:: Asian Journal of Andrology ::

Contraceptive effect of Curcuma longa (L.) in male albino rat

Purohit Ashok, Bhagat Meenakshi

Reproductive Physiology Section, Department of Zoology, JNV University, Jodhpur (Raj.) 342 005, India

Asian J Androl 2004 Mar; 6: 71-74

Keywords: Leydig cells; Curcuma longa; seminiferous tubules; antiandrogens; spermatozoa; male contraception

Abstract

Aim: To study the contraceptive effect of the crude extracts of Curcuma longa in male albino rats. Methods: Rats were fed orally with Curcuma longa aqueous and 70 % alcoholic extract for 60 days (500 mg·kg^-1·day^-1). Results: A reduction in sperm motility and density was observed in both the treated groups. Conclusion: Curcuma longa may have affected the androgen synthesis either by inhibiting the Leydig cell function or the hypothalamus pituitary axis and as a result, spermatogenesis is arrested.

1 Introduction

Curcuma longa (family Zingiberaceae), commonly known as 'Haldi', is a perennial herb cultivated throughout India and is widely used as an antibiotic in folk medicines and as spices. C. longa also possesses antimutagenic and anticarcinogenic properties [1]. The aqueous and alcoholic extracts of C. longa have been shown to completely inhibit the fertility with reduction in sperm count and motility [2]. We have reported [3] its effect on testicular cell population dynamics in male rats. The present work was designed to study the possible mechanism of the male contraceptive effect of this plant.

2 Materials and methods

2.1 Plant and preparation

The fresh rhizomes of Curcuma longa were purchased from the market and was identified by the Department of Botany of this University. The aqueous extract was prepared as follows: the shade-dried rhizomes were cut into pieces of around 1 cm long and boiled in distilled water (500 g in 2000 mL) with slow fire to reach a final volume of 1000 mL (500 mg/mL). The 70 % ethyl alcohol extract was prepared by Soxhlet extraction in 70 % ethanol (1:3) for 18 h. The ethanol was evaporated under reduced pressure to obtain the residue, which was then suspended in distilled water at a concentration of 500 mg plant in one mL of water. The dose (500 mg/kg for both extracts) was 1/10 of the LD50 for both extracts.

2.2 Animals and groups

Adult proven fertile male Wistar rats weighing 150g-250 g were obtained from the animal house of this University. Animals were kept under controlled environmental conditions with a commercial diet and tap water ad libitum. They were randomly divided into 3 groups of 10 animals each as follows.
Group 1: Control (1 mL distilled water per day)
Group 2: Aqueous extract (500 mg·kg^-1·day^-1)
Group 3: Alcoholic extract (500 mg·kg^-1·day^-1)

2.3 Observations

After 55 days of treatment, fertility test was performed and 24 h after the last dose at day 60, the animals were scarified using light ether anaesthesia. Blood was collected through cardiac puncture and serum was separated. The RBC and WBC count, glucose, haemoglobin and haemtocrite were estimated with routine methods. The serum was also analyzed for cholesterol[4], HDL-cholesterol [5], phospholipid [6] and triglycerides [7]. The testes and accessory sex organs (epididy-mis, seminal vesicle and ventral prostate) were dis-sected, freed form fat and connective tissue were weighed. The organs were fixed in Bouin's solution, embedded in paraffin, sectioned and stained with haemotoxylin and eosin for histological studies. Sperm motility was estimated in cauda epididymis and sperm density in cauda epididymis and testes by the method of Pradad et al [8].

2.4 Data processing

Data were expressed in meanSEM. The Student's t test was used to analyze the significance of difference and a probability of P<0.05 was considered significant.

3 Results

3.1 Routine examination

The weights of testes and epididymides were significantly lower (P0.01) in both the extract treated groups than in the controls. The weights of the seminal vesicle and ventral prostate of the treated groups were also decreased, but statistically insignificant (Table 1). The sperm motility in cauda epididymides was decreased by 60.13 % and 60.39 % in the aqueous and alcoholic extract groups, respectively. A highly significant (P 0.01) reduction in sperm density in the testes and cauda epididymides was observed in both the extract treated groups as compared with the controls. Fertility test showed zero fertility in both treated groups (Table 2). No significant change was observed in blood and serum parameters (data not presented). Two months after cessation of drug treatment, the sperm count and motility were recovered.

Table 1. Body and organ weights of rats (meanSEM).^cP< 0.01, compared with controls.

Group (n=5)	Body weight (gm)		Testis	Epididymis	Seminal Vesicle	Ventral Prostate
Group (n=5)	Initial	Final	mg/100gm body weight
Control (Gr.A)	211.338.17	237.539.51	1209.051.01	560.0015.63	618.6058.60	279.6867.42
Aqueous extract treated (Gr. B)	162.58.92	188.07.12	669.0158.71^c	378.0822.42^c	494.0344.20	196.253.75
Alcoholic extract treated (Gr. C)	156.54.38	170.06.40	678.6962.18^c	384.0067.59^*	410.6073.73	191.0514.32

Table 2. Sperm density and motility and fertility test in rats (meanSEM). ^cP<0.01, compared with controls.

Group (n=5)	Sperm Density (10⁶/mL)		Sperm Motility (%)	Fertility test (No. of rats pregnant)
Group (n=5)	Testis	Cauda	Sperm Motility (%)	Fertility test (No. of rats pregnant)
Control (Gr.A)	4.810.08	57.312.51	79.323.73	5
Aqueous extract treated (Gr. B)	0.590.49^*	3.060.69^*	19.196.37^*	0
Alcoholic extract treated (Gr. C)	1.110.22^*	9.561.72^*	18.935.42^*	0

3.2 Histological observation

The seminiferous tubules of testes decreased in size and underwent destrophy. The number of spermatogenic elements like spermatogonia, spermatocytes and spermatids decreased significantly in both extract treated groups. The Leydig cells were scattered within the interstitial space. Both extracts affected mainly the secondary spermatocytes and spermatids. The seminiferous tubule and Leydig cell nuclei diameters were significantly reduced as compared to the controls (Figures 1-3). The lumen was devoid of spermatozoa. The Leydig cells were disintegrated. The epithelial cell height of the caput and cauda epididymides was significantly reduced and the lumen was devoid of spermatozoa (Figures 4- 9).

Figure 1. Testis: control rat.

Figure 2. Testis: aqueous extract treated rat.

Figure 3. Testis: alcoholic extract treated rat.

Figure 4. Caput epididymides: control rat.

Figure 5. Caput epididymides: aqueous extract treated rat.

Figure 6. Caput epididymides: alcoholic extract treated rat.

Figure 7. Cauda epididymides: control rat.

Figure 8. Cauda epididymides: aqueous extract treated rat.

Figure 9. Cauda epididymides: alcoholic extract treated rat.

4 Discussion

An antiandrogenic effect of the extracts is suggested by the reduced sperm motility in cauda epididymides. Reduction in Leydig cell nuclei diameter and disintegration of Leydig cells always lead to reduced androgen synthesis [9]. A decrease in the weights of accessory sex structure and reduction in epithelial cell height of the caput and cauda epididymides also support the view of decreased androgen level as these organs are androgen-dependant [10]. The principal cells of epididymal epithelium synthesize proteins, which are androgen-dependant and form one of the constituents that ensures the maturation of spermatozoa [11]. A decrease in the testis weight may reflect a reduction in the number of spermatogenic elements and spermatozoa [12].

Results of the present study indicate that C. longa extracts may have impaired androgen synthesis either by inhibiting Leydig cell function or hypothalamus pituitary axis, resulting in infertility. Normal blood and serum parameters indicate a low systemic toxicity of the plant material.

References

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Correspondence to: Dr. Ashok Purohit, Chairman, Department of Zoology, JNV University, Jodhpur (Raj.) 342 005, India.
Tel: +91-291-720 839
E-mail: purohit1411@yahoo.com, meenakshi1910@yahoo.com
Received 2003-06-24 Accepted 2003-09-28