Study On Antimicrobial And Contraceptive Pessum Preparations

Objective and background: In recent years,barrier Contraception are gaining importance as it has contraceptive and STD inhibiting properties .Safe sexual behavior adovacated by some people ,namely persisting on use of condom and reducing sexual partners ,can prevent the spread of STD in some degree ,while data shows that only when safe sexual behavior is widespread ,could it exhibits efficacy in worldwide epidemiology . STDs and AIDS are gaining significant importance at present due to rapid spread ,high cost of treatment ,and the increased risk of transmission of other STDs and AIDS .Current therapies available for symptomatic treatment of STDs and AIDS are quite expensive beyond the reach of common man and are associated with emergence of drug resistance .Medicinal plants have a long history of use and their use is widespread in both developing and developed countries .Herbal medicines provide rational means for the treatment of many diseases that are obstinate and in other systems of medicine .They are gaining popularity because of several advantages such as often fewer side effects ,better patient tolerance ,and relatively less expensive .Medicinal effects of plants tend to normalize physiological function and correct the underlying cause of the disorder Also ,plants are often less prone to the emergence of drug resistance . Our homeland is abundant in herbal medicine resources ,so we could take advantage of the clinical experience accumulated in thousand years ,and applying modern scientific technology to it.Traditional Chinese medicines could be developed into a safe and economic method ,being an alternertive approach to the prevention of STDs and AIDS . Gonococcus ,Candida albicans, Trichomonas vaginalis and human papilloma virus are the most frequent pathogens in the female's urogenital system. We had screened a complex prescription included six herbal medicines from prophase test.The complex prescription can actively inhibiting gonococcus,Candida albicans and Trichomonas vaginalis.It is certified that the complex prescription has many effects of antimicrobial in vivo test on rats. Based on the prophase test and not cutting down the effect of antimicrobial,we want to screen a complex prescription included three to four herbal medicines that it can effective inhibiting gonococcus, Candida albicans, Trichomonas vaginalis and human papilloma virus. We intend to combine N-9 and herbal medicines to formulate a pessum preparations that can prevent infection and unwanted pregnancy ,providing a contraceptive approach controlled women. Methods: 1. Based on the prophase test, screen a complex prescription effective inhibiting gonococcus, Candida albicans, Trichomonas vaginalis and human papilloma virus. (1) In vitro isolation and cultivation of gonococcus, Candida albicans, Trichomonas vaginalis. (2) Ethanol extract of the water extract of every tested herbal medicines were obtaind . (3) Disk agar diffusion method was employed in screening herbs with inhibiting effect on gonococcus,Candida albicans .MIC were determined by broth dilution method .The inhibitory activity against Trichomonas were measured by the death rate of Trichomonas.Polymerase chain reaction technique was applied to detect the human papilloma virus DNA in the lesion of condyloma acuminatum by herbal medicines. (4) After determined the complex prescriptio, design by uniform distribution and screen the compounding that it had the best effective. 2. Quality control of the pessum (1) To prepare the pessum with glycogelatin, complex prescriptio of herbal medicines and nonoxynol-9. To evaluate the quality of the pessum. (2) TLC was employed to perform the identity of Rhizoma coptidis, Rhizoma polygoni cuspidate .Coated silica gel plates were used. (3) To establish the quality standard for the pessum .Qualitative and quantitative analyses were carried out for berberine hydrochlorid.The berberine hydrochlorid content in pessum was determined by TLC-UV spectrotometer method . (4) Quantitative analyses were carried out for nonoxynol-9.The nonoxynol-9 content in pessum was determined by UV spectrotometer method . 3. Evaluation of the anti-infection and spermicidal effect of the homemade pessum in vitro (1) MIC were determined by broth dilution method to evaluate the effect on inhibitinggonococcus,Candida albicans.The inhibitory activity against Trichomonas were measured by the death rate of Trichomonas.Polymerase chain reaction technique was applied to detect the HPV DNA in the lesion of condyloma acuminatum by homemade pessum. (2) Use the method of the improved Sander-Cramer to evaluate the spermicidal effect of homemade pessum. 4. Evaluation of the irritative effect of the homemade pessum on rat's vagina We had bunged the rat's vagina with the pessum once a day for ten days,and then anatomized rats,obtain effective the vaginal tissue and observed the pathologic slices.. Results: 1. In vitro isolation and cultivation of the pathogens and screening the herbal medicines (1) The identity of gonococcus was confirmed by facts: kidney shaped diplococcus in pairs ,gram-negative, oxidase activity test positiver. The identity of Candida albicans was confirmed by facts: gram positive, having mycelial threads, obvious growth on Sabouraud's media. The Trichomonas vaginalis can be recognized by its motility ,being a little larger than leucocytes ,and if stained can be shown to have flagella ,with an undulating membrane at the side. (2) Rhizoma coptidis, Rhizoma coptidis + Rhizoma polygoni cuspidate has the best effect of against gonococcus, MIC of them is 12.5mg/ml.MIC of Rhizoma coptidis + Rhizoma polygoni cuspidate against Candida albicans is 25mg/ml,it is the lowest. Under the identical experimental condition, Radix pulsatillae result in 94.3% death rate of Trichomonas vaginalis after two hours.It is better than Agrimoniae and Radix pulsatillae + Agrimoniae. Each of Rhizoma coptidis, Rhizoma polygoni cuspidate, Radix pulsatillae, Rhizoma coptidis + Rhizoma polygoni cuspidate + Radix pulsatillae may exert an inhibiting effect on HPV-DNA in CA in vitro. (3) We choose Rhizoma coptidis + Rhizoma polygoni cuspidate + Radix pulsatillae as the complex prescription from these antimicrobial tests and screen the best effective compounding that the rate of Rhizoma coptidis-Rhizoma polygoni cuspidate-Radix pulsatillae is 3:1:2. 2. Quality control of the pessum Prepared the pessum with glycogelatin, complex prescriptio of herbal medicines andnonoxynol-9.The quality evaluation of the homemade pessum consistent with the regulation. (2) TLC was employed to perform the identity of Rhizoma coptidis, Rhizoma polygoni cuspidate .Coated silica gel plates were used. (3) UV spectrum showed Berberine hydrochlorid absorbed peaks at 430nm among 380nm to 700nm. The standard curve has good response linearity.The recovery was 97.63% and relative standard deviation (RSD) was 1.88%, with good reproducibility. (4) UV spectrum showed nonoxynol-9 absorbed peaks at 276nm. The standard curve has good response linearity. 3. Evaluation of the anti-infection and spermicidal effect of the homemade pessum in vitro (1) The homemade pessum can actively inhibiting Candida albicans,gonococcus,and Trichomonas vaginalis in vitro.It may exert a containment effect on HPV-DNA in CA in vitro. (2) It is feasible to using homemade pessums as a spermicide.The minimal concentration was 0.1953mg/ml, coincidence with nonoxynol-9 pessums. 4. Evaluation of the irritative effect of homemade pessums on rat's vaginaFolling the accepted Eckstein meteyard,the effect score of homemade pessums on rat's vagina was 6.75,and by compared with Nonoxynol-9 pessum's score(6.25),there is no significant difference in the result of t-test. CONCLUSIONS: 1. Rhizoma coptidis + Rhizoma polygoni cuspidate has the conspicuous effect of against gonococcus, Candida albicans. Radix pulsatillae has the best effect of against Trichomonas vaginalis. Each of them may exert an inhibiting effect on HPV-DNA in CA in vitro.We screened the best effective of antimicrobial compounding that the rate of Rhizoma coptidis -Rhizoma polygoni cuspidate -Radix pulsatillae is 3:1:2. 2. The quality evaluation of the homemade pessum consistent with the regulation. TLC-scanning determine the content of Berberine hydrochlorid in homemade pessums.The method is simple, accurate and available for quality control of homemade pessus,with good reproducibility and recovery. UV spectrum determine the content of Nonoxynol-9 in the homemade pessums,it consistent with the regulation.3. The tests in vitro confirmed that homemade pessums has better effect of antimicrobial and spermicidal effect. 4. Homemade pessums has some vaginal irritative effect on rats,but it is acceptable. 5. Homemade pessum is a good contraception pessum with antimicrobial from above.