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Antifertility Effect of Chronically Administered Tabernaemontana Divaricata Leaf Extract on Male Rats

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Antifertility Effect of Chronically Administered Tabernaemontana Divaricata Leaf Extract on Male Rats Asian Pacific Journal of Tropical Medicine (2012)547-551 547 Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Medicine journal homepage:www.elsevier.com/locate/apjtm Document heading doi: Antifertility effect of chronically administered Tabernaemontana divaricata leaf extract on male rats Sachin Jain*, Ankit Jain, Pritesh Paliwal, Shailendra Singh Solanki 1Department of Pharmacognosy, College of Pharmacy, IPS Academy, Rajendra Nagar, Indore (MP) India 452012 ARTICLE INFO ABSTRACT Article history: Objective: Tabernaemontana To investigate the antifertility effect of chronically administered Received 22 December 2011 divaricata (T. divaricata) Methods: leaf extract on male rats. The effect of 50% ethanol extract Received in revised form 15 April 2012 T. divaricata T Accepted 18 July 2012 of leaves on reproduction was studied on male rats. he study was divided into Available online 20 July 2012 four groups of five animals each. The first groups (I) received vehicle alone to serve as control. ( ) The second, third and fourth groupsp.o II, II and IV of animals werep.o administered the leaf extract daily at 50 mg/kg body weight, .,100 mg/kg body weight, ., and 200 mg/kg body weight, Keywords: p.o Results: Tabernaemontana divaricata ., respectively, for a period of 60 days. Significant decreases in the weight of testes, epididymis, seminal vesicle and ventral prostate were observed. A dose related reduction in Anti-fertility the testicular sperm count, epididymal sperm count and motility, number of fertile male, ratio Male rats between delivered and inseminated females and numbers of pups were observed. The testis 50% ethanol extract showed a clear correlation between the dose and severity of lesions of seminiferous epithelium. Reproduction In general, the seminiferous tubules appear reduced in size with a frequently filled eosinophilic Hormone levels material. Spermatogenesis arrested at the secondary spermatocyte stage. Pachytene spermatocytes were undergoing degeneration. Disorganigation and sloughing of immature germ cell were visible. Leydinf cells were atrophied. No morphological changes were observed in Sertoli cells. Significant reduction in serum concentration of luteinizing hormone and testosterone were observed. No distinct change in serum FSH concentration was recorded. The final body weights of all groups Conclusiowere elevatedns: markedly. No alterations were recorded in anyT. divaricata hematologiocal parameters. It is concluded that the 50% ethanol extract of leaf produced dose related effect on male reproduction without altering general body metabolism. [8] 1. Introduction antifertility properties, was undertaken in our laboratory . As part of this research programme, we present in this paper Tabernaemontana antifertility efficacy of leaves of the plant divaricata (T. divaricata) The quest for the oral contraceptive agent that can control . A glabrous, evergreen shrub human fertility is as old as recorded history. Although 1.8-2.4 m in height with silvery grey bark and milky latex. [1] a wide variety of synthetic contraceptive agents are Leaves are simple, opposite, elliptic or elliptic-lanceolate, available, these cannot be used continuously due to their smooth, glossy green, acuminate and wavy margins; flowers [2, 3] severe side effects . Hence people are looking back to are white, sweetly fragrant in 1-8 flowered cymes at the age old tradition of using herbal medicines, which have bifurcations of the branches, lobes of corolla overlapping minimum side effects. India in general and Western Ghat to right in the bud[16]. It is used as thermogenic, anodyne, region in particular has enormous wealth of medicinal astringent, vermifuge, odonalgia and in treatment of plants. Presently, a major programme on systematic strangury, paralysis, arthralgia and melalgia. Flower juice investigation of medicinal plants for their phytochemical, mixed with oil alleviates burning sensation, cures eye biological and pharmacological properties, including sore and skin diseases, leaves juice applied to wounds to *Corresponding author: Sachin Jain, Department of Pharmacognosy, College of prevent inflammation and used in opthalmia,[3]. Flower Pharmacy, IPS Academy, Rajendra Nagar, AB Road, Indore MP INDIA 452012 Email: [email protected] contains Dregamine, 20-epiervatamine, tabernaemontanine, Tel: +919424443869, +917697177502 vobasine, voacangine, voacamine, flavonoid aglycones, Fax: +91 0731-4041627 Sachin Jain et al./Asian Pacific Journal of Tropical Medicine (2012)547-551 548 p.o. flavonol glycosides; isovoacristine, voaphylline- body weight/day; for 60 days. hydroxyindolenine, janetine (tetrahydrolivadine), N-methyl- A suspension of the extract was prepared in sterile distilled voaphylline (hecubine). Kaempferol, and leaves contains water (100 mg/mL) before administration. The required drug Dregamine, 20-epiervatamine, tabernaemontanine, was administered orally with a glass syringe fitted with a vobasine, voacangine, voacamine, flavonoid aglycones, feeding needle. flavonol glycosides, isovoacristine, -amyrin, lupeol and 毩 2.4. Sacrification schedule their acetates, 毬-sitosterol, coronaridine, apparicine, ervaticine (2-acyl indole derivative), ervatinine, hyderabadine, lahoricine, mehranine, stapfinine, voacristine, Twenty-four hours after their last dose, the rats were voharine and a dimeric alkaloid, conophylline 17-毬 weighed and sacrificed under light ether anesthesia. oestrdiol. A literature survey reveals that no systematic 2.5. Parameter approach has been made to study the antifertility activity of leaves of this plant. Therefore the present preliminary 2.5.1 Sperm motility and count investigation reports the reproduction effect of 50% ethanol T. divaricata extract of leaves on male rats. Cauda epidymal sperm motility and count, and testicular sperm count (maturing spermatozoa with head and tail) were made[12]. One hundred milligram of each tissue was minced 2. Materials and methods in 1 mL of physiological saline. For sperm motility, one drop of evenly mixed sample was applied to a glass slide under 2.1. Animal model the cover glass. The percent motility was determined by counting both motile and immotile spermatozoa per unit Ractus Twenty colony bred adult male albino rats ( area epididymal and testicular sperm count were also made norvegicus – 3 , Sprague Dawley strain), 3-5 months old expressed as million/mm of suspension. and weighing between 175 and 250 g with proved fertility 2.5.2. Fertility test were marked properly and housed two or three animals in polypropylene cages under controlled environmental Male rats were introduced to female, 200-300 g (male: conditions (12- h light: 12- h dark). They were fed pelleted female ratio, 1:2) at 17:00 h after 55 days of treatment. The standard rat feed (Ashirwad Food, Chandigarh, India) successful mating was confirmed in the forthcoming morning supplemented with soaked gram and wheat and allowed free from 56 to 61 day by vaginal plug and spermatozoa in the access to water. vaginal smear. The inseminated female were separated and allowed to deliver at term, and the number of pups delivered 2.2. Plant material and their characteristics were noted. T. divaricata 2.5.3. Body and organ weights One collection of the leaves from Indore, Madhya Pradesh India. The leaves were shade dried, The initial and final body weights of the animals were powered and soxhlated with ethanol (50% v/v) at 55-60 曟 recorded. The testes, epididymides, seminal vesicle and for 36 h. the solvent was distilled off under petroleum ether, ventral prostate were dissected out, freed from adherent benzene, chloroform and acetone to remove impurities left, tissues and blood, and weighed to the nearest milligram. if any during extraction. Thus the resulting mass was dried 2.5.4. Testicular histology under vacuum and kept at 4 曟. One (right) of the two testes of each animal was fixed in 2.3. Treatment protocol ’ Bouin s fixative solution, dehydrated in graded ethanol, cleared in xylene and embedded in paraffin wax. Section Animals were equally distributed into four treatment were cut at 5 micrometer, stained with Harris hematoxylin groups containing five in each. and eosin and observed under a light microscope. — Group I Animals in this group were given vehicle (Sterile p.o 2.5.5. Radioimmunoassay of hormones distilled water) alone . for 60 days to serve as vehicle — treated control; Group II Animals in this group were Blood sample were also collected for estimations of serum T. divaricata treated with leaf (50% EtOH) extract at the gonadotropins and testosterone by radioimmunoassay (RIA). p.o dose of 50 mg/kg body weight/day; . for 60 days; Groups Serum sample were separated by standard procedure and — T. divaricata – III Animals in this groups received leaf (50% stored at 20 for subsequent analysis. p.o 曟 EtOH) extract at the dose of 100 mg/kg body weight/day; . 2.5.6. Hematology for 60 days; Group IV-- Animals in this groups received T. divaricata leaf (50% EtOH) extract at the dose of 200 mg/kg The counts of RBC and WBC, hemoglobin, hematocrit, Sachin Jain et al./Asian Pacific Journal of Tropical Medicine (2012)547-551 549 and standard hematological indices (color index, mean Spermatozoa with shortened and thinned flagella were corpuscular hemoglobin, mean corpuscular hemoglobin present in the semen found in the vaginal smears of females, concentration and mean corpuscular volume) were which were cohabited with the treated males. All delivered determined from the blood collected
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