Natural products in regulation of male fertility.

Medicinal plants may prove useful in developing plant-based strategies for regulation of male fertility. The present review describes the antifertility potential of certain medicinal plants, viz. Azadirachta indica, Curcuma longa, Allamanda cathartica and Bacopa monnieri in Parkes (P) male mice. The results suggested that treatment with the aqueous extracts of these plants caused reversible suppression of spermatogenesis and fertility in P mice and that there were no signs of detectable toxicity in treated mice. Further research needs to be done to develop plant-based strategies for control of male fertility.

Introduction

According to the World Population Prospects (2017), the world population is around 7.6 billion, and with the present trend, it is anticipated to rise to 8.6 billion by 2030, 9.8 billion by 2050 and 11.2 billion by 21001. The population of our country has increased by >181 million during 2001-20112. Both government and non-government organizations are making all efforts to control the human population, but the outcome has not been very satisfactory. One of the possible reasons could be the limited availability of contraceptive choices3. Women are the main users of the contraceptives. Contraceptives developed for females are effective in preventing unplanned pregnancy; however, because of side effects, some women cannot use these contraceptives on health ground45. Therefore, the development of male contraceptive will help in planning family6.

The male contraceptives act by blocking meeting of sperm to the egg either by physical barriers (condoms, vasectomy and experimental vas occlusion methods) or by inhibiting spermatogenesis (hormonal and non-hormonal methods)6. Approximately 30 per cent of couples currently depend on condom and vasectomy as male methods of fertility regulation, although both of these methods have their own limitations6. The major drawbacks of condom and vasectomy are their high failure rates and lack of complete reversibility after the reversal operation, respectively7. A contraceptive that is safe, effective, reversible and rapid in action should be considered acceptable for use in men. Besides, it should not affect other androgen-dependent functions. In addition, the application mode should be easy and price considerably low.

In Indian traditional medical system of Ayurveda, many herbal extracts have been used for the treatment of a variety of ailments and that these extracts have also been used in regulating as well as improving fertility8. Several compounds derived from natural herbs in different phases of clinical development signify natural products as sources of new drug candidates9.

Several studies were conducted to develop herbal contraceptives10111213. The success in search of a plant-based male contraceptive is best illustrated with the discovery of gossypol by Chinese scientists, which is regarded as a major breakthrough in male contraception1415. This gave a great impetus to researches on gossypol and considerable amount of work has been carried out on the antifertility properties of this compound in both animals and humans16171819202122. In clinical studies, however, gossypol produced two major side effects: occasional occurrence of hypokalaemia and variable differences in reversibility of male fertility23. The potential of low dose of gossypol together with steroid hormones has also been investigated for use in male contraception2425. Efforts are being continued to explore a suitable plant product for use in the regulation of male fertility (Table). The present review describes the antifertility potential of four plants, viz. Azadirachta indica, Curcuma longa, Allamanda cathartica and Bacopa monnieri in Parkes (P) strain male laboratory mouse, which has been used as an animal model in our laboratory32374243.

Table

List of plants exhibiting antifertility properties in male rats and mice

Plant	Type of extract and part of plant	Route of administration, dose and duration	Animal model	Effects	References
Abrus precatorius	Ethanolic extract of seed	Intraperitoneal; 20, 40 and 60 mg/kg BW for 20 day	Mouse	Suppression of spermatogenesis; decreased serum testosterone and decreased sperm count	26
Aegle marmelos	Aqueous extract of leaves	Oral; 100, 200 and 300 mg/kg BW for 60 days	Rat	Decreased reproductive organs weight; decreased serum testosterone; and anti-spermatogenic and antifertility effects	27
Allium sativum	Crude extract of bulb Aqueous extract of bulb	Feed; 5, 10 and 15 per cent for 30 day Oral; 500 and 1000 mg/kg BW for 28 days	Rat Rat	Germ cell apoptosis; and inhibition of Leydig cell steroidogenesis Increased morphologically abnormal spermatozoa and decreased sperm concentration	28 29
Andrographis paniculata	Alcoholic extract of leaf	Oral; 250 and 500 mg/kg BW for 30 and 60 days	Rat	Decreased weights of testis and epididymis; reduced size of seminiferous tubules; and degeneration of spermatozoa	30
Citrus limon	Ethanolic extract of leaf	Oral; 500 and 1000 mg/kg BW for 35 days	Mouse	Anti-spermatogenic and antifertility effects; reduced serum testosterone; and reversibility after 56 days of treatment withdrawal	31
Coccinia indica	Ethanolic extract of leaf	Oral; 200 and 500 mg/kg BW for 35 days	Mouse	Anti-spermatogenic and antifertility effects; reduced serum testosterone; and reversibility 56 days after treatment withdrawal	32
Dalbergia sissoo	Aqueous extract of leaf	Oral; 50 and 100 mg/kg BW for 35 days	Mouse	Anti-spermatogenic and antifertility effects; reduced serum testosterone; and reversibility after 56 days of treatment withdrawal	33
Enicostemma axillare	Ethanolic extract of leaf	Intragastric; 375 and 750 mg/kg BW for 55 days	Rat	Inhibited spermatogenesis and steroidogenesis; and reversibility 55 days after treatment withdrawal	34
Juniperus phoenicea	Ethanolic extract of cones	Intraperitoneal; 400 or 800 mg/kg BW for 21 days	Rat	Anti-spermatogenic and antifertility effects	35
Madhuca indica	Alcoholic extract of leaves	Oral; 200 mg/kg BW for 20 days	Rat	Decreased body weight; decreased reproductive organs weight; regressed seminiferous tubules; and decreased serum testosterone	36
Mimusops elengi	Aqueous extract of fruit	Oral; 200, 400 and 600 mg/kg BW for 35 days	Mouse	Anti-spermatogenic and antifertility effects; and reversibility 56 days after treatment withdrawal	37
Opuntia dillenii	Methanolic extract of phylloclade	Oral; 50 mg/kg BW for 30 days	Rat	Reduced serum testosterone level; decreased sperm count and motility; and reduced fertility	3
Tabernaemontana divaricata	Ethanolic extract of leaf	Oral; 50, 100 and 200 mg/kg BW for 60 days	Rat	Decreased reproductive organs weight; decreased sperm count and motility; spermatogenic arrest; and reduced serum testosterone and fertility	38
Taraxacum officinale	Aqueous extract of whole plant	Oral; 1.065 and 2.130 g/kg BW for 60 days	Rat	Decreased testis weight; decreased sperm count and motility; spermatogenic arrest and reduced fertility	39
Terminalia chebula	Aqueous-ethanolic (1:1 v/v) extract of fruits	Oral; 60 mg/0.5 ml distilled water for 28 days	Rat	Affected spermatogenesis; decreased activities of androgenic key enzymes and decreased plasma testosterone	40
Trachyspermum ammi	Ethanolic extract of fruit	Oral, 100, 200 and 400 mg/kg BW for 60 days	Rat	Reduced testis weight; decreased sperm number and motility; increased production of abnormal sperm; and reversibility after 120 days of treatment withdrawal	41
Urena lobata	Ethanolic extract of root	Intragastric; 300 and 600 mg/kg BW for 55 days	Rat	Inhibition of spermatogenesis and steroidogenesis and reversibility 55 days after treatment withdrawal	34

BW, body weight

Azadirachta indica

A. indica L. (family, Meliaceae), known commonly as neem, is a medicinal plant and this is found in semi-tropical and tropical climates in countries such as India, Pakistan and Bangladesh4445. Extracts of different parts of this tree have been found useful in the treatment of ulcer, malaria, liver disease, cancer, high blood sugar, dermatological disease, intestinal worms, fever, eye problem, urinary disorder, etc4647. Azadirachtin is the most important and active constituent of neem, while others include nimbolinin, nimbin, nimbidin, nimbidol, sodium nimbinate, gedunin, salannin and quercetin48.

: Upadhyay et al49 reported that a single administration (50 μl) of neem oil into the lumen of the vas deferens on each side in male rats induced a long-term inhibition of fertility, without affecting the libido. Neem oil impaired spermatogenesis up to nine months after the treatment, while the serum level of testosterone was not affected and also there was no increase in the anti-sperm antibodies. Joshi et al50 reported that Azadirachta treatment in rats caused a decrease in diameter of the seminiferous tubules, with atrophy of the spermatogenic elements and the Leydig cells. They suggested that the cessation of the spermatogenic process after neem treatment was caused probably because of the deficiency in androgen production. There was a gradual recovery in anti-androgenic action of the A. indica in male albino rats 8, 16 and 24 days after withdrawal therapy. Parshad et al51 showed no effect of aqueous extract of neem leaf on spermatogenesis and on the litter size and fertility index.

In P mice, neem treatment (50, 100 and 200 mg/kg body weight/day for 28 days) did not affect body weight and also the weights of testis, epididymis and seminal vesicle42. The testis of control mice showed normal histological features. By contrast, testes in neem-treated mice exhibited both affected and normal seminiferous tubules in the same sections. The affected seminiferous tubules showed degenerative changes such as presence of vacuoles in the germinal epithelium, loosening of epithelium, marginal condensation of chromatin in round spermatids, formation of giant cells and mixing of germ cell types in stages of spermatogenesis. Further, the neem treatment also affected sperm parameters in the epididymis and fructose level in the seminal vesicle. The litter size in females impregnated by neem-treated males was also affected. By six weeks of treatment withdrawal, however, the changes caused in the reproductive organs recovered to control levels42.

The mechanism by which A. indica causes impairment in spermatogenesis is not properly understood. It is reported that the antifertility effect of neem is not associated with alterations in the serum level of testosterone4951. In P mice, vacuoles were often noticed in the epithelium in affected seminiferous tubules in the testis after neem treatment. Intraepithelial vacuoles have also been noticed in affected seminiferous tubules in rat testis after gossypol treatment, and such vacuoles are reported to occur primarily in the Sertoli cells52. Thus, it is probable that in P mice, neem treatment causes suppression of spermatogenesis by acting through Sertoli cells42.

Curcuma longa

C. longa L. (family, Zingiberaceae) a perennial herb, is grown throughout India. Curcumin is the active ingredient in turmeric and this exhibits protective and preventive properties against several diseases such as cancer and autoimmune, neurological, metabolic, lung, liver and cardiovascular diseases5354. Besides curcumin, turmeric also contains sesquiterpenes (turmerone, atlantone, zingiberone, turmeronol, germacrone and bisabolene), carbohydrates, protein, resins and caffeic acid55.

: Aqueous rhizhome extract of C. longa in a dose of 500 mg/kg body weight for 60 days caused a decrease in the weight of the epididymis, seminal vesicle, ventral prostate and testis56. The treatment also caused a reduction in sperm count and motility and a reduction in the number of germ cells and hence decreased fertility; the Leydig cells were also adversely affected. These effects of the treatment were attributed to the anti-androgenic nature of the extract. These authors further reported return of sperm count and motility in the epididymis of Curcuma-treated rats two months after withdrawal of treatment56.

In our study in P mice, Curcuma treatment (600 mg/kg body weight/day, for 56 and 84 days) had no effect on body weight but caused a marked depression in weights of the testis, epididymis and seminal vesicle43. The treatment also had adverse effects on sperm parameters in the cauda epididymidis, on levels of sialic acid and fructose in the epididymis and seminal vesicle, respectively, and on serum level of testosterone. Further, fertility of Curcuma-treated males was also affected. Histologically, testes in Curcuma-treated mice exhibited degenerative changes in the seminiferous tubules although normal tubules were also seen in sections. The diameter of the seminiferous tubules and height of the germinal epithelium in testes of Curcuma-treated mice were also decreased. By 56 days of withdrawal therapy, however, the changes noted in the reproductive indices recovered to control levels43.

The mechanism by which Curcuma treatment induces anti-spermatogenic effects in mice testis is not properly understood. In immature male rat, it is suggested that Curcuma comosa (an another species of Curcuma) acts directly on the testis or indirectly inhibits gonadotropin secretion, thereby lowers testosterone production, or acts at both the levels57. It is known that testosterone is essential for sustenance of spermatogenesis58. The observation in P mice that Curcuma treatment caused reduction in the serum level of testosterone suggested that the Curcuma-induced suppression of spermatogenesis in mice testes was probably caused because of the deficiency of testosterone. The curcumin analogues are also shown to interfere with 17β-hydroxysteroid dehydrogenase isoform 3 activity and that this enzyme plays an important role in testosterone biosynthesis in the Leydig cells59.

Allamanda cathartica

A. cathartica L. (family, Apocynaceae) known commonly as the golden trumpet, yellow bell or the buttercup flower, exhibits various pharmacological properties such as anticancer, anti-inflammatory, antimicrobial, antifungal, anti-leukaemic, wound healing, antibiotic, anti-dermatophytic and anti-hypertensive60. The roots of A.cathartica contain iridoid lactone, allamandin and two other iridoids, allamandicin and allamdin; leaves and stem contain sesquiterpenes, ursolic acid, β-amyrin and β-sitosterol andursolic acid, β-amyrin and β-sitosterol, respectively, and flowers contain kaempferol, quercetin and hesperitin61.

: In P mice, A. cathartica treatment (150 mg/kg body weight/day for 14, 28 and 42 days) did not affect the body weight or the weights of testis and seminal vesicle, although epididymal weight was markedly decreased in treated mice62. Sperm parameters (motility, viability and number) in the cauda epididymidis and fertility were also affected in treated males62. Fructose level in the seminal vesicle and level of serum testosterone were not affected. In Allamanda-treated mice, marked histological changes were observed in the testis, and both affected and normal seminiferous tubules were seen in the same section. In testes of mice dosed with A. cathartica leaf extract, the affected seminiferous tubules exhibited diverse degenerative changes62. Germinal epithelial height and tubular diameter were also decreased in treated mice. Testis in treated mice showed high percentage of affected seminiferous tubules than in controls. By 56 days of withdrawal therapy, however, the changes caused in the reproductive organs recovered to control levels62.

In P mice, treatment with A. cathartica caused inhibition of spermatogenesis and this inhibition did not appear to be mediated via Leydig cells as no differences could be detected in serum testosterone level between treated mice and controls62. It is, therefore, likely that the treatment may act directly on the seminiferous tubules, resulting into the suppression of spermatogenesis. The observation that the intraepithelial vacuoles were frequently noticed in the seminiferous tubules showing degenerative changes in testes of Allamanda-treated mice support the above contention5262.

Bacopa monnieri

The plant B. monnieri (L.) Wettst. (family, Plantagenaceae) known as Brahmi grows in damp soils and marshes throughout the subcontinent6364. Pharmacological effects of Brahmi have also been evaluated in laboratory studies and results show many beneficial actions/properties including memory boosting, anti-Parkinson, anti-stroke, anticonvulsant, antidepressant, anti-anxiety, antioxidant, gastrointestinal and hepatoprotective, antimicrobial and anti-inflammatory activities65. Bacopa contains Brahmine, nicotinine, herpestine, des-saponin glycosides-triterpenoid saponins such as bacosides A and B66.

: In P mice, Bacopa treatment (250 mg/kg body weight/day for 28 and 56 days) did not affect the body weight or the weights of the testis, epididymis and seminal vesicle, but weight of epididymis was significantly decreased in mice treated with the plant for 28 days compared to controls67. Sperm parameters (motility, viability and number) in the cauda epididymidis and fructose level in the seminal vesicle were also adversely affected by the treatment; further, fertility of males was also affected as females mated to Bacopa-treated males did not show live implants67. The serum level of testosterone, however, remained unaltered in treated mice. Histologically, alterations were noticed in testes of Bacopa-treated mice, while testes in controls showed normal features. The testis in treated mice showed both affected and normal seminiferous tubules in the same section. The affected seminiferous tubules in both the dosage groups showed exfoliation of germ cells, loosening of germinal epithelium, presence of vacuoles in the epithelium and formation of giant cells67. Further, height of the germinal epithelium and diameter of the seminiferous tubules were also decreased in testes of mice treated with Bacopa compared to controls. By 56 days of treatment withdrawal, however, the changes induced in the reproductive organs returned to control levels67.

In P mice, Bacopa treatment did not influence testosterone secretion by the Leydig cells as no differences could be noted in serum testosterone level between treated mice and controls67. It may, therefore, be hypothesized that the Bacopa acts directly on the seminiferous tubules. Sertoli cells are well known to play an important role in maintenance of spermatogenesis and that any damage to these cells would cause suppression of spermatogenesis. The occurrence of intraepithelial vacuoles in the affected seminiferous tubules in testes of treated mice suggested that the anti-spermatogenic action of Bacopa in P mice was mediated via Sertoli cells5267.

Conclusion

Treatment with A. indica, C. longa, A. cathartica and B. monnieri in P mice produced reversible inhibition of the spermatogenic process and fertility, thereby advocating viability of these plants in male contraception. One of the major issues associated with plant-based research for fertility regulation is that the results show much variation, with 0-100 per cent activity with the same plant, and further, a herbal contraceptive practiced by humans may not be effective in animal models11. Hence, a better approach would be to assess the efficacy of the plants in humans themselves, after evaluation of their safety in animal models. Further, time and place of collection, proper identification, standard protocol for extraction and schedule of administration should also be considered while interpreting the results. It should also be remembered that many phytomedicines are extracts of the whole plants and synergistic interactions between components of the plants are essential for their efficacies; in many cases, it has been found that a total herb extract has exhibited a better outcome than an isolated compound68.

Financial support & sponsorship:

This work was supported by grants (3/1/2/13/01-RHN; 3/1/2/15/04-RHN) from the Indian Council of Medical Research, New Delhi, and the University Grants Commission, New Delhi, to the Centre of Advanced Study in Zoology, Banaras Hindu University, Varanasi.

Conflicts of Interest:

None.