Abstract:
The present invention relates to microbicidal and spermicidal compositions, methods and topical pharmaceutical compositions containing N-cocoyl amino acid pyrrolidone carboxylates (N-cocoyl amino acid pyrrolidone salts, CAPS) as active ingredients. The invention is for the prevention and control of sexually transmitted diseases and for the prevention of pregnancy. The microbicidal spectrum of the invention includes various STD pathogens, HIV viruses as well as gram positive and negative bacteria, and yeasts. Spermicidal barriers containing the compositions are also provided. 
     The N-cocoyl amino acid pyrrolidone salts is represented by the formula: 
     
       
                 
         
             
             
         
       
     
     Wherein A is an amino acid residue and R is a C.sub.1O-C.sub.14 fatty acid residue

Description:
RELATED APPLICATION 
       [0001]    This application is based on, and claims benefit of, U.S. Provisional Application Ser. No. 61/001,936, filed on Nov. 7, 2007. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relate to the topical application of a novel composition containing N-cocoyl amino acid pyrrolidone salts for preventing or reducing the transmission of sexually transmitted diseases (STDs) in sexually active individuals. This invention also relates to compositions and methods for preventing conception and/or reducing the risk of conception in sexually active females. The present compositions and methods are especially effective for preventing of sexually transmitted diseases during male/female sexual intercourse. It can, however, be used by heterosexual, homosexual, and bisexual individuals to reduce the risk of being infected by, or of transmitting, a sexually transmitted disease through sexual contact. 
         [0003]    The method of this invention comprises application of an effective amount of the microbicidal formulation within the vagina or anorectic region with choice of different dosage form applications. The invention also relates to methods of inhibiting the activity of other pathogens whose mode of transmission is nonsexual. The compositions of use in the invention are also effective in the inhibition of bacteria and fungi, which coexist with viruses or viral infections. 
       BACKGROUND OF THE INVENTION 
       [0004]    Sexually transmitted diseases (STDs) are among the most prevalent and communicable diseases, and continue to be a significant public health problem. It is estimated that more than 250 million people worldwide, and close to 3 million people in the United States, are infected annually by gonorrhea. Annual worldwide incidence of syphilis is estimated at 50 million people, with 400,000 in the United States annually needing treatment. The human immunodeficiency virus (HIV), resulting in fatal acquired immunodeficiency syndrome (AIDS), has spread rapidly in both homosexual and heterosexual groups. For many sexually transmitted infections, vaccines do not exist, and therapeutic agents are only partially effective, expensive, and difficult to distribute. In addition, female partners in many relationships do not control pregnancy or STI. One approach to the general control of STDs is the use of topically applied, female controlled microbicides that inactivate the relevant pathogens. 
         [0005]    It is thought that certain bacteria known to cause STD&#39;s may aid in HIV transmission. In persons who have been exposed to HIV, certain bacteria that cause STD&#39;s often fail to respond to therapies that are otherwise highly effective. HIV infection may help the spread of a bacterial STD that in turn helps to spread HIV. STD&#39;s pathogens such as Chlamydeous, syphilis, genital herpes and gonorrhea that cause ulcerations of the genital skin seem to increase the risk of acquiring or transmitting HIV infection sexually. 
       DESCRIPTION OF THE PRIOR ART 
       [0006]    NONOXYL-9 (N-9) is present in a large number of condoms and other spermicidal agents. Recent research, however, has found that N-9 can disrupt epithelial cells in the vagina, thereby increasing the risk of HIV infection. Frequent use of N-9 is also positively correlated with bacterial vaginosis, genital ulcers and Vulvitis, vaginal Candidiasis, toxic shock syndrome, and epithelial disruption of the cervix and the vagina. The final phase III multi-centre randomised placebo-controlled trial of N-9, undertaken by the United Nations Joint Programme on HIV/AIDS in 2002, showed that N-9 had no efficacy in preventing HIV transmission. Indeed, the transmission rate was marginally higher in the N-9 group. In 2003 the World Health Organization (WHO) recommended that people not use N-9 for protection against HIV/AIDS or other STIs. (Van Damme L, et al. (2002) Effectiveness of COL-1492, a nonoxynol-9 vaginal gel, on HIV-1 transmission in female sex workers: A randomised controlled trial. Lancet 360:971-977.; Stephenson J (2000) Widely used spermicide may increase, not decrease, risk of HIV transmission. Jama 284(8): 949.) 
         [0007]    U.S. Pat. No. 5,004,757 is directed to a method of deactivating viruses on surfaces by applying a three-part composition containing glutaraldehyde. The composition also contains hydrogen-bonded glycol molecules to eliminate aldehyde odor, and an anionic surfactant such as sodium dodecyl sulfate (SDS) as a potentiator of the virucidal activity of the glutaraldehyde component. The patent indicates that SDS has limited virucidal activity on its own, but has a synergistic effect when combined with gluteraldehyde. Due to the fact that gluteraldehyde is a well-known mutagen, the invention is not useful with direct contact of human epithelium tissues. 
         [0008]    U.S. Pat. Nos. 6,063,773, 7,078,392 presented a method for treating and preventing various infections, including papilloma virus and fungal and parasitic infections. The inventions relate to use sulfated polysaccharide, such as cellulose sulfate (a high molecular weight carboxymethyl cellulose-based polymer), for treating and preventing various sexual infections. The cellulose sulfate formulations were tried clinically for preventing HIV transmissions. In 2007, World Health Organization and UNAIDS has prematurely stopped the clinical trial of using cellulose sulfate to prevent HIV transmission in women because of a higher number of HIV infections in the active group compared with the placebo group. (Lut Van Damme et al. Lack of Effectiveness of Cellulose Sulfate Gel for the Prevention of Vaginal HIV Transmission, N Engl J med 359:463-472; Statement by the world Health Organization and UNAIDS: Cellulose sulfate microbicide trial stopped, 31 Jan. 2007) 
         [0009]    U.S. Pat. Nos. 5,314,917 and 6,297,278 provided an antimicrobial composition for inhibiting the activity of enveloped viruses, treatment of viral infections and method for inactiviting sperm. The composition comprises: a mixtures of a betaine, an amino oxide and a protonating agent to adjust the pH of the overall composition to about 5.5 or below (C31G). Clinical trials showed that C31G was associated with higher reporting of reproductive adverse events. The data were insufficient to conclude whether C31G is effective at preventing HIV infection relative to placebo. (Feldblum P J, et al. (2008) SAVVY Vaginal Gel (C31G) for Prevention of HIV Infection: A Randomized Controlled Trial in Nigeria. PLoS ONE 3(1): e1474. Peterson L, et al. (2007) SAVVY(R) (C31G) Gel for Prevention of HIV infection in Women: A Phase 3, Double-Blind, Randomized, Placebo-Controlled Trial in Ghana. PLoS ONE 2(12): e1312.) 
         [0010]    U.S. Pat. No. 5,617,877 introduced an acidic buffer gel contained within a flexible dome device positioned over the uterine cervix for controlling pH in the vagina for in-activating both sperm and STIs. The device is made of a dome and the absorbent acidic buffer gel is contained within the dome. The buffer gel (BufferGel™) is reported to be a negatively charged, non-absorbable high molecular weight polymer gel, designed to maintain vaginal pH below 5 in the presence of semen. BufferGel relies on a polymer comprised of carboxylated monomers to control the vaginal pH. BufferGel is placed over the uterine cervix that allowing the STD-causing microbes to readily migrate throughout the lower genital tract. The use of devices in combination with BufferGel requires significant skill and motivation by the user to obtain, and maintain, proper placement of the device. It is important to mention that some of the pathogens will survive the low pH environment. The efficacy of the acidic gel is solely depends on the acidity of the gel, and it might not guarantee a desirable effect on all the STI pathogens. 
         [0011]    U.S. Pat. Nos. 7,226,914 and 7,235,536 relates to the treating, inhibiting or preventing of certain infectious agents including papilloma virus and various vaginitis-causing microbes by employing cellulose sulfate and other sulfated polysaccharides. Carraguard is used as a microbicidal gel containing a sulfated polysaccharide. Resent clinical trial with this sulfated polysaccharides composition as microbicide did not demonstrate that the compound were effective in preventing male-to-female HIV transmission during vaginal intercourse (Microbicide Trials Network: Statement on Carraguard phase III clinical trial findings, Feb. 18, 2008). 
         [0012]    U.S. Pat. No. 6,417,144 relate to a solution for cleaning contact lenses. The solution comprises an amino acid derivative and at least one nonionic surfactant. The amino acid derivative is used as a component to increase the cleaning power together with a nonionic surfactant. The solution for contact lenses has an excellent cleaning effect and an adequate safety for the eye. This invention did not mention of preventing HIV and STDs. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0013]    The present invention relates to a novel composition and method for the prevention and control of sexually transmitted diseases and for the prevention of pregnancy. The invention also relates to treatment of viral infections. More particularly, the invention relates to a method for inhibiting the development of diseases and infections caused by viruses and some sexually transmitted pathogens whose major mode of transmission is sexual. In other aspects the invention also relates to methods of inhibiting the activity of enveloped viruses and other pathogens whose mode of transmission is nonsexual. The compositions of use in the invention are also effective in the inhibition of bacteria and fungi, which coexist with viruses or viral infections. Additionally, the invention relates to a prevention or treatment for virus related diseases, particularly sexually transmitted diseases related to AIDS, and to diseases related to this and other opportunistic infections of the immune-compromised host. 
         [0014]    Transmission of HIV is often associated with the co-transmission of other viral and/or microbial pathogens. HIV may not be the sole agent responsible for AIDS (Duesberg, P. H. (1991) Proc. Natl. Acad. Sci. 88:1575-1579; Lemaitre, M., Guetard, D., Henin, Y., Montagnier, L. and Zerial, A. (1990). Res. Virol. 141:5-16). For this reason, antimicrobial agents, such as those described in this invention, with a broad spectrum of activities against viruses, bacteria, and yeasts may be of particular value in the prevention and treatment of Acquired Immune Deficiency Syndrome (AIDS). It is thought that certain bacteria known to cause STD&#39;s may aid in HIV transmission. In persons who have been exposed to HIV, certain bacteria that cause STD&#39;s often fail to respond to therapies that are otherwise highly effective. HIV infection may help the spread of a bacterial STD that in turn helps to spread HIV. STD&#39;s pathogens that cause ulcerations of the genital skin seem to increase the risk of acquiring or transmitting HIV infection sexually. The compositions described in the invention may also be of use to inactivate other viruses, including vaccinia, varicella, herpes zoster, cytomegalovirus, influenza, mumps and measles. 
       APPLICATIONS OF THE INVENTION 
       [0015]    The present invention provides a novel composition and method for the prevention and control of sexually transmitted diseases and for the prevention of pregnancy. The invention also relates to treatment of viral infections. More particularly, the invention relates to a method for inhibiting the development of diseases and infections caused by viruses and some sexually transmitted pathogens whose major mode of transmission is sexual. In other aspects the invention also relates to methods of inhibiting the activity of pathogens whose mode of transmission is nonsexual. The compositions of the invention are also effective in the inhibition of bacteria and fungi, which coexist with viruses or viral infections. 
         [0016]    The compositions can be formulated in the form of a gel, liquid, aerosol, mist, sponge, spray, foam, gel, cream, salve, jelly, suppository and film. The compositions can be applied to the external genital organs, vagina, anorectic region and rectum in different dosage forms with appropriate apparatus, for example the gel could be applied to vagina or rectum through an applicator or syringe. The compositions of the invention can also be incorporated into douches. The compositions can also be incorporated into wipes. 
         [0017]    The invention also provides a method for preventing and the treatment of certain skin diseases. Such as ringworm and other fungi infectious skin diseases. 
         [0018]    The invention also provides a broad spectrum and highly efficient microbicidal composition used for reducing the risk of transmission of STD-causing organisms to health care providers and laboratory personnel (or other persons) who may come in contact with biological samples and specimens 
         [0019]    The compositions of the present invention can also be used as spermicides. These compositions can be used alone, with other known spermicides and with or incorporated into contraceptive devices such as condoms, sponges, vaginal inserts, contraceptive films, diaphragms, suppositories, contraceptive patches or sustained release devices. For use as spermicides, these compositions of the invention can be applied alone; with other microbicides; and with or incorporated into the contraceptive devices described above. 
         [0020]    The composition can also be used in animals as disinfecting or antiseptic agent. 
       Synthesis and Preparation of the N-cocoyl Amino Acid Pyrrolidone Salt 
       [0021]    The microbicidal composition of the present invention consisting of an amino acid derivative that is modified with containing a C.sub.10-14 alkyl group at the N-terminus, particularly preferably N-cocoyl group is preferred. The amino acid residue bears a positive charge and the negative counter ion is organic molecule, preferably pyrrolidone salts. There are no halogen elements, such as chloride or bromide, in the structure of the amino acid compound. One general example is an amino acid ethyl ester acylated at the N-terminus with a coconut oil fatty acid residue, and having DL-pyrrolidone carboxylic acid added thereto. 
         [0022]    The N-cocoyl amino acid pyrrolidone salts represented by the formula: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    Wherein A is an amino acid residue and R is a C.sub.10-C.sub.14 fatty acid residue. In the structure, the amino acid residue could be from that of an arginine, lysine, and histidine; homolysine, or an unnatural amino acid residue bearing a positive charge, and other di or tri-peptide bearing a positive charge group. 
         [0023]    For example, the N-cocoyl Arginine pyrrolidone salts is prepared with the following procedures: 
         [0024]    Preparation solution A: Mix 1.2 equivalent N,N′-Dicyclohexylcarbodiimide (DCC) with 1 equivalent myristic acid (CH 3 (CH 2 ) 10 COOH) in DMF and stir for 45 minute to 2 hours, Add in 1.2 equivalent N-Hydroxylsuccinimide (NHS) dissolved in CH 2 Cl 2 , keep stirring until precipitate forms, which is the undesired side product dicyclohexane urea. Filter the reaction mixture to remove the precipitate urea, and save the solution as solution A. 
         [0025]    Preparation solution B: L-Arginine ethyl ester dihydrochloride and 1 equivalent of NaOH at 0 degree C. in DMF or CH 2 Cl 2  for 30 minutes, and then add 1.3 equivalent of triethyl amine (Et 3 N). Mix to a homogeneous solution and save as solution B. 
         [0026]    Preparation of the amino salt: Mix solution A and B together at room temperature for 2 hours. Remove most of the solvent by vacuum. Dissolve the product in methanol and add one equivalent of NaOH, and extract the product with a mixture of water and ether/EtOAc. Save and dry the organic phase under vacuum. Re-precipitate the product by using solvent hexane or water or methanol, or a mixture of methanol and water. Re-dissolve the product in methanol, and add one equivalent of DL-pyrrolidone carboxylic acid, and then remove the methanol under vacuum to obtain the final product. 
         [0027]    In the formulation of the present invention the N-cocoyl amino acid pyrrolidone salts is contained usually in a proportion of from 0.003 to 3.0% (w/w). 
         [0028]    The N-cocoyl amino acid pyrrolidone salts are soluble in water and highly safe with respect to acute toxicity (LD.sub.50). It has no irritation to skin and ophthalmic mucosa. Moreover, it has good biodegradable characteristic and can rapidly be decomposed in wastewaters. 
       Preparation of the Compositions 
       [0029]    In the microbicide compositions of the present invention a natural plant polysaccharides could be incorporated in the formula to enhance the antibacterial effect and protect the epithelium tissues. The plant polysaccharides may, for example, be Aloe Vera polysaccharides. 
         [0030]    In the microbicide compositions of the present invention an ampholytic surfactant may be added and incorporated so long as it does not inhibit the effect obtained by the N-cocoyl amino acid pyrrolidone salts, in an amount which does not impair the antimicrobial effect. The ampholytic surfactant may, for example, be an amine oxide such as alkyl dimethyl amine oxide. 
         [0031]    In the present invention, in addition to the specific active ingredient, one or more additive components conventionally used may further be incorporated in a conventional amount, as the case requires. The additive can be used including natural polysaccharides, natural plant acid and their salts, fragrance, colorant, flavor, plasticizer, stabilizing agent, emulsifier or moisturizer. Such an additive component is preferably one, which has a high safety for the human body, which is adequately compatible with active ingredients, and which has no influence over the effectiveness and stability of the composition. 
         [0032]    In order to facilitate a further understanding of the invention, the following examples are presented primarily for the purposes of illustrating more specific details thereof. The invention is not to be deemed as limited thereby except as defined in the claims. 
       EXAMPLE 1 
       [0033]    The liquid microbicide composition of the present invention is prepared by adding and incorporating the active ingredients in proper amounts into a proper aqueous medium in an optional order in a conventional method: 
         [0000]    
       
         
               
             
               
               
               
             
               
             
               
               
               
             
           
               
                   
               
             
             
               
                 Mix the following ingredients (w/w) to obtain a homogeneous solution 
               
             
          
           
               
                   
                 N-cocoyl amino acid pyrrolidone salts: 
                 0.003-3.0, 
               
               
                   
                 Water: 
                  10.0-99.997. 
               
             
          
           
               
                 When plant polysaccharides (powder of freeze dried  Aloe Vera  gel) 
               
               
                 is added in the composition, add the following ingredients (w/w) 
               
               
                 and mix to obtain a homogeneous solution 
               
             
          
           
               
                   
                 Plant polysaccharides: 
                 0.001-4.0 
               
               
                   
                 N-cocoyl amino acid pyrrolidone salts: 
                 0.003-3.0 
               
               
                   
                 Water: 
                  10.0-99.995 
               
               
                   
                   
               
             
          
         
       
     
         [0034]    The gel microbicide composition of the present invention is prepared by the following procedures:
       A. Mix the following ingredients (w/w) and heat to 60° C.-95° C. with stirring to obtain a homogeneous solution       
 
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 Ampholytic surfactant: 
                 0.0016-3.0 
               
               
                   
                   Aloe Vera : 
                  0.001-4.0 
               
               
                   
                 Water 
                  10.0-99.999 
               
               
                   
                   
               
             
          
         
       
       
         
           
             B. Add Hydroxypropyl ethyl cellulose (w/w) 0.010-5.0 to the 60° C.-95° C. homogeneous solution, stir for more than 30 minutes, stops heating, slowly reduces temperature to 50° C. 
             C. Add the N-cocoyl amino acid pyrrolidone salts 0.003-3.0 to the above solution, 
           
         
       
     
         [0038]    Keep stirring until the mixture&#39;s temperature dropped to room temperature to obtain a colorless transparent gel. 
         [0039]    An appropriated amount (3-5 ml) of gel was filled into a syringe. The filled syringe was sealed within an aluminum foil bag. At use, the user open the foil bag and insert the syringe to vagina or rectum to apply the material. 
         [0040]    Table 1 listed 37 formulations. They are prepared following the above procedures to make liquid or gel preparations. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 FORMULATIONS OF MICROBICIDES (W/W) 
               
             
          
           
               
                 Formulation 
                 AV 
                 C1 
                 H 2 O 
                 CAPS 
                 HP 
               
               
                   
               
             
          
           
               
                 1 
                 1.00 
                 1.00 
                 94.50 
                 1.50 
                 2.00 
               
               
                 2 
                 0.00 
                 0.00 
                 99.40 
                 0.60 
                 0.00 
               
               
                 3 
                 0.25 
                 0.00 
                 99.08 
                 0.67 
                 0.00 
               
               
                 4 
                 0.0013 
                 0.002 
                 99.995 
                 0.0034 
                 0.01 
               
               
                 5 
                 0.25 
                 0.33 
                 96.75 
                 0.67 
                 2.00 
               
               
                 6 
                 0.006 
                 0.008 
                 99.92 
                 0.017 
                 0.05 
               
               
                 7 
                 0.013 
                 0.017 
                 99.84 
                 0.034 
                 0.10 
               
               
                 8 
                 0.003 
                 0.003 
                 99.9 
                 0.007 
                 0.02 
               
               
                 9 
                 0.005 
                 0.007 
                 99.9 
                 0.013 
                 0.04 
               
               
                 10 
                 0.025 
                 0.033 
                 99.7 
                 0.067 
                 0.200 
               
               
                 11 
                 0.003 
                 0.004 
                 99.96 
                 0.008 
                 0.025 
               
               
                 12 
                 1.50 
                 0.50 
                 50.00 
                 1.00 
                 0.50 
               
               
                 13 
                 2.00 
                 0.0016 
                 90.0 
                 3.00 
                 5.0 
               
               
                 14 
                 0.20 
                 0.25 
                 95.50 
                 0.50 
                 0.30 
               
               
                 15 
                 0.00 
                 0.00 
                 10.00 
                 0.003 
                 0.00 
               
               
                 16 
                 0.00 
                 0.00 
                 99.995 
                 0.003 
                 0.00 
               
               
                 17 
                 0.00 
                 0.00 
                 98.50 
                 1.5 
                 0.00 
               
               
                 18 
                 0.00 
                 0.00 
                 97.00 
                 3.0 
                 0.00 
               
               
                 19 
                 4.0 
                 0.00 
                 96.00 
                 0.003 
                 0.00 
               
               
                 20 
                 1.8 
                 0.00 
                 97.00 
                 1.2 
                 0.00 
               
               
                 21 
                 0.001 
                 0.00 
                 97.00 
                 3.0 
                 0.00 
               
               
                 22 
                 0.00 
                 3.0 
                 97.00 
                 0.003 
                 0.00 
               
               
                 23 
                 0.00 
                 1.7 
                 97.00 
                 1.3 
                 0.00 
               
               
                 24 
                 0.00 
                 0.0016 
                 97.00 
                 3.0 
                 0.00 
               
               
                 25 
                 0.00 
                 0.00 
                 95.00 
                 0.003 
                 5.0 
               
               
                 26 
                 0.00 
                 0.00 
                 96.30 
                 1.4 
                 2.3 
               
               
                 27 
                 0.00 
                 0.00 
                 96.99 
                 3.0 
                 0.01 
               
               
                 28 
                 0.001 
                 0.00 
                 97.00 
                 0.003 
                 0.00 
               
               
                 29 
                 2.0 
                 1.5 
                 94.90 
                 1.6 
                 0.00 
               
               
                 30 
                 4.0 
                 0.0016 
                 93.00 
                 3.0 
                 0.00 
               
               
                 31 
                 0.00 
                 3.0 
                 96.99 
                 0.0033 
                 0.01 
               
               
                 32 
                 0.00 
                 1.3 
                 94.50 
                 1.7 
                 2.5 
               
               
                 33 
                 0.00 
                 0.00 
                 92.00 
                 3.0 
                 5.0 
               
               
                 34 
                 0.001 
                 0.00 
                 99.995 
                 0.0033 
                 0.01 
               
               
                 35 
                 2.2 
                 0.00 
                 93.20 
                 1.8 
                 2.8 
               
               
                 36 
                 4.0 
                 0.00 
                 88.00 
                 3.0 
                 5.0 
               
               
                 37 
                 0.0 
                 0.0 
                 99.5 
                 0.5 
                 0.0 
               
               
                   
               
               
                 Note: 
               
               
                 AV = Freeze dried  Aloe Vera  gel powder; 
               
               
                 C1 = Alkyldimethylamine oxide; 
               
               
                 H 2 O = Water; 
               
               
                 CAPS = N-cocoyl Arginine pyrrolidone salts; 
               
               
                 HP = Hydroxypropyl methyl cellulose. 
               
             
          
         
       
     
       EXAMPLE 2 
       [0041]      
         [0000]    
       
         
               
             
               
               
               
             
           
               
                   
               
               
                 MICROBICIDE COMPOSITION OF SUPPOSITORIES 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 CAPS 
                 4.8 g 
               
               
                   
                 Alkyldimethylamine oxide 
                 2.4 g 
               
               
                   
                   Aloe Vera  powder 
                 1.8 g 
               
               
                   
                 Sodium phytate 
                 1.8 g 
               
               
                   
                 Glycerogelatin 
                 900.0 g  
               
               
                   
                 Glycerin 
                 20.0 g  
               
               
                   
                 Water 
                 80.0 g  
               
               
                   
                   
               
               
                   
                 Dissolve N-cocoyl Arginine pyrrolidone salts (CAPS), sodium phytate and freeze dried  Aloe Vera  powder in water, add glycerin and mix to a homogeneous solution, Add the above to Glycerogelatin, mix at 45° C. to homogeneous, and injected to molds, cooled and ejected for packing. Each one weights 4 gm. 
               
             
          
         
       
     
       EXAMPLE 3 
       [0042]      
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                   
               
               
                 MICROBICIDE COMPOSITIONS OF MIST AND SPRAY 
               
             
          
           
               
                   
                 Ingredients 
                 Spray 
                 Mist 
               
               
                   
                   
               
               
                   
                 CAPS 
                 0.6 kg 
                 0.8 g 
               
               
                   
                 Alkyldimethyl amine oxide 
                 0.0 kg 
                 0.4 g 
               
               
                   
                 Isopropylene 
                 5.0 kg 
                 5.0 g 
               
               
                   
                 Dimethyl Ether 
                 0.0 kg 
                 20.0 g  
               
               
                   
                 Water 
                 94.4 kg  
                 80.0 g  
               
               
                   
                   
               
               
                   
                 Spray: Dissolve N-cocoyl Arginine pyrrolidone salts (CAPS) in water, slowly add isopropylene and mix to homogeneous solution.; package in container with spray device. 
               
               
                   
                 Mist: Dissolve N-cocoyl Arginine pyrrolidone salts (CAPS) and Alkyldimethyl amine oxide in water, slowly add isopropylene and mix to homogeneous solution; add Dimethyl Ether and fill in container with spray device. 
               
             
          
         
       
     
       EXAMPLE 4 
       [0043]      
         [0000]    
       
         
               
             
               
               
               
             
           
               
                   
               
               
                 MICROBICIDE COMPOSITION OF FILM 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 CAPS 
                 0.80 kg 
               
               
                   
                 
                   Aloe Vera 
                 
                 0.16 kg 
               
               
                   
                 Gelatin 
                 6.20 kg 
               
               
                   
                 Hydroxyethyl cellulose 
                 0.50 kg 
               
               
                   
                 Glycerin 
                 31.00 kg  
               
               
                   
                 Water 
                 25.50 kg  
               
               
                   
                   
               
               
                   
                 Triturate Gelatin with Hydroxyethyl cellulose and glycerin. Mix thoroughly to form a slurry. Dissolve N-cocoyl Arginine pyrrolidone salts (CAPS) in warm water to obtain a homogeneous solution. Add the solution to the above slurry. Heat the mixture at 40° C. and mix until cellulose and gelatin are completely hydrated. Pour the solution on polyethylene sheet to cast a film of about 2 mm thick to be cut after cooling at size 3.9 × 3.9 cm. 
               
             
          
         
       
     
       EXAMPLE 5  
     Effects of the Microbicide on  Ureaplasma Urealyticum    
     1. Materials 
       [0000]    
       
         
           
             1) Testing strain:  Ureaplasma Urealyticum : clinical strain 
             2) Culture medium: High efficiency  Ureaplasma  culture medium; 
             3) Testing sample: Microbicide solution prepared as Example 1, formulation 3.
 
2. Testing Methods and Standards:          Technical Standard for Disinfection          (2002 Edition) Issued by “The Health Department of China”
 
             1) The tests followed the “Standard” with consideration of the characteristics of  Ureaplasma urealyticum    
             2) Testing Temperature: 20° C.˜25° C. 
             3) Concentration of sample dilution: 1:100; 1:200; 1:500; 1:1000 
             4) Concentration of  Ureaplasma Urealyticum:  10 7    
             5) Treatment:  Ureaplasma Urealyticum  suspension:diluted sample=1:9 
             6) Reacted time: 1 min.; 3 min.; 5 min.; 
             7) Diluting methods were used to remove the residue of the sample 
             8) Tests repeated for two times
 
3. Results: The Killing Effects of sample to  Ureaplasma Urealyticum  (Table 2)
 
           
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 The Effects of Testing Sample on  Ureaplasma Urealyticum   
               
             
          
           
               
                 time 
                 Dilution of Sample 
                   
               
             
          
           
               
                 (min.) 
                 1:100 
                 1:200 
                 1:500 
                 Control 
               
               
                   
               
               
                 1 
                 − 
                 + 
                 + 
                 + 
               
               
                 3 
                 − 
                 − 
                 + 
                 + 
               
               
                 5 
                 − 
                 − 
                 + 
                 + 
               
               
                   
               
               
                 Note: 
               
               
                 Control:  Ureaplasma Urealyticum  growth observed; 
               
               
                 +:  Ureaplasma Urealyticum  growth observed; 
               
               
                 −: No  Ureaplasma Urealyticum  growth observed. 
               
             
          
         
       
     
       4. Conclusion 
       [0055]    The testing results showed: Testing Sample diluted for 100 times and reacted with  Ureaplasma Urealyticum  suspension for 1 minute killed  Ureaplasma Urealyticum.    
       EXAMPLE 6  
     Effects of the Microbicide on  Neisseria Gonorrhoeae    
     1. Materials 
       [0000]    
       
         
           
             1) Testing strain:  Neisseria Gonorrhoeae , clinical strain 
             2) Culture medium: 10% blood dish; 
             3) Testing sample: Microbicide solution prepared as Example 1, formulation 3.
 
2. Testing Methods and Standards:          Technical Standard for Disinfection          (2002 Edition) Issued by “The Health Department of China”
 
             1) The tests followed the “Standard” with consideration of the characteristics of  Neisseria gonorrhoeae    
             2) Temperature: 25° C.˜28° C. 
             3) Concentration of sample prepared: 1:25; 1:50; 1:100 
             4)  Neisseria gonorrhoeae  suspension: 10 7    
             5) Treatment:  Neisseria gonorrhoeae  suspension:diluted sample solution=1:1 
             6) Treatment time: 1 min.; 3 min.; 5 min.; 
           
         
       
     
       3. Results 
       [0065]    The Killing Effect of Testing Sample on  Neisseria Gonorrhoeae  (Table 3) 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Effects of Testing Sample on  Neisseria Gonorrhoeae   
               
             
          
           
               
                 React time 
                 Dilution of Sample 
                   
               
             
          
           
               
                 (min.) 
                 1:25 
                 1:50 
                 1:00 
                 Control 
               
               
                   
               
               
                 1 
                 − 
                 − 
                 − 
                 + 
               
               
                 3 
                 − 
                 − 
                 − 
                 + 
               
               
                 5 
                 − 
                 − 
                 − 
                 + 
               
               
                   
               
               
                 Note: 
               
               
                 +: growth of  Neisseria gonorrhoeae  observed 
               
               
                 −: No growth of  Neisseria gonorrhoeae   
               
             
          
         
       
     
       4. Conclusion 
       [0066]    The testing results showed: Testing Sample diluted for 100 times and reacted with  Neisseria gonorrhoeae  suspension for 1 minute, killed  Neisseria gonorrhoeae.    
       EXAMPLE 7  
       [0067]    Effects of the Microbicide on  Trichomonas vaginalis    
       1. Materials 
       [0000]    
       
         
           
             1) Testing strain: Clinical isolated  Trichomonas vaginalis  strain 
             2) Culture medium: liver infusion medium; 
             3) Testing sample: Microbicide solution prepared as Example 1, formulation 3.
 
2. Testing Methods and Standards:          Technical Standard for Disinfection          (2002 Edition) Issued by “The Health Department of China”
 
           
         
       
     
       3. Results 
       [0071]    The Killing effect of testing sample to  Trichomonas vaginalis  (Table 4) 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Effects of Testing Sample on  Trichomonas Vaginalis   
               
             
          
           
               
                 time 
                 Dilution of sample 
                 Control 
               
             
          
           
               
                 (minutes) 
                 1:50 
                 1:100 
                 1:200 
                 1:50 
               
               
                   
               
               
                 1 
                 − 
                 − 
                 + 
                 + 
               
               
                 3 
                 − 
                 − 
                 + 
                 + 
               
               
                 5 
                 − 
                 − 
                 + 
                 + 
               
               
                   
               
               
                 Note: 
               
               
                 +: Active  Trichomonas vaginalis   
               
               
                 −:  Trichomonas vaginalis  were not active and were dissolved 
               
             
          
         
       
     
       4. Conclusion 
       [0072]    The testing results showed: Testing Sample diluted for 100 times and reacted with  Trichomonas vaginalis  suspension for 1 minute killed  Trichomonas vaginalis.    
       EXAMPLE 8 
     Effects of the Microbicide on HIV-1 
     1. Materials 
       [0000]    
       
         
           
             1) Human Immunodeficiency Virus: HIV-1 IIIB strain; 
             2) Cell: MT 4 cell; 
             3) Testing sample: Microbicide solution prepared as Example 1, formulation 2.
 
2. Testing Methods and Standards:          Technical Standard for Disinfection          (2002 Edition)
 
             1) Diluted the Testing sample to 1:20; 1:40; 1:80 and 1:160 solutions with distilled water. 
             2) Added 0.9 ml sample (1:20; 1:40; 1:80, 1:160) to 0.1 ml HIV suspension solution respectively 
             3) The mixtures were reacted for 1 min.; 3 min.; 5 min. at room temperature 
             4) Take 0.1 ml each of the above and dilute with culture fluid to 1:1000 
             5) The virus concentration were titrated (96 well plate, each well contains 0.1 ml of 50000/ml MT4 cell) 
             6) Incubated for 7 days (37° C., 5% CO 2 ) 
             7) Added 0.1 ml of fresh culture fluid to each well at the third day 
             8) Observe the cell growth everyday 
             9) Evaluate the existence of Virus with CPE 
           
         
       
     
       3. Experimental Groups 
       [0000]    
       
         
           
             1) Testing group 
             2) Drug (sample) removed control group (1000 times diluted Virus+1000 times diluted sample) 
             3) Normal cell control group 
             4) Virus control group (Virus+culture fluid) 
           
         
       
     
       4. Results 
       [0089]    HIV-1 titration was 10 7  TCID 50 . 
         [0090]    The “drug removed control group” showed that the dilution could stop the effect of sample to the virus. 
         [0091]    The Testing sample at 1:20 and 1:40 dilution reacted with HIV-1 for 1 min, titrated and cultured with MT4 cell, no cell abnormality were observed 
         [0092]    The Testing sample at 1:80 dilution reacted with HIV-1 for 3 min., titrated and cultured with MT4 cell, no cell abnormality were observed. 
         [0093]    The Testing sample at 1:160 dilution reacted with HIV-1 for 5 min., titrated and cultured with MT4 cell, no cell abnormality were observed. 
       5. Conclusion 
       [0094]    Testing sample diluted at 1:20 (Example 1, formulation 15) and 1:40 solutions reacted with HIV-1 suspension for 1 min; diluted at 1:80 solution reacted with HIV-1 suspension for 3 min; diluted at 1:160 solution reacted with HIV-1 suspension for 5 min, all the kill rates were 100%. 
       EXAMPLE 9  
       [0095]    The Minimum Bactericide Concentration (MBC) of Testing Sample to  Candida albicans    
       1. Materials 
       [0000]    
       
         
           
             1) Testing strain:  Candida albicans  ATCC 10231, the 4 th  generations. 
             2) Testing sample: Microbicide solution prepared as example 1, formulation 2 
           
         
       
     
       2. Testing Method 
       [0098]    Nutrient broth dilution method, Temperature 21±1° C. 
       3. Testing Results (Table 5) 
       [0099]      
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 The MBC of Testing sample to  Candida albicans   
               
             
          
           
               
                   
                   
                   
                   
                 Bacterial 
               
               
                   
                 MBC of Sample dilutions 
                 Positive 
                 Negative 
                 concentration 
               
             
          
           
               
                 test 
                 1 
                 2 
                 4 
                 8 
                 16 
                 32 
                 64 
                 128 
                 256 
                 control 
                 control 
                 (CFU/ml) 
               
               
                   
               
               
                 1 
                 − 
                 − 
                 − 
                 − 
                 − 
                 + 
                 + 
                 + 
                 + 
                 + 
                 − − 
                 2.13 × 10 6   
               
               
                 2 
                 − 
                 − 
                 − 
                 − 
                 − 
                 + 
                 + 
                 + 
                 + 
                   
                   
                 1.89 × 10 6   
               
               
                   
               
               
                 Note: 
               
               
                 “+” bacterial growth observed; 
               
               
                 “−” no bacterial growth observed. 
               
             
          
         
       
     
       4. Conclusion 
       [0100]    To  Candida albicans  the MBC of testing sample is it&#39;s 16 times dilution (2 min). 
       EXAMPLE 10  
     Effects of the Microbicide on HIV-1 
     1. Materials 
       [0000]    
       
         
           
             1) Human Immunodeficiency Virus: HIV-1 IIIB strain; 
             2) Cell: MT 4 cell; 
             3) Testing sample: Microbicide gel prepared as Example 1, formulation 5.
 
2. Testing Methods and Standards:          Technical Standard for Disinfection          (2002 Edition) Issued by The Health Department of China.
 
             1) HIV Virus deactivation Tests, Page: 38 
             2) Diluted the Testing sample to 1:20; 1:40 and 1:80 solutions with distilled water. 
             3) Added 0.9 ml sample (1:20; 1:40; 1:80) to 0.1 ml HIV suspension solution respectively 
             4) The mixtures were reacted for 1 min.; 3 min.; 5 min. and 10 min. at room temperature 
             5) Take 0.1 ml each of the above and dilute with culture fluid to 1:1000 
             6) The virus concentration were titrated (96 well plate, each well contains 0.1 ml of 50000/ml MT4 cell) 
             7) Incubated for 7 days (37° C., 5% CO 2 ) 
             8) Added 0.1 ml of fresh culture fluid to each well at the third day 
             9) Observe the cell growth everyday 
             10) Evaluate the existence of Virus with CPE 
           
         
       
     
       3. Experimental Groups: 
       [0000]    
       
         
           
             1) Testing group 
             2) Drug (sample) removed control group (1000 times diluted Virus+1000 times diluted sample) 
             3) Normal cell control group 
             4) Virus control group (Virus+culture fluid) 
           
         
       
     
       4. Results 
       [0000]    
       
         
           
             1) HIV-1 titration was 10 7  TCID 50 . 
             2) The “drug removed control group” showed that the dilution could stop the effect of sample to the virus. 
             3) The testing sample at 1:20 and 1:40 dilution reacted with HIV-1 for 1 min, titrated and cultured with MT4 cell, no cell abnormality were observed. 
             4) The testing sample at 1:80 dilution reacted with HIV-1 for 3 min., titrated and cultured with MT4 cell, no cell abnormality were observed 
           
         
       
     
       5. Conclusion 
       [0122]    Testing sample diluted at 1:20 (Example 1, formulation 7) and 1:40 solutions (Example 1, formulation 6) reacted with HIV-1 suspension for 1 minutes, the kill rate was 100%. 
         [0123]    Testing sample diluted at 1:80 solution reacted with HIV-1 suspension for 3 min, the kill rate was 100%. 
       EXAMPLE 11  
     The Effects of the Microbicide on  Treponema Pallidum    
     1. Materials 
       [0000]    
       
         
           
             1)  Treponema Pallidum : Nichols strain 
             2) Testing sample: Microbicide gel prepared as of Example 1, formulation 5
 
2. Testing Methods and Standards          Guidelines for Pre-clinical and Clinical Research of New Drugs          (1993 Edition): “The Experimental Methods and Procedures for Anti- Treponema Pallidum” 
 
             1) Temperature: 20-25° C. 
             2) Sample Dilutions: 1:5; 1:10; 1:20; 1:40; 1:80; 1:100 
             3)  Treponema Pallidum  Suspension: each field≧30  Treponema Pallidum    
             4) Reacting time: 1 min.; 3 min.; 5 min.; 10 min. 
             5) Sample diluted with distilled water 
             6) No terminating agent was employed in the tests 
             7) The deactivating effects for  Treponema Pallidum  were recorded at different time intervals. 
             8) The tests repeated two times 
           
         
       
     
       3. Results 
       [0134]      
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 The deactivating Effect of Testing Sample to  Treponema Pallidum   
               
             
          
           
               
                 
                   Treponema 
                 
                 React time 
                 Sample dilution 
               
             
          
           
               
                 
                   Pallidum 
                 
                 (min.) 
                 1:5 
                 1:10 
                 1:20 
                 1:40 
                 1:80 
                 1:100 
               
               
                   
               
             
          
           
               
                 Nichols strain 
                 1 
                 − 
                 − 
                 + 
                 + 
                 + 
                 + 
               
               
                 Nichols strain 
                 3 
                 − 
                 − 
                 − 
                 + 
                 + 
                 + 
               
               
                 Nichols strain 
                 5 
                 − 
                 − 
                 − 
                 − 
                 + 
                 + 
               
               
                 Nichols strain 
                 10 
                 − 
                 − 
                 − 
                 − 
                 − 
                 + 
               
               
                   
               
               
                 Note: 
               
               
                 −: deactivated 
               
               
                 +: not deactivated. 
               
             
          
         
       
     
       4. Conclusion 
       [0135]    Testing sample diluted at 1:10 (Example 1, formulation 10) deactivated  Treponema Pallidum  within 1 min. 
         [0136]    Testing sample diluted at 1:20 (Example 1, formulation 7) deactivated  Treponema Pallidum  within 3 min. 
         [0137]    Testing sample diluted at 1:40 (Example 1, formulation 6) deactivated  Treponema Pallidum  within 5 min. 
         [0138]    Testing sample diluted at 1:80 (Example 1, formulation 1 1) deactivated  Treponema Pallidum  within 10 min. 
       EXAMPLE 12  
       [0139]    The Effects of the Microbicide on  Chlamydia trachomatis    
       1. Materials 
       [0000]    
       
         
           
             1)  Chlamydia  strain:  Chlamydia trachomatis  E Bour type (the 10 th  generation) 
              (Provided by the CDC of USA) 
             2) Cells: McCoy cell; 
             3) Culture medium: RPMI-1640 culture medium; 
             4) Testing sample: Microbicide gel prepared as of Example 1, formulation 5 
             5) Neutralizer: Diluting neutralize methods were employed, the diluents was 1640 medium with 10% bovine serum.
 
2. Testing Methods and Standards:          Technical Standard for Disinfection          (2002 Edition) Issued by “The Health Department of China”
 
           
         
       
     
         [0145]    The tests followed the “Standard” with consideration of the characteristics of  Chlamydia trachomatis.  
       1) Temperature: 25° C.   2) Concentration tested: 1:20; 1:50; 1:100; 1:200; 1:500   3) Concentration of  Chlamydia trachomatis  suspension: 10 5      4) Reacted time: 1 min.; 3 min.; 5 min.; 10 min.   5) Diluting methods were used to remove the residue of sample.       
 
       3. Results 
       [0151]    Killing Effects of Testing sample to  Chlamydia trachomatis  (Table 7) 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 The Killing Effects of Testing sample on  Chlamydia trachomatis   
               
             
          
           
               
                   
                 time 
                 Dilution of Testing sample 
                   
               
             
          
           
               
                 strain 
                 (min.) 
                 1:20 
                 1:50 
                 1:100 
                 1:200 
                 1:500 
                 control 
               
               
                   
               
             
          
           
               
                 E 
                 1 
                 − 
                 − 
                 + 
                 + 
                 + 
                 + 
               
               
                 Bour 
                 3 
                 − 
                 − 
                 − 
                 + 
                 + 
                 + 
               
               
                 strain 
                 5 
                 − 
                 − 
                 − 
                 + 
                 + 
                 + 
               
               
                   
                 10 
                 − 
                 − 
                 − 
                 − 
                 + 
                 + 
               
               
                   
               
               
                 Note: 
               
               
                 Control:  Chlamydia trachomatis  growth observed; 
               
               
                 +: there are  Chlamydia trachomatis  inclusion in the cell; 
               
               
                 −: there was no  Chlamydia trachomatis  inclusion in the cell 
               
             
          
         
       
     
       4. Conclusion 
       [0152]    Testing sample diluted at 1:50 (example 1, formulation 9) and reacted with  Chlamydia trachomatis  suspension for 1 minute killed  Chlamydia trachomatis.    
       EXAMPLE 13  
     The Effects of the Microbicide on  Ureaplasma Urealyticum    
     1. Materials 
       [0153]    Testing strain:  Ureaplasma Urealyticum : international standard strain; used 6 th  generation 
         [0154]    Culture medium: High efficiency  Ureaplasma  culture medium; 
         [0155]    Testing sample: Microbicide gel prepared as of Example 1, formulation 5. 
         [0000]    2. Testing Methods and Standards:          Technical Standard for Disinfection          (2002 Edition) Issued by “The Health Department of China” 
         [0156]    The tests followed the “Standard” with consideration of the characteristics of  Ureaplasma Urealyticum  
       1) Testing Temperature: 25° C.˜28° C.   2) Concentration of sample dilution: 1:100; 1:200; 1:500; 1:1000   3) Concentration of  Ureaplasma Urealyticum:  10 7      4) Treatment:  Ureaplasma Urealyticum  suspension:diluted sample=1:9   5) Reacted time: 1 min.; 3 min.; 5 min.; 10 min.   6) Diluting methods were used to remove the residue of drug   7) Tests repeated for two times       
 
       3. Results 
       [0164]    The Killing Effect of Microbicide gel to  Ureaplasma Urealyticum  (Table 8) 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 The Effects of Testing sample on  Ureaplasma Urealyticum   
               
             
          
           
               
                   
                 time 
                   
                 Dilutions of Testing Sample 
                   
               
             
          
           
               
                   
                 (min.) 
                 1:100 
                 1:200 
                 1:500 
                 1:1000 
                 control 
               
               
                   
                   
               
             
          
           
               
                   
                 1 
                 − 
                 − 
                 + 
                 + 
                 + 
               
               
                   
                 3 
                 − 
                 − 
                 + 
                 + 
                 + 
               
               
                   
                 5 
                 − 
                 − 
                 + 
                 + 
                 + 
               
               
                   
                 10 
                 − 
                 − 
                 + 
                 + 
                 + 
               
               
                   
                   
               
               
                   
                 Note: 
               
               
                   
                 Control:  Ureaplasma Urealyticum  growth observed; 
               
               
                   
                 +:  Ureaplasma Urealyticum  growth observed; 
               
               
                   
                 −: No  Ureaplasma Urealyticum  growth observed. 
               
             
          
         
       
     
       4. Conclusion 
       [0165]    The testing results showed: Testing sample diluted for 200 times (Example 1, formulation 4) and reacted with  Ureaplasma Urealyticum  suspension for I minute, killed  Ureaplasma Urealyticum.    
       EXAMPLE 14  
       [0166]    The Effects of the Microbicide on  Neisseria gonorrhoeae    
       1. Materials 
       [0000]    
       
         
           
             1) Testing strain: Standard strain G, provided by WHO 
             2) Culture medium: 10% blood dish; 
             3) Testing sample: Microbicide gel prepared as of Example 1, formulation 5.
 
2. Testing Methods and Standards:          Technical Standard for Disinfection          (2002 Edition) Issued by “The Health Department of China”
 
           
         
       
     
         [0170]    The tests followed the “Standard” with consideration of the characteristics of  Neisseria gonorrhoeae  
       1) Temperature: 25° C.˜28° C.   2) Concentration of sample prepared: 1:25; 1:50; 1:100; 1:250; 1:500   3)  Neisseria gonorrhoeae  suspension: 10 7      4) Treatment:  Neisseria gonorrhoeae  suspension:diluted sample solution=1:1   5) Treatment time: 1 min.; 3 min.; 5 min.; 10 min.   6) Residue drug were removed with diluting methods   7) Tests repeated for three times       
 
       3. Results 
       [0178]    The Killing Effect of Microbicide gel to  Ureaplasma Urealyticum  (Table 9) 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 The Killing Effects of Testing Sample to  Neisseria gonorrhoeae   
               
             
          
           
               
                   
                 React time 
                   
                 Dilutions of Testing sample 
                   
               
             
          
           
               
                   
                 (min.) 
                 1:50 
                 1:100 
                 1:200 
                 1:500 
                 control 
               
               
                   
                   
               
             
          
           
               
                   
                 1 
                 − 
                 − 
                 + 
                 + 
                 + 
               
               
                   
                 3 
                 − 
                 − 
                 + 
                 + 
                 + 
               
               
                   
                 5 
                 − 
                 − 
                 + 
                 + 
                 + 
               
               
                   
                 10 
                 − 
                 − 
                 − 
                 + 
                 + 
               
               
                   
                   
               
               
                   
                 Note: 
               
               
                   
                 +: growth of  Neisseria gonorrhoeae  observed 
               
               
                   
                 −: No growth of  Neisseria gonorrhoeae   
               
             
          
         
       
     
       4. Conclusion 
       [0179]    The testing results showed: Testing sample diluted for 100 times (example 1, formulation 8) and reacted with  Neisseria gonorrhoeae  suspension for 1 minute killed  Neisseria gonorrhoeae.    
       EXAMPLE 15  
       [0180]    The Effects of the Microbicide on  Trichomonas vaginalis    
       1. Materials 
       [0000]    
       
         
           
             1) Testing strain: Clinical isolated  Trichomonas vaginalis  strain 
             2) Culture medium: liver infusion medium; 
             3) Testing sample: Microbicide gel prepared as of Example 1, formulation 5.
 
2. Testing Methods and Standards:          Technical Standard for Disinfection          (2002 Edition) Issued by “The Health Department of China”
 
           
         
       
     
         [0184]    The tests followed the “Standard” with consideration of the characteristics of  Trichomonas vaginalis  
       1)  Trichomonas vaginalis  suspension: 10 5 /ml   2) Concentration of sample prepared: 1:50; 1:100; 1:200   3) Treatment:  Trichomonas vaginalis  suspension:diluted sample solution=1:1   4) Reaction time: 1 min.; 3 min.; 5 min.; 10 min.   5) Temperature: 20˜25° C.   6) Residue drug were removed with diluting methods   7) Tests repeated for two times       
 
       3. Results 
       [0192]      
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 The Killing Effects of Testing Sampleto  Trichomonas vaginalis   
               
             
          
           
               
                 React time 
                 Dilutions of Testing sample 
                   
               
             
          
           
               
                 (min.) 
                 1:100 
                 1:200 
                 1:400 
                 control 
               
               
                   
               
             
          
           
               
                 1 
                 − 
                 ± 
                 + 
                 + 
               
               
                 3 
                 − 
                 ± 
                 + 
                 + 
               
               
                 5 
                 − 
                 ± 
                 + 
                 + 
               
               
                 10 
                 − 
                 − 
                 + 
                 + 
               
               
                   
               
               
                 Note: 
               
               
                 +: Active  Trichomonas vaginalis   
               
               
                 ±: Most  Trichomonas vaginalis  were killed, a few active left 
               
               
                 −:  Trichomonas vaginalis  were not active and were dissolved 
               
             
          
         
       
     
       4. Conclusion 
       [0193]    The testing results showed: Testing sample diluted for 100 times (example 1, formulation 8) and reacted with  Trichomonas vaginalis  suspension for 1 minute, killed  Trichomonas vaginalis.    
       EXAMPLE 16  
     The Effects of the Microbicide on Herpes Simplex Virus 
     1. Materials 
       [0000]    
       
         
           
             1) Testing strain: Herpes Simplex type II strain. Standard international strain, provided by the Drug and bio-product office of the Health Department of China. 
             2) Cell: Vero cell strain, introduced by the Cell biology Institute of Shanghai 
             3) Culture medium: 1640 culture medium 
             4) Testing sample: Microbicide gel prepared as of Example 1, formulation 5.
 
2. Testing Methods and Standards:          Technical Standard for Disinfection          (2002 Edition) Issued by “The Health Department of China”
 
           
         
       
     
         [0198]    The tests followed the “Standard” with consideration of the characteristics of Herpes Simplex
       1) Virus suspension: 5×10 4 /ml   2) Concentration of sample prepared: 1:20; 1:40; 1:50   3) Reaction time: 1 min.; 3 min.; 5 min.; 10 min.   4) Temperature: 20-25° C.   5) Residue drug were removed with diluting methods (2% bovine serum culture medium as diluents)   6) Tests repeated for two times       
 
       3. Results 
       [0205]    The Killing Effect of Microbicide gel to Herpes Simplex Virus (Table 11) 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 The Effects of Testing Example on Herpes Simplex Virus 
               
             
          
           
               
                 React time 
                 Dilutions of Testing sample 
                   
               
             
          
           
               
                 (min.) 
                 1:20 
                 1:40 
                 1:50 
                 Control 
               
               
                   
               
             
          
           
               
                 1 
                 − 
                 ± 
                 + 
                 + 
               
               
                 3 
                 − 
                 ± 
                 + 
                 + 
               
               
                 5 
                 − 
                 ± 
                 + 
                 + 
               
               
                 10 
                 − 
                 ± 
                 + 
                 + 
               
               
                   
               
               
                 Note: 
               
               
                 +: Virus growth observed, infected cell &gt; 50% 
               
               
                 ±: Virus growth observed, infected cell &lt; 10% 
               
               
                 −: No virus growth 
               
             
          
         
       
     
       4. Conclusion 
       [0206]    The testing results showed: Testing sample diluted for 20 times (Example 1, formulation 7) and reacted with Herpes Simplex Virus. Herpes Simplex Virus was killed with 20 times dilution of the sample in 1 minute. 
       EXAMPLE 17  
       [0207]    The Effects of the Microbicide on  Staphylococcus aureus    
       1. Materials 
       [0000]    
       
         
           
             1) Testing strain:  Staphylococcus aureus  ATCC6538, 5-6 th  generations, provided by the reserve center of China microbial reservation committee. 
             2) Testing medium carrier: Sterilized filter paper 
             3) Testing sample: Microbicide gel prepared as of Example 1, formulation 5.
 
2. Testing Methods and Standards:          Technical Standard for Disinfection          (2002 Edition)
 
           
         
       
     
         [0211]    GB 15979-2002          Technical Standard for Testing Disinfection Product          “Procedures for testing bactericide effect” 
       3. Results 
       [0212]    The bactericide effect of testing sample to  Staphylococcus aureus    
         [0213]    At 20±1° C. temperature, three repeated tests showed: The testing sample reacted with  Staphylococcus aureus  for 1 minute the average bactericide rate was 95.70%. (table 12) 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                 The Bactericide Effect of Testing Sample to  Staphylococcus aureus   
               
             
          
           
               
                   
                   
                 Average 
                   
               
               
                 Time 
                 Bactericide rates of three tests 
                 bactericide 
                 Control 
               
             
          
           
               
                 (min.) 
                 First test 
                 Second test 
                 Third test 
                 rate (%) 
                 (CFU/pc.) 
               
               
                   
               
             
          
           
               
                 1 
                 92.13 
                 96.87 
                 98.10 
                 95.70 
                 1.28 × 10 4   
               
               
                 3 
                 99.64 
                 100.00 
                 99.58 
                 99.74 
                 1.25 × 10 4   
               
               
                 5 
                 99.72 
                 100.00 
                 99.95 
                 99.89 
                 1.23 × 10 4   
               
               
                   
               
             
          
         
       
     
       4. Conclusion 
       [0214]    Testing sample (Example 1, formulation 5) reacted with  Staphylococcus aureus  for 1 minute the average bactericide rate was 95.70%. 
       EXAMPLE 18  
       [0215]    The Effects of the Microbicide on  Candida albicans    
       1. Materials 
       [0000]    
       
         
           
             1) Testing strain:  Candida albicans  ATCC 10231, the 5-6 generations; Provide by the “Reserve Center of Chinese Microbial Reservation Committee”. 
             2) Testing sample: Microbicide gel prepared as of Example 1, formulation 5.
 
2. Testing Methods and Standards:          Technical Standard for Disinfection          (2002 Edition) “Procedures for testing bactericide effect” GB15979-2002          Technical Standard for Testing Disinfection Product         
 
           
         
       
     
         [0218]    Temperature: 20±1° C. 
       3. Results 
       [0219]    The bactericide effect of Testing sample to  Candida albicans  ATCC10231 At 20±1° C. temperature, three repeated tests showed: Testing material reacted with  Candida albicans  ATCC10231 for 1 minutes, the average bactericide rate was 95.47%. (Table 13) 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 13 
               
             
             
               
                   
               
               
                 The bactericide effect of Testing Sample to  Candida albicans   
               
             
          
           
               
                   
                   
                 Average 
                   
               
               
                 Time 
                 Bactericide rates of three tests 
                 bactericide 
                 Control 
               
             
          
           
               
                 (min.) 
                 First test 
                 Second test 
                 Third test 
                 rate (%) 
                 (CFU/pc.) 
               
               
                   
               
               
                 1 
                 97.95 
                 93.81 
                 94.65 
                 95.47 
                 3.07 × 10 4   
               
               
                 3 
                 99.66 
                 99.96 
                 99.30 
                 99.53 
                 2.99 × 10 4   
               
               
                 5 
                 99.91 
                 99.88 
                 99.75 
                 99.84 
                 2.96 × 10 4   
               
               
                   
               
             
          
         
       
     
       4. Conclusion 
       [0220]    Testing sample reacted with  Candida albicans  for 1 minutes, the average bactericide rate was 95.47%. The Microbicidal Gel has bactericide effect to  Candida albicans.    
       EXAMPLE 19  
       [0221]    The Effects of the Microbicide on  Eschetichia coli    
       1. Materials 
       [0000]    
       
         
           
             1) Testing strain:  Eschetichia coli  8099, the 5-6 generations; provided by the “Reserve Center of Chinese Microbial Reservation Committee”. 
             2) Testing sample: Microbicide gel prepared as of Example 1, formulation 5.
 
2. Testing Methods and Standards:          Technical Standard for Disinfection          (2002 Edition) “Procedures for testing bactericide effect” GB15979-2002          Technical Standard for Testing Disinfection Product         
 
           
         
       
     
       3. Results 
       [0224]    The bactericide effect of Testing sample to  Eschetichia coli:    
         [0225]    At 20±1° C. temperature, three repeated tests showed: the sample gel reacted with  Eschetichia coli  for 1 minutes, the average bactericide rate was 99.97%. (Table. 14) 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                 The Bactericide Effect of Testing Sample to  Eschetichia coli   
               
             
          
           
               
                   
                   
                 Average 
                   
               
               
                 Time 
                 Bactericide rates of three tests 
                 bactericide 
                 Control 
               
             
          
           
               
                 (min.) 
                 First test 
                 Second test 
                 Third test 
                 rate (%) 
                 (CFU/pc.) 
               
               
                   
               
             
          
           
               
                 1 
                 99.96 
                 100.00 
                 99.95 
                 99.97 
                 1.30 × 10 4   
               
               
                 3 
                 100.00 
                 100.00 
                 100.00 
                 100.00 
                 1.22 × 10 4   
               
               
                 5 
                 100.00 
                 100.00 
                 100.00 
                 100.00 
                 1.20 × 10 4   
               
               
                   
               
             
          
         
       
     
       4. Conclusion 
       [0226]    Testing sample reacted with  Eschetichia coli  for 1 minutes, the average bactericide rate was 99.97%. 
       EXAMPLE 20  
       [0227]    Spermicidal Effects of the Microbicide Solution (in vitro). 
       1. Materials 
       [0000]    
       
         
           
             1) Human sperm: Normal sperms obtained from three health Adults Testing 
             2) Sample: Microbicide solution prepared as of Example 1, formulation 37. 
           
         
       
     
       2. Methods 
       [0000]    
       
         
           
             1) Testing sample was tested undiluted (1:1), diluted 5 times (1:5) and 10 times (1:10). Mix the prepared samples with equal volume of fresh sperm (0.5 ml sample and 0.5 ml sperm) 
             2) Determine the minimum concentration and time of demobilize all the sperms 
             3) Use saline as negative control. 
             4) Repeat the tests for three times. 
           
         
       
     
       3. Results 
       [0234]    The undiluted solution reacted with sperms for 30 seconds. The survival rate of sperms was zero. Table 15, 16 and 17: 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 15 
               
               
                   
               
               
                 Testing Results on Sperm Sample 1: 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 sample 
               
               
                   
                 concentration 
               
             
          
           
               
                   
                 Time 
                   
                 1:1 
                 1:5 
                 1:10 
               
               
                   
                   
               
               
                   
                 30 sec 
                 Sperm count (million/ml) 
                 23 
                 23 
                 23 
               
               
                   
                   
                 Active rate (%) 
                 0 
                 50 
                 60 
               
               
                   
                  1 min 
                 Sperm count (million/ml) 
                 23 
                 22 
                 22 
               
               
                   
                   
                 Active rate (%) 
                 0 
                 45 
                 45 
               
               
                   
                  2 min 
                 Sperm count (million/ml) 
                 22 
                 22 
                 22 
               
               
                   
                   
                 Active rate (%) 
                 0 
                 42 
                 45 
               
               
                   
                  3 min 
                 Sperm count (million/ml) 
                 23 
                 23 
                 23 
               
               
                   
                   
                 Active rate (%) 
                 0 
                 40 
                 40 
               
               
                   
                   
               
             
          
           
               
                   
                   
                 Before test 
                 Control/saline 
               
               
                   
                   
               
               
                   
                 Sperm count (million/ml) 
                 45 
                 23 
               
               
                   
                 Active rate (%) 
                 82 
                 72 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 16 
               
               
                   
               
               
                 Testing Results on Sperm Sample 2: 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 sample 
               
               
                   
                 concentration 
               
             
          
           
               
                   
                 Time 
                   
                 1:1 
                 1:5 
                 1:10 
               
               
                   
                   
               
               
                   
                 30 sec 
                 Sperm count (million/ml) 
                 13.5 
                 13.5 
                 13.5 
               
               
                   
                   
                 Active rate (%) 
                 0 
                 50 
                 50 
               
               
                   
                  1 min 
                 Sperm count (million/ml) 
                 13.5 
                 13.4 
                 13.4 
               
               
                   
                   
                 Active rate (%) 
                 0 
                 50 
                 50 
               
               
                   
                  2 min 
                 Sperm count (million/ml) 
                 13.3 
                 13.4 
                 13.5 
               
               
                   
                   
                 Active rate (%) 
                 0 
                 45 
                 50 
               
               
                   
                  3 min 
                 Sperm count (million/ml) 
                 13.2 
                 13.2 
                 13.3 
               
               
                   
                   
                 Active rate (%) 
                 0 
                 40 
                 50 
               
               
                   
                   
               
             
          
           
               
                   
                   
                 Before test 
                 Control/saline 
               
               
                   
                   
               
               
                   
                 Sperm count (million/ml) 
                 25 
                 13.5 
               
               
                   
                 Active rate (%) 
                 62 
                 62 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 17 
               
               
                   
               
               
                 Testing Results on Sperm Sample 3 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 sample 
               
               
                   
                 concentration 
               
             
          
           
               
                   
                 Time 
                   
                 1:1 
                 1:5 
                 1:10 
               
               
                   
                   
               
               
                   
                 30 sec 
                 Sperm count (million/ml) 
                 29.7 
                 29.5 
                 29.4 
               
               
                   
                   
                 Active rate (%) 
                 0 
                 60 
                 61 
               
               
                   
                  1 min 
                 Sperm count (million/ml) 
                 29.6 
                 29.0 
                 29.0 
               
               
                   
                   
                 Active rate (%) 
                 0 
                 58 
                 59 
               
               
                   
                  2 min 
                 Sperm count (million/ml) 
                 29.0 
                 29.0 
                 29.0 
               
               
                   
                   
                 Active rate (%) 
                 0 
                 58 
                 58 
               
               
                   
                  3 min 
                 Sperm count (million/ml) 
                 28.5 
                 28.5 
                 28.5 
               
               
                   
                   
                 Active rate (%) 
                 0 
                 58 
                 58 
               
               
                   
                   
               
             
          
           
               
                   
                   
                 Before test 
                 Control/saline 
               
               
                   
                   
               
               
                   
                 Sperm count (million/ml) 
                 58 
                 30 
               
               
                   
                 Active rate (%) 
                 75 
                 73 
               
               
                   
                   
               
             
          
         
       
     
       4. Test Results 
       [0235]    The undiluted solution reacted with equal volume sperm for 30 sec, 1 min, 2 min, 3 min. The sperm survive rate was zero. The spermicidal rate of testing microbicide solution was 100% in 30 seconds (in vitro). Saline reacted with equal volume sperm, the sperm count and active rate was not influenced. 
       5. Conclusion 
       [0236]    The spermicidal rate of testing sample was 100% in 30 seconds (in vitro). 
       EXAMPLE 21  
       [0237]    Spermicidal Effects of the Microbicide Gel (in vitro) 
       1. Materials 
       [0000]    
       
         
           
             1) Human sperm: Normal sperms obtained from three health Adults 
             2) Testing sample: Microbicide gel prepared as of Example 1, formulation 5. 
           
         
       
     
       2. Methods 
       [0000]    
       
         
           
             1) Dilute the Testing sample to different dilute concentrations (1:10, 1:5, 1:1) 
             2) Mix the diluted samples with equal volume fresh sperm (0.5 ml sample and 0.5 ml sperm) 
             3) Determine the minimum concentration and time of demobilize all the sperms 
             4) Use saline as negative control 
             5) Repeat the tests for three times 
           
         
       
     
       3. Results 
       [0245]    The undiluted gel reacted with sperms for 30 seconds, the survive rate of sperms was zero; 
         [0246]    The gel diluted at 1:1, 1:5 and reacted with equal volume sperm for 30 sec., 1 min., 2 min. and 3 min. The sperm survive rate was zero; 
         [0247]    The gel diluted at 1:10 and reacted with equal volume sperm for 30 sec. The sperm survive rate was 10%; 
         [0248]    The 1:10 gel dilution reacted with sperm for 1 min., 2 min. and 3 min. respectively the sperm survive rate was zero. 
         [0249]    The 1:10 dilution deactivated all the sperms for 60 seconds. The survive test showed the sperm survive rate was zero. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
             
           
               
                 TABLE 18 
               
             
             
               
                   
               
               
                 Testing Results on Sperm Sample 1: 
               
             
          
           
               
                   
                 sample dilution 
               
             
          
           
               
                 Time 
                   
                 gel 
                 1:1 
                 1:5 
                 1:10 
               
               
                   
               
             
          
           
               
                 30 
                 sec 
                 Sperm count (million/ml) 
                 12.3 
                 11.4 
                 11.0 
                 10.1 
               
               
                   
                   
                 Active rate (%) 
                 0 
                 0 
                 0 
                 10 
               
               
                 1 
                 min 
                 Sperm count (million/ml) 
                 — 
                 10.0 
                 9.8 
                 9.7 
               
               
                   
                   
                 Active rate (%) 
                 — 
                 0 
                 0 
                 0 
               
               
                 2 
                 min 
                 Sperm count (million/ml) 
                 — 
                 9.6 
                 9.8 
                 9.5 
               
               
                   
                   
                 Active rate (%) 
                 — 
                 0 
                 0 
                 0 
               
               
                 3 
                 min 
                 Sperm count (million/ml) 
                 — 
                 9.5 
                 9.4 
                 9.5 
               
               
                   
                   
                 Active rate (%) 
                 — 
                 0 
                 0 
                 0 
               
               
                   
               
             
          
           
               
                   
                 Before test 
                 Control/saline 
               
               
                   
               
               
                 Sperm count (million/ml) 
                 20.1 
                 12.5 
               
               
                 Active rate (%) 
                 65 
                 64.5 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
             
           
               
                 TABLE 19 
               
             
             
               
                   
               
               
                 Testing Results on Sperm Sample 2: 
               
             
          
           
               
                   
                 Sample concentration 
               
             
          
           
               
                 Time 
                   
                 gel 
                 1:1 
                 1:5 
                 1:10 
               
               
                   
               
             
          
           
               
                 30 
                 sec 
                 Sperm count (million/ml) 
                 20.0 
                 19.2 
                 19.0 
                 18.9 
               
               
                   
                   
                 Active rate (%) 
                 0 
                 0 
                 0 
                 5 
               
               
                 1 
                 min 
                 Sperm count (million/ml) 
                 — 
                 18.4 
                 19.0 
                 18.7 
               
               
                   
                   
                 Active rate (%) 
                 — 
                 0 
                 0 
                 0 
               
               
                 2 
                 min 
                 Sperm count (million/ml) 
                 — 
                 18.6 
                 18.6 
                 18.5 
               
               
                   
                   
                 Active rate (%) 
                 — 
                 0 
                 0 
                 0 
               
               
                 3 
                 min 
                 Sperm count (million/ml) 
                 — 
                 18.0 
                 18.0 
                 17.9 
               
               
                   
                   
                 Active rate (%) 
                 — 
                 0 
                 0 
                 0 
               
               
                   
               
             
          
           
               
                   
                 Before test 
                 Control/saline 
               
               
                   
               
               
                 Sperm count (million/ml) 
                 36.3 
                 20.9 
               
               
                 Active rate (%) 
                 70 
                 69 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
             
           
               
                 TABLE 20 
               
             
             
               
                   
               
               
                 Testing Results on Sperm Sample 3: 
               
             
          
           
               
                   
                 Sample concentration 
               
             
          
           
               
                 Time 
                   
                 gel 
                 1:1 
                 1:5 
                 1:10 
               
               
                   
               
             
          
           
               
                 30 
                 sec 
                 Sperm count (million/ml) 
                 14.3 
                 14.2 
                 14.2 
                 14.0 
               
               
                   
                   
                 Active rate (%) 
                 0 
                 0 
                 0 
                 15 
               
               
                 1 
                 min 
                 Sperm count (million/ml) 
                 — 
                 13.8 
                 13.7 
                 14.0 
               
               
                   
                   
                 Active rate (%) 
                 — 
                 0 
                 0 
                 0 
               
               
                 2 
                 min 
                 Sperm count (million/ml) 
                 — 
                 13.6 
                 13.5 
                 13.3 
               
               
                   
                   
                 Active rate (%) 
                 — 
                 0 
                 0 
                 0 
               
               
                 3 
                 min 
                 Sperm count (million/ml) 
                 — 
                 13.2 
                 13.1 
                 13.1 
               
               
                   
                   
                 Active rate (%) 
                 — 
                 0 
                 0 
                 0 
               
               
                   
               
             
          
           
               
                   
                 Before test 
                 Control/saline 
               
               
                   
               
               
                 Sperm count (million/ml) 
                 23.2 
                 15.1 
               
               
                 Active rate (%) 
                 75 
                 75 
               
               
                   
               
             
          
         
       
     
       4. Conclusion 
       [0250]    The spermicidal rate of testing microbicide gel was 100% in 30 seconds (in vitro), the minimum effective dilution was 1:5. 
         [0251]    The spermicidal rate of testing microbicidal gel was 100% in 60 seconds (in vitro), the minimum effective dilution was 1:10.