Effect of Stephania hernandifolia leaf extract on testicular activity in rats

D. Jana, R. Maiti, D. Ghosh

Asian J Androl 2003 Jun; 5: 125-129             

Keywords: Stephania hernandifolia; androgens; enzymes; testosterone; germ cells

Abstract

Aim: The testicular inhibitory effect of the aqueous fraction of methanol extract of Stephania hernandifolia leaf was studied in male Wistar rats. Methods: The supernatent and the precipitate part of aqueous fractions of the methanol extract of the leaf were gavaged separately to rat at a similar dose of 200 mg/mL per 100 g body weight per day for 28 days. After cessation of treatment, various observations were conducted. Results: In both treated groups, there were significant decreases in the relative weights of the sex organs, the testicular key androgenic enzymes activities, the plasma level of testosterone, the number of different germ cells at stage VII of seminiferous epithelial cell cycle and the seminiferous tubular diameter in comparison to the controls. Neither of the parts had somatic, renal and hepatic toxicity. This study suggested that the active molecules present in the aqueous fraction of methanol extract of Stephania hernandifolia leaves might be steroids as indicated by thin layer chromatography using specific staining substance for steroid molecules. Conclusion: In rats, the aqueous fraction of methanol extract of the S. hernandifolia leaves possesses certain testis-inhibitory substances, which may be steroid-like agents.

1 Introduction

Many plants are known to possess antispermatogenic or male antifertility effect [1-3]. In West Bengal state of India, the folk women use the crude aqueous extract of Stephania hernandifolia (family Menispermaceae) leaves for the prevention of pregnancy. This plant grows maximally in the rainy season in the plain as well as hilly areas. We previously reported that the extract of S. hernandi-folia leaves inhibited ovarian gametogenesis [4] and testicular steroidogenesis and gametogenesis [5]. It is our intent to screen the active substance(s) present in the leaves of S. hernandifolia. The present experiment had been conducted to study the testicular inhibitory effect of the precipitate and the supernatant parts of the aqueous fraction of methanol extract of S. hernandifolia leaves.

2 Materials and methods

Fresh green leaves of S. hernandifolia were dried in an incubator for 2 days~3 days at 40 and powdered using electrical grinder. The powdered plant material (150 g) was extracted with 500 mL of methanol for 18 h at 25 in a Soxhlet apparatus and a deep green methanol extract was obtained. The solvent of the extract was removed under reduced pressure to yield a dark green residue. The residue was dissolved in 200 mL distilled water and heated at 50 for 12 h under reflux on a steam bath to form a deep brown aqueous fraction of methanol extract. It was then mixed with absolute alcohol at a ratio of 1:2, shaked vigorously for 2 h and the container was allowed to stand over night at 12 ~ 14 . The supernatant and the precipitate parts were separated and dried separately in a lyophilizer. The dried supernatant was dissolved in olive oil and the precipitate, in distill water, both at a concentration of 200 mg/mL.

2.2 Animals and treatment

Twenty four healthy male Wistar rats (Joy Tara Traders, Kolkata, India), 3 months of age, weighing 95 g ?5 g were selected for acclimation for 15 days in this laboratory (12 h dark and 12 h light, 28 2 ) prior to experimentation. They were provided with standard animal chew and water ad libitum. Animals were divided at random into 4 groups of 6 animals each. Animals of the Group 1 were gavaged with the olive oil solution at a dose of 200 mg/mL per 100 g body weight per day for 28 days, Group 2 with the water solution at a similar dose and duration and Group 3 (gavaged with olive oil) and Group 4 (gavaged with water) served as the controls. At day 29, animals were sacrificed by light ether anesthesia after taking the body weight. Blood was collected using heparinized syringe and the plasma was separated and kept at -20 for testosterone assay. Both testes, seminal vesicles, epididymis, prostate, liver and kidney were dissected out, trimmed off the attached tissue and the wet weights recorded. The liver, kidney and right testis of each animal were kept at -20 for enzymatic studies. The left testis of each animal was placed in Bouin's fluid for histological studies.

2.3 Observations

Testicular D5,3b-hydroxysteroid dehydrogenase (HSD) and 17b-HSD activities were measured according to the method of Talalay [6] and Jarabak et al. [7], respectively. One unit of enzyme activity was the amount causing a change in absorbance of 0.001/min at 340 nm.

The liver and kidney tissues were homogenized separately in a Potter Elvijhem homogenizer using ice cold homogenizing medium (0.22 mol/L Tris-HCl buffer pH 7.5) at a tissue concentration of 20 mg/mL for estimation of alkaline phosphatase (ALP) and acid phosphatase (ACP) activities according to the methods of Malamy and Horecker [8] and Vanha-Pertulla and Nikkanen [9], respectively. Glutamate oxaloacetate transminase (GOT) and glutamate pyruvate transaminase (GPT) activities in these organs were also estimated according to the method of Goel [10]. Plasma level of testosterone was measured by the immunoenzymatic method by ELISA (Merck, Japan) [11].

Paraffin blocks of testis were serially cut at 5 mm thickness and stained with haematoxyline and eosin. Quantitative analysis of gametogenesis was carried out at stage VII of seminiferous epithelial cell cycle according to the method of Leblond and Clermont [12]. The prepared slides were placed under high power objective in a phase contrast microscope and with the help of the occular micrometer; the seminiferous tubular diameter (STD) was measured.

The presence of steroid or steroid-like compounds was evaluated by thin layer chromatographic study using specific stain for steroid bio-molecules [13].

2.4 Statistical analysis

Data were expressed in meanSEM. For statistical analysis of data, ANOVA followed by multiple two-tail comparison t-test was used and P<0.05 was considered significant.

There was no significant difference of body growth among these four groups. The relative testis, prostate, seminal vesicle and epididymal weights were significantly decreased in both treated groups in comparison to the controls, which were significantly more marked in the supernatant than those in the precipitate fraction treated group. The relative hepatic and renal weights were not significantly changed in both treated groups (Table 1).

Testicular D5,3b-HSD and 17b-HSD activities were decreased significantly in the supernatant treated group in respect to the precipitate treated group as well as to the control group, although the said enzymes activities were also significantly decreased in the precipitate treated group in comparison to the controls (Table 1).

The ACP, ALP, GOT and GPT activities in liver and kidney were not significantly altered in both treated groups compared with the control groups (Table 2).

Table 2. Effect of supernatant and precipitate parts of aqueous fraction of methanol extract of S. hernandifolia leaf on number of germ cells at stage VII of seminiferous cycle (n), STD (m100), ACP and ALP (mg of PNP/mg of tissue/h), GOT and GPT activities (unit/mg of tissue) in liver and kidney in rats (n=6). ^bP<0.05, compared with the corresponding controls, ^e P<0.05, compared with the corresponding controls and precipitate treated group.

Plasma level of testosterone was decreased significantly in both the treated groups as compared with the controls and the decrease was significantly more marked in the supernatant treated group than that in the precipitate treated group (Table 1).

Quantitative study of germ cells at stage VII of seminiferous epithelial cell cycle revealed that treatment with precipitate fraction resulted a significant diminution in the number of ASg, pLSc, mPSc and 7Sd in respect to their control. After supernatant fraction treatment there was a significant reduction in the number of ASg, pLSc, mPSc and 7Sd in comparison to their controls as well as to the precipitate fraction group (Figure 1~Figure 4) (Table 2). Histrometric studies indicated that STD was decreased significantly in both treated groups and was even significantly more marked in the supernatant than that in the precipitate treated group (Figure 1~Figure 4) (Table 2).

Figure 1. Testicular cross-section in distilled water control at stage VII seminiferous cycle showing normal spermatogenesis with normal STD (400).

Violet spots were shown on thin layer chromatographic plates, which indicated the presence of steroid-like substances in both the supernatant and precipitated fractions. The color intensity of violet spots was more prominent in plates containing the supernatant part in comparison to the precipitate part.

4 Discussion

The decreased weights of testis and the secondary sex organs and the inhibition of testicular D⁵, 3b-HSD and 17b-HSD activities by the plant extracts may be the result of pituitary inhibition [14]. These findings were in accordance with the fact that the plasma testosterone levels were depressed in both treated groups.

Quantification of different germ cells at stage VII was carried out, as in this stage all varieties of germ cells were present and the cellular association was composed of elements positioned equidistant in the entire process of spermatogenesis [12]. The reduction in different variety germ cells in both treated groups may also be due to pituitary inhibition [15]. The diminution in STD, which is one of the indicators of plasma testosterone [5], in both treated groups supported the presence of a low plasma testosterone level.

Both parts of the aqueous fraction of methanol extract of the S. hernandifolia leaves did not exert toxic effect on body growth; there was no significant alteration in the hepatic and renal weights as well as the hepatic and renal ACP, ALP, GOT and GPT activities in both treated groups, suggesting that the plant extract was practically without metabolic toxicity [16].

Our result indicated that the supernatant fraction contained more bioactive substance having anti-testicular activity than the precipitate. In TLC analysis with a specific stain for steroids, it was shown that both parts of the aqueous fraction of methanol extract displayed steroid positive molecules and the color intensity for steroid was denser in the supernatant than that in the precipitate part. In conclusion, in rats the aqueous fraction of methanol extract of the S. hernandifolia leaves possesses testis-inhibitory substance, which may be a steroid-like agent. Further research is necessary to disclose the nature of the bioactive substance.

The authors gratefully acknowledge the financial assistance from the Indian Council of Medical Research (ICMR), New Delhi, Project No. 5/10/33/99/RHN to DG.

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Correspondence to: Dr. Debidas Ghosh, Reproductive Endocrinology and Family Welfare Research Unit, Department of Human Physiology with Community Health, Vidyasagar University, Midnapore, 721 102 West Bengal, India.
Tel: +91-322-260 558 ext. 450/454, Fax +91-322-262 329
E-mail: debidasghosh@yahoo.com
Received 2003-11-04   Accepted 2003-02-27