Title of Invention	"PROCESS FOR THE PREPARATION OF AN IMPROVED ANTIMICROBIAL AND SPERMICIDAL COMPOSITION"
Abstract	The present invention provides a process for the preparation of an improved antimicrobial and spernucidal composition comprising 1 to 40% by weight of Praneem, 0.01 to 10% by weight of purified saponins from Sapmndus mukerossi, between 0.01 to 20% by weight Meniha citrafa oil and U to 05% by weight of zinc acetate dispersed in suitable carrier or vehicle. The process cumprising air drying leaves of Azadirachfa indica, powdering said air dried leaves and subjecting them to extraction with water under hot conditions to obtain the leaf extract, dissolving lt in water, subjecting it to decolourisation, and recovering the Praneem; preparing uiirilied saponins by subjecting air dried pericarp of Sapindus mukerossi to extraction, cuncentration to a syrupy residue, subjecting said residue to further extraction and then precipitation; dispersing the above Praneem and purified saponins in a conventional carrier; adding citrata oil to said mixture; adding zinc acetate, and other conventional ingredients if desired; converting the final composition into the required shape and form, if desired.

Title of Invention

"PROCESS FOR THE PREPARATION OF AN IMPROVED ANTIMICROBIAL AND SPERMICIDAL COMPOSITION"

Abstract

The present invention provides a process for the preparation of an improved antimicrobial and spernucidal composition comprising 1 to 40% by weight of Praneem, 0.01 to 10% by weight of purified saponins from Sapmndus mukerossi, between 0.01 to 20% by weight Meniha citrafa oil and U to 05% by weight of zinc acetate dispersed in suitable carrier or vehicle. The process cumprising air drying leaves of Azadirachfa indica, powdering said air dried leaves and subjecting them to extraction with water under hot conditions to obtain the leaf extract, dissolving lt in water, subjecting it to decolourisation, and recovering the Praneem; preparing uiirilied saponins by subjecting air dried pericarp of Sapindus mukerossi to extraction, cuncentration to a syrupy residue, subjecting said residue to further extraction and then precipitation; dispersing the above Praneem and purified saponins in a conventional carrier; adding citrata oil to said mixture; adding zinc acetate, and other conventional ingredients if desired; converting the final composition into the required shape and form, if desired.

Full Text	Field of the invention 1 he present invention relates to a process for the preparation of an improved antimicrobial and spermicidal composition and a composition prepared thereby. The composition of the present invention is capable of use for prevention of AIDS, other sexually transmitted infections and curing of leucorrhea due to reproductive tract infections. The composition of the invention is also capable of potent spermicidal action and prevention of unplanned pregnancy by intravaginal application prior to mating. Background of the invention Sexually transmitted diseases including AIDS and unplanned pregnancies continue to be a serious problem. Improved barrier methods and potent safe spermicides with antibacterial, antifungal and antiviral properties are in demand. The World Health Organization reported the occurrence of 333 million new cases of Sexually Transmitted Diseases (STDs) in only one year excluding those of AIDS (World Health Organization. Report on fighting disease fostering development, Geneva, 1996.). AIDS due to HIV virus is also spreading fairly rapidly globally, in India, the figures of those positive for HIV rose from none in 1986 to 3-4 million in early 1999,are expected to double by the year 2000. The transmission of AIDS virus, besides through contaminated blood, is mostly by sex with unsuspecting partners presenting no obvious symptoms. It is further observed that a large number of women in India and elsewhere have abnormal vaginal discharge (leucorrhea) caused due to pathogenic microorganisms in the vagina which include aerobic and anaerobic microbes, fungi such as Candida albicans and Candida uopicalis, Trichomonas and Gardnerella. These infections are collectively represented as RTIs - Reproductive Tract Infections. A study by the Indian Council of Medical Research (ICMR) reported 27 6% of women below 35 years of age having abnormal vaginal discharge caused by pathogens, bacteria, fungi or viruses (Bhujwala et al. Ind. J. Med Res. 1985; 81: 251-6.). Bang et al (Lancet 1989, 1: 85-6.) found that incidence of RTIs was as high as 50% in rural Maharashtra. Although specific drugs and antibiotics, for example Metronidazole (for iiichomonasis), tetracycline or Doxycycline (for uncomplicated gonorrhea infections). Nystatin or Clotrimazole or Miconazole or Ketoconazole (for vaginal candidasis), Acyclovir (for genital herpes) and Benzathine penicillin (for other RTIs) are available, they have side effects like local irritation, burning sensation, nausea, vomiting, diarrhoea, dizziness, headache, skin rashes and phototoxicity. No single wide spectrum composition is available for intravaginal use that can cure or prevent infections due to commonly encountered vaginal pathogens without any side effects. In addition, microorganisms are increasingly becoming resistant to one or more antibiotics. From 1994-1997, the proportion of Neisseria gonorrhoeae highly resistant to ciprofloxacin (MIC>/=4µg/ml) increased substantially among female sex workers in the Philippines (Klausner et al. J. Infect. Dis. 1999 Mar; 179(3): 729-33). Therefore, prevention of sexually transmitted infections including AIDS becomes as important to the public health as is finding a cure for them. Another problem that is of tremendous concern particularly for developing countries like India is overpopulation. The problems associated with overpopulation are well-known - high population density in urban centres leading to epidemics, inadequate sanitation, excess pressure on available resource use, etc. While a number of methods of family planning through birth control are available, no single method suits all nor does the same method suit the same individual at different stages of reproductive life. Spermicidal preparations have the advantage of topical applicability (not involving systemic intake) and can be used as and when the need arises. Furthermore, in contrast with condoms, the control is with the female partner Therefore, spermicidal preparations have found wider acceptability due to the convenience factor. Spermicides available in the art employ Nonoxynol-9 (N-9) as the active ingredient which was reported to provide protection against chlamydia and gonorrhea (Rosenberg et al. Am. J. Public Health 1992; 82: 669-74), Trichomonas vaginalis (Bolch et al. Am J. Obstet. Ciynecol. 1973; 115: 1145-8.; Singh et al. Contraception 1972; 5: 401-11.), Staphylococcus aureus (Snelton et al. Contraception 1981; 24: 631-4.), genital herpes and feline immunodeficiency virus in animal models (Whaley et al. J. Infec. Dis. 1993; 168:1009-11.; Moench et al. AIDS 1993; 7: 797-802.). High frequency use of N-9, however, results in vaginal irritation, inflammation, genital ulceration and allergic vaginitis which may enhance susceptibility to sexually transmitted infections (Lee et al. J. Pharm. Sci. 1996, 85(1) 91-5.). Possibly because of these properties a clinical trial in Kenya indicated as enhanced transmission rather than prevention of HIV in professional sex workers using Nonoxynol based spermicides i Kreiss et al. JAMA 1992; 265: 477-82.). A variety of alternative synthetic microbicides (Neurath et al. Lancet 1996; 347: 1703-4.; Baba et al Antimicrob. Agents Chemother. 1988, 32: 1742-5.; Gonzalez et al. Antimicob. Agents Chemother. 1987; 31: 1388-93.; Nisbet et al. Antimicrob Agents Chemother 1979; 16: 855-7.; Louis et al. Contraception 1985, 32: 199-205., Hansen et al. Scand J. Infect. Dis. 1991, 23 425-30.-. Ito et al. Am. J. Virol. 1974; 13: 1312-8., Petronio et al. Chemotherapy 1997; 43 211-7.. Oehniger et al. Fertil. Steril. 1991; 55: 165-9., Bourinbaiar et al. Contraception 1994; 49: 211;-7 , Shubair et al. Gynecol. Obstet. Invest. 1990, 29: 67-70., Malkovsky et al. Lancet 1988, 19. o45.; Asculai et al. Antimicrob. Agents Chemother. 1978; 13: 686-90., Amortegui et al Contraception 1984; 30: 135-41.; Foldesy et al. Fertil. Steril. 1986, 45: 550-5., Amstey et al. Obstet. Gynecol. 1975; 46: 528-9.; Sokal et al. Sex Transm. Dis. 1995; 22: 22-4.; Zamora et al. Am J Surg. 1986; 151: 400-6.) have demonstrated activity against sperm and sexually transmitted pathogens in vitro, but few have been tested in vivo. For these reasons, there is need for vaginal compositions which are free of irritation and which are active in vivo against sexually transmitted diseases/ AIDS and unplanned pregnancy. A substantial number of prior art compositions are available only in the form of pessaries and some of them require wetting before insertion into the vagina. The failure to wet or improper wetting of these pessaries before insertion is a recurring phenomenon either due to lack of knowledge or simple memory failure on the part of the female member. An object of this invention is the provision of a composition that does not require wetting, with high spermicidal action and wide spectrum inhibitory action on genital pathogens including those transmitted by sex. In addition, the composition must be capable of use by women intravaginally to cure leucorrhea caused due to reproductive tract infections The composition should also be capable of antimicrobial action on microbes resistant to one or more drugs Furthermore the use of the composition before sexual intercourse should prevent the transmission of a variety of sexual transmitted infections, such as gonorrhea, herpes, chlamydia and AIDS, besides others, from, the partner and also safeguard women against unwanted pregnancy by exercising spermicidal action. The composition should also be in a form which is easy to use and totally free of irritation. The applicant's efforts towards the achievement of such subjects have led them to study ancient and indigenous plants such as neem (Azadirachta indica), reetha (Sapindus imikerossn and Mentha citrata already well known for their utility in other areas. The neem tree (Azadirachta indica) found throughout India is used in indigenous and household medicine for different purposes. Almost every part of the tree has been utilized for treatment of various disorders and diseases. Neem leaves are used to mitigate the pruritic sensation in smallpox, chicken pox and measles. Extracts from various parts of the neem tree are used because of their bactericidal properties in indigenous tooth powders, toothpaste and toilet soaps, its antibacterial activity has been demonstrated by many. Chatterjee and Roy (Ind. 3 Med. Res. 1917; 5: 656) demonstrated the in vivo antibacterial activity of margosates obtained from Neem oil. Murthy et al. (Indian J. Pharmacol. 1958; 2: 387-96 and 456-461.) showed that nimbidin, one of the active principles of Neem has inhibitory properties against Mycobacterium tuberculosis. Singh et al. (Ind. J. Med. Res. 1974; 9: 65.) showed that Neem oil has antibacterial activity against E.coli in vitro. Chaurasia et al. (Indian J. Hospital Pharm 1978; 15: 166.) showed that Neem oil has antibacterial activity against various bacteria potentially pathogenic to plants and animals. Rao et al. (Ind. J. Med. Res. 1986; 84: 314-16.) showed that Neem oil has significant antibacterial properties against pathogenic Gram negative bacilli such as Ps. aeruginosa, E.coli, Proteus group and K. aerugenes and against gram positive cocci such as Siaph. pyogenes. Although many investigators (Sinha et al. Ind. J. Med. Res. 1984; 79: 131-36.; Bardhan et al. Indian J Physiol. Pharmacol. 1991; 35(4):278-80.; Sharma & Saksena. Ind. J. Med. Sci. 1959; 13: 1038.; Sharma & Saksena. Ind. J. Med. Res.1959; 47: 322.) have reported the spermicidal activity of Neem oil and its components, however, their findings reveal a rather low spermicidal activity of Neem oil if used as such. Furthermore Neem oil has very strong pungent odour. Riar et al. (Contraception 1990 Oct.; 42(4): 479-87.) extracted the volatile, odorous traction of neem oil coded as Nirn-76 by steam distillation which exercises spermicidal activity ai 25 mg/ml concentration on human sperm. Though better than the native neem oil, activity remains still very low. Another major disadvantage of Nim-76 is its strong pungent odour that would dissuade for all practical purposes the use by choice of such preparations for vaginal insertion before intercourse. US Patent No. 5,196,197; 1993 overcomes the problem of low spermicidal efficacy of Neem oil, by potentiating the spermicidal efficacy synergistically by combination with purified saponins from Sapindus mukerossi (reported to have spermicidal activity by Khanna - Indian Patent No. 150335; 1982). Our Indian Patent Application 1351/Del/97 dated May 20, 1997 overcomes the problems of acceptability and unpleasant odor and color of Neem oil by use of purified neem leaf extract (Praneem) in place of Neem oil. Spermicidal activity is further enhanced by addition of Mentha cnrata oil (Mentha citrata is a hybrid variety of Mentha spicata & Mentha aquatica. Wealth of India, Shastri ed. New Delhi:CSIR, 1988; Vol. VI (L-M):338.) and zinc acetate (reported to have antiviral and spermicidal activity by Fahim & Wang. Contraception. 1996; 53: 231-36.) Indian Patent Application No. 1351/Del/97 relates to a composition for prevention of unwanted pregnancy which comprises a major amount of conventional carriers, from 1 to 40% by weight of said composition, Praneem, from 0.01-10% by weight of purified saponins, from 0.5-5% of zinc acetate, from 0.5% to 20% by weight of Mentha citrata oil and the balance if any comprising of conventional additives. This composition had shown in vitro activity against urinary tract E.coli and N.gonorrhoeae. It also showed virucidal activity against HIV-1 and HSV-2. Composition was effective in killing 100% of human sperms in 20 seconds in vitro up to iO times dilution. However, it prevented pregnancy in 100% of rabbits tested, at l/6th of human dose. Further dilutions were not effective. Also the zinc acetate component causes skin irritation (Lansdown. Food Chem Toxicol 1991 Jan; 29(1): 57-64). Accordingly, it is necessary to obtain a composition which results in the obviation of all trie drawbacks enumerated above - those of efficacy, odor, staining, irritation and mucosal damage. Objects of the invention The main object of the present invention is to provide a process for the preparation of an iinproved antimicrobial and spermicidal composition, which prevents the transmission of sexually transmitted pathogens such as N. gonorrhoeae, herpes, Chlamydia trachomatis and AIDS It is another object of the present invention to provide a process for the preparation of a ^ate antimicrobial and non-irritant spermicidal composition containing a very negligible amount wi'oi no zinc acetate. „ It is a further object of the present invention to provide a process for the preparation of an improved antimicrobial and spermicidal composition, which is a cure for leucorrhea caused by reproductive tract infections. It is another object of the present invention to safeguard women against unwanted pregnancy. it is another object of the invention to provide a method of fertility control, which is completely safe and free of side effects. Yet another object of the present invention is to provide a method of fertility control which can be restricted only to the occasions when the need arises, which does not require systemic intake and which is affordable. Yet a further object of the present invention is to provide a process for the preparation of an improved contraceptive composition for use in the above method which has dual properties of contraception and prevention of a large number of genital tract infections. It is another object of the present invention to provide a process for the preparation of a contraceptive or spermicidal composition in the form of a foaming tablet that is easy to use and is under control of woman in contrast to condom which is under control of the male partner Summary of the invention The spermicidal activity of zinc acetate is well documented in the art. This being so, no prior art would teach away from the use of zinc acetate in a spermicidal composition. However, it has been surprisingly found that the reduction or even total elimination of zinc acetate from a spermicidal composition especially, the composition claimed by copending Indian Patent Application No. 1315/Del/97 not only does not affect the efficacy thereof but totally avoids the problems of irritation associated with zinc acetate completely. It is known that a neem extract by itself will not have the same level of spermicidal effect as that of zinc acetate. Yet, even in the absence of zinc acetate, the composition of the present invention described herein exhibits greater spermicidal activity than neem extract alone or zinc acetate alone and exhibits spermicidal activity of equal efficacy as compared to the composition claimed in copending Indian Patent Application 1315/Del/97. Accordingly, the present invention provides an procgss for the preparation of an improved antimicrobial and spermicidal composition charatersing taht it compare1 to 40% by weight of Praneem,. 0.01 to 10% by weight of purified saponins from Sapindus mukerossi, between 0.01 to 20% by weight Mentha citrata oil and 0 to 0.5% by weight of zinc acetate dispersed in a convenmtion carrier or vehicle, said process comprising air drying leaves of Azadirachta indica, powdering .said air dried leaves and subjecting them to extraction with water under hot conditions to obtain the leaf extract, dissolving it in water, subjecting it to decolourisation, and recovering the Praneem; preparing purified saponins by subjecting air dried pericarp of Sapindus mukerossi to extraction, concentration to a syrupy residue, subjecting said residue to further extraction and then precipitation, dispersing the above Praneem and purified saponins in a conventional carrier; adding citrata oil to said mixture; adding zinc acetate, and optiopnally other conventional ingredients and, optionally converting the final composition into the required shape and form, if desired.. The composition prepared by the process of the present invention is not a mere admixture of ingredients resulting in an aggregate of the properties thereof, but is a synergistic admixture evincing biological activity. in one embodiment of the present invention, the amount of zinc acetate is less than 0.5 to, preferably, 0 % by wt. In another embodiment of the present invention, Praneem is present in an amount of 5-60% by weight of said composition. In another embodiment of the present invention, purified saponins are present in an amount of 1- 5% by weight of said composition. In another embodiment of the present invention, Mentha citrata oil is present in an amount of 1- 10% by weight of said composition. In another embodiment of the present invention, the conventional additives are selected from preservatives, antioxidants, effervescent mixtures, gelling agents, polysaccharides, disintegrants, diluents and lubricants. In another embodiment of the present invention, the carrier is a diluent selected from .actose, starches, sucrose, mannitol, sorbitol, microcrystalline cellulose, polyethylene glycol, coca butter, Tweens and Spans. In a further embodiment of the invention, the polyethylene glycol carrier is replaced by sodium alginate and calcium gluconate. In a still further embodiment of the invention, the composition is produced in the form of it foaming tablet that does not need wetting before insertion into the vagina. In a further embodiment of the present invention, the preservative is selected from the group consisting of phenol, chloro cresol, o-phenyl phenol, methyl parabens, propyl paraben and their sodium salts, benzylkonium chloride, boric acid and benzoic acid. In another embodiment of the present invention, antioxidant is selected from the group consisting of ascorbic acid, propyl gallate, L-tocopherol, butylated hydroxyl anisole and butylated hydroxl toluene. In yet another embodiment of the present invention, the disintegrant is selected from the group consisting of sodium carboxy methyl cellulose, guar gum, agar, starch and Tween. In another embodiment of the present invention, the gelling agent is selected from the group consisting of calcium gluconate, calcium lactate and calcium carbonate. In a further embodiment of the present invention, the polysaccharide is selected from the group consisting of sodium/ potassium alginates or carrageenan (iota/ kappa). In another embodiment of the present invention, the lubricant is selected from the group consisting of light liquid paraffin, talc, aerosol, magnesium'stearate and sodium stearate. In another embodiment of the present invention, the composition prepared by the process of the invention is in the form of a pessary, cream, gel, soft gelatin capsules, foaming tablets, loams, spray or aerosol. The invention also relates to an improved antimicrobial and spermicidal composition which comprises 1 to 40% by weight of Praneem, 0.01 to 10% by weight of purified saponins horn Sapindus mukerossi, between 0.01 to 20% by weight Mentha citrata oil and 0 to 0.5% by weight of zinc acetate dispersed in suitable carrier or vehicle. According to a further feature, the present invention also provides a spermicidal composition comprising a dispersion of neem leaf extracts, purified saponins, zinc acetate and/ or citrata oil in a pharmaceutical^ acceptable carrier or base therefore. In a further embodiment of the invention, polyethylene glycol is replaced by ;.odium/potassium alginates and/ or carrageenan (iota/kappa). Brief description of the accompanying drawings Figure 1 is the HPLC profile of Praneem. Figure 2 is a gas chromatogram of Citrata oil. Detailed description of the invention It is found that reduction in concentration or complete removal of zinc acetate from the composition does not in anyway effect the spermicidal and antimicrobial properties of the composition and in fact eliminates the irritation observed with the use of higher concentrations of zinc acetate. In-vivo and clinical data clearly show that the present composition with or without zinc acetate is equally effective clinically. The present composition inhibits the growth in culture of urinary tract Kcoli (including multidrug resistant strains) and N. gonorrhoeae (including strains resistant to penicillin). The composition manifested virucidal activity against HIV-1 at >2% and 50% dilutions on contact tor 1-2 minutes with the virus. Intravaginal inoculation of the present composition before inoculation of the pathogen prevented lesions and vaginal transmission of HSV-2 and (Itkunydia trachomatis in progestin sensitized mice. Composition when used in women suffering from abnormal vaginal discharge due to various pathogens completely cured the clinical symptoms in 100% of subjects who used the composition for 7 consecutive days. The present composition although killed 100% of human sperms in 20 seconds in vitro up to 8 times dilution, there was no change in the in-vivo activity. At one sixth of human dose to rabbits of proven fertility, 15-60 minutes prior to mating, it prevented pregnancy in 15 out of 15 rabbits tested. The effect was further potentiated when the present composition was delivered in ibrm of pessary or vaginal tablet. In post coital tests as per WHO protocol it was found effective in 8 out of 9 women tested. The composition of the present invention therefore, provides a method for blocking reversibly the fertility of a female without impairing ovulation by applying a predetermined amount of a contraceptive composition based on neem leaf extracts, purified saponins, citrata oil and or zinc acetate to the vagina of the female subject. Under the process of this invention, it has been established that the compositions made containing the following ingredients; - Praneem (purified Neem (Azadirachta indica) leaf extract) - Purified saponins from Sapindus mukerossi -Mentha citrata oil and/ or - Zinc acetate dispensed in the described vehicles, have potent spermicidal action and at the same time have a wide range of antimicrobial activity against a number of vaginal pathogens. 1) Praneem - In accordance with this invention there is provided a new antimicrobial and spermicidal fraction from the Neem leaves obtained by the extraction, purification and decolonisation of Neem leaves. The air-dried leaves are coarsely powdered to pass through 20 mesh sieve and then exhaustively extracted with water under hot condition to obtain the extract. The extract is concentrated four times under vacuum. The concentrated material is spray dried. The spray dried aqueous alcoholic extract of Neem leaves is dissolved in 20 times water and decolourized with activated charcoal by boiling. Charcoal is removed by filtration The filtrate is reduced in volume four times under vacuum. The concentrated material is freeze dried to obtain Praneem. J he specifications of purified extract 'Praneem' are; colourless to off-white powder, the pH of 1 % w/v solution ranges between 7.7-9.5, and contains bitters>5% w/w. The finger print profile Dy HPLC is as per Fig.-l. Nimbin is used as the marker to standardize the Praneem. At 15% solution in isotonic buffer, it kills all the sperms within 20 seconds. 2). Purified saponins - For preparation of purified saponins, pericarp of air-dried Sapindus mukerossi is powdered and exhaustively extracted with 90% aqueous-ethanol by soxhlation. The extract is concentrated to thick syrupy residue under vacuum below 50°C. The residue is then extracted with isopropyl alcohol under cold conditions. The iso-propyl alcohol is distilled under vacuum below 40°C and residue dried under high vacuum to obtain light cream coloured hygroscopic powder. An alternate method of purifying the alcohol extracts of Sapindus mukerossi pericarp is to extract the residue successively with petroleum ether, ether, carbon tetrachloride, chloroform and acetone. The residue is dissolved in absolute ethanol and filtered. The solution is added drop wise to ether, when a light brown precipitate is obtained. This step is repeated several times. The specifications of the purified saponins are as follows: Melting range 90-118°C; soluble in water, alcohol and chloroform, pH of 1% w/v solution in water is 3-5, total saponins content as per Indian pharmacopoeia is not less than 30% w/w; at 0.25% w/v solution in isotonic buffer it kills 100% of human sperm within 20 seconds. 3, Mentha ciirata - is a hybrid of Men/ha spicala and Mentha ac/itat/aa. Citrata oil is obtained from leaves of Mentha citrata oil and has the following specifications: characteristic odor, acid value 0.6 +l0%, kills 100% human sperms within 20 seconds at 5% w/v concentration. Gas liquid chromatogram is shown in Fig.-2. 4). Zinc acetate - Zinc acetate which is commercially easily available has following specifications: pH of aqueous solution is about 5-6, kills 100% human sperm within 20 seconds at 1 % vv/v concentration. The Praneem, purified saponins, citrata oil and/ or zinc acetate of the present invention, for practical application, can be utilized in the form of composition or compositions. In these composition and compositions, the active ingredients are mixed with pharmaceutically acceptable base as is well known in the art. If desired, adjuvants such as antioxidants, stabilizers, preservatives, effervescent mixtures may also be added. Examples of the compositions and compositions according to the invention are those known to one skilled in the art and include Pessaries and Cream, Gel, Soft gelatin capsules, foaming tablets, foams, spray and other aerosol compositions. The final composition and compositions are prepared in a manner known to one skilled in the art, for example by extending ihe active compound with conventional dispersible liquid diluent carriers and/ or dispersible solid carriers optionally with the use of carrier vehicle assistants, e.g., conventional surface active agents, including emulsifying agents and/ or dispersing agent and/ or gelling agents. it is observed that when polyethylene glycol carrier is replaced by sodium/potassium alginates or carrageenan, it is easy to obtain a foaming tablet that does not require wetting before insertion into the vagina. Thus the irritation observed in the case of some prior art pessaries due to the failure in several cases to properly wet the pessary before insertion into the vagina is avoided. Also, the control over the use of the composition killing sperms is with the female partner Diluents such as lactose, starches, sucrose, mannitol, sorbitol and microcrystalline cellulose can be used in the composition to increase the bulk. Furthermore, polyethylene glycols, coca butter, Tweens and Spans, water soluble polysaccharide can be used as the base. Lubricants such as magnesium stearate, sodium stearate, colloidal silica, stearic acid and talc can be used at the final stage for lubrication of the granules. The Praneem, purified saponins, and/ or zinc acetate, citrata oil of the present invention may be employed and/ or with such solid and/ or liquid dispersible carrier vehicles and/ or with oihe: known compatible active agents, especially spermicidal agents and antimicrobial agents etc 'i' desired, or in the form of particular dosage preparations for specific application made [herefrom, such as pessary, cream, gel, aerosol, soft gelatin capsule, foam tablets, bioadhesive table. mucoadhesive tablet, sustained release tablet, controlled release tablet or effervescent tablets which are thus ready for use. As commercially marketed preparations, these generally contemplate carrier composition mixtures in which the active ingredient Praneem of this invention is present in an amount substantially between about 1% and 40% by weight and preferably between about 5% and 30% by weight, of the mixture. Purified saponins are present in an amount substantially between 0.01% and 10% by weight and preferably between 1 to 5% by weight of the mixture, Mentha citrata oil is present in an amount substantially between 0.01% and 20% by weight and preferably between about 1% and 10% and zinc acetate is present in an amount substantially between 0 to 0.5% by weight. The following examples are presented to illustrate the invention are not to be construed as limiting in scope. A. Composition for Pessary Active ingredients of present invention can be dispensed in cocoa butter/hydrogenated \ egetable oils/glycerinated gelatin/polyethylene glycols/Tweens/Spans. The preservatives that can be used include phenol, chlorocresol, o-phenyl phenol, methyl juuabens, propyl paraben, sodium salts of parabens, Benzalkonium chloride, boric acid, benzoic ..ud The antioxidants that can be used are selected from the group comprising ascorbic acid, propyl gallate, L. tocopherol, butylated hydroxy anisole, butylated hydroxy toluene. The effervescent mixtures include citric acid/ tartaric acid/ sodium bicarbonate. Disintegrants are generally selected from sodium carboxy methyl cellulose, guar gum, agar, starch, Tween. Lubricants are generally selected from light paraffin, talc, and sodium -learate. Example -1 Praneem Polyethylene glycol (PEG)-6000 Polyethylene glycol (PEG)-1450 Polvethylene glycol (PEG)-400 Methyl paraben sodium (mpNa) Percentage (w/w) 20 8 18 26 0.05 Propyl paraben sodium (ppNa) 0 15 Mentha citrata oil 5(v/w) Purified saponins 3 1 ween -80 1 (.'in'ic acid monohydrate 10 sodium bicarbonate 7 f iyhi liquid paraffin 0.8 ^ lethod of Preparation i) PLG's are melted at 60°C and Tween-80, light liquid paraffin are added to it. 2) Praneem is passed through 60 mesh sieve and added to the mixture of step-1. i) Purified saponins, methyl paraben sodium, propyl paraben sodium, are passed through 60 mesh sieve separately and added to the above mixture. 4) Citric acid monohydrate is powdered and passed through 60 mesh sieve and added to the above mixture and finally sodium bicarbonate is added and the mixture is heated with stirring until the effervescence ceases. s) The mixture is cooled to 50°C and citrata oil added, mixture is stirred and poured into the mold and cooled. Example - 2 Percentage (w/w) 30 8 18 26 0.05 0.15 l(v/w) Praneem Polyethylene glycol (PEG) 6000- Poiyethylene glycol (PEG) 1450- Polyethylene glycol (PEG) 400- Viethyl paraben sodium (mpNa) Propyl paraben sodium (ppNa) Mentha citrata oil Purified saponins 1 i'ween-80 1 Citric acid monohydrate 7 Sodium bicarbonate 7 Light liquid paraffin 0.8 Method of Preparation i) PEG's are melted at 60°C and Tween-80 and light liquid paraffin are added to it. : Praneem is passed through 60 mesh sieve and added to the mixture of step-1. .1' Purified saponins, mpNa, ppNa, are passed through 60 mesh sieve separately and added to the above mixture. 4) Citric acid monohydrate is powdered and passed through 60 mesh sieve and added to the above mixture and finally sodium bicarbonate is added and the mixture is heated with stirring until the effervescence ceases. 5) The mixture is cooled to 50°C and citrata oil added, mixture is then stirred and poured into the moid and cooled. Example - 3 Percentage (w/w) Praneem 5 1 olyethylene glycol (PEG) 6000- 10 Polyethylene glycol (PEG) 1450- 20 Polyethylene glycol (PEG) 400- 26 Methyl paraben sodium (mpNa) 0.05 Propyl paraben sodium (ppNa) 0.15 1 kmhn citrata oil 10 (v/w) Purified saponins 5 i'ween--80 1 Citric acid monohydrate 12 Sodium bicarbonate 10 Light liquid paraffin 0.8 Method of Preparation i ) PEG's are melted at 60°C and Tween-80 light liquid paraffin are added to it. 2) Praneem is passed through 60 mesh sieve and added to the mixture of step-1. i) Purified saponins, mpNa, ppNa, are passed through 60 mesh sieve separately and added to the abo\e mixture. 4) Citric acid monohydrate is powdered and passed through 60 mesh sieve and added to the above mixture and finally sodium bicarbonate is added and the mixture is heated with stirring until the effervescence ceases. 5) 'The mixture is cooled to 50°C and citrata oil added, mixture is stirred and poured into the moid and cooled. AN 11M1CROBIAL ACTIVITY 1) Antiviral activity to "trucidat action on HIV-1 Potent virucidal action against AIDS virus HIV-1 has been established in two nuernationally recognised laboratories employing two different standard methods. In one center, a known aliquot of the virus HIV-1 was exposed to a dispersion of the polyherbal composition for 1 or 10 minutes and the viable virus checked for growth in activated i inphocytes. All appropriate controls were employed and the polyherbal composition was tested at more than one dilution. These studies demonstrated very high virucidal activity of the composition as shown in Tables la and lb. At another center, a cell free HIV inactivation assay was employed. 10 ulitre of concentrated virus stock was mixed with 90 ulitre of the polyherbal composition solution for 2 minutes, diluted with 0.9% NaCl solution and transferred to 96 well plate preseeded with MT cells at 1 x104 /10 µlitres medium. Cultures were incubated at 37°C and scored microscopically ror virus induced cytopathic effect and agent induced cytotoxicity finally on day 7. Nonoxynol-9 was used as positive control. the results are given in Table - 2. b) In >-vi\>o virucidal action on human Herpes Simplex Virus-2 (HSV-2) Susceptibility of female mice to HSV-2 was increased by giving a subcutaneous injection of progestin for 7 days prior to viral inoculation. On the day of inoculation, Praneem composition dissolved in phosphate buffered saline was delivered to the vaginas of mice ioliowed by HSV-2. Coitus was mimicked by moving the pipette in and out four times, i niection was assessed by culturing vaginal lavages taken three days after the viral inoculum and observing the animals for lesions. Table-3 shows that mice pretreated with the Praneem composition before inoculation with HSV-2, then neither developed lesions characteristic of herpes nor showed the presence of v'irus in their vaginal lavages. (i. Anti-fungal activity Anti-candida activity was assayed by the agar diffusion test with the modification that instead of using drug soaked discs, the drug was spotted directly on the growth media (SDA-Difco) containing specific concentration of the inoculum. The Candida inoculum was prepared by suspending a single isolated colony in sterile distilled water. Cell suspension was matched with 0.5 McFarland tube which is equivalent to cell density of 108cells/ml. 100 ul of this suspension was added to sterile petri plates. Autoclaved Sabourauds dextrose agar was cooled to 41- 42°C before adding 10 \|u.l to the inoculum containing plates. Plates were rocked gently, to uniformly distribute the inoculum in the growth media. Agar was allowed to set at room trnperature and plates dried at 37°C for 15 minutes. The polyherbal compositions were diluted o 50% with sterile distilled water and 20 µl spotted on the dried plates. An equivalent volume of sterile distilled water was spotted in control plates. Plates were incubated at 37°C for 48 hours, fluconazole was used as positive control. The test was carried out in triplicate and the average diameter of inhibition was recorded. III) Antibacterial activity a/ Action against N. gonorrhoeae Polyherbal composition was dispersed in water and spotted on N. gonorrhoeae ! noculation plates. Penicillin disc was used as standard. Plates were incubated at 37°C for 18-24 ■icurs and zone of inhibition was recorded. Table 4 shows that it inhibits not only the growth of normal clinical isolates, but also those of penicillin resistant strains of iV.gonorrhoeae. b> I ciion against E. coli Table 5 shows that growth was inhibited by dispersion of the polyherbal composition in ali strains including several multidrug resistant strains. i in-vivo antibacterial action on Chlamydia trachomatis Female mice were given a subcutaneous injection of progestin for 7 days prior to inoculation o\~Chlamydia trachomatis (Ct -D) to increase their susceptibility to infection. On the day of inoculation, Praneem tablet dispersed in phosphate buffered saline (PBS) was delivered to ihe vagina of mice and stirred. Approximately 20 seconds later, 10 ulitre of lO5 inclusion forming units of Ct -D were delivered and stirred. Swabs were taken on days 4 and 8 post inoculation and plated onto McCoy target cell monolayers and incubated at 37°C for 72 hours. Only 14% mice pretreated with the Praneem composition before inoculation of 10 ID50 of Ct -D became infected as compared to 86% in the PBS control group. Results are shown in Table 6. IV) Evidence for the efficacy of the Praneem Polyherbal composition for curing leucorrhea due to reproductive tract infections. Women of reproductive age group between 18 to 45 years suffering from complains of excessive vaginal discharge were examined by experienced clinical experts at the Postgraduate Institute of Medical Education and Research, Chandigarh. The investigations done on each patient include a complete blood count, urine analysis, VDRL, pap smear, random sugars and vaginal swab cultures. Swabs were taken to conduct microbiological cultures and microscopic examination for the presence of pathogens causing clinically abnormal vaginal discharge. These cultures are done before and after the use of Praneem pessary. The women were directed to use for 7 days one Praneem pessary every night before going to bed. The pessary (wetted with water) could be inserted with washed fingers deep in the vagina On the 10th day after start of the treatment repeat clinical examination was carried out and swabs were taken for microbiological cultures. These investigations carried out in twenty-eight women revealed that intravaginal treatment with Praneem pessary was effective in curing clinical symptoms of leucorrhoea in all the patients who used the drug for stipulated time. Remission was recorded in all women investigated. As regards the microbiology, the aerobic and anaerobic microbial flora was drastically reduced in every case. The pessary was also effective against Gardnerella vaginalis and Candida albicans. Results are shown in Table 7. CONTRACEPTIVE EFFICACY In evaluating the spermicidal activity of the individual ingredients and final composition of this invention the Sander Cramer Slide test is employed. The results are given in Table 8. The contraceptive efficacy is determined in New Zealand White rabbits. The results are given in Table 9. B: Composition of the Foaming Tablets The Praneem polyherbal composition is preferably dispensed in form of a tablet, which can be inserted easily in the vagina with washed fingers. The tablet withstands the high environmental room temperatures prevalent in most parts of India without any deterioration for several months. It starts dispersing immediately after insertion in the vagina and its foaming gel formation continues and persists for several hours. This forms a lubricating bio-adhesive lining of the vaginal mucosa, which provides additional physical protective barrier to the passage of pathogenic microorganisms into the vaginal cells over and above the direct microbicidal action of various constituents in the tablet. The tablet does not cause irritation of the vaginal tissue nor mucosal damage on repeated use. Active ingredients of the present invention can be dispensed with water soluble polysaccharides such as sodium/ potassium alginates or carrageenan (iota/ kappa), gelling agents such as calcium gluconate/ calcium lactate/ calcium carbonate etc., an effervescent mixture containing a base and an acid that initiates foam and helps in disintegrating trie tablet. The base can be sodium or potassium carbonate or bicarbonate, whereas the acid can be any fruit acid such as tartaric, citric, fumaric and malic etc. Diluents such as lactose, starches, sucrose, mannitol and microcrystalline cellulose can be used to increase the bulk. Example- 4 Percent w/w 'Tanccm 18.3 Punned saponins 3.7 \ leutha citrata oil. 6.1 v/w VKiium alginate 20 •sodium bicarbonate 15 ' aleium gluconate 8 ; uctose 10.6 Magnesium Slearate 1.3 .e;o:>ol J v!ei hod of Preparation All the solid materials are passed through 60-mesh sieve and dried at 50°C for 2 hours. all the ingredients are mixed and compressed in a tabletting machine to make slugs or passed through compactor or granulated with isopropyl alcohol and dried. Slugs are screened / milled, mass are separated and slugged again, screened/ milled once more or alternatively compacted mass is passed through sieve (10#) to obtain granules. Granules are lubricated and compressed into tablets. Example - 5 Percent w/w r'raiieeni 20 I'urified saponins 4 Mentha citrata oil 6 v/w -'me Acetate 0.5 vxiium alginate 20 Sodium bicarbonate 15 'uric acid monohydrate 15 ( aleium gluconate 8 i aciose 8.2 Magnesium Stearate ],3 Verosol 3 Method of Preparation \\[ the solid materials are passed through 60-mesh sieve and dried at 50°C for 2 hours. All the ingredients are mixed and compressed in a tabletting machine to make slugs or passed through compactor or granulated with isopropyl alcohol and dried. Slugs are screened / milled, lines are separated and slugged again, screened / milled once more or alternatively compacted mass is passed through sieve (10#) to obtain granules. Granules are lubricated and compressed into tablets. EVIDENCE FOR THE EFFICACY OF THE PRANEEM TABLET FOR CURING LEUCORRHOEA DUE TO REPRODUCTIVE TRACT INFECTIONS Same protocol was adopted for clinical trials as with Praneem pessary, the only dilference was the tablet didn't require the initial wetting with water and could be used as such tor uisertion in the vagina. The investigation was carried out in twenty-five women, results are shown in the Table 10, whereby 100% remission was obtained in women who took the complete course of the tablets. spermicidal activity Sander Cramer Slide test was employed. Results are given in Table 11. Table 1 a. Virucidal Activity of the control samples on HIV-1. (Table Removed) Tabie 1 b. Virucidal Activity of Praneem Pessary on HIV-1. (Table Removed) Keduction in virus titre indicates the anti HIV potency of the compound. Table 2, Inhibition of HIV-I-CF induced cytopathic effect. (Table Removed) Concentrations are expressed in % w/w except for N-9 (nonoxynol-9) in which case percents indicate w/v. Initial testing concentration 0.5/2.5g. TCTD50: Tissue culture toxic dose (50%). *** Log reduction in virus titre indicates the anti HIV potency of the compound. Titre of the virus control: 6.5-logioTdD5o/0.1 ml. t N-9 is the standard positive control. Table 3. In vivo protection from Herpes-Simplex Virus-2 vaginal transmission. (Table Removed) Table 4. Antibacterial Activity against Standard strains and clinical isolates of Neisseria (Table Removed) Table 5. Antibacterial activity against E.colL (Table Removed) Resistant to Ciprofloxacin, Cephalexin, Gentamycin, Cefatoxime, .Amikacin, Augmentin. ; Penicillin (0.5 IU) was used as positive control. Table 6 vivo protection front Chlamydia trachomatis vaginal transmission. (Table Removed) Table 7: Clinical response of Praneem Pessary in women suffering from abnormal vaginal discharge. (Table Removed) Praneem Pessary was used after wetting. * Panaal response was noted in patients who did not use the drug for the stipulated time. Table 8. Synergistic spermicidal activity of the ingredients of the Praneem Pessary. (Table Removed) Determined by Sander Cramer test on seven different seamier samples with sperm count aging from 60x106/ml to 90xlO6 /7ml and 60-80% motility. Table 9. Contraceptive efficacy in Rabbits. (Table Removed) Today commercially available nonoxynol-9 pessary, sold by Ortho Johnson. Table 10: Clinical response of Praneem Tablet in women suffering from abnormal vaginal discharge. (Table Removed) Martial response was noted in patients who did not use the drug for the stipulated time. We claim: A process for the preparation of an improved antimicrobial and spermicidal composition characterised in that it comprises ito 40% by weight of Praneem, 0.01 to 10% by weight of purified saponins from Sapindus mukerossi, between 0.01 to 20% by weight Mentha citrate oil and up to 0.5% by weight of zinc acetate dispersed in a conventional carrier or vehicle, said process comprising air drying leaves of Azadirachta indica, powdering said air dried leaves and subjecting them to extraction with water under hot conditions to obtain the leaf extract, dissolving it in water, subjecting it to decolourisationm, and recovering by conventional methods, the Praneem; preparing purified saponins by subjecting air dried pericarp of Sapindus mukerossi to extraction, concentration to a syrupy residue, subjecting said residue to further extraction and followed precipitation; dispersing by conventional methods the above Praneem and purified saponins in a conventional carrier; adding citrate oil to said mixture; adding zinc acetate, and optionally, other conventional additives; and optionally, converting the final composition into the required shape and form. A process as claimed in claim 1 wherein said Praneem is present in an amount of 5-30% by weight of said composition. 3. A process as claimed in claim 1 or 2 wherein said purified saponins are present. in an amount of 1-5% by weight of said composition. 4. A process as claimed in any preceding claim wherein said Mentha citrate oil is present in an amount of 1-10% by weight of said composition. 5. A process as claimed in any preceding claim wherein said conventional additives are selected from preservatives, antioxidants, effervescent mixtures, gelling agents, polysaccharides carriers, disintegrants, diluents and lubricants. 6. A process as claimed in claim 5 wherein said carrier is a diluent selected from lactose, starches, sucrose, Mannitol, sorbitol, microcrystalline cellulose. polyethylene glycol, cocoa butter, Tweens and Spans. H. 7. A process as claimed in claim 5 wherein said preservative is selected from the group consisting of phenol, chloro cresol, 0-phenyl, methyl parabens, propyl paraben and their sodium. salts, benzylkonium chloride, boric acid and benzoic acid 8. A process as claimed in claim 5 wherein said antioxidant is selected from the group consisting of ascorbic acid, propyl gallate, L-tocopherol, butylated hydroxyl anisole and butylated hydroxl toluene. 9. A process as claimed in claim 5 wherein the said disintegrant is selected from the group consisting of sodium carboxy methyl cellulose, guar um, agar, starch and Tween. 10. A process as claimed in claim 5 wherein said gelling agent is selected from the group consisting of calcium gluconate, calcium lactate and calcium carbonate. 11. A process as claimed in claim 5 wherein the said polysaccharide is selected from the group consisting of sodium alginate, potassium alginate or carrageenan (iota/kappa).. 12. A process as claimed in claim 5 wherein the said lubricant is selected from the group consisting of light liquid paraffin, talc, aerosol, magnesium stearate, and sodium stearate. 13. A process as claimed in any preceding claim wherein the said composition is in the form of a pessary, cream, gel, soft gelatin capsules, foaming tablets, foams, spray or aerosol. 14. A process for the preparation of an improved antimicrobial and spermicidal composition substantially as herein described and as illustrated with reference to the examples.

Full Text

Field of the invention
1 he present invention relates to a process for the preparation of an improved antimicrobial and spermicidal composition and a composition prepared thereby. The composition of the present invention is capable of use for prevention of AIDS, other sexually transmitted infections and curing of leucorrhea due to reproductive tract infections. The composition of the invention is also capable of potent spermicidal action and prevention of unplanned pregnancy by intravaginal application prior to mating.
Background of the invention
Sexually transmitted diseases including AIDS and unplanned pregnancies continue to be a serious problem. Improved barrier methods and potent safe spermicides with antibacterial, antifungal and antiviral properties are in demand. The World Health Organization reported the occurrence of 333 million new cases of Sexually Transmitted Diseases (STDs) in only one year excluding those of AIDS (World Health Organization. Report on fighting disease fostering development, Geneva, 1996.). AIDS due to HIV virus is also spreading fairly rapidly globally, in India, the figures of those positive for HIV rose from none in 1986 to 3-4 million in early 1999,are expected to double by the year 2000. The transmission of AIDS virus, besides through contaminated blood, is mostly by sex with unsuspecting partners presenting no obvious symptoms.
It is further observed that a large number of women in India and elsewhere have
abnormal vaginal discharge (leucorrhea) caused due to pathogenic microorganisms in the vagina
which include aerobic and anaerobic microbes, fungi such as Candida albicans and Candida
uopicalis, Trichomonas and Gardnerella. These infections are collectively represented as RTIs -
Reproductive Tract Infections. A study by the Indian Council of Medical Research (ICMR)
reported 27 6% of women below 35 years of age having abnormal vaginal discharge caused by
pathogens, bacteria, fungi or viruses (Bhujwala et al. Ind. J. Med Res. 1985; 81: 251-6.). Bang et
al (Lancet 1989, 1: 85-6.) found that incidence of RTIs was as high as 50% in rural
Maharashtra. Although specific drugs and antibiotics, for example Metronidazole (for
iiichomonasis), tetracycline or Doxycycline (for uncomplicated gonorrhea infections). Nystatin
or Clotrimazole or Miconazole or Ketoconazole (for vaginal candidasis), Acyclovir (for genital
herpes) and Benzathine penicillin (for other RTIs) are available, they have side effects like local
irritation, burning sensation, nausea, vomiting, diarrhoea, dizziness, headache, skin rashes and
phototoxicity. No single wide spectrum composition is available for intravaginal use that can
cure or prevent infections due to commonly encountered vaginal pathogens without any side effects. In addition, microorganisms are increasingly becoming resistant to one or more antibiotics. From 1994-1997, the proportion of Neisseria gonorrhoeae highly resistant to ciprofloxacin (MIC>/=4µg/ml) increased substantially among female sex workers in the Philippines (Klausner et al. J. Infect. Dis. 1999 Mar; 179(3): 729-33). Therefore, prevention of sexually transmitted infections including AIDS becomes as important to the public health as is finding a cure for them.
Another problem that is of tremendous concern particularly for developing countries like India is overpopulation. The problems associated with overpopulation are well-known - high population density in urban centres leading to epidemics, inadequate sanitation, excess pressure on available resource use, etc. While a number of methods of family planning through birth control are available, no single method suits all nor does the same method suit the same individual at different stages of reproductive life. Spermicidal preparations have the advantage of topical applicability (not involving systemic intake) and can be used as and when the need arises. Furthermore, in contrast with condoms, the control is with the female partner Therefore, spermicidal preparations have found wider acceptability due to the convenience factor.
Spermicides available in the art employ Nonoxynol-9 (N-9) as the active ingredient which was reported to provide protection against chlamydia and gonorrhea (Rosenberg et al. Am. J. Public Health 1992; 82: 669-74), Trichomonas vaginalis (Bolch et al. Am J. Obstet. Ciynecol. 1973; 115: 1145-8.; Singh et al. Contraception 1972; 5: 401-11.), Staphylococcus aureus (Snelton et al. Contraception 1981; 24: 631-4.), genital herpes and feline immunodeficiency virus in animal models (Whaley et al. J. Infec. Dis. 1993; 168:1009-11.; Moench et al. AIDS 1993; 7: 797-802.). High frequency use of N-9, however, results in vaginal irritation, inflammation, genital ulceration and allergic vaginitis which may enhance susceptibility to sexually transmitted infections (Lee et al. J. Pharm. Sci. 1996, 85(1) 91-5.). Possibly because of these properties a clinical trial in Kenya indicated as enhanced transmission rather than prevention of HIV in professional sex workers using Nonoxynol based spermicides i Kreiss et al. JAMA 1992; 265: 477-82.).
A variety of alternative synthetic microbicides (Neurath et al. Lancet 1996; 347: 1703-4.; Baba et al Antimicrob. Agents Chemother. 1988, 32: 1742-5.; Gonzalez et al. Antimicob. Agents Chemother. 1987; 31: 1388-93.; Nisbet et al. Antimicrob Agents Chemother 1979; 16: 855-7.; Louis et al. Contraception 1985, 32: 199-205., Hansen et al. Scand J. Infect. Dis. 1991, 23 425-30.-. Ito et al. Am. J. Virol. 1974; 13: 1312-8., Petronio et al. Chemotherapy 1997; 43 211-7.. Oehniger et al. Fertil. Steril. 1991; 55: 165-9., Bourinbaiar et al. Contraception 1994; 49:
211;-7 , Shubair et al. Gynecol. Obstet. Invest. 1990, 29: 67-70., Malkovsky et al. Lancet 1988, 19. o45.; Asculai et al. Antimicrob. Agents Chemother. 1978; 13: 686-90., Amortegui et al Contraception 1984; 30: 135-41.; Foldesy et al. Fertil. Steril. 1986, 45: 550-5., Amstey et al. Obstet. Gynecol. 1975; 46: 528-9.; Sokal et al. Sex Transm. Dis. 1995; 22: 22-4.; Zamora et al. Am J Surg. 1986; 151: 400-6.) have demonstrated activity against sperm and sexually transmitted pathogens in vitro, but few have been tested in vivo. For these reasons, there is need for vaginal compositions which are free of irritation and which are active in vivo against sexually transmitted diseases/ AIDS and unplanned pregnancy.
A substantial number of prior art compositions are available only in the form of pessaries and some of them require wetting before insertion into the vagina. The failure to wet or improper wetting of these pessaries before insertion is a recurring phenomenon either due to lack of knowledge or simple memory failure on the part of the female member.
An object of this invention is the provision of a composition that does not require wetting, with high spermicidal action and wide spectrum inhibitory action on genital pathogens including those transmitted by sex. In addition, the composition must be capable of use by women intravaginally to cure leucorrhea caused due to reproductive tract infections The composition should also be capable of antimicrobial action on microbes resistant to one or more drugs
Furthermore the use of the composition before sexual intercourse should prevent the transmission of a variety of sexual transmitted infections, such as gonorrhea, herpes, chlamydia and AIDS, besides others, from, the partner and also safeguard women against unwanted pregnancy by exercising spermicidal action. The composition should also be in a form which is easy to use and totally free of irritation.
The applicant's efforts towards the achievement of such subjects have led them to study ancient and indigenous plants such as neem (Azadirachta indica), reetha (Sapindus imikerossn and Mentha citrata already well known for their utility in other areas.
The neem tree (Azadirachta indica) found throughout India is used in indigenous and household medicine for different purposes. Almost every part of the tree has been utilized for treatment of various disorders and diseases. Neem leaves are used to mitigate the pruritic sensation in smallpox, chicken pox and measles. Extracts from various parts of the neem tree are used because of their bactericidal properties in indigenous tooth powders, toothpaste and toilet soaps, its antibacterial activity has been demonstrated by many. Chatterjee and Roy (Ind. 3 Med. Res. 1917; 5: 656) demonstrated the in vivo antibacterial activity of margosates obtained
from Neem oil. Murthy et al. (Indian J. Pharmacol. 1958; 2: 387-96 and 456-461.) showed that nimbidin, one of the active principles of Neem has inhibitory properties against Mycobacterium tuberculosis. Singh et al. (Ind. J. Med. Res. 1974; 9: 65.) showed that Neem oil has antibacterial activity against E.coli in vitro. Chaurasia et al. (Indian J. Hospital Pharm 1978; 15: 166.) showed that Neem oil has antibacterial activity against various bacteria potentially pathogenic to plants and animals. Rao et al. (Ind. J. Med. Res. 1986; 84: 314-16.) showed that Neem oil has significant antibacterial properties against pathogenic Gram negative bacilli such as Ps. aeruginosa, E.coli, Proteus group and K. aerugenes and against gram positive cocci such as Siaph. pyogenes.
Although many investigators (Sinha et al. Ind. J. Med. Res. 1984; 79: 131-36.; Bardhan et al. Indian J Physiol. Pharmacol. 1991; 35(4):278-80.; Sharma & Saksena. Ind. J. Med. Sci. 1959; 13: 1038.; Sharma & Saksena. Ind. J. Med. Res.1959; 47: 322.) have reported the spermicidal activity of Neem oil and its components, however, their findings reveal a rather low spermicidal activity of Neem oil if used as such. Furthermore Neem oil has very strong pungent odour. Riar et al. (Contraception 1990 Oct.; 42(4): 479-87.) extracted the volatile, odorous traction of neem oil coded as Nirn-76 by steam distillation which exercises spermicidal activity ai 25 mg/ml concentration on human sperm. Though better than the native neem oil, activity remains still very low. Another major disadvantage of Nim-76 is its strong pungent odour that would dissuade for all practical purposes the use by choice of such preparations for vaginal insertion before intercourse.
US Patent No. 5,196,197; 1993 overcomes the problem of low spermicidal efficacy of Neem oil, by potentiating the spermicidal efficacy synergistically by combination with purified saponins from Sapindus mukerossi (reported to have spermicidal activity by Khanna - Indian Patent No. 150335; 1982).
Our Indian Patent Application 1351/Del/97 dated May 20, 1997 overcomes the problems of acceptability and unpleasant odor and color of Neem oil by use of purified neem leaf extract (Praneem) in place of Neem oil. Spermicidal activity is further enhanced by addition of Mentha cnrata oil (Mentha citrata is a hybrid variety of Mentha spicata & Mentha aquatica. Wealth of India, Shastri ed. New Delhi:CSIR, 1988; Vol. VI (L-M):338.) and zinc acetate (reported to have antiviral and spermicidal activity by Fahim & Wang. Contraception. 1996; 53: 231-36.) Indian Patent Application No. 1351/Del/97 relates to a composition for prevention of unwanted pregnancy which comprises a major amount of conventional carriers, from 1 to 40% by weight of said composition, Praneem, from 0.01-10% by weight of purified saponins, from 0.5-5% of zinc acetate, from 0.5% to 20% by weight of Mentha citrata oil and the balance if any
comprising of conventional additives. This composition had shown in vitro activity against urinary tract E.coli and N.gonorrhoeae. It also showed virucidal activity against HIV-1 and HSV-2. Composition was effective in killing 100% of human sperms in 20 seconds in vitro up to iO times dilution. However, it prevented pregnancy in 100% of rabbits tested, at l/6th of human dose. Further dilutions were not effective. Also the zinc acetate component causes skin irritation (Lansdown. Food Chem Toxicol 1991 Jan; 29(1): 57-64).
Accordingly, it is necessary to obtain a composition which results in the obviation of all trie drawbacks enumerated above - those of efficacy, odor, staining, irritation and mucosal damage. Objects of the invention
The main object of the present invention is to provide a process for the preparation of an iinproved antimicrobial and spermicidal composition, which prevents the transmission of sexually transmitted pathogens such as N. gonorrhoeae, herpes, Chlamydia trachomatis and AIDS
It is another object of the present invention to provide a process for the preparation of a ^ate antimicrobial and non-irritant spermicidal composition containing a very negligible amount wi'oi no zinc acetate. „
It is a further object of the present invention to provide a process for the preparation of an improved antimicrobial and spermicidal composition, which is a cure for leucorrhea caused by reproductive tract infections.
It is another object of the present invention to safeguard women against unwanted pregnancy.
it is another object of the invention to provide a method of fertility control, which is completely safe and free of side effects.
Yet another object of the present invention is to provide a method of fertility control which can be restricted only to the occasions when the need arises, which does not require systemic intake and which is affordable.
Yet a further object of the present invention is to provide a process for the preparation of an improved contraceptive composition for use in the above method which has dual properties of contraception and prevention of a large number of genital tract infections.
It is another object of the present invention to provide a process for the preparation of a contraceptive or spermicidal composition in the form of a foaming tablet that is easy to use and is under control of woman in contrast to condom which is under control of the male partner
Summary of the invention
The spermicidal activity of zinc acetate is well documented in the art. This being so, no prior art would teach away from the use of zinc acetate in a spermicidal composition. However, it has been surprisingly found that the reduction or even total elimination of zinc acetate from a spermicidal composition especially, the composition claimed by copending Indian Patent Application No. 1315/Del/97 not only does not affect the efficacy thereof but totally avoids the problems of irritation associated with zinc acetate completely. It is known that a neem extract by itself will not have the same level of spermicidal effect as that of zinc acetate. Yet, even in the absence of zinc acetate, the composition of the present invention described herein exhibits greater spermicidal activity than neem extract alone or zinc acetate alone and exhibits spermicidal activity of equal efficacy as compared to the composition claimed in copending Indian Patent Application 1315/Del/97.
Accordingly, the present invention provides an procgss for the preparation of an
improved antimicrobial and spermicidal composition charatersing taht it compare1 to 40% by
weight of Praneem,. 0.01 to 10% by weight of purified saponins from Sapindus mukerossi, between 0.01 to
20% by weight Mentha citrata oil and 0 to 0.5% by weight of zinc acetate dispersed in a convenmtion
carrier or vehicle, said process comprising air drying leaves of Azadirachta indica, powdering
.said air dried leaves and subjecting them to extraction with water under hot conditions to obtain
the leaf extract, dissolving it in water, subjecting it to decolourisation, and recovering the
Praneem;
preparing purified saponins by subjecting air dried pericarp of Sapindus mukerossi to extraction,
concentration to a syrupy residue, subjecting said residue to further extraction and then
precipitation,
dispersing the above Praneem and purified saponins in a conventional carrier;
adding citrata oil to said mixture;
adding zinc acetate, and optiopnally other conventional ingredients and,
optionally converting the final composition into the required shape and form, if desired..
The composition prepared by the process of the present invention is not a mere admixture of ingredients resulting in an aggregate of the properties thereof, but is a synergistic admixture evincing biological activity.
in one embodiment of the present invention, the amount of zinc acetate is less than 0.5 to, preferably, 0 % by wt.
In another embodiment of the present invention, Praneem is present in an amount of 5-60% by weight of said composition.
In another embodiment of the present invention, purified saponins are present in an amount of 1- 5% by weight of said composition.
In another embodiment of the present invention, Mentha citrata oil is present in an amount of 1- 10% by weight of said composition.
In another embodiment of the present invention, the conventional additives are selected from preservatives, antioxidants, effervescent mixtures, gelling agents, polysaccharides, disintegrants, diluents and lubricants.
In another embodiment of the present invention, the carrier is a diluent selected from .actose, starches, sucrose, mannitol, sorbitol, microcrystalline cellulose, polyethylene glycol, coca butter, Tweens and Spans.
In a further embodiment of the invention, the polyethylene glycol carrier is replaced by sodium alginate and calcium gluconate.
In a still further embodiment of the invention, the composition is produced in the form of it foaming tablet that does not need wetting before insertion into the vagina.
In a further embodiment of the present invention, the preservative is selected from the group consisting of phenol, chloro cresol, o-phenyl phenol, methyl parabens, propyl paraben and their sodium salts, benzylkonium chloride, boric acid and benzoic acid.
In another embodiment of the present invention, antioxidant is selected from the group consisting of ascorbic acid, propyl gallate, L-tocopherol, butylated hydroxyl anisole and butylated hydroxl toluene.
In yet another embodiment of the present invention, the disintegrant is selected from the group consisting of sodium carboxy methyl cellulose, guar gum, agar, starch and Tween.
In another embodiment of the present invention, the gelling agent is selected from the group consisting of calcium gluconate, calcium lactate and calcium carbonate.
In a further embodiment of the present invention, the polysaccharide is selected from the group consisting of sodium/ potassium alginates or carrageenan (iota/ kappa).
In another embodiment of the present invention, the lubricant is selected from the group consisting of light liquid paraffin, talc, aerosol, magnesium'stearate and sodium stearate.
In another embodiment of the present invention, the composition prepared by the process of the invention is in the form of a pessary, cream, gel, soft gelatin capsules, foaming tablets, loams, spray or aerosol.
The invention also relates to an improved antimicrobial and spermicidal composition which comprises 1 to 40% by weight of Praneem, 0.01 to 10% by weight of purified saponins
horn Sapindus mukerossi, between 0.01 to 20% by weight Mentha citrata oil and 0 to 0.5% by weight of zinc acetate dispersed in suitable carrier or vehicle.
According to a further feature, the present invention also provides a spermicidal composition comprising a dispersion of neem leaf extracts, purified saponins, zinc acetate and/ or citrata oil in a pharmaceutical^ acceptable carrier or base therefore.
In a further embodiment of the invention, polyethylene glycol is replaced by ;.odium/potassium alginates and/ or carrageenan (iota/kappa).
Brief description of the accompanying drawings
Figure 1 is the HPLC profile of Praneem. Figure 2 is a gas chromatogram of Citrata oil.
Detailed description of the invention
It is found that reduction in concentration or complete removal of zinc acetate from the composition does not in anyway effect the spermicidal and antimicrobial properties of the composition and in fact eliminates the irritation observed with the use of higher concentrations of zinc acetate. In-vivo and clinical data clearly show that the present composition with or without zinc acetate is equally effective clinically.
The present composition inhibits the growth in culture of urinary tract Kcoli (including multidrug resistant strains) and N. gonorrhoeae (including strains resistant to penicillin). The composition manifested virucidal activity against HIV-1 at >2% and 50% dilutions on contact tor 1-2 minutes with the virus. Intravaginal inoculation of the present composition before inoculation of the pathogen prevented lesions and vaginal transmission of HSV-2 and (Itkunydia trachomatis in progestin sensitized mice. Composition when used in women suffering from abnormal vaginal discharge due to various pathogens completely cured the clinical symptoms in 100% of subjects who used the composition for 7 consecutive days.
The present composition although killed 100% of human sperms in 20 seconds in vitro up to 8 times dilution, there was no change in the in-vivo activity. At one sixth of human dose to rabbits of proven fertility, 15-60 minutes prior to mating, it prevented pregnancy in 15 out of 15 rabbits tested. The effect was further potentiated when the present composition was delivered in ibrm of pessary or vaginal tablet. In post coital tests as per WHO protocol it was found effective in 8 out of 9 women tested.
The composition of the present invention therefore, provides a method for blocking reversibly the fertility of a female without impairing ovulation by applying a predetermined
amount of a contraceptive composition based on neem leaf extracts, purified saponins, citrata oil and or zinc acetate to the vagina of the female subject.
Under the process of this invention, it has been established that the compositions made containing the following ingredients; - Praneem (purified Neem (Azadirachta indica) leaf extract) - Purified saponins from Sapindus mukerossi -Mentha citrata oil and/ or - Zinc acetate dispensed in the described vehicles, have potent spermicidal action and at the same time have a wide range of antimicrobial activity against a number of vaginal pathogens.
1) Praneem - In accordance with this invention there is provided a new antimicrobial and spermicidal fraction from the Neem leaves obtained by the extraction, purification and decolonisation of Neem leaves.
The air-dried leaves are coarsely powdered to pass through 20 mesh sieve and then exhaustively extracted with water under hot condition to obtain the extract. The extract is concentrated four times under vacuum. The concentrated material is spray dried. The spray dried aqueous alcoholic extract of Neem leaves is dissolved in 20 times water and decolourized with activated charcoal by boiling. Charcoal is removed by filtration The filtrate is reduced in volume four times under vacuum. The concentrated material is freeze dried to obtain Praneem. J he specifications of purified extract 'Praneem' are; colourless to off-white powder, the pH of 1 % w/v solution ranges between 7.7-9.5, and contains bitters>5% w/w. The finger print profile Dy HPLC is as per Fig.-l. Nimbin is used as the marker to standardize the Praneem. At 15% solution in isotonic buffer, it kills all the sperms within 20 seconds.
2). Purified saponins - For preparation of purified saponins, pericarp of air-dried Sapindus mukerossi is powdered and exhaustively extracted with 90% aqueous-ethanol by soxhlation. The extract is concentrated to thick syrupy residue under vacuum below 50°C. The residue is then extracted with isopropyl alcohol under cold conditions. The iso-propyl alcohol is distilled under vacuum below 40°C and residue dried under high vacuum to obtain light cream coloured hygroscopic powder.
An alternate method of purifying the alcohol extracts of Sapindus mukerossi pericarp is to extract the residue successively with petroleum ether, ether, carbon tetrachloride, chloroform and acetone. The residue is dissolved in absolute ethanol and filtered. The solution is added drop wise to ether, when a light brown precipitate is obtained. This step is repeated several times.
The specifications of the purified saponins are as follows: Melting range 90-118°C; soluble in water, alcohol and chloroform, pH of 1% w/v solution in water is 3-5, total saponins content as per Indian pharmacopoeia is not less than 30% w/w; at 0.25% w/v solution in isotonic buffer it kills 100% of human sperm within 20 seconds.
3, Mentha ciirata - is a hybrid of Men/ha spicala and Mentha ac/itat/aa. Citrata oil is obtained from leaves of Mentha citrata oil and has the following specifications: characteristic odor, acid value 0.6 +l0%, kills 100% human sperms within 20 seconds at 5% w/v concentration. Gas liquid chromatogram is shown in Fig.-2.
4). Zinc acetate - Zinc acetate which is commercially easily available has following specifications: pH of aqueous solution is about 5-6, kills 100% human sperm within 20 seconds at 1 % vv/v concentration.
The Praneem, purified saponins, citrata oil and/ or zinc acetate of the present invention, for practical application, can be utilized in the form of composition or compositions. In these composition and compositions, the active ingredients are mixed with pharmaceutically acceptable base as is well known in the art. If desired, adjuvants such as antioxidants, stabilizers, preservatives, effervescent mixtures may also be added.
Examples of the compositions and compositions according to the invention are those known to one skilled in the art and include Pessaries and Cream, Gel, Soft gelatin capsules, foaming tablets, foams, spray and other aerosol compositions. The final composition and compositions are prepared in a manner known to one skilled in the art, for example by extending ihe active compound with conventional dispersible liquid diluent carriers and/ or dispersible solid carriers optionally with the use of carrier vehicle assistants, e.g., conventional surface active agents, including emulsifying agents and/ or dispersing agent and/ or gelling agents.
it is observed that when polyethylene glycol carrier is replaced by sodium/potassium alginates or carrageenan, it is easy to obtain a foaming tablet that does not require wetting before insertion into the vagina. Thus the irritation observed in the case of some prior art pessaries due to the failure in several cases to properly wet the pessary before insertion into the vagina is avoided. Also, the control over the use of the composition killing sperms is with the female partner
Diluents such as lactose, starches, sucrose, mannitol, sorbitol and microcrystalline cellulose can be used in the composition to increase the bulk. Furthermore, polyethylene glycols, coca butter, Tweens and Spans, water soluble polysaccharide can be used as the base.
Lubricants such as magnesium stearate, sodium stearate, colloidal silica, stearic acid and talc can be used at the final stage for lubrication of the granules.
The Praneem, purified saponins, and/ or zinc acetate, citrata oil of the present invention
may be employed and/ or with such solid and/ or liquid dispersible carrier vehicles and/ or with
oihe: known compatible active agents, especially spermicidal agents and antimicrobial agents
etc 'i' desired, or in the form of particular dosage preparations for specific application made
[herefrom, such as pessary, cream, gel, aerosol, soft gelatin capsule, foam tablets, bioadhesive table. mucoadhesive tablet, sustained release tablet, controlled release tablet or effervescent tablets which are thus ready for use.
As commercially marketed preparations, these generally contemplate carrier composition mixtures in which the active ingredient Praneem of this invention is present in an amount substantially between about 1% and 40% by weight and preferably between about 5% and 30% by weight, of the mixture.
Purified saponins are present in an amount substantially between 0.01% and 10% by weight and preferably between 1 to 5% by weight of the mixture, Mentha citrata oil is present in an amount substantially between 0.01% and 20% by weight and preferably between about 1% and 10% and zinc acetate is present in an amount substantially between 0 to 0.5% by weight.
The following examples are presented to illustrate the invention are not to be construed as limiting in scope.
A. Composition for Pessary
Active ingredients of present invention can be dispensed in cocoa butter/hydrogenated \ egetable oils/glycerinated gelatin/polyethylene glycols/Tweens/Spans.
The preservatives that can be used include phenol, chlorocresol, o-phenyl phenol, methyl juuabens, propyl paraben, sodium salts of parabens, Benzalkonium chloride, boric acid, benzoic ..ud
The antioxidants that can be used are selected from the group comprising ascorbic acid, propyl gallate, L. tocopherol, butylated hydroxy anisole, butylated hydroxy toluene.
The effervescent mixtures include citric acid/ tartaric acid/ sodium bicarbonate.
Disintegrants are generally selected from sodium carboxy methyl cellulose, guar gum, agar, starch, Tween. Lubricants are generally selected from light paraffin, talc, and sodium -learate.
Example -1
Praneem
Polyethylene glycol (PEG)-6000 Polyethylene glycol (PEG)-1450 Polvethylene glycol (PEG)-400 Methyl paraben sodium (mpNa)
Percentage (w/w)
20 8 18 26
0.05

Propyl paraben sodium (ppNa) 0 15
Mentha citrata oil 5(v/w)
Purified saponins 3
1 ween -80 1
(.'in'ic acid monohydrate 10
sodium bicarbonate 7
f iyhi liquid paraffin 0.8
^ lethod of Preparation
i) PLG's are melted at 60°C and Tween-80, light liquid paraffin are added to it.
2) Praneem is passed through 60 mesh sieve and added to the mixture of step-1.
i) Purified saponins, methyl paraben sodium, propyl paraben sodium, are passed through 60
mesh sieve separately and added to the above mixture.
4) Citric acid monohydrate is powdered and passed through 60 mesh sieve and added to the
above mixture and finally sodium bicarbonate is added and the mixture is heated with stirring
until the effervescence ceases.
s) The mixture is cooled to 50°C and citrata oil added, mixture is stirred and poured into the
mold and cooled.
Example - 2

Percentage (w/w)
30 8 18 26 0.05 0.15 l(v/w)
Praneem
Polyethylene glycol (PEG) 6000-
Poiyethylene glycol (PEG) 1450-
Polyethylene glycol (PEG) 400-
Viethyl paraben sodium (mpNa)
Propyl paraben sodium (ppNa)
Mentha citrata oil
Purified saponins 1
i'ween-80 1
Citric acid monohydrate 7
Sodium bicarbonate 7
Light liquid paraffin 0.8
Method of Preparation
i) PEG's are melted at 60°C and Tween-80 and light liquid paraffin are added to it.
: Praneem is passed through 60 mesh sieve and added to the mixture of step-1.
.1' Purified saponins, mpNa, ppNa, are passed through 60 mesh sieve separately and added to the
above mixture.
4) Citric acid monohydrate is powdered and passed through 60 mesh sieve and added to the
above mixture and finally sodium bicarbonate is added and the mixture is heated with stirring
until the effervescence ceases.
5) The mixture is cooled to 50°C and citrata oil added, mixture is then stirred and poured into the moid and cooled.
Example - 3
Percentage (w/w)
Praneem 5
1 olyethylene glycol (PEG) 6000- 10
Polyethylene glycol (PEG) 1450- 20
Polyethylene glycol (PEG) 400- 26
Methyl paraben sodium (mpNa) 0.05
Propyl paraben sodium (ppNa) 0.15
1 kmhn citrata oil 10 (v/w)
Purified saponins 5
i'ween--80 1
Citric acid monohydrate 12
Sodium bicarbonate 10
Light liquid paraffin 0.8
Method of Preparation
i ) PEG's are melted at 60°C and Tween-80 light liquid paraffin are added to it. 2) Praneem is passed through 60 mesh sieve and added to the mixture of step-1. i) Purified saponins, mpNa, ppNa, are passed through 60 mesh sieve separately and added to the abo\e mixture.
4) Citric acid monohydrate is powdered and passed through 60 mesh sieve and added to the above mixture and finally sodium bicarbonate is added and the mixture is heated with stirring until the effervescence ceases.
5) 'The mixture is cooled to 50°C and citrata oil added, mixture is stirred and poured into the moid and cooled.
AN 11M1CROBIAL ACTIVITY 1) Antiviral activity
to "trucidat action on HIV-1
Potent virucidal action against AIDS virus HIV-1 has been established in two nuernationally recognised laboratories employing two different standard methods. In one center, a known aliquot of the virus HIV-1 was exposed to a dispersion of the polyherbal composition for 1 or 10 minutes and the viable virus checked for growth in activated i inphocytes. All appropriate controls were employed and the polyherbal composition was tested at more than one dilution. These studies demonstrated very high virucidal activity of the composition as shown in Tables la and lb.
At another center, a cell free HIV inactivation assay was employed. 10 ulitre of concentrated virus stock was mixed with 90 ulitre of the polyherbal composition solution for 2 minutes, diluted with 0.9% NaCl solution and transferred to 96 well plate preseeded with MT cells at 1 x104 /10 µlitres medium. Cultures were incubated at 37°C and scored microscopically ror virus induced cytopathic effect and agent induced cytotoxicity finally on day 7. Nonoxynol-9 was used as positive control. the results are given in Table - 2. b) In >-vi\>o virucidal action on human Herpes Simplex Virus-2 (HSV-2)
Susceptibility of female mice to HSV-2 was increased by giving a subcutaneous injection of progestin for 7 days prior to viral inoculation. On the day of inoculation, Praneem composition dissolved in phosphate buffered saline was delivered to the vaginas of mice ioliowed by HSV-2. Coitus was mimicked by moving the pipette in and out four times, i niection was assessed by culturing vaginal lavages taken three days after the viral inoculum and observing the animals for lesions.
Table-3 shows that mice pretreated with the Praneem composition before inoculation with HSV-2, then neither developed lesions characteristic of herpes nor showed the presence of v'irus in their vaginal lavages. (i. Anti-fungal activity
Anti-candida activity was assayed by the agar diffusion test with the modification that instead of using drug soaked discs, the drug was spotted directly on the growth media (SDA-Difco) containing specific concentration of the inoculum. The Candida inoculum was prepared by suspending a single isolated colony in sterile distilled water. Cell suspension was matched with 0.5 McFarland tube which is equivalent to cell density of 108cells/ml. 100 ul of this suspension was added to sterile petri plates. Autoclaved Sabourauds dextrose agar was cooled to
41- 42°C before adding 10 |u.l to the inoculum containing plates. Plates were rocked gently, to uniformly distribute the inoculum in the growth media. Agar was allowed to set at room trnperature and plates dried at 37°C for 15 minutes. The polyherbal compositions were diluted o 50% with sterile distilled water and 20 µl spotted on the dried plates. An equivalent volume of sterile distilled water was spotted in control plates. Plates were incubated at 37°C for 48 hours, fluconazole was used as positive control. The test was carried out in triplicate and the average diameter of inhibition was recorded.
III) Antibacterial activity
a/ Action against N. gonorrhoeae
Polyherbal composition was dispersed in water and spotted on N. gonorrhoeae ! noculation plates. Penicillin disc was used as standard. Plates were incubated at 37°C for 18-24 ■icurs and zone of inhibition was recorded.
Table 4 shows that it inhibits not only the growth of normal clinical isolates, but also those of penicillin resistant strains of iV.gonorrhoeae. b> I ciion against E. coli
Table 5 shows that growth was inhibited by dispersion of the polyherbal composition in ali strains including several multidrug resistant strains. i in-vivo antibacterial action on Chlamydia trachomatis
Female mice were given a subcutaneous injection of progestin for 7 days prior to inoculation o\~Chlamydia trachomatis (Ct -D) to increase their susceptibility to infection. On the day of inoculation, Praneem tablet dispersed in phosphate buffered saline (PBS) was delivered to ihe vagina of mice and stirred. Approximately 20 seconds later, 10 ulitre of lO5 inclusion forming units of Ct -D were delivered and stirred. Swabs were taken on days 4 and 8 post inoculation and plated onto McCoy target cell monolayers and incubated at 37°C for 72 hours.
Only 14% mice pretreated with the Praneem composition before inoculation of 10 ID50 of Ct -D became infected as compared to 86% in the PBS control group. Results are shown in Table 6.
IV) Evidence for the efficacy of the Praneem Polyherbal composition for curing leucorrhea
due to reproductive tract infections.
Women of reproductive age group between 18 to 45 years suffering from complains of excessive vaginal discharge were examined by experienced clinical experts at the Postgraduate Institute of Medical Education and Research, Chandigarh. The investigations done on each patient include a complete blood count, urine analysis, VDRL, pap smear, random sugars and vaginal swab cultures. Swabs were taken to conduct microbiological cultures and microscopic
examination for the presence of pathogens causing clinically abnormal vaginal discharge. These cultures are done before and after the use of Praneem pessary.
The women were directed to use for 7 days one Praneem pessary every night before going to bed. The pessary (wetted with water) could be inserted with washed fingers deep in the vagina On the 10th day after start of the treatment repeat clinical examination was carried out and swabs were taken for microbiological cultures.
These investigations carried out in twenty-eight women revealed that intravaginal treatment with Praneem pessary was effective in curing clinical symptoms of leucorrhoea in all the patients who used the drug for stipulated time. Remission was recorded in all women investigated. As regards the microbiology, the aerobic and anaerobic microbial flora was drastically reduced in every case. The pessary was also effective against Gardnerella vaginalis and Candida albicans. Results are shown in Table 7.
CONTRACEPTIVE EFFICACY
In evaluating the spermicidal activity of the individual ingredients and final composition of this invention the Sander Cramer Slide test is employed. The results are given in Table 8.
The contraceptive efficacy is determined in New Zealand White rabbits. The results are given in Table 9.
B: Composition of the Foaming Tablets
The Praneem polyherbal composition is preferably dispensed in form of a tablet, which can be inserted easily in the vagina with washed fingers. The tablet withstands the high environmental room temperatures prevalent in most parts of India without any deterioration for several months. It starts dispersing immediately after insertion in the vagina and its foaming gel formation continues and persists for several hours. This forms a lubricating bio-adhesive lining of the vaginal mucosa, which provides additional physical protective barrier to the passage of pathogenic microorganisms into the vaginal cells over and above the direct microbicidal action of various constituents in the tablet. The tablet does not cause irritation of the vaginal tissue nor mucosal damage on repeated use. Active ingredients of the present invention can be dispensed with water soluble polysaccharides such as sodium/ potassium alginates or carrageenan (iota/ kappa), gelling agents such as calcium gluconate/ calcium lactate/ calcium carbonate etc., an effervescent mixture containing a base and an acid that initiates foam and helps in disintegrating trie tablet. The base can be sodium or potassium carbonate or bicarbonate, whereas the acid can
be any fruit acid such as tartaric, citric, fumaric and malic etc. Diluents such as lactose, starches, sucrose, mannitol and microcrystalline cellulose can be used to increase the bulk.
Example- 4
Percent w/w
'Tanccm 18.3
Punned saponins 3.7
\ leutha citrata oil. 6.1 v/w
VKiium alginate 20
•sodium bicarbonate 15
' aleium gluconate 8
; uctose 10.6
Magnesium Slearate 1.3
.e;o:>ol J
v!ei hod of Preparation
All the solid materials are passed through 60-mesh sieve and dried at 50°C for 2 hours.
all the ingredients are mixed and compressed in a tabletting machine to make slugs or passed through compactor or granulated with isopropyl alcohol and dried. Slugs are screened / milled, mass are separated and slugged again, screened/ milled once more or alternatively compacted mass is passed through sieve (10#) to obtain granules. Granules are lubricated and compressed
into tablets.
Example - 5
Percent w/w
r'raiieeni 20
I'urified saponins 4
Mentha citrata oil 6 v/w
-'me Acetate 0.5
vxiium alginate 20
Sodium bicarbonate 15
'uric acid monohydrate 15
( aleium gluconate 8
i aciose 8.2
Magnesium Stearate ],3
Verosol 3
Method of Preparation
\\[ the solid materials are passed through 60-mesh sieve and dried at 50°C for 2 hours. All the ingredients are mixed and compressed in a tabletting machine to make slugs or passed through compactor or granulated with isopropyl alcohol and dried. Slugs are screened / milled, lines are separated and slugged again, screened / milled once more or alternatively compacted mass is passed through sieve (10#) to obtain granules. Granules are lubricated and compressed into tablets.
EVIDENCE FOR THE EFFICACY OF THE PRANEEM TABLET FOR CURING LEUCORRHOEA DUE TO REPRODUCTIVE TRACT INFECTIONS
Same protocol was adopted for clinical trials as with Praneem pessary, the only dilference was the tablet didn't require the initial wetting with water and could be used as such tor uisertion in the vagina. The investigation was carried out in twenty-five women, results are shown in the Table 10, whereby 100% remission was obtained in women who took the complete course of the tablets. spermicidal activity
Sander Cramer Slide test was employed. Results are given in Table 11.
Table 1 a. Virucidal Activity of the control samples on HIV-1.
(Table Removed)
Tabie 1 b. Virucidal Activity of Praneem Pessary on HIV-1.
(Table Removed)
Keduction in virus titre indicates the anti HIV potency of the compound.
Table 2, Inhibition of HIV-I-CF induced cytopathic effect.
(Table Removed)
Concentrations are expressed in % w/w except for N-9 (nonoxynol-9) in which case
percents indicate w/v. Initial testing concentration 0.5/2.5g.
TCTD50: Tissue culture toxic dose (50%). *** Log reduction in virus titre indicates the anti HIV potency of the compound. Titre of the
virus control: 6.5-logioTdD5o/0.1 ml.
t N-9 is the standard positive control.

Table 3. In vivo protection from Herpes-Simplex Virus-2 vaginal transmission.
(Table Removed)

Table 4. Antibacterial Activity against Standard strains and clinical isolates of Neisseria
(Table Removed)

Table 5. Antibacterial activity against E.colL
(Table Removed)
Resistant to Ciprofloxacin, Cephalexin, Gentamycin, Cefatoxime, .Amikacin, Augmentin. ; Penicillin (0.5 IU) was used as positive control.
Table 6 vivo protection front Chlamydia trachomatis vaginal transmission.
(Table Removed)
Table 7: Clinical response of Praneem Pessary in women suffering from abnormal vaginal discharge.
(Table Removed)
Praneem Pessary was used after wetting. * Panaal response was noted in patients who did not use the drug for the stipulated time.
Table 8. Synergistic spermicidal activity of the ingredients of the Praneem Pessary.
(Table Removed)

Determined by Sander Cramer test on seven different seamier samples with sperm count aging from 60x106/ml to 90xlO6 /7ml and 60-80% motility.

Table 9. Contraceptive efficacy in Rabbits.

(Table Removed)
Today commercially available nonoxynol-9 pessary, sold by Ortho Johnson.
Table 10: Clinical response of Praneem Tablet in women suffering from abnormal vaginal
discharge.
(Table Removed)
Martial response was noted in patients who did not use the drug for the stipulated time.

We claim:

A process for the preparation of an improved antimicrobial and spermicidal composition characterised in that it comprises ito 40% by weight of Praneem, 0.01 to 10% by weight of purified saponins from Sapindus mukerossi, between

0.01 to 20% by weight Mentha citrate oil and up to 0.5% by weight of zinc acetate dispersed in a conventional carrier or vehicle, said process comprising air drying leaves of Azadirachta indica, powdering said air dried leaves and subjecting them to extraction with water under hot conditions to obtain the leaf extract, dissolving it in water, subjecting it to decolourisationm, and recovering by conventional methods, the Praneem;

preparing purified saponins by subjecting air dried pericarp of Sapindus mukerossi to extraction, concentration to a syrupy residue, subjecting said residue to further extraction and followed precipitation;

dispersing by conventional methods the above Praneem and purified saponins in a conventional carrier;

adding citrate oil to said mixture;

adding zinc acetate, and optionally, other conventional additives;

and optionally, converting the final composition into the required shape and form.

A process as claimed in claim 1 wherein said Praneem is present in an amount of 5-30% by weight of said composition.

3. A process as claimed in claim 1 or 2 wherein said purified saponins are present. in an amount of 1-5% by weight of said composition.

4. A process as claimed in any preceding claim wherein said Mentha citrate oil is present in an amount of 1-10% by weight of said composition.

5. A process as claimed in any preceding claim wherein said conventional additives are selected from preservatives, antioxidants, effervescent mixtures, gelling agents, polysaccharides carriers, disintegrants, diluents and lubricants.

6. A process as claimed in claim 5 wherein said carrier is a diluent selected from lactose, starches, sucrose, Mannitol, sorbitol, microcrystalline cellulose. polyethylene glycol, cocoa butter, Tweens and Spans.

H.
7. A process as claimed in claim 5 wherein said preservative is selected from the group consisting of phenol, chloro cresol, 0-phenyl, methyl parabens, propyl paraben and their sodium. salts, benzylkonium chloride, boric acid and benzoic acid

8. A process as claimed in claim 5 wherein said antioxidant is selected from the group consisting of ascorbic acid, propyl gallate, L-tocopherol, butylated hydroxyl anisole and butylated hydroxl toluene.

9. A process as claimed in claim 5 wherein the said disintegrant is selected from the group consisting of sodium carboxy methyl cellulose, guar um, agar, starch and Tween.

10. A process as claimed in claim 5 wherein said gelling agent is selected from the group consisting of calcium gluconate, calcium lactate and calcium carbonate.

11. A process as claimed in claim 5 wherein the said polysaccharide is selected from the group consisting of sodium alginate, potassium alginate or carrageenan (iota/kappa)..

12. A process as claimed in claim 5 wherein the said lubricant is selected from the group consisting of light liquid paraffin, talc, aerosol, magnesium stearate, and sodium stearate.

13. A process as claimed in any preceding claim wherein the said composition is in the form of a pessary, cream, gel, soft gelatin capsules, foaming tablets, foams, spray or aerosol.

14. A process for the preparation of an improved antimicrobial and spermicidal composition substantially as herein described and as illustrated with reference to the examples.

Documents:

999-del-2003-abstract.pdf

999-del-2003-assignment.pdf

999-del-2003-claims.pdf

999-del-2003-complete specification (granted).pdf

999-del-2003-correspondence-others.pdf

999-del-2003-correspondence-po.pdf

999-del-2003-description (complete).pdf

999-del-2003-drawings.pdf

999-del-2003-form-1.pdf

999-del-2003-form-13.pdf

999-del-2003-form-19.pdf

999-del-2003-form-2.pdf

999-del-2003-form-3.pdf

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Patent Number

217525

Indian Patent Application Number

999/DEL/2003

PG Journal Number

38/2008

Publication Date

19-Sep-2008

Grant Date

27-Mar-2008

Date of Filing

14-Aug-2003

Name of Patentee

TALWAR RESEARCH FOUNDATION

Applicant Address

E-6, NEB VALLEY, NEB SERAI, SAINIK FARMS, NEW DELHI-110068, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	TALWAR, GURSARAN PRASHAD	TALWAR RESEARCH FOUNDATION, E-6 NEB SERAI, SAINIK FARMS, NEW DELHI-110068, INDIA.
2	RAGHUVANSHI, POONAM	TALWAR RESEARCH FORNDATION , E-6, NEB SERAI, SAINIK FARMS, NEW DELHI-110068, INDIA.

PCT International Classification Number

A61K 31/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA