Antifertility effects of methanolic pod extract of Albizzia lebbeck (L.) Benth in male rats
R. S. Gupta, J. B. S.Kachhawa, R. Chaudhary
Reproduction Physiology Section, Department of Zoology, University of Rajasthan, Jaipur-302 004, India
Asian J Androl 2004 Jun; 6: 155-159
Keywords: Albizzia lebbeck; sertoli cells; spermatogonia; spermatocytes; leydig cells
Aim: To evaluate the antifertility activity of the methanolic pod extract of Albizzia lebbeck (L.) Benth in male albino rats. Methods: The methanolic pod extract of Albizzia lebbeck was administrated orally for 60 days at 50, 100 and 200 mg·kg-1·day-1 to male albino rats. Sperm motility and density in cauda epididymides were assessed. Biochemical and histological analysis were performed in blood samples and reproductive organs. Results: A. lebbeck pod extract brought about a significant decrease in the weights of testis, seminal vesicles, epdidymis and ventral prostate. The sperm motility and density were significantly reduced. There was a marked reduction in the numbers of primary spermatocytes, secondary spermatocytes and spermatids. The Sertoli cell count as well as its cross sectional surface area were significantly decreased. The Leydig cell nuclear area and the number of mature Leydig cells were also significantly decreased. The protein, glycogen and cholesterol content of the testis, the fructose in the seminal vesicles and protein in the epididymis were significantly decreased. The RBC and WBC counts, haemoglobin, haematocrit and blood sugar were within the normal range. Conclusion: The methanolic extract of A. lebbeck pods causes spermatogenic arrest in male albino rats.
Albizzia lebbeck (L.) Benth (Mimosoideae), commonly called Indian Siris or East Indian walnut, is one of the most promising fodder trees for semi-arid regions. The tree is used in folk remedies in bolus, enemas, ghees or powders for abdominal tumors. Reported to be pectoral astringent, rejuvenant and tonic, the siris tree is a folk remedy for boils, cough, flu and eye and lung ailments. The seed oil is used for leprosy and the powdered seed in scrofulous swelling. The ethanolic extracts of A. lebbeck leaves exhibited anticonvulsant activity . Albizzia julibrissin Durazz is reported to have sedative activity . The total alkaloidal fraction of Albizzia inopinata leaves has been shown to act on the central nervous system . The studies on the male antifertility effects of various medicinal plants have aroused much interest [4-7]. The present investigation was designed to evaluate the antifertility effect of the methanolic pod extract of A. lebbeck in male albino rats.
2 Materials and methods
2.1 Plant collection and extraction
The pods of Albizzia lebbeck were collected from Rajasthan University Campus from April to September 2001. It was identified and authenticated by Dr. N. J. Sarana, Associate Professor, Department of Botany, University of Rajasthan.
The shade dried pods (300 g) were powdered and extracted with 70 % methanol (b.p. 60 - 80 ) in a soxhlet for 24 hours. The methanol was removed under reduced pressure to obtain a viscous brown material, which, after washing with petroleum ether, turned to be a dark brown solid. Three hundred gram of the dried pods yielded 27 g of dark brown solid.
Male albino rats of Wistar strain, 16-18 weeks old weighing between 150 g -160 g obtained from Jamia Hamdard, Hamdard University, New Delhi. The rats were housed in plastic cages under standardized condition (12 h light/12 h dark; 25 3 and 35 % - 60 % humidity). Rat feed (Hindustan Lever Ltd.) and tap water provided ad libitum. Body weight of each animal in all groups was measured weekly to see the possible body weight loss throughout the experiments.
2.3 Experimental design
Male rats of proven fertility were divided into 4 groups of 10 rats each. The daily dose of the plant extract was freshly dissolved in 0.5 mL of distilled water and administered to each treated animal every morning for 60 days.
Group 1: Control rats received 0.5 mL/day
of the vehicle.
2.4 Fertility test
The mating tests were performed from day 55 to 60. The male rats were cohabited with proestrus females at a ratio of 1:3. The presence of vaginal plug and sperm in the vaginal smear in the next morning were considered positive mating. The mated females were sacrificed at day 16 of pregnancy to note the implantation sites.
2.5 Autopsy schedule
Twenty-four hours after the last dosing, the males were weighed and autopsied under ether anaesthesia and the reproductive organs i.e. testes, epididymides, seminal vesicles and ventral prostate were removed, cleared off fats and connective tissues, weighed and kept at -20 .
2.6 Sperm density and motility
The sperm density was assessed in cauda epididymides and testes: the tissues were mashed in physiological saline (0.9 % NaCl) and the sperm were counted in a Neubauer's counting chamber. Epididymal spermatozoa were obtained through a puncture at the cauda with a disposable hypodermic needle, dispersed in saline solution and the motility was determined with a haemocytometer.
2.7 Biochemical studies
Testicular tissues were assayed for protein, sialic acid, glycogen and cholesterol. Fructose was estimated in the seminal vesicle. Blood was analyzed for RBC and WBC counts, haemoglobin, haematocrit and sugar. Serum protein, cholesterol, triglycerides, phospholipids and HDL-Cholesterol were determined.
2.8 Histophathological studies
Tissues were fixed in Bouin's fluid, passed through ascending series of ethanol and then through xylene and embedded in paraffin. Tissues were sectioned at 5 m and stained with haematoxylin and eosin for the discrimination of the stages of spermatogenesis.
2.9 Cell population
The evaluation of the cell population was based on the calculation made for each cell type per cross section of the seminiferous tubule. The Sertoli cells, spermatogonia, primary spermatocytes (preleptotene and pachytene), secondary spermatocytes and round spermatids were counted under high magnification. These crude counts were corrected by using the Abercrombie's correcting factor. Percent of tubule containing step-19 spermatid was counted. Interstitial cell types such as fibroblast and mature and degenerating Leydig cells were estimated applying a differential count which were statistically varified by the bionomial distribution. Mean seminiferous tubular diameters were determined by measuring and tracing an average of 100 selected seminiferous tubules. The Leydig cell nuclei area and Sertoli cell area were measured at 800 magnification.
2.10 Statistical analysis
Data are expressed as meanSEM and are analyzed by using the Student's t-test.
3.1 Body and organ weights
The oral administration of A. lebbeck pod extract at all dose levels did not cause significant change in the body weight, but the weights of testes, epididymides, seminal vesicle and ventral prostate were significantly reduced (P < 0.01) (Table 1).
Table 1. Effect of Albizia lebbeck pod extract on body & organ weights and testis histometry. cP < 0.01 vs control.
3.2 Sperm motility, density and fertility
The sperm motility in cauda epididymides was decreased by 65.87 %, 71.45 % and 78.11 % in 50, 100 and 200 mg/kg dose levels, respectively, in comparison to the control (P < 0.01). The extract reduced the fertility of male rats by 100 % at all three dose levels (Table 2).
Table 2. Effect of Albizia lebbeck pod extract on sperm motility, density and fertility in rats. cP<0.01 vs control.
Protein contents of testes, cauda epididymides, seminal vesicle and ventral prostate were significantly reduced (P < 0.01) and the content of sialic acid showed a significant decrease in testes, cauda epididymides, seminal vesicle and ventral prostate (P < 0.01). The testicular glycogen and cholesterol levels and the seminal vesicle fructose level were also significantly reduced (P < 0.01) (Table 3). The RBC and WBC counts, the haemoglobin, the haematocrit, the blood sugar and the serum protein, cholesterol, triglycerides, phospholipids and HDL-cholesterol levels were within the normal range.
Table 3. Effect of Albizia lebbeck pod extract on biochemical parameters in male rats. cP < 0.01 vs control.
3.4 Cell population
A. lebbeck pod extract brought about a significant reduction in most of the seminiferous tubular cell types. The spermatogonia was decreased by 48.25 %, 46.13 % and 50.49 % at 50, 100 and 200 mg.kg-1.day-1 dose levels, respectively. The preleptotene and pachytene spermatocytes were reduced significantly (P < 0.01). The secondary spermatocytes also showed diminished counts by 46.77 %, 52.40 % and 53.93%, respectively. Rounded spermatid cell population was markedly decreased by 53.22 %, 59.60 % and 51.41 %, respectively. The number of Sertoli cells as well as it cross sectional surface area showed a notable depletion (P < 0.01) at all three dose levels. The diameter of seminiferous tubules reduced markedly (P < 0.01). The step-19 spermatid stage containing tubules were reduced by 58.69 %, 68.71 % and 72.28 %, respectively as compared with controls. The number of mature Leydig cells decreased significantly (P < 0.01), whereas the fibroblast and degenerating Leydig cells remained unaffected at all the dose levels (Tables 1 and 4).
Table 4. Effect of Albizia lebbeck pod extract on testicular cell population in rats. cP < 0.01 vs control.
Methanolic pod extract of A. lebbeck significantly affected the male reproduction. In the present study, the reduced testicular and accessory sex organ weights, the decreased seminiferous tubular diameter and the spermatogenic arrest indicate a wide spread damage , which could be due to reduced protein contents in these organs. Similar results have been observed with Semecarpus anacardium fruits  and Carica papaya [5, 10, 11].
The protein synthesis and concentration in the accessory sex organs are androgen dependent. Administration of antiandrogens caused a significant decrease in the protein concentration of epididymis, which was restored to normal levels after testosterone therapy . Reduced sialic acid contents of the testis and accessory organs may alter the structural integrity of acrosomal membrane, which ultimately affects the metabolism, motility and fertilizing capacity of spermatozoa . The low glycogen content in the testis after A. lebbeck administration is possibly due to the inhibition of phosphorylase activation or the depletion of certain other enzymes which could block androgen synthesis . Cholesterol is the precursor in the synthesis of steroid hormone and the requirement of cholesterol for normal testicular activity has been well established. Reduction in the fructose concentration in the seminal vesicle might be the result of a decreased secretory activity .
Analysis of stage-specific concentration of androgen and androgen binding proteins suggests that stage VII and stage VIII (step-19 spermatid containing stages) of the seminiferous epithelial cycle are particularly androgen-dependent . In the present study, it was shown that Sertoli cells were quantitatively and morphologically altered, which might be the results of androgens lack . The control of spermatogenic cycle is mediated by Sertoli cells which regulate cell cycle kinetics and influence the differentiation of primordial germ cells . In conclusion, the methanolic pod extract of A. Lebbeck suppresses spermatogenesis and alters the structure and activity of the Sertoli and Leydig cells in a dose-dependent manner.
The authors are thankful to the Head, Department of Zoology, Prof. N.K.Lohiya Coordinator SAP, Department of Zoology, University of Rajasthan, Jaipur for providing necessary facilities and CSIR, New Delhi, India for financial support.
VS, Chopde CT, Deshmukh VK. Anticonvulsive activity of Albizzia lebbeck,
Hibiscus rosa sinesis and Butea monosperma in experimental
animals. J Ethnopharmacol 2000;71: 65-75.
Correspondence to: Dr.
R. S. Gupta, TR-03 Teachers Hostel, University of Rajasthan, Jaipur-302