Spermicidal & contraceptive properties of Praneem polyherbal pessary
Raghuvanshi, Poonam
Background & objectives: Though a number of barrier methods and potent spermicides are available, most of these have nonoxynol-9 (N-9) as the active ingredient which is observed to cause inflammation and genital ulceration on repeated use. The present study was undertaken to develop a safe spermicide with conjoint microbicidal properties.
Methods: A polyherbal pessary was formulated with purified ingredients from neem (Azadirachta indica) leaves, Sapindus mukerossi (pericarp of fruit) and Mentha citrata oil. Spermicidal action on human sperm was tested by Sander-Cramer slide test in vitro and by post coital tests in vivo. Contraceptive action was tested in rabbits.
Results: The combination of the three herbal ingredients resulted in the potentiation of the spermicidal action by 8-folds. The post coital tests confirmed the spermicidal properties of the Praneem polyherbal pessary (PPP) in women with high cervical mucous score around mid estrus. It also prevented in most women the migration of sperm into the cervical mucous. In 15 rabbits studied pregnancy was prevented by the intravaginal administration of PPP, whereas 13 of the 15 animals in the control group became pregnant.
Interpretation & conclusion: The Praneem polyherbal pessary has potent spermicidal action on human sperm in vitro and in vivo. When applied in the vagina before mating, it prevented rabbits from becoming pregnant.
Key words Azadirachta indica – post-coital tests – rabbits – saponins.
The Cairo conference on Population and Development1 enunciated women’s demand for contraceptives that are fully under their control, do not require systemic intake and can protect them from sexually transmitted infections in addition to preventing an unwanted pregnancy. Improved barrier methods and potent safe spermicides with antibacterial, antifungal and antiviral properties are in demand. Most of the available spermicides employ nonoxynol-9 (N-9) as the active ingredient and though it is a powerful detergent with high spermicidal properties, its repeated use has been observed to cause inflammation and genital ulceration2. Possibly because of these properties a clinical trial in Kenya indicated an enhanced transmission rather than prevention of HlV in professional sex workers-using such spermicides3.
The present communication reports results of studies on a Praneem polyherbal formulation dispensed as pessary which employs purified ingredients from 3 plants, each exercising spermicidal action, which gets pronounced synergistically when these are used in combination. This formulation inhibits a wide range of microbial and viral pathogens of the genital tract4.
Material & Methods
Physico-chemical characteristics of active ingredients : The Praneem polyherbal formulation consists of three components: Praneem, purified saponins and Mentha citrata oil. The composition of the pessary is described elsewheres.
Praneem is a purified fraction of the spray dried aqueous alcoholic extract of neem (Azadirachta indica) leaves. It is completely devoid of odour and colour. Praneem migrates as a single spot on visualization with UV on thin layer chromatography (TLC) employing iso-propyl alcohol: toluene: glacial acetic acid: water (25:20:10:10) as solvent system. However, on staining with vanillin sulphuric acid, other spots characteristic of the fraction are visible. These serve as a finger print profile for standardization of the preparation. The physico– chemical characteristics of the fraction are supplemented by bioactivity tested by quantitative determination of the spermicidal activity of the preparation.
Saponins are purified from the aqueous ethanolic extracts of the air-dried pericarp of fruits of Sapindus mukerossi. Its finger print profile was standardized by TLC. Mentha citrata oil migrates predominantly (86%) as a single peak with retention time of 18.5 min in gas liquid chromatogram.
The Praneem polyherbal pessary (PPP) was packed in triple laminated pouches. The shelf life at room temperature (25 deg C) of the pessary in the final pack was determined by accelerated stability studies at two conditions i.e. 30 deg C and 60 per cent relative humidity (RH); and at 40 deg C and 75 per cent relative humidity.
Dispersion studies: The time for dissolution of PPP in distilled water was determined in a water bath at 37 deg C. The dispersion in monkey vagina (5 monkeys) was conducted by the cold-light technique of visual inspection. The dispersion in human vagina was investigated in 5 women who volunteered for the study by inspection by the resident doctor at various time intervals after insertion.
Spermicidal activity : The spermicidal activity was determined by slide test employing a modified version of the protocol originally described by Sander and Cramer6, which measures the minimum concentration of a spermicidal agent required to kill 100 per cent of sperms within 20 sec. 50 (mu)lof the solution of the test ingredient at two-fold serial dilutions, was mixed with 20(mu)l of sperm suspension containing 60 million sperms per ml. The mixture was observed under the microscope for 20 sec at 10X and read for motile sperm. If any motile sperms were seen, the dilution was recorded as `not effective’. All the test solutions, which passed the test were mixed with 250(mu)l of Baker’s buffer (glucose 3%; Na2HPO4, 2H2O 0.31%; NaCl 0.2%; KH2PO4 0.01%) and incubated in a 370 deg C water bath for at least 60 min. The solution was slowly vortexed and observed again after 60 min for presence of any motile sperm. The concentration of the compound was recorded as effective if both tests indicated the absence of motile sperm. Semen samples were obtained from normal healthy volunteers by masturbation. Each test was perfomed on four different semen samples with sperm count ranging from 60X 10^sup 6^ /ml to 90X 10^sup 6^, /ml sperms with 60-80 per cent motility. Spermicidal activity of individual ingredients and combined formulation was determined by making their solution in Baker’s buffer.
M.citrata oil was tested as oil in water emulsion, prepared using polyvinyl alcohol (molecular weight 40,000) as emulsifier. Baker’s buffer was used as control in all experiments except citrata oil where 2 per cent polyvinyl alcohol in Baker’s buffer was used.
In vivo contraceptive action in rabbits These studies were conducted in the animal house of the International Center for Genetic Engineering and Biotechnology, New Delhi.
In vivo contraceptive action was tested in 45 New Zealand rabbits, 15 each for the Praneem polyherbal pessary, TODAY a standard marketed pessary, and the controls. The pessary (2.7g) was diluted 1:6 in physiological saline and 1 ml (equivalent to one sixth the human dose) administered by tuberculin syringe in the vagina of rabbits of proven fertility. At different intervals i.e. 15,30,60 min after the application, rabbits were mated with males of proven fertility. Rabbits were checked for sperm positivity and observed for delivery of pups, if any. In the control group, 1 ml of physiological saline was given. Comparison of contraceptive efficacy between the control and experimental groups was computed by 2-tail Fisher’s exact test of significance.
Post coital tests (PCTs) in women : PCTs with PPP and a standard commercially marketed pessary (TODAY) were conducted as per WHO protocol7 in women of reproductive age, who had elective tubal ligation. Ethical approval was obtained from the Institutional Ethics Committee of the Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh, India for the study and subjects were enrolled by informed consent. Only women with high cervical scores (> 10 as per WHO norms) as determined by viscosity, ferning or crystallization, spinnbarkeit, cellularity and quantity and whose husband’s semen parameters were good were selected for the study with the test formulations. Screening of women with acceptable cervical mucous score was done in the month previous to the one in which the formulation was tested. The tests were conducted in the mid cycle. The subjects were asked to insert the pessary approximately 30 min prior to intercourse. These women reported to the clinic within 12 h of intercourse for vaginal examination. In tests where no live sperm was detectable in posterior vaginal specimens, nor any live or dead sperm detectable in cervical mucus, the effect of the pessary was concluded as ‘effective’. In women where all sperms were noted as killed in posterior vaginal samples but in whom occasional dead sperms were noted in the cervical mucous, the results were graded as `partially effective’. In the situation where live sperms were detected, the PCTs were graded as `not effective’.
Results
Physical characteristics of the Praneem polyherbal pessary : PPP retained full spermicidal activity for 6 months of observation period at 30 deg C and 60 per cent RH and for 3 months at 40 deg C and 75 per cent RH. The shelf life of PPP at room temperature is thus computed to be 24 months. It starts dispersing immediately in aqueous phase (at 37 deg C) in vitro with complete dispersion achieved in 15 to 17 min. In vivo, its dispersion time in monkey vagina was found to be 4 to 6 min. In the five women who volunteered for the dispersion studies, the pessary started dispersing soon after insertion, in 45 min 90-95 per cent dispersion was noted and complete dispersion was observed in all women tested within 60 min.
Spermicidal properties in vitro : Each ingredient of the formulation had spermicidal activity, their individual concentrations for causing 100 per cent kill of human sperms in 20 sec are given in Table 1. As a combined formulation, a synergistic spermicidal effect was obtained with full spermicidal activity on human sperms in vitro at 8 times dilution (Table 1). Contraceptive efficacy : Female rabbits receiving the dispersion of the pessary in physiological saline at one sixth dose did not conceive on mating with males of proven fertility. The contraceptive effect persisted for 60 min of observation time after the application (Table II). The fertility of control animals in the colony was 86.6 per cent with 13 of the 15 animals carrying pregnancy to term and delivering healthy pups. The commercial pessary was fully effective till 30 min after intravaginal application with the contraceptive activity diminishing at 60 min. The difference between the experimental and control groups was statistically significant at each of the three time points (P
Post coital tests (PCTs) in women : These were conducted at PGIMER, Chandigarh, in nine women with the Praneem pessary. Observations are recorded in Table III. PCTs with the commercially marketed pessary TODAY were carried out in seven women (Table IV). These investigations showed that the Praneem pessary was fully effective in five women, partially effective in three and ineffective in one. In contrast TODAY was not found to be fully effective in any of the seven women tested and was partially effective in five and ineffective in two as per the WHO criteria described in methods section.
Discussion
A pessary constituting purified ingredients from three plant sources has been made with dual properties of potent spermicidal action and action against a wide spectrum of bacteria and viruses. Amongst others, it inhibits the growth of Neisseria gonorrhoeae, urinary tract Escherichia coli and HIV-1, and prevents the transmission of Herpes simplex virus and Chlamydia trachomati.s by the vaginal route4. Finger print profiles were developed to assure preparation of pessaries of reproducible physicochemical characteristics and biological properties.
Praneem polyherbal pessary and cream have undergone preclinical acute, sub-acute and chronic toxicology studies. These studies have demonstrated that the cream formulation is free from toxicity and does not produce sensitization and irritation8. Phase-I clinical trials with the PPP were conducted in 23 women in three major institutions in India. Daily intravaginal use of this pessary for seven days had no undesirable effect on vaginal cytology, metabolic and organ functions (data submitted to Drug Regulatory Authority but not published). The Praneem polyherbal formulations dispensed as cream or pessary are thus devoid of local and systemic reactions.
Each ingredient of the formulation had individual spermicidal action. Their combination had synergistic action with eight times amplification of the spermicidal effect on human sperms in vitro by the standard Sander-Cramer test. This is consistent with the observations on a previous PPH cream with slightly different composition9. In vivo the pessary demonstrated high contraceptive efficacy in rabbits of proven fertility. The effect was manifest at least up to one hour after application of the dispersed pessary. Post coital tests conducted in women with high cervical mucous scores and whose husbands had good sperm counts and motility showed that the PPP was at least as effective a spermicide, if not more, as the presently marketed nonoxynol-9 containing pessary ‘TODAY’. Of interest was the observation that PPP prevented the migration of sperm into the cervical mucous in 5 of 9 women, whereas this did not happen in any one of the seven women on whom PCTs were carried out with the pessary TODAY. PCTs do not assess the contraceptive efficacy of the pessaries in humans, for which the Phase-II efficacy trials on a statistically large number of the subjects would be required. These do however provide valuable information in situ on not only spermicidal action but also on blocking the passage of live sperm into the cervical mucous in mid esterus cervical mucous.
Acknowledgment
These studies were supported by grants from the Rockefeller Foundation, New York and the Talwar Research Foundation, New Delhi. Authors are thankful to the International Centre for Genetic Engineering and Biotechnology, New Delhi for animal and laboratory facilities.
References
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Poonam Raghuvanshi, Rashmi Bagga^, Diljot Malhotra^, Sarala Gopalan^ & G.P. Talwar
Talwar Research Foundation, New Delhi & ^Postgraduate Institute of Medical Education & Research, Chandigarh, India
Received September 20, 2000
Copyright Indian Council of Medical Research Apr 2001
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