Activity of Alkaloid Extract of Carica papaya. Seeds on Reproductive Functions in Male Wistar Rats

ABSTRACT

Activity of the alkaloid extract of Carica papaya. Linn seed on male reproductive physiology was investigated. Male rats were divided into two groups. Each group of 20 male rats was further divided into 4 subgroups of 5 rats. Each rat was treated with C. papaya. seed extract (10, 50, and 150 mg kg⁻¹day⁻¹) daily for 3 days for fecundity study, semen analysis, and testis histopathology, respectively. Twenty male rats treated with C. papaya. seed extract (10, 50, and 150 mg kg⁻¹ day⁻¹), abstained from sex for 7 days and divided into 4 groups, were mated with fertile female rats: another set of 30 male rats, divided into 4 groups, treated with the seed extract (10, 50, and 150 mg kg⁻¹ day⁻¹), respectively, was used for semen analysis and testis histopathology. The results showed that oral administration of C. papaya. seed extract prevented ovum fertilization, reduced sperm cell counts, revealed sperm cell degeneration, and induced testicular cell lesion. These observations led to the conclusion that C. papaya. seed extract oral administration could induce reversible male infertility and therefore could be used for pharmaceutical development of a male contraceptive.

• KEYWORD
• Carica papaya
• male fertility
• semen
• testis

Introduction

A fruit tree, Carica papaya. Linn (Caricaceae), is commonly grown in the southern region of Nigeria. It is popularly known as paw-paw in most parts of the world. In Nigeria traditional medicine, the extracts of unripe fruits of C. papaya. are used for the treatment of hypertension and malaria/fever. The dry seeds of the ripe paw-paw fruits are also used for the treatment of typhoid fever, diabetes mellitus, and hypoglycemia (Udoh et al., 2004b). Lohiya and Goyal (1994) reported that the seed extract of C. papaya. induced suppression of spermatogenesis, which could influence fertility in male rats. Udoh et al. (2004a) observed that the histology of thyrotrophs (TSH cells) of anterior pituitary of male rats treated with the extract of C. papaya. showed progressive hypertrophy and degranulation. Following the above facts about the activity of C. papaya. seeds, it became necessary to undertake this research.

Materials and Methods

Plant material

Ripe fruits of C. papaya. were obtained from the University of Calabar vicinity around the months of January and March 2002. The plant was scientifically identified by Frank Apejoye of the Botany Department, University of Calabar, Nigeria, and voucher specimen no. 73 was deposited in the Herbarium, Department of Botany, University of Calabar, Nigeria.

Preparation of extract

C. papaya. seeds were isolated from the ripe fruits of paw-paw. The seeds were washed in tap water, sun-dried for 100 h, and oven-dried at 40°C for 24 h. The dry-seeds were ground into powder using an electric blender (Inaulimex, Sussex, UK).

The powder sample (500 g) of C. papaya. seeds was wrapped in a thimble and placed in a 1000 cm³ Soxhlet extractor and extracted first in petroleum ether for 8 h to remove the fat. The petroleum ether residues were re-extracted in ethanol for 72 h. The ethanol extract was evaporated into a paste form. The paste extract, 10 g, was weighed and dissolved in 10 ml of distilled water to give a concentration of 1 g/ml. The stock solution of C. papaya. was kept frozen at −20°C for use.

Treatment

Sexually mature Wistar rats of both sexes obtained from University of Calabar Animal House, weighing between 150 and 200 g, were used. Adult male rats abstained from sex for 7 days were divided into 4 groups of 5 rats per group for fecundity study. Another set of 20 adult male rats were divided into 4 groups of 5 rats per group for sperm cell integrity, sperm cell count, and testiscular cells histology. Group 1 rats of each set were trusted orally, 0.5 ml of corn oil; group 2 to 4 of each set received orally, C. papaya. seeds extract, 10, 50, and 150 mg kg⁻¹ day⁻¹ for 3 days, respectively.

Fecundity study

The first set of 20 male rats weighing between 150 and 200 g were divided into 4 groups of 5 rats each. The adult male rats were abstained from sex for 7 days before mating with adult female rats in the same weight range. The adult female rats were artificially brought into estrus by exposing both male and female rats to each other without physical contact for 4 days. Induction of the estrus cycle was confirmed by microscopic examination of the vaginal smear following clinical laboratory procedure. On the fourth day of the estrus cycle, the male rats, nontreated as control and those treated with the extract of C. papaya. (10, 50, and 150 mg kg⁻¹ day⁻¹), were mated with fertile female rats in the ratio 1:1. Mating was confirmed by a vaginal plug observed after 24 h of mating. The female rats mated were kept under observation for pregnancy over a period of 5 gestations.

Sperm cell morphology, integrity study

C. papaya. seed extract was given orally to 4 groups of male rats of 5 rats/group. Group 1 receive corn oil as control; groups 2 to 4 were administered C. papaya. extract, 10, 50, and 150 mg kg⁻¹day⁻¹ for 3 days. Twenty-four hours after treatment, the male rats were sacrificed, their right testes isolated for sperm cell integrity and morphology study, and the left testes were fixed in 10% formal-saline for histopathology.

For determination of sperm cell counts, semen samples were collected from the left testes through surgical operation. The collected semen was diluted with normal saline (1:200), fixed with MIF fixative stain, 1 drop per ml (Sapero & Lawless, 1953; Ufearo et al., 1996). The prepared sample was examined under a microscope using a hemocytometer chamber (Buerker, Berlin, Germany). Spermatozoa concentration was determined and showed differential cell count which revealed physical abnormalities. The value was expressed as a percentage of the total sperm cells count, that is, percentage abnormality = total sperm cell count minus active cell count divided by total sperm cell count × 100.

The morphology of the sperm cell was microscopically examined after fixing in 70% alcohol and stained with hematoxylin (Sigma, St. Louis, MO, USA).

Statistical analysis

Student's t.-test was used to evaluate significant difference in the mean of number of liters per group, mean sperm count, and the percentage of abnormal spermatozoa. Correlations between mean sperm cell integrity, sperm cell count, and the percentage of abnormal sperm were estimated using linear regression analysis.

Results

Fecundity

Adult female Wistar rats mated with male rats (1:1) after treatment with C. papaya. (50 and 150 mg/kg) daily for 3 days showed no pregnancy after 3 gestation observations (Table 1), whereas female rats mated with male rats treated with corn oil (n = 5) delivered an average of 9 litters after a 21 day gestation period (Table 1). However, one of the female rats mated with male rats (n = 5) treated with C. papaya., 10 mg kg⁻¹day⁻¹ daily for 3 days delivered only 4 litters out of 9 average litters expected.

Table 1 C. papaya. ethanol extract (CPEE, 10, 50, and 150 mg kg⁻¹day⁻¹) treatment for 3 days on the fecundity of male rats.

Group	Experiment	n	Dosage (mg/kg)	NPR/NRM	Average litters
1	Control (corn oil)	5	0.5 ml/rat	5/5	9
2	CPEE	5	10	1/5	1 < 1
3	CPEE	5	50	0/5	0
4	CPEE	5	150	0/5	0

n, number of rats; NPR, number of pregnant rats; NRM, number of rats mated.

Semen analysis

C. papaya. extract, (10, 50, and 150 mg kg⁻¹day⁻¹) administered to male rats (n = 5) daily for 3 days decreased sperm cell counts (Tables 2 and 3). Comparatively, the control male rat semen analysis showed normal cell count of 62 × 10 cell/mm³, whereas semen samples of male rats (n = 5) treated with C. papaya. (10, 50, and 150 mg kg⁻¹day⁻¹) daily for 3 days showed a dose-related decrease in the sperm cell counts with a percentage abnormality of between 65 and 90 compared to control, p < 0.1 and r = 0.4 (positive correlations between sperm cell count and integrity) (Tables 2 and 3).

Table 2 C. papaya. ethanol extract (CPEE 10, 50, and 150 mg kg⁻¹ day⁻¹) treatment for 3 days on the sperm cell integrity in male rats.

Group	Experiment	n	Dosage (mg/kg)	X¯BW (g)	X¯CC (c/mm^3)	X¯%AB
1	Control (corn oil)	5	0.5 ml/rat	205.6±2	62 × 10^6±1	40±1
2	CPEE	5	10	147±1	38 × 10^6±2	65±3
3	CPEE	5	50	170±3	25 × 10^6±2	84±1
4	CPEE	5	150	187.3±2	15 × 10^6±3**	90±1

*p < 0.1

**p < 0.05 by Student's t.-test.

X¯BW, mean body weight; X¯CC, mean cell count; X¯%AB, mean percent of abnormal cell count; n, number of rats.

Table 3 C. papaya. ethanol extract (CPEE 10, 50, and 150 mg kg⁻¹day⁻¹) treatment for 3 days on the sperm cell count in male rats.

Group	Experiment	n	Dosage (mg/kg)	X¯BW (g)	X¯CC (c/mm^3)	X¯%AC
1	Control (corn oil)	5	0.5 ml/rat	205.6±2	62 × 10^6±1	37.2±0.1
2	CPEE	5	10	147.5±1	38 × 10^6±2	13.3±0.1
3	CPEE	5	50	170.6±3	25 × 10^6±1	3.8±1.0**
4	CPEE	5	150	187.3±2	15 × 10^6±3**	1.5±0.15**

*p < 0.1

**p < 0.05 by Student's t.-test compared to control.

n, number of rats; X¯BW, mean body weight per group; X¯ACC, mean active cells counted per group; X¯%AC, mean percent of active cells.

Testis histology

C. papaya. extract (10, 50, and 150 mg kg⁻¹day⁻¹) in male rats caused some pathological changes in the testes morphology (Table 4). The pathologic effects observed were dose-related and ranged from mild atrophy of seminiferous tubules to severe Leydig and Sertoli cell metaplasia to degeneration of spermatozoa (Table 4; Figs. 1-4).

Figure 1Photomicrograph of the cross section of rat testis treated with corn-oil alone as control (× 160).

Figure 2Photomicrograph of the cross section of rat testis treated with C. papaya. seeds extract, 10 mg kg⁻¹ day⁻¹ for 3 days. Arrows showing (a) mild atrophy of seminiferous tubules and (b) spermatogenesis arrest (× 160).

Figure 3Photomicrograph of the cross section of rat testis treated with 50 mg kg⁻¹ day⁻¹ for 3 days. Arrows showing (a) spermatogenesis arrest, (b) Leydig cell metaplasia, and (c) seminiferous tubule atrophy (× 160).

Figure 4Photomicrograph of the cross section of rat testis treated with 150 mg kg⁻¹ day⁻¹ for 3 days. Arrows showing (a) sperm cell degeneration, (b) Leydig cell metaplasia, (c) seminiferous tubules atrophy, and (d) spermatogenesis arrest (×160).

Table 4 C. papaya. ethanol extract (CPEE, 10, 50, and 150 mg kg⁻¹day⁻¹) treatments for 3 days on the morphology of testes.

Group	Experiment	Dosage	Observation
1	Control (corn oil)	0.5 ml/rat (mg kg^−1 day^−1)	Normal morphology
2	CPEE	10	A mild degeneration of spermatozoa and spermatogenesis arrest.
3	CPEE	50	Spermatogenesis arrest, Leydig cell metaplasia, and seminiferous tubule atrophy.
4	CPEE	150	Severe spermatogenesis arrest at spermatogonia stage, sperm cell degeneration, seminiferous tubule atrophy, Leydig and Sertoli cell metaplasia.

Discussion

The results of this study showed that the extract of C. papaya. seeds administered to male rats (50 and 150 mg kg⁻¹day⁻¹) for 3 days interfered with fertility. Fecundity study showed that repeated oral administration of C. papaya. (50–150 mg kg⁻¹day⁻¹) for 3 days to male rats mated with adult female rats (1:1) prevented pregnancy in the female rats. However, the female rats mated with male rats treated with corn oil only delivered an average number of 9 litters after 21 days gestation period, while the female rats mated with male rats treated with (10 mg kg⁻¹day⁻¹) delivered only 4 litters. This observation revealed that treatment of male rats with appropriate doses of the extract could inhibit male reproductive functions.

Semen analysis of those male rats treated with (10, 50, and 150 mg kg⁻¹day⁻¹) for 3 days showed a reduced active sperm cell count in a dose-related manner. This observation showed a significant decrease of active sperm cell counts in the male rats treated with 50 or 150 mg kg⁻¹day⁻¹ of the extract as compared to controls. This effect could be responsible for the failure of ovum fertilization. The finding was similar to the report that low sperm count and inactive spermatozoa in semen were always responsible for infertility in rats (Gupta & Wambebe, 1990; Ufearo et al., 1996; Udoh & Kehinde, 1999; Udoh & Ekpenyoung, 2000; Braide et al., 2003; Ladeji & Udoh, 2004; Udoh et al., 2004a).

Testis morphology of male rats treated with the extract revealed Leydig and Sertoli cell metaplasia and degeneration of spermatozoa. The net effects of extract treatment on male rats therefore could result in male reproductive dysfunction. Microscopic observation of the sperm cells showed that administration of extract altered the morphology of sperm cells pathologically. This observation was similar to that reported by Ufearo et al. (1996) that abnormal sperm morphology caused infertility in the fertile subjects. A similar effect of the seeds was also observed by Ekanem and Okoronkwo (2003) and Ekanem and Bassey (2003) on the fertility of male and female Nile tilapia, respectively.

This investigation gave a clue into the mechanism of action of the extract and its possible pharmacologic effect on male reproduction. However, the net observation from this study allowed the conclusion that the extract might interfere with male reproductive functions.