Evaluation of contraceptive efficacy of Cassia angustifolia and Kigelia pinnata fruits extract in male albino ratsINTRODUCTIONUncontrolled growth of human population is a threat to human existence, especially in developing countries like India. The current growth rate of population in India exhibit that India will be the most popular country in the near future (Khan ME et at, 1992; Umadevi et al, 2013 ) Unintended pregnancies creates extra burden on the community and causes poverty socio-economic problems and pollution. Therefore fertility control is the only means to maintain sustainable development (Aladakatti et al, 2010). Although, many contraceptive methods are available to control fertility these includes natural, chemical, hormonal barrier and immunological. However, yet no suitable method has emerged which is effective and free from side-effects (Gupta et at, 2003). The oral containing estrogen and progesterone have been used effectively in women, since a long, have some risk factors, also triggered the need to develop a new safe method. A large number of plants species have been explored around the world including India and China in search to develop a reversible male contraceptive agent since man relied on plant products as sources of therapeutic agents without causing any side effects (Deshpande et al, 1980; Mathur, 1985; Griffin, 1996; Lampiao, 2013; Muthulakshmi et al, 2013 Natarajan et al 2014).REVIEW OF LITERATUREThere is a great need to support individuals in family-planning since uncontrolled worlds population creates negative impact on environment, economic growth and poverty especially in underdeveloped countries (Aladakatti et al., 2010). About 90% of the worlds contraceptive users are women. Though considerable progress has been made in the development of highly effective, acceptable and reversible methods of contraception in females, progress and possibilities on males are still slow and limited (Beckman et al 1996; Mali et al 2011) World health organizations and pharmaceutical companies continue to financially support to develop a suitable `methods for contraception to check unintended pregnancies and side effects (El-Kashoury et al., 2009; Shweta et al., 2011).The ethanolic extract of Maytenus emarginata was found to have reversible contraceptive efficacy in male albino rats (Chaudhary et al., 2010). The methanolic extract of Piper betle was also found effective for its antifertility activity in female albino rats (Ranjan et al., 2013). A large number of natural plant products have been explosed to investigate effects on spermatogenesis (Ghosh et at, 2002). Since the existing male contraceptives have side effects or drawbacks in uses (Hiremath et al, 2000; Gupta et al, 2006) and the development of a method for fertility control in male can provide tremendous social and public health benefits. Yanhe et al, (1998) observed decreased sperm count, and changes in seminiferous tubules of the testis. Carica papaya have been used by female to check pregnancies possess antifertility effects (Natarajan et al, 2014). For centuries herbal potions and pessaries have been concocted with the goal of preventing, and or disrupting pregnancy. However, taking herbal contraceptives may risk exposure to health concerns, not always 100% effective, and should not be taken with prescribed medication or having an existing health problem. Taking herbal contraceptives long term may or may not cause a health concerns (Shweta et al., 2011).On the basis of survey of literature of medicinal importance following two medicinal plants has been selected for the present study: 1. Cassia angustifolia, also known as Senna, Indian senna (Family Caesalpinaceae ). It is cultivated in dry lands of southern and western India, and indigenous to Arabia. Useful parts of this plant are leaves, fruit and pod. It is useful in habitual costiveness. Cassia angustifolia leaves extract with effective concentration and administration has potential uses of antifertility activity with the advantage of no toxicity. It is used as expectorant, wound dresser, antidysentric, carminative and laxative. It is also Useful in loss of appetite, hepatomegaly, spleenomegaly, indigestion, malaria, skin diseases, jaundice and anaemia. Leaves of plant are made into a paste, and applied to various skin diseases. The effects of C.angustifolia established in histological preparation and also were observed in seminiferous tubules of testis, seminiferous epithelium underwent extensive vaculation due to premature developmental cells (Dhanapal et al, 2012). 2. The Kigelia pinnata (Family Bignoniaceae) also known as K. africana Sausage Tree, Worsboom,etc characteristically shaped fruits, has a variety of traditional medicinal uses throughout Africa, India and the Middle East has been widely cultivated .The plant Kigelia africana extract has its phytochemical effects on microorganisms and cancer cells.It is used to cure many ailments including skin problems and cancer, gynaecological problems, wound healing, malaria, and others. (Saini et al., 2009 ).hypothesis It is supposed that the plant extract of Cassia angustifolia and Kigelia pinnata may be used in male fertility control in albino rats.

aim and Objectives of the study The present investigation is planned to evaluate safety and reversible contraceptive efficacy of methanolic extracts of Cassia angustifolia and Kigelia pinnata to develop an orally effective and reversible male contraceptive with the following objectives.1. Effects of the drug on morphology and functions of testicular and epididymal sperms in rats.2. To isolate Sertoli cells and Leydig cells to evaluate the morphological changes if any with the treatment in rats.

3. To screening of the toxic effects, if any, following drug administration.1.Identification of plantsSpecimens voucher of Cassia angustifolia and Kigelia pinnata will be collected around the Jaipur district, India deposited at Herbarium of Department of Botany, University of Rajasthan, Jaipur for identification and herbarium number.

2.Preparation of extractsFresh fruits of Cassia angustifolia and Kigelia pinnata will be collected and shade dried, grind in a mixture soaked in 50% methanol for overnight, boiling it at 55 C - 60C for 24 hours and finally filtered with gauze. The filtrate will concentrated under the reduce pressure at 50 5 C to obtained crystal powder to use for experiment (WHO 1983).

3. Animal Model Colony-bred, healthy adult (4-5 months old) male albino rats (Rattus norvegicus) of the Sprague-Dawley strain, weighing between 150- 200 g will be used. The animals will be housed in polypropylene cages, measuring 430270150 mm, under controlled environmental conditions with provision of a 12 h light: 12 h dark regimen. The animals will be fed a platted standard rat chow supplemented with soaked gram and wheat, water will be provided ad libitum.4. Treatment ProtocolRats of similar body weight, size and age will be grouped in five groups two main. Experiments will be carried out during the course of study, to observe antifertility effect mode of action/effects nature of the extract and reversibility effects. EXPERIMENT- IAntifertility study of Cassia angustifolia fruits

In this experiment antifertility activity of methanol extract will be carried out. The animals will be divided into five treatment groups, each consisting of 10 animals in each.Group-A: The animals of this group will be given vehicle (sterile distilled water) alone orally for 60 days to serves as vehicle controls.

Group-B: The animals of this group will be treated with Cassia angustifolia extract 50 mg/kg.b.wt./day for 60 days.

Group-C: The animals of this group will be treated with Cassia angustifolia extract 100 mg/kg.b.wt./day for 60 days.

Group-D The animals of this group will be treated with Cassia angustifolia extract 200 mg/kg.b.wt. /day for 60 days.Group-E: The animals of this group will be administrated 100 mg/kg.b.wt./day for 60 days and will be kept for a recovery period of 30 days. EXPERIMENT- II

Antifertility study of Kigelia africana fruitsIn this experiment antifertility activity of methanol extract will be carried out. The animals will be divided into five treatment groups, each consisting of 10 animals in each.

Group-A: The animals of this group will be given vehicle (sterile distilled water) alone orally for 60 days to serves as vehicle controls. Group-B: The animals of this group will be treated with Kigelia pinnata extract 50 mg/kg.b.wt./day for 60 days. Group-C: The animals of this group will be treated with Kigelia pinnata extract 100 mg/kg.b.wt./day for 60 days. Group-D The animals of this group will be treated with Kigelia pinnata extract 200 mg/kg.b.wt. /day for 60 days. Group-E: The animals of this group will be administrated 100 mg/kg.b.wt./day for 60 days and will be kept for a recovery period of 30 days. 5. Schedule of SacrificeAfter 24 hrs of from the last dosing/recovery for each group, the animals will be weighed and sacrificed.6. Study parameters (1) Hematology The counts of RBC and WBC (Lynch et al.,1960) hemoglobin, hematocrit and standard hematological indices, namely color index, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) will be recorded . (2) Serum Biochemistry Serum alanine amino transaminase ( SGOT) (Reitman and Frankel, 1957),Aspartate amino transaminase (SGPT) (Reitman and Frankel,1957),Acid Phosphatases (Gutman and Gutman, 1940) and Alkaline Phosphatases (Fiske and Subbarow, 1925).(3) Tissue Biochemistry

The testis, cauda epididymis, seminal vesicles and ventral prostate will be dissected out, freed from adherent tissues and weighted at nearest milligram. Protein (Lowry et al, 1951), Glycogen (Montgomery, 1957). Cholesterol (Mann, 1964), Sialic acid (Warren, 1959), Ascorbic acid (Roe and Kuether, et al,1943) and Fructose (Foreman et al, 1973) will be estimated in right side of testis and other accessory reproductive organs.(4) Hormone Assay Blood samples will be also collected and serum will be separated for the estimation of FSH, LH and testosterone by radioimmunoassay (WHO, 2003) .(5) Body and Organ Weights The initial and final body weights of the animals will be recorded. Testes, cauda epididymis, seminal vesicles and ventral prostrate will be dissected out, freed from adherent tissues, and weighed to the nearest milligram on an electronic balance.(6) Fertility Test Successful mating will be carried out with all the animals 5 days prior to sacrifice (male female ratio 1:2). The mated females will be allowed to complete the gestation. The litter size will be recorded and percent fertility will be calculated (WHO, 1983).(7) Sperm Motility and Density Sperm motility in cauda and density in cauda and testes will be measured by routine procedure and express as millions/mm3 suspension (WHO, 1983).(8) Histology

Contralaterial side of the testis, epididymis, vas deferens, seminal vesicle, ventral prostate, will be fixed in Bouin's fluid, dehydrated in graded ethanol, cleared in xylene, and embedded in paraffin wax. Sections are cut at 5 will be stained with Harris's hematoxylin and eosin and observed under a light microscope.TEM will be prepared to observe effects on the cellular level.(9) Isolation of Sertoli cells and Leydig cellsSertoli cells from testes of rats will be isolated according to the procedure of Welsh and Wiebe (1975) and will be used for morphological comparison with Sertoli cells and Leydig cells of control treated vehicles (Sharma et al; 2006).7. Ethical AspectsThe study will be carry out under the supervision of the ethical committee of the Department of Zoology, University of Rajasthan, Jaipur, India and CPCSEA, (2006) guidelines will be followed for the maintenance of experimental animals. 8. Statistical analysis Data are expressed as mean S.E. and analyze for statistical significance by using student's t test. The data will be considered as significant and highly significant at p 0.001, respectively (Gupta, 1978). Significans of the workThe present investigation is planned to evaluate safety and reversible contraceptive efficacy of 50%methonolic extracts of Cassia angustifolia and Kigelia pinnata fruits with the following objectives

1. To establish research and development priorities to develop a new methods for regulation of fertility in males.

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