ABSTRACT- Objective: In this experiment adult male albino rats were treated with 50% ethanolic extract of Tephrosia purpurea fruits at the dose levels of 50, 100 and 200 mg/kg body weight for 60 days, to evaluate antifertility effects in search of a reversible male contraceptive agent from medicinal plants.
Materials and Methods: Body and organs weight of all treated animals was recorded, blood and serum were analyzed for hematological indices and clinical biochemistry. To observe effects on reproductive system of animal’s protein, fructose, sialic acid, ascorbic acid, and glycogen contents were estimated in their testes and sex accessory organs. The treated male rats were mated with proestrous females and sperm motility, sperm density was determined and FSH, LH and testosterone hormones were measured to evaluate the effects on fertility. For histopathological observation testes were fixed in Bouin's fluid, sections were cut at 6 µ and stained with Harris's Haematoxylin and eosin.
Results: Analysis of blood and serum revealed no significant effect after 60 days of the extract treatment. Body weight of the extract treated rat had no significant alteration, whereas the weight of reproductive organs was decreased significantly as compared to animals of control group. Protein, sialic acid, fructose contents and level of LH and testosterone hormones was decreased significantly after treatment in extract treated rats as compared to control.
Conclusions: The fertility, sperm density and motility were declined significantly in rats treated with the ethanolic extract of Tephrosia purpurea fruits. It is concluded that it might be due to androgen inhibition effects.
Key-words- Antifertility, Tephrosia purpurea, Rat, Testosterone

INTRODUCTION
Rapidly increasing population now becomes a global concern since it creates negative impact on social, economic development and health of human being. Uncontrolled population is the major reason behind poverty, unemployment and environmental pollution1. However, different types of contraceptives are available to control human fertility.
Currently available contraceptive are failed to check population and also have side effects². Various adverse effects like hormonal imbalance, headache, depression, weight gain have been reported by different contraceptive users^3-4. This situation demands the search of safe, cheap, orally effective and reversible new contraceptives.
The plants have been a source of folk medicine since ancient times^5-6. In last decades several plant species have been explored for the antifertility activities in many animal models including non human primates to develop a safe reversible male contraceptive agent for human use^7-9. The plant Tephrosia purpurea also known as ‘sharpunkha’ has been used for treatment of various human diseases like, bilious febrile attack, bronchitis, boils, pimples, diarrhea, gonorrhea, heart and spleen diseases^10-12 but no intention has been paid on use of fertility regulating effects of Tephrosia purpurea, therefore, the present investigation was designed to observe effects on the reproductive functions and general body metabolism of the ethanolic extract of the plant.

MATERIALS AND METHODS
Identification of the plant test material: Specimen voucher of Tephrosia purpurea was submitted to the taxonomist for the identification of the plant at the Department of Botany, University of Rajasthan, Jaipur (RUBL 211331).

Preparation of plant test material: The fruits of plant were shade dried and then crushed mechanically. Their 50% ethanolic extract was prepared according to the WHO protocol CG-04¹³.

Experimental animal model: Colony-bred healthy fertile male Wistar rats (Rattus novergicus) in the weight range of 150-200 gm were selected to the study. The animals were housed in polypropylene cages, measuring 430×270×150 mm. They were maintained under laboratory condition of temperature, humidity (60% ±1%) and 12 h light/ dark cycle. They fed rat pallated rats feed and water was provided ad libitum.

Ethical aspects: The CPCSEA (2006) and Ethical committee of Department of Zoology, University of Rajasthan, Jaipur guidelines were followed for the maintenance and experiments on animals¹⁴.

Experimental design: The animals were randomly divided into five treatment groups each consisting of 8 animals. Group- I served as a control and treated with distilled water for 60 days. The three animal Groups- II, III, IV were given extract at dose levels of 50, 100 and 200 mg/kg/body wt/day respectively for 60 days dissolved in distill water. Animals of Group-V were given the extract 100mg/kg/body wt/day dissolved in distilled water for 60 days followed by 30 days of recovery period. This group served as recovery group.

Sperm motility and density: To determine sperm motility and density the cauda epididymis was immediately removed after the autopsy. The results were determined by counting both motile and immotile sperm in Neubaur chamber. The sperm density was calculated in testes, epididymides and expressed in million per ml¹⁵.

Fertility Test: To check the fertility of all rats the fertility test was performed prior to the experiment and during 55 to 60 days. Male rats were cohabited with proestrous females in ratio of 1:2. The female rats were allowed to complete gestation period. Their vaginal smears were checked for positive mating. The inseminated female rats were separated and the numbers of litters delivered were recorded and litter size, fertility percentage was calculated.

Body and Organ Weights: The initial and final body weights of the animals were recorded. Reproductive and vital organs viz, liver, kidney, heart were dissected out, freed from adherent tissue and weighed accurately up to milligram level.

Histopathology: The testis was fixed in Bouin's fluid and processed, sectioned at 6 µ and stained with Harris's Haematoxylin and eosin and observed under a light microscope.

Serum Biochemistry: Serum was separated and stored at -20° C for total cholesterol16, serum alanine amino transaminase¹⁷, aspartate amino transaminase¹⁷, acid phosphatases¹⁸ and alkaline phosphatases¹⁹ analysis. FSH, LH and testosterone hormones level were assayed by radioimmunoassay²⁰.

Tissue Biochemistry: The testis, epididymis, seminal vesicles and ventral prostrate were dissected out and analyzed for Protein21, glycogen²², cholesterol²³, sialic acid²⁴, ascorbic acid²⁵ and fructose²⁶ contents.

STATISTICAL ANALYSIS
The data obtained from the above experiments were expressed in terms of mean ± SEM. The data were analyzed statistically by using Student’s “t” test and the significance of the differences was set as significant at p<0.05 and highly significant at p<0.001.

RESULTS
The blood hematology and serum biochemistry showed no significant changes which marks the non-toxic action of the extract treatment on metabolism of treated rats.

Effect on the body and reproductive organs weight: No dose regimen showed any significant change in the body weight of the rats in comparisons to control (Group-I) animals. However, weight of reproductive organs was decreased significantly while vital organs and body weight showed no significant changes (non significant data are not shown). The weight of body and organs found normal in the rat of recovery groups (Table-1).

Table I- Effects of Tephrosia purpurea on Body and Organ weight on treated male rats

Treatment	Body Weight (gram)		Organ Weight (mg/100 gm.b.wt.)
	Initial	Final	Testes	Seminal Vesicle	Cauda	Caput
Group-I	134.37±2.39	159.37± 2.74	778.75±6.70	446.50±5.43	66.45±2.65	76.75±2.11
Group-II	138.75±2.63 ns	162.50± 2.50 ns	661.87±5.17***	406.37±2.57***	62.86±1.90 ns	70.50±2.42
Group-III	147.90±2.83*	169.37± 3.07 ns	594.50± 2.27***	383.25± 2.19***	47.60±1.02***	65.12±0.95**
Group-IV	138.12±1.87 ns	165.62±2.57 ns	588.91±2.21***	372.59±1.32***	42.93±0.88***	60.91±0.61***
Group-V	137.50± 2.11 ns	155.00± 2.31 ns	784.74± 4.54 ns	442.49± 4.11 ns	62.87±0.69 ns	75.95±1.96 ns

(Mean ± SEM ) Group II, III, IV and V Compared with Group I.
***=Highly significant (p=0.001)**=Significant (p=0.01),*=Significant (p=0.05 ),ns= Non significant

Effect on sperm motility and density: The sperm density and motility decreased significantly (p<0.001) after treatment of the dose of plant. They were found normal after recovery period in recovery group (Fig. 1-2).

                         
                                                        Fig-1                                                                                                                                                 Fig-2

Biochemical changes: The ethanolic extract treatment of Tephrosia purpurea decreased levels of protein (p<0.001), sialic acid (p<0.001), fructose (p<0.001), glycogen (p<0.001) and cholesterol (p<0.05) levels in reproductive organs however, no significant change observed in vital organs. There was no significant change observed in recovery group (Table 2).

Table II- Effects of Tephrosia purpurea on tissue biochemistry on treated male rats

Treatment	Protein (mg/gm)		Sialic Acid (mg/gm)		Cholesterol (mg/gm)	Fructose (mg/gm)	Ascorbic Acid (mg/gm)	Glycogen (mg/gm)
	Testis	Cauda	Testis	Cauda	Testis	Seminal Vesicle	Adrenal	Testes
Group-I	244.16±7.60	269.81±7.70	5.86±0.12	5.97±0.10	7.80±0.41	5.29±0.13	5.32±0.66	7.40± 0.52
Group-II	217.74±1.45*	238.99±3.80**	4.33±0.20***	5.32±0.13**	7.82±0.45 ns	4.94±0.26ns	5.95±0.54ns	6.86± 0.51 ns
Group-III	197.83±1.41***	236.17±2.05**	3.73±0.11***	4.46±0.13***	7.76±0.50 ns	4.54±0.26*	5.32±0.24ns	7.39±0.38 ns
Group-IV	190.45±0.67***	230.18±0.93**	3.49±0.13***	4.35±0.08***	6.52±0.23*	4.30±0.10***	5.23±0.04ns	6.23± 0.15 ns
Group-V	242.82±3.73 ns	268.24±0.65 ns	5.78±0.06 ns	5.68±0.15 ns	7.57±0.45 ns	5.31±0.06 ns	5.20±0.06 ns	6.76± 0.62 ns

(Mean ± SEM ) Group II, III, IV and V Compared with Group I.
***=Highly significant (p=0.001),**=Significant (p=0.01),*=Significant (p=0.05 ), ns= Non significant

Blood and Serum profile of animals after the treatment: No significant change was observed in total cholesterol, serum alanine amino transaminase, aspartate amino transaminase, acid phosphatases and alkaline phosphatases in serum of all rats after the treatment at different dose levels in comparison to control rats (data are not shown).

Changes in Hormones level: The extract treatment caused significantly low level of testosterone hormone (p<0.05) and LH (p<0.05) in dose dependent manner. However, no significant change was observed in rats after the treatment in FSH as compared to control. The hormone levels were found normal in rats of recovery group (Fig: 3-5).


                                       Fig-3                                                                               Fig-4                                                                               Fig-5

Effects on Histology of testes and spermatogenesis: Histopathological observations of the testis after the Tephrosia purpurea treatment showed degenerated germinal epithelium of seminiferous tubules and reduced number of sperms in dose dependent manner. The histological study of control animals showed all successive stages of spermatogenesis in control animals (Photomicrograph-1).The lumen were filled with sperm, Leydig cells were present in between the tubules. Tephrosia purpurea treatment at 50 mg/kg (Photomicrograph-2) showed a few lesions affecting in tubules, while rats treated with 100 and 200 mg/kg/body wt/day (Photomicrograph-3,4) affected almost all tubules, however, spermatogenesis alters up to normal level in rats of recovery Groups-V (Photomicrograph-5) after recovery period.

DISCUSSION
The weight of testes and accessory reproductive organs was significantly decreased by the treatment of ethanolic fruit extract of Tephrosia purpurea as compared to control rats. In tissue biochemistry, the level of sialic acid was found significantly decreased. Sialic acid is essential for the structural integrity of acrosomal membrane of sperm. Therefore, the significantly decreased levels of sialic acid might affect the structure of spermatozoa and this may be the reason of decreased motility and fertilizing ability of sperm. The significantly declined glycogen content in testis reflects possibly decreased number of post meiotic germ cells, reflects reduced number of mature sperm in lumen. Similar results have been reported in rats earlier by²⁷ with different plant extract treatment.
A marked reduction in sperm motility and density was observed in treated rats when compared to control animals. In mating experiments the fertility of male rats was reduced and this might be due to decreased sperm motility and density of treated rats. The decreased level of protein in testis and other reproductive organs indicate suppressed male hormones level especially of androgens. A decreased level of cholesterol indicates the low synthesis of cholesterol in reproductive organs. This may be the reason behind the decreased synthesis of testosterone in testis after the treatment with different doses of plant.
Since testosterone is the most crucial for initiation, continuation of spermatogenesis and also to maintain accessory sex organs. The decreased level of testosterone indicates that the treatment suppress the synthesis of androgen level in treated animals. The testosterone level and spermatozoa production are regulated by LH and FSH. The testosterone is main androgen produced by Leydig cells under the influence of LH. LH together with testicular autocrine and paracrine factors responsible for the regulation and production male sex hormone and spermatogenesis in testis²⁸. Since testosterone hormone play key role in male reproductive system therefore, decreased level of testosterone in rats after 60 days of Tephrosia purpurea treatment suggests antiandrogenic effects of the treatment resulted decreased no. of mature sperm due to degenerative changes in germinal epithelium and germ cells. The decreased level of testosterone in rats followed with extract treatment possibly responsible to reduced proteins, fructose and sialic acid contents^29-31 in testis, epididymis and seminal vesicle; and inhibition of spermatogenesis can occur due to altered Leydig cell functions³². These results are similar with the results of with the treatment of Withanolide- A in adult male albino rats³³.

CONCLUSIONS
It can be concluded that oral administration of 50% ethanolic extract of Tephrosia purpurea decreased fertility of male rats might be due to the decreased level of proteins, fructose and sialic acid contents; and decreased level of testosterone and LH hormones leads to degenerative changes in testis and accessory reproductive organs resulted inhibition of sperm production and motility. Further study is needed in higher animal models to observe effects and to develop a male contraceptive from Tephrosia purpurea.

ACKNOWLEDGMENT
The Head and Coordinator, Centre for Advanced Studies, Department of Zoology, University of Rajasthan, Jaipur for laboratory facilities and UGC, New Delhi for financial support are greatly acknowledged.

REFERENCES

1. Mali PC, Chaturvedi M and Ansari AS. (Induction of reversible antifertility with a crude ethanol extracts of Citrullus colocynthis Schrad fruit in male rats). Pharmacology, 2003; 4(68): 38-48.
2. Mali PC. (Control of fertility in male wistar rats treated with hydro alcoholic extract of Withania somnifera fruits). Int J Pharm Bio Sci, 2013; 5: 13-21.
3. M Chaturvedi, PC Mali and VP Dixit. (Fertility regulation in male rats with the help of Echinops echinatus (Roxb.) root extract). J Phytol Res, 1995; 8: 115-118.
4. Mali PC, Singh AR and Verma MK. (Contraceptive effects of Withanolide- an in adult male albino rats). Adv Pharmacol Toxicol, 2015; 16: 31-44.
5. Luhadia G, Sharma DK and Soni PK. (Exploration of traditional medicinal plants for antifertility effects: A Review). Adv Pharmacol Toxicol, 2015; 16: 65-71.
6. Soni PK, Luhadia G and Sharma DK. (Antifertility activates of traditional medicinal plants in male with emphasis on their mode action: A Review). J Global Biosci, 2015; 4: 1165-1179.
7. Unny R, Chauhan AK and Joshi YC. (A review on potentiality of medicinal plants as the sources of new contraceptive principles). Phytomedicine, 2003; 10: 233-260.
8. Rajesh R, Chitra K and Padmaa MP. (Evaluation of in vivo Antioxidant Potential of the Aerial Parts of Aerva lanata Linn Juss in Streptozotocin Induced Oxidative Stress Rats). Medicinal Plant Research, 2016; 6: 15-21.
9. Sharma DK, Luhadia G, Soni PK and Mali PC. (Traditionally used Indian medicinal plants exhibits contraceptive activities: A Review). Int J Pharmacol Bio ScI, 2015; 9: 39-45.
10. Sangeetha B and Krishnakumari S. (Tephrosia purpurea (Linn.) pers: a folk medicinal plant ameliorates carbon tetrachloride induced hepatic damage in rats). Int J Pharma Bio Sci, 2010; 1: 98-104.
11. Dalwadi PP, Patel LJ and Patani V. (Tephrosia purpurea Linn (Sharpunkha, Wild Indigo): A Review on Phytochemistry and Pharmacological Studies). Indian J Pharm Biol Res, 2014; 2: 108-121.
12. Negi N, Kalia NA and Brar S. (Therapeutic potential of Tephrosia purpurea). J Chem Pharma Res, 2015; 7: 574-580.
13. WHO. Annual technical Report 1998 Special programme of Research Development and Research Training in Human reproduction, Geneva, World Health Organization.
14. CPCSEA. Committee for the Purpose of Control and Supervision on Experiments on animals, New Delhi, 2006: Indian Council of Medical Research.
15. WHO. Annual technical Report 1998 Special programme of Research Development and Research Training in Human reproduction. World Health Organization, Geneva 1983.
16. Zlatkis A, Zak B and Boyle AJ. (A new method for determination of serum cholesterol). J Lab Clin Med, 1953; 41: 486-492.
17. Reitman S and Frankel SA. (A Colometric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminase). Am J Clin Pathol,1957; 28: 56-63.
18. Gutman EB and Gutman AB. (Estimation of acid phosphatase activity in blood serum). J Biol Chem, 1940; 136: 201-205.
19. Fiske CH and Subbarao Y. (Colorimetric determination of phospholipids). J Biol Chem, 1925; 66: 375-400.
20. WHO, Guidelines for the Assessment of Herbal Medicine Programme on traditional Medicine doc. WHO/TRM/91.4. WHO, Geneva. Published by World Health Organization, 2003.
21. Lowry OH, Rosenbrough NJ and Far AL. (Protein measurements with the folin phenol reagents). J Biol Chem, 1951; 193: 265-275.
22. Montgomery R. (Determination of glycogen). Arch Biochem Biophys, 1957; 73: 378-389.
23. Mann T. The biochemistry of semen and the male reproductive tract, London UK, Mc Theun, 239.
24. Warren L. (The thiobarbituric acid assay of sialic acid). J Biochem, 1959; 8: 1971-1975.
25. Roe JH and Kuther CA. (Detection of ascorbic acid in whole blood and urine through the 2, 4, DNPH derivative of dehydroascorbic acid). J Biochem, 1943; 147: 399-401.
26. Foreman D. (A modification of the Roe procedure for determination of fructose in tissues with increase specificity). Analyt Biochem, 1973; 56: 584-590.
27. Chaturvedi M, Mali, PC and Dixit VP. (Antifertility effects of the roots of Echinops echinatus (Roxb.) in male rats). J Environ and Pollu, 1995; 2: 153–157.
28. Sharpe RM. (Testosterone and spermatogenesis). J Endocrinol, 1987; 113: 1-2.
29. Huang HFS, Pogach LM and Nathan E. (Synergistic effects of follicle stimulating hormone and testosterone on maintenance of spermiogenesis in hypophysectomized rats: relationships with androgen-binding protein status). Endocrinology, 1991; 128: 3152-3161.
30. Hermo L, Oko R and Morales CR. (Secretion and endocytosis in the male reproductive tract: a role in sperm maturation). Int. Rev. Cytol, 1994; 54: 106-189.
31. O’Donnell L, McLachlan RI and Wreford NG. (Testosterone promotes the conversion of round spermatids between stages VII and VIII of the rat spermatogenic cycle). Endocrinology, 1994; 135: 2608–2614.
32. Brooks DE. (Metabolic activity in the epididymis and its regulation by androgens). Physiol Rev, 1981; 61: 515-517.
33. Mali PC, Singh AR and Verma MK (Contraceptive effects of Withanolide- a in adult male albino rats). Adv Pharmacol Toxicol, 2015; 16: 31-44.

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