1 Introduction

2 Materials and methods

2.1 Test materials

The seeds of Carica papaya (Caricaceae,Voucher No. RUBL 16590) of honey dew variety, were examined using the extraction procedures described earlier^[6]. Briegly, the seeds of Carica papaya were shade-dried, powdered and soxhalated in chloroform at 58 for 123 hours. The soxhalated material was concentrated under reduced pressure and further purified by silica gel column chromatography using benzene, chloroform and ethyl acetate as eluents. Following partial purification, the benzene chromatographic fraction, which showed significant effect on semen parameters^[4,6], was subjected to methanol and ethyl acetate sub-fractionation and the residues after evaporation of the solvents were used for further purification by Thin Layer Chromatography (TLC). Two compounds were obtained from each sub-fraction on TLC viz., MCP 1 & 2 and ECP 1 & 2, respectively. The compounds were scraped, extracted with the corresponding solvents, evaporated to dryness under reduced pressure and the residues were separated. The chloroform extract, the benzene chromatographic fraction of the chloroform extract and its methanol and ethyl acetate sub-fractions and the isolated compounds MCP 1 & 2 and ECP 1 & 2 which showed significant effects on sperm parameters by oral administration in rats and rabbits^[3,5,6], were used in the present investigation for in vitro sperm motility assessment. The extract, fractions and compounds were dissolved in Biggers Whitten and Whittingham (BWW) medium, supplemented with sodium bicarbonate (210 mg/L), glucose (100 mg/L), sodium pyruvate (3 mg/mL), bovine serum albumin (300 mg/L), sodium lactate (0.37 mL), benzyl penicillin (10,000 U/mL) and HEPES buffer (2 mL) at concentrations 2%, 1%, 0.5% and 0.1%.

2.2 Sample preparation

Semen samples (n=15) from normal subjects after 48 hours of sexual abstinence were subjected to routine semen analysis^[8] following liquefaction at 37. Sperm counts above 50 million/mL with normal morphology, rapid and linear and progressive motility, and viability above 50% was considered for the in vitro test. WHO's strict evaluation criteria were followed for assessing sperm morphology^[8]. Spermatozoa free of seminal plasma were obtained by centrifugation, washed once in BWW media and adjusted to the final concentration of 50 million/mL.

2.3 Experimental procedure

Prior to experiment, semen samples and the seed extract/product preparations were prewarmed to 37; 250 L semen sample containing 15 million spermatozoa was added to 250 L of the extract preparation at various concentrations. Sperm motility (a, b, c & d grades)^[8], was assessed immediately after addition of the semen samples and thereafter every 5 min for up to 30 min in a phase contrast microscope along with placebo control. Following completion of the experiment, sperm viability and morphological assessments were carried out by nigrosin-eosin and papanicolaou staining methods, respectively^[8].  

2.4 Ultrastructural studies

Following completion of motility assessment, the spermatozoa were separated by centrifugation, washed with phosphate buffer (0.1 mol/L; pH 7.2) and pelleted by centrifugation. The pellets were immediately fixed in 2.5% glutaraldehyde in phosphate buffer for 30 min and washed thrice in phosphate buffer. For scanning electron microscopy (SEM), a thin film of spermatozoa was smeared on an SEM stub with silver paint, air dried, sputter coated with gold and observed under the scanning electron microscope (Leo SEM 435 VP). For transmission electron microscopy (TEM), the spermatozoa pellet was post-fixed in OsO₄ for 30 min, washed in phosphate buffer, followed by distilled water, dehydrated in acetone, embedded in low viscosity spur medium and polymerized at 60 for 48 h. The ultrathin sections were stained with uranyl acetate and lead citrate and observed under the transmission electron microscope (Philips model CM-10).

2.5 Sperm revival test

Following completion of the experiment, the spermatozoa were washed twice with BWW medium and incubated once again in the same medium free of extract/products at 37 for 30 min to observe revival of sperm  motility, if any. Immotile spermatozoa showing vibratory movement to progressive motility after incubation were considered revived.

2.6 Statistical analysis

3 Results

Sperm motility from its initial level of 55.07%3.30% dropped immediately following the addition of the chloroform extract, benzene chromatographic fraction and its sub-fractions as well as the isolated compounds, ranged between 8.3%0.7% and 31.2%1.5%. The effects were dose-dependent and more pronounced effects were observed with the compounds isolated from the ethyl acetate sub-fractions (ECP 1 & 2). Although many of the spermatozoa showed only vibratory movement after 5 min of addition of the drugs, particularly at 2% concentration, total inhibition of motility was observed after 10 min in the chloroform extract, benzene chromatographic fraction of the chloroform extract and ECP 1 & 2 treated samples. None of the spermatozoa showed motility, 25 min after addition of the extract/fractions/compounds (Tables 1 & 2).

The viability of spermatozoa, following completion of the experiment, fell significantly (P<0.001; Range 22.8%0.6% to 45.4%1.2% as against 54.9%1.2% in control) and the effects were more pronounced in the ECP 1 & 2 treated samples at 2% concentration. Sperm abnormality showed a significant increase in its pattern (P<0.001; Range 26.7%1.1% to 46.9%1.3% as against 16.6%1.8% in  control samples). Most abnormalities were found in MCP 1 & 2 treated samples at 2% concentration (Table 3). 

Table 1.Human sperm motility (%) following incubation with chloroform extract, bezene chromatographic fraction and its sub-fractions of the chloroform extract of the seeds of Carica papaya in vitro (meanSEM of 15 samples)^*.

4 Discussion

The mechanism of action of many of the spermicidal compounds of plant origin seems to be by surface action, disrupting the plasma membrane of the spermatozoa^[1]. The currently used vaginal spermicide, nonoxynol-9 acts in a similar manner. It produces disruption of lipids within the sperm membrane, particularly on the acrosome and mid-piece, causing disruption and rapid loss of sperm motility^[9,10]. Praneem, a polyherbal cream  containing purified neem seed extract, quinine hydrochloride and the reetha saponins extracted from the  pericarp of Sapindus mukorossi, at 10% concentration has high potency to modify the mucus and inhibit the penetration of sperm^[15]. Inhibition of the sperm specific enzymes acrosin and hyaluronidase, which play  an important role in the fertilization process by plant derivatives, has also been reported^[1]. It is believed that the flavonoids and their derivatives, flavonones and flavonols, contain hyaluronidase inhibitory activity^[1].    

Farnsworth and Waller^[1] have screened a large number of plants for spermicidal  and reported that a majority of plant-derived spermicides are triterpene saponins of several structural types, flavonoids and phenol compounds. The saponins of Cyclomen persicum, Primula vulgaris and Gypsophyla paniculata have been reported  to produce instant immobilization of human spermatozoa within 20 seconds^[11]. Acacic acid from the bark of Acacia concinna, oleanolic acid and porceric acid from the roots of Albizza procera and anagalligenone from the whole plant Anagallis arvensis also produce instant sperm immobilization within one minute in human spermatozoa^[12]. An instant spermicidal activity of pittoside A & B, the sapogenins isolated from the plant Pittosporum nilghirense has also  been reported^[13].

In the present study the chloroform extract, the benzene chromatographic fraction of the chloroform extract and its methanol and ethyl acetate sub-fractions and the isolated compounds, ECP 1 & 2 and MCP 1 & 2, which exerted their systemic action on the sperm parameters in our earlier studies, have also shown a sperm immobilizing effect on human spermatozoa in vitro. The effect is spermicidal and not spermiostatic as there is no revival of motility after incubation with BWW medium free extracts. The isolated compounds ECP 1 & 2 were found to be more effective, lowering the motility to less than 10%, immediately after addition of the compound at 2% concentration. SEM and TEM of spermatozoa after completion of incubation  showed membrane damage in the head as well as mid-piece suggesting that the mode of action appears similar to that of nonoxynol-9^[9,10] and most other herbal spermicidal agents^[1]. Although not specific, the membrane damage in the sperm head could cause acrosin and hyaluronidase inhibition as well, as reported for other herbal spermicides^[1].

The sperm immobilizing effect was dose-dependent. However, instant immobilization of spermatozoa has not been observed in the present study, although the compounds ECP 1 & 2 drastically affected the sperm motility to less than 10% and most of them showed slow progressive to vibratory movement that may imply infertility in fertility trials^[8]. A still higher concentration may induce instant immobilization as in CONSAP, a herbal cream formulation containing saponins of fruit pericarp of Sapindus mukorossi where 2.5% concentration has been tested in woman volunteers^[14].

A possibly useful approach may be a combination of Carica papaya products with other spermicides as in praneem, where the combination of purified neem seed extract, reetha saponins and quinine HCl dispensed in a water washable  cream base has been tried, for a better applicability^[15]. Such a combination approach should, in addition to the  instant sperm immobilization action, contain an antimicrobial action too, as in praneem which reportedly contains  antimicrobial action against the vaginal pathogens Chlamydia trachomatis, Candida albicans and Gardeneralla vaginalis tested in vitro in phase I clinical trial^[15]. Importantly, the leaves/seeds/root/fruit extracts of Carica papaya are reported to possess antibacterial, antiviral and antiyeast activities^[16,17]. These extracts were particularly sensitive to Candida albicans, tobacco mosaic virus, ring spot virus and a number of bacteria which includes Salmonella, Staphylococcus and Pseudomonas species^[16]. This would be an added advantage for any Carica papaya-based vaginal contraceptive. These extracts were particularly advantageous over nonoxynol-9, that it does not cause inflammatory reaction and irritation of vaginal epithelium upon repeated use (unpublished observations, Lohiya et al). Further studies are in progress with a combination approach and its possible antimicrobial action for a better applicability.

5 Acknowledgements

References

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[3] Lohiya NK, Goyal RB. Antifertility investigations on the crude chloroform extract of Carica papaya Linn. seeds in male albino rats. Ind J Exp Biol 1992; 30: 1051-5. 
[4] Pathak N, Mishra PK, Manivannan B, Lohiya NK. Sterility due to inhibition of sperm motility by oral administration of benzene chromatographic fraction of the chloroform extract of the seeds of Carica papaya in rats. Phytomedicine  (In press).
[5] Lohiya NK, Pathak N, Mishra PK, Manivannan B. Reversible contraception with choloroform extract of Carica papaya Linn. seeds in male rabbits. Reprod Toxicol 1999; 13: 59-66. 
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[8] WHO Laboratory manual for the examination of human semen and sperm-cervical mucus interaction. 4th ed. Cambridge: Cambridge University Press; 1999.
[9] Schill WB, Wolff HH. Ultrastructure of human spermatozoa in the presence of the spermicide nonoxynol-9 and a vaginal contraceptive containing nonoxynol-9. Andrologia 1981; 13: 42-9.
[10] Wilborn WH, Hahn DW, McGuire JJ. Scanning electron microscopy of human spermatozoa after incubation with the spermicide nonoxynol-9. Fertil Steril 1983; 39: 717-9.
[11] Primorac M, Sekulovic D, Antonic S. In vitro determination of the spermicidal activity of plant saponins. Pharmazie 1985; 40: 585.
[12] Kamboj VP, Dhawan BN. Research on plants for fertility regulation in India. J Ethnopharmacol 1982; 6: 191-226.
[13] Jain GK, Pal R, Khanna NM. Spermicidal saponins from Pittosporum nilghirense Wight et Apnott. Ind J Pharm Sci 1980; 42: 12-3.
[14] Kamboj VP, Dhawan BN. Fertility regulating plants on Indian scene-an update. Contraceptive Research Today and Tomorrow. New Delhi: Indian Council of Medical Research 1989. p 115-25. 
[15] Talwar GP, Garg S, Dhar V, Chabra R, Ganju A, Upadhyay SN. Praneem polyherbal cream and pessaries with dual properties of contraception and alleviation of genital infections. Curr Sci 1995; 68: 437-40.
[16] Ross IA. Carica papaya L. In: Ross IA, editor. Medicinal Plants of the World. Totowa, NJ: Humana Press Inc; 1999.
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