Curcuma longa and Azadirachta indica: Herbals for Fertility Control

 

Rahul Shivarkar1* and Swati Patil2

1Sinhgad College of Pharmacy (Poly), Vadgaon (Bk), Pune-411041.

2Prin. K. M. Kundnani College of Pharmacy, Mumbai-400005.

 

ABSTRACT:

The increment of population raises many sufferings like lack of food, water, energy and raw material supply. India's population is growing fast, posing a threat to the country's sustained development.  Most of the currently used methods of birth control are associated with certain risk and side effects. Herbal medicines are being used by about 80% of the world population primarily in the developing countries for primary health care. They have stood the test of time for their safety, efficacy, cultural acceptability and lesser side effects. In the present investigation the ethanolic extract of Curcuma longa rhizomes and Azadirachta indica leaves alone and in combination were evaluated for reversible antifertility activity in male wistar rats. Administration of extracts alone and in combination for four weeks significantly affects the sperm count, viability, motility and serum testosterone level. Whereas, combination of extracts showed most significant results in both the treatment and recovery groups. Therefore, it may be concluded that the combination of extract posses significant reversible antifertility activity.

 

KEYWORDS: population, antifertility, sperm count, testosterone

 

 

INTRODUCTION:

The population explosion is one of the major problems of present era in the world1. Herbal drugs constitute only those traditional medicines which primarily use medicinal plant preparations for therapy. Thus, the quest for developing an antifertility agent from a plant source in the regulation of male fertility appears to be an attractive proposition. Therefore there is a need to explore plants for their antifertility potential in the male, with the hope of developing a contraceptive for use in men.

 

Curcuma longa (C. longa) belonging to family Zingiberaceae has been used for thousands of years as consumable herb and spice in India2. Curcumin has a selective sperm-immobilizing effect and this compound may have potential clinical applications as a novel intra-vaginal spermicidal agent for contraception and HIV prevention3.C. longa treatment causes marked alterations in the male reproductive organs and that the alterations are reversible after cessation of treatment and may have a potential in the regulation of fertility in the male4.  Azadirachta indica (A. indica) belonging to family Meliaceae has been used in our traditionally as general antiseptics, antimicrobial, treatment of urinary disorders, fever and bronchitis, skin diseases, septic sores, infected burns, hypertension and inflammatory diseases5.As an antifertility agent it appears to have potential both including male infertility and as a vaginal and oral contraceptive, where it also prevents ovum implantation6,7. Aqueous extracts of old and tender leaves of Azadirachta indica have potent spermicidal effect8.

In spite of the continuing need no safe and reversible chemical agent is known that can adequately control fertility without producing toxic effect9. Hence, there is a need for additional reversible male contraceptive methods, especially in the developing world, where population overgrowth is a problem. New male reversible methods are considered to be an additional tool to reduce over population. Herbal drugs are effective as well as free from side effects having a wide potential as antifertility agent. Literature survey revealed the plants like C. longa and A.indica individually can act as newer option for fertility control. Therefore, the present research work was planned to evaluate reversible male antifertility activity of an oral suspension containing combination of ethanolic extract of Curcuma longa and Azadirachta indica.

 

MATERIALS AND METHODS:

Collection of plant material

The rhizomes of C. longa were purchased from local market Mumbai, India, while the leaves of A. indica were collected from, Pune, India. The C. longa rhizomes and A.indica leaves were authenticated at the Agharkar Research Institute, Pune (Auth09-84 andAuth09-83 respectively).

 

Preparation of extracts and suspension:

Shade dried rhizomes and leaves were coarsely powered and extraction of the powdered plant materials was subjected to continuous hot extraction method by using  90% ethanol as solvent and  successive extraction in Soxhlet extractor using different solvents in the order of their increasing polarity namely petroleum ether, benzene, chloroform, acetone, ethanol and water. The ethanolic extract of C. longa rhizomes (EECI) and A.indica (EEAI) was concentrated in vacuum under reduced pressure using rotary evaporator (Buchi, Flawil, Switzerland) and stored in the refrigerator until used. Suspension of each extract and their combination was prepared in 2% Carboxy methyl cellulose (CMC).

 

Animals

Male wistar rats (150-200 g) were used for experimental study. The animals were maintained under standard laboratory conditions at temperature 23 ± 20C with relative humidity 55 ± 10 % and 12 hrs light and dark cycle throughout all the experiments. Animals had free access to food and water ad libitum. All the experimental procedures and protocols used in this study were reviewed and approved (Protocol no.091013) by the Institutional Animal Ethics Committee.

 

Chemicals:

Sodium Carboxy methyl cellulose was procured from Jineshwar Pharma Chem, Mumbai and Cadmium Chloride was procured from Bliss Pharmaceutical Pvt. Ltd, Mumbai.

 

Preliminary phytochemical screening

The extracts of C. longa and A. indica screened for the presence of alkaloids, tannins volatile oils, resins, phenolic, triterpens, glycosides, fatty acids, saponins, flavonids and carbohydrates.

 

Evaluation of male reversible anti fertility activity:

The rats were divided into eight groups; n=6. All the rats were treated daily for four weeks as follows:

Group 1 served as control (normal group) and was daily administered 2 % CMC.

Group 2 served as standard and was daily administered cadmium at a dose of 1mg/kg/i.p.

Group 3 treated with EECL and was daily administered at a dose of 500 mg/kg/p. o.

Group 4 treated with EEAI and was daily administered at a dose of 50 mg/kg/p. o.

Group 5 treated with a 2 % suspension of EECL (250 mg/kg) and EEAI (25mg/kg) and was daily administered  (p. o)

Group 6 treated as recovery group for group 3after EECL treatment.

Group 7 treated as recovery group for group 4 after EEAI treatment.

Group 8 treated as recovery group for group 5 after treatment of suspension of EECL and EEAI.

 

Sperm collection and analysis:

At the end of 4th week of treatment of group1 to 5 and after recovery period of group 6 to 8 the final body weight of male rats were recorded and rats were sacrificed. At the time of euthanasia, spermatozoa were obtained from caudae epididymides and vasdeferens of male rats from each group and placed in Phosphate buffered saline maintained at 37°C. (pH7.4). Sperm count, motility and viability were assessed as per the WHO guidelines for semen analysis10.

 

Estimation of serum testosterone:

The blood of sample of male rats was collected by cardiac puncture method at the end of 4th week treatment of treatment and after the end of recovery period. Serum level of testosterone was measured by radioimmunoassay using commercial kit (Immunotech, Marseille, France) as per manufacturer's instruction11.

 

RESULTS:

The extracts were screened for preliminary phytochemical tests. The C. longa extract showed presence of volatile oils, resins and phenolic compounds while the A. indica leaves extract showed presence of triterpens, glycosides, fatty Acids, saponins, flavonids and carbohydrates.

 

The results showed that the oral treatment of suspension containing both the C. longa and A. indica extract causes greater reduction in sperm count, viability, motility, serum testosterone level of male rats as compare to normal group and that the alterations were found to be reversible after cessation of treatment. The results demonstrate that the oral suspension containing C. longa and A. indica extract has the potential for development as a male antifertility agent.

 

 

Table no. 1. Sperm count, motility and viability of male rats of treated and recovered groups:

Groups

Semen analysis

Sperm Count

mill/ml

Viability

(%)

Motility (%)

Fast (%)

Sluggish (%)

Local Oscillatory (%)

1

42.0+0.68

96.75+0.75

63.0+1.08

27.5+1.04

13.00+0.70

2

10.03+0.77

0.00

0.00

0.00

0.00

3

31.48+1.059

75.25+1.88

19.25+0.47

8.75+1.78

7.50+1.04

4

22.43+1.45

56.25+3.26

31+1.35

18.25+2.28

6.26+4.78

5

15.38+1.05

37.75+2.25

14.0+0.19

11.00+1.35

5.00+0.40

6

35.63+081

83.00+1.32

54.00+1.82

19.75+0.62

6.25+1.04

7

36.03+.054

58.00+2.12

40.00+0.921

13.50+1.19

6.26+1.22

8

36.68+0.64

83.25+0.94

56.00+1.08

22.25+1.09

9.950+0.47

 

 

Sperm Analysis:

Table no. 1 showed, the oral treatment of combination of EECL and EEAI for male rats of group 5 causes significant reduction in sperm count, viability and motility when compared with normal. By 56 days of treatment withdrawal, however, the altered sperm parameters recovered to normal.

 

Statistical evaluation was carried out using one way ANOVA followed by Dennett’s test. The p values <0.01 were considered statistically significant.

 

Treatment of the suspension containing both EECL and EEAI to group 5 showed significant reduction in sperm count, motility, and viability when compared with group 1.

 

Figure1: Sperm count in rats (n=6) after treatment of cadmium chloride (standard), C. longa, A .indica suspension of C. longa and A. indica and following treatment withdrawal.

 

Figure 1 demonstrate that, the EECL and EEAI in combination form (oral suspension) causes greater reduction in sperm count when compared with normal and individual extract treatment and the reduction was regained after withdrawal of treatment.

 

Figure 2 demonstrate that EECL and EEAI in combination form (oral suspension) causes greater reduction in sperm viability and motility when compared with normal and the reduction was regained after withdrawal of treatment.

 

Figure2: Sperm viability (%) and motility (%) after  treatment of cadmium chloride (standard), C. longa, A. indica, suspension of both C. longa and A. indica extract on sperm viability and motility in rats (n=6) and following treatment withdrawal.

 

Serum Testosterone:

Significant reduction was noted in serum level of testosterone in suspension of EECL and EEAI treated rats compared to controls. By 56 days of treatment withdrawal, however, the testosterone level increased (Table no. 2 and Fig. 3).

 

Table no. 2. Serum testosterone levels of male rats of treated and recovered groups:

Groups

Serum Testosterone ng/dl

1

433+2.327

2

0.00

3

390.5+8.85

4

258.5+8.855

5

229.08+6.13

6

358.80+13.63

7

358.8+10.31

8

338.0+4.262

 

Figure3: Serum testosterone levels in rats (n=6) after treatment of cadmium chloride (standard), C. longa, A. indica, suspension of both C. longa and A. indica extracts and following treatment withdrawal.

 

DISCUSSION:

The results of the present study indicate that the combined treatment of C. longa and A. indica extracts showed greater reduction in sperm count, viability, and motility and testosterone levels as compared to the normal and individual treatment of both the extract. Previous studies was conducted by using individual aqueous extract of C. longa and A. indica which demonstrated spermicidal effect4, 7,12. Present study conducted by using the combination of ethanolic extracts of C. longa and A. indica which indicate significant alterations of sperm parameters and serum testosterone and these alterations were recovered with withdrawal of treatment.

In conclusion, Curcuma longa and Azadirachta indica showed significant reversible male antifertility effects. The oral treatment of both the extract in an oral suspension form to male rats probably induces deficiency of testosterone and causes suppression of spermatogenesis in rat testes which further leads to decrease in sperm count, motility and viability.

Today when the world demand of herbal medicine is on rise, these plants may have potential for fertility control and may overcome the side effects of currently available contraceptives. Thus provide an alternative strategy in the search of new herbal contraceptives which will help to reduce over population.

 

REFERENCES:

1.       Unny R., Chauhan A., Joshi Y., Dobha M., Gupta R.,A review on potentiality of medicinal plants as the source of new contraceptive principles, Phytomedicine 10, 2003 233–260.

2.       Yogeshwaran, Turmeric: Traditional herb par excellence, Herbal Tech Industry 2, 2005, 12-18.

3.       Tara R., Manoj M., Mahadevan R., Curcumin : a potential vaginal contraceptive Contraception, 68,2003, 219-223.

4.       Mishra R., Singh S., Reversible antifertility effect of aqueous rhizome extract of Curcuma longa in male laboratory mice, Contraception, 79, 2009, 479-487.

5.       Yogeshwaran, Medicinal properties of Neem, Herbal Tech Industry1, 2005, 32.

6.       Trease, Evans, Aspects of Asian Medicine and its Practice in the West, Pharmacognosy,15, 2002, 470

7.       Khillare B., Shrivastav T., Spermicidal activity of Azadirachta indica (neem) leaf extract, Contraception, 68, 2003, 225–229.

8.       Mishra R., Sing K., Effect of aqueous Extract of Azadirachta indica on reproductive on reproductive organs in male mice, Indian Journal of Experimental Biology, 43, 2005, 1093-1103.

9.       John Lye R., Petra S., Patrick V., Andrea W., Male contraception-a topic with many facets, Molecular and Cellular Endocrinology, 216, 2004, 75-82.

10.     World Health Origination, Sperm motility and sperm vitality, WHO laboratory manual for the examination and processing of human semen 5, 21-32.

11.     WHO. Method manual   Programme for the provision of matched assay reagents for the radioimmunoassay of hormones in reproductive physiology fifth editor. Geneva: World Health Organisation: 1987.

12.     Sathiyaraj K., Sivaraj A., Vinoth kumar P., Spermicidal Activity of Azadirachta indica (Neem) Aqueous Leaf Extract on Male Albino Rats, International Journal of Pharm Tech Research 2 (1), 588-591.

 

 

Received on 24.01.2011

Accepted on 28.02.2011     

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 3(3): May- June 2011, 124-127