was paid to its possible effect on the reproductive system of mammals. In the present study the male antifertility activity of Alstonia scholaris bark extract was investigated.

2 Material and methods

2.1 Plant material

Alstonia scholaris bark was harvested in Sep. 2000 from Jaipur region (India) and authenticated by Dr. Navjyot Sarana, Associate Professor, Department of Botany, University of Rajasthan, Jaipur (India).

2.2 Extraction and isolation

Shade dried and powdered stem bark of Alstonia scholaris was extracted with ethanol for about 46-48 h. The filtrate obtained was concentrated under reduced pressure and yielded a dark brown semi-solid mass. The extract so obtained was washed with petroleum ether for the removal of the fat portion. This fat free part was extracted with benzene. A part of this final extract was employed in the study on its biological activity and the rest, for the isolation of constituent compounds by means of chromatographic separation over Si-gel column.

2.3 Animals

Wistar rats, weighing 150-160 g, obtained from Jamia Hamdard, Hamdard University, New Delhi were used. Animals were housed in steel cages and maintained under standard conditions (12 h light/ 12 h dark cycle; 253; 35 %~60 % relative humidity). Rat feed (Hindustan Lever Ltd.) and tap water were provided ad libitum.

2.4 Study protocol

Male rats of proven fertility were divided into two groups of 10 each. Group 1: Controls, receiving vehicle (distilled water, 0.5 ml/day) for 60 days. Group 2: Treated, receiving the extract (200 mg/day) for 60 days.

On day 61, rats were sacrificed and the testes, epididymides, seminal vesicles, ventral prostate and liver were removed, cleared of fat and connective tissue, weighed and kept at -20 until assayed for total protein, glycogen and sialic acid [4,5,6]. Sperm concentration and motility was determined by the method of Prasad, et al [7].

2.5 Histological preparations

Tissues were fixed in Bouin's fluid and paraffin sections were made and stained with hematoxylin and eosin or periodic acid Shiff reagent (PAS) to discriminate the stages of spermatogenesis [8].

The evaluation of cell population dynamics was based on the counts of each cell type per cross-tubular sections. Various cell components were quantitatively analysed using spherically appearing sections. Abercrombie's correcting factor was introduced [9] to correct for the better chance a big cell has to be counted. Mean tubular diameters were determined by tracing and measuring an average of 100 selected seminiferous tubules. The diameter of Leydig cell nuclei were measured at 800

2.7 Fertility test

The mating tests were performed from day 55 to day 60. The male rats were cohabited with proestrus females at a ratio of 1: 3. The vaginal plug and the presence of sperm in the vaginal smear the next morning were checked as the indices for positive mating. The mated females were separated to note the implantation sites on day 16 of pregnancy through laparotomy.

2.8 Statistical analysis

Data were expressed in meanSEM. The Student's t-test was used to compare the significance of differences and P<0.05 was set as significant.

3.1 Body and organ weights

Alstonia scholaris bark extract did not cause any significant change in the body weight of treated rats. However, the weights of testes, epididymides, seminal vesicle and ventral prostate were significantly (P<0.01) reduced when compared with the controls (Table 1).

The number of step 19 spermatids was reduced by 79.6 %, spermatogonia by 55.8 %, preleptotene spermatocytes by 61.9 % and pachytene spermatocytes by 60.1 %. The total number of Sertoli cells were also reduced after Alstonia extract feeding (Table 2).

Table 2. Effect of A. scholaris on testicular cell population in rats. MeanSEM, n=10. cP<0.01 vs control. Percent variations vs control in parentheses.

The seminiferous tubular diameter and the Leydig cell nuclear area were reduced significantly (P<0.01) when compared to controls (Table 1).

3.3 Biochemical parameters

As shown in Table 3, the protein contents of testes, epididymides, seminal vesicles and ventral prostate were reduced significantly (P<0.01) in the treated group. The sialic acid contents of the testes, cauda epididymides and ventral prostate were depleted. The glycogen contents of testes were decreased significantly (P<0.01). The fructose content in seminal vesicle was also lowered, whereas the testicular cholesterol was significantly elevated (P<0.01).

Table 3. Effect of A. scholaris on tissue biochemistry in rats. All units in mg/g. MeanSEM, n=10. ^aP>0.05; ^cP<0.01 vs control.

3.4 Sperm indices

As shown in Table 4, the treated rats showed a significant (P<0.01) reduction in the sperm concentration of testes and cauda epididymides. The sperm motility of the cauda epididymides was also reduced significantly (P<0.01). Alstonia treatment reduced the fertility of male rats by 100 %.

Table 4. Effect of A. scholaris on sperm motility and concentration and fertility in rats. MeanSEM; ^cP<0.01 vs control.

3.5 Isolation of compounds

The following compounds were isolated from the final ethanol extract by means of chromatographic separation over Si-gel column. By eluting the column with pet ether:benzene (3:1), b-amyrin and lupiol acetate were obtained. Further elution of column with pet ether:benzene (2:2) yielded a-amyrin, with benzene:chloroform (3:1) yielded venenative, with benzene:chloroform (1:3) yielded rhazine and with chloroform (100 %) yielded yohimbine.

4 Discussion

Oral administration of Alstonia scholaris bark exrelated to the number of spermatids and spermatozoa present in the organ. The reduced testicular weights and shrunken seminiferous tubular dimension indicate a wide spread testicular damage [10].

A. scholaris bark extract exerted a strong inhibitory effect on epididymides, seminal vesicle and prostate gland as evinced by decrease in their weights. A reduction in the weight of accessory reproductive organs suggested the reduced availability of androgens [11]. Rat prostate glands contain some androgen receptors which are the direct target of androgen action [12] and mainly dependent on testicular androgens [13]. Russell and Clermont [14] found that the regression of seminiferous tubular epithelium after depletion of the pituitary hormone was due to the discriminate degeneration of mid-pachytene spermatocytes and step 19 spermatids in stage VII. In the mammalian spermatogenic cycle, normally step-19 spermatids are found in stage-VII and VIII and are particularly androgen dependent [15,16]. A reduction in the number of Sertoli cells will adversely affect spermatogenesis as sertoli cells provide all or most nutritional and physical support for the developing germ cells[15]. Sperm count and motility, which were markedly reduced in the present study, show statistically significant correlation with the fertility [17,18].

It is evident that testicular function would be altered by reduced protein content [19]. Sialic acid acts as a lubricant?to facilitate the downward movement of sperm and to reduce friction among spermatozoa [20]. Mukherjee et al.[11] studied the effect of flutamide on testes and accessory organs of male rats and also found a reduction in sialic acid contents. Cholesterol is involved in steroidogenesis in testes. Increased level of cholesterol in testes is attributed to decreased androgen concentration, which resulted in impaired spermatogenesis [21]. Depletion in testicular glycogen was possibly attributed to the decreased number of post-meiotic germ cells [22]. Reduction in the fructose concentration in the seminal vesicle might be the result of a decreased secretory activity [23].

5 Acknowledgements

The authors are thankful to the Head, Dept. of Zoology and Chemistry; Prof. N. K. Lohiya, Coordinator, SAP, Dept. of Zoology, University of Rajasthan and Principal, S.S. Jain Subodh P. G. College, Jaipur (India) for providing necessary facilities and University Grants Commission, Regional Office, Bhopal (MP) India for financial support.

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Correspondence to: Dr. R.S. Gupta, Reproduction Physiology Section, Department of Zoology, University of Rajasthan, Jaipur-302004, India.

Received 2001-11-06    Accepted 2002-05-10