Study On Anti-fungal Activity For Skin Pathogenic Fungi Of The Extract From Plant Amedica Jatropha Curcas L.

Fungi has wide varieties of genus,recorded more than100,000 kinds of them. However,it is pathogenic to humans about 400 kinds.According to encroach on different tissues and organs,clinically,divide them into superficial and deep fungi.Superficial mycosis,usually called dermatomycosis is a group of contagiosity diseases caused by superficial fungi infecting epidermisor appendages of skin. China from north to south across the temperate,subtropical and tropical zone. Superficial fungal pathogen species have certain change.However,The overall picture is Trichophyton rubrum dominated in our country.This kind of disease is mainly caused by dermatophytes such as Trichophyton,Epidermophyton,Microsporum. When they encroach on proteinum of cornified epidermis,pelage,fingernail or toenail,the dermatophytes multiply heavily on topical skin,and then cause topical inflammatory reaction and pathological changes of skin due to mechanical stimulation and metabolite.Forceful scratch or daubing intensively irritant drug always impair topical organism and lead to secondary infection caused by bacterium. The better situation is local diapyetic.However,the worse one is that secondary infection inducing necrotic organism can induce lymphangitis,lymphadenitis,abscess, or septicemia.Moreover,these superficial dermatomycosis is characterized by stubborn,easiness to relapse,long treatment cycle and infectiousness.The commonly used clinical antifungal drugs and some new kinds of antifungal agents,such as azoles,propylene amines,morpholines,have certain curative effect for these superficial dermatomycosis,but can’t get satisfactory results in the aspects of high-performance and low toxicity,drug resistance which makes the drug-resistant strains increasing.Therefore,the treatment of fungal infections is still facing many challenges.Especially in recent years,due to widely used broad-spectrum antibioticand,immunosuppressive agents,as the rising immunodeficiency disease ratio and any other factors,it shows the upward trend in the incidence of superficial mycosis.Therefore,searching and development of new antifungal drug with high-performance and low toxicity have been one of focus areas of medicine research all over the world.Traditional Chinese medicines has attracted increasing attention in the study of antifungal agents because of its advantage of safety and fewer side effects.Many researchers found that Jatropha curcas L.,a common used Traditional Chinese medicines recorded in the "Chinese Materia Medica",belonging to one of the traditional medicinal plants has a great commercial value for development.According to folk practic,it can be used for the treatment of scabies, ringworm and athlete’s foot with satisfactory effect.Domestic and foreign scholars have done extensive researches on the chemical compositions and pharmacological activities of Jatropha curcas,and the results indicate that Jatropha curcas L shows excellent antifungal activity and possess a potential wide range of medicinal values. Take some plant pathogenic fungi as test objects,such as Pyriculariaoryzae Cav. Pestalotia funerea,Rhizoctonia solani kuha Sclerotinia sclerotiorum(Lib) de Bary.Indoor activity test results show curcin that is extracted and separated from Jatropha curcas L.can significantly inhibit the growth of mycelial and production of spores when the concentration reaches 5mg/mL.As the concentration improved to 50mg/mL,Pyriculariaoryzae Cav.can’t generate spores and the inhibition ratio for spores formation of Pestalotia funereal reaches 83.3%.In addition,the n-hexane, ethyl acetate,chloroform and methanol extracts exhibit strong antibacterial activity against Bacillus subtilis,Staphylococcus aureus,Staphylococcus epidermidis and Sarcina lutea.Moreover,the inhibitory concentration is less than 1.5mg/mL. Furthermore,Jatropha curcas L.is large area planted and wildly distributed in our country.Therefore,it can provides enough resource for deep development of the medicinal value of Jatropha curcas L.Based on the past antifungal researches on the antifungal activity of Jatropha curcas L.and current require of the treatment of superficial dermatomycosis,this project study systematically the antifungal activity against frequent skin pathogenic fungi.using the extracts from the leaf of Jatropha curcas L.Objectives1.Compare the antifungal activity of three types of extracts from the leaf of Jatropha curcas L using different solvents(methanol,petroleum ether and purified water).Select the extract with the best antifungal activity.2.Carry out sensitivity test against common skin pathogenic fungi for the selected extract from the leaf of Jatropha curcas L.Carry out a quantitative study for the antifungal activity to find out the most sensitive kind of common skin pathogenic fungi.3.Carry out animal experiments with selected extract and strain to comprehensively evaluate the antifungal activity of the extract of Jatropha curcas L..Methods1.Transfer the strains of standard and clinically isolated Trichophyton rubrum, Trichophyton mentagrophytes,Microsporum gypseum onto Sadouraud solid medium twice,then choose the growth of colonies to get bacteria suspension with 0.5MacFarland standard turbidity followed by the dilution of the obtained bacteria suspension with the liquid medium RPMI1640 by 1:10 mixture ratio.Carry out the bacteriostatic test in vitro,set up three types of solvent extracts as the drug group, three types of solvent as solvent group and blank control group,and each group contains 10 petri dishes.Drug groups and solvent groups are inoculated with the aformentioned bacteria suspensions 100μl respectively,and set into constant temperature incubators at 27℃after blending.Observe the formation of colonies every day and keep observation for two weeks.Then compare the number of colonies generated to find the antibacterial activities of different extracts.2..According to NCCLS disk diffusion method and constant broth dilution method (M38-P) recommended by the United States Commission on Clinical Laboratory Standards(NCCLS) and newly improved MTT method,carry out drug susceptibility test for selected extracts and a quantitative study for the antifungal activity.3.Use trichophyton rubrum to form animal models of guinea pigs.After successful modeling,treat them with the selected extract in three ways,high,middle and low dosages respectively,and than evaluate the antifungal activity for methanol extract from Jatropha curcas L.by comparison of the result with the positive group,healthy group and negative control group.4.Statistical Analysis:Employ the spss13.0 statistical software for data processing. Comparison the OD values of absorbance of each strain at different drug concentrations.Using completely randomized design analysis of variance(One—Way ANOVA).Homogeneity of variance,Between the concentration of different drugs compared using LSD method,if it is not satisfied homogeneity of variance,using Dunnett T3 method.During zoology anti-fungal infection experiments,pre-treatment and treatment of one week and two weeks later,compare each group of guinea pig model of tinea corporis of the level of lesions score,using repeated measure analysis of variance.As P＜0.05,while for further comparison between groups,time points using the LSD(homogeneity of variance) or Dunnett’sT3 method(heterogeneity of variance).After treatment for one week and two weeks in each group,the comparison of the basis of clinical symptoms to determine the efficacy evaluation and check negative ones based on mycology,both use K-independent samples of non-parametric test((Kruskal-Wallis H).If P＜0.05,use two independent samples of non-parametric test(Mann-Whitbey U) between each treatment group.The comparison of differences from fungi tested between two ways of Sabouraud culture and direct microscopic examination,use two independent samples of non-parametric test(Mann-Whitbey U),too.The comparison of each group between the cure rates use K-independent samples of non-parametric test.Results1.Through solid medium bacteriostatic test in vitro,record the colonies of all standard and clinical isolated strains of skin pathogenic fungi under the action of the three solvents extracts.It is methanol extract from Jatropha curcas L.leaves having the strongest inhibition for skin pathogenic fungi by the statistics.The inhibition strength is methanol extract from Jatropha curcas L.leaves stronger than petroleum ether extract and distilled water extract.2.According to NCCLS recommend a constant dilution method(M38-P) and the modified MTT method,detect the minimum inhibitory concentration of methanol extracts Jatropha curcas L.leaves for each strain.The concentration of the extracts is 2.5mg/ml,then serial dilution by two proportion,the concentrations are 1250.00mg/mL、625.00mg/mL、312.50mg/mL、156.25mg/mL、78.13mg/mL、39.06mg/mL、19.53mg/mL、9.77mg/mL、4.88mg/mL respectively.Constant dilution method measuring the scopes of MICs of the strains are 9.77～39.06mg/mL for Trichophyton rubrum,39.06～78.13mg/mL for Trichophyton mentagrophytes and 39.06～78.13mg/mL for Microsporum gypseum.Modified MTT method measuring the scopes of the MICs for all the strains are 39.06～156.25mg/mL for Trichophyton rubrum,156.25mg/mL for Trichophyton mentagrophytes and 78.13～156.25mg/mL for Microsporum gypseum.Through the comparison of minimum inhibitory concentration(MICs),Trichophyton rubrum is the most sensitive for methanol extracts from Jatropha curcas L.leaves.3.After the success of forming animal models of guinea pigs,it is no significant difference for score of skin lesion of guinea pigs with the ketoconazole positive control group After the treatment of two weeks,by sabouraud cultivate and direct microscopy methods for mycology test,there is negative number for high middle low doses of methanol extract from Jatropha curcas L.Moreover,with the increased dose and increased the number of negative,especially,it is no significant difference between high dose group and the ketoconazole group.Through the treatment of methanol extract from Jatropha curcas L.and ketoconazole,Observe the pathological section of the skin with integrity of the epithelial structure and clear staining of dermal nuclei.However,Negative control group and methanol solvent group with serious damage of epithelial structure and swoleen dermis nuclei and severe interstitial edema.Conclusions1.Among the extracts of Jatropha curcas L.leaves.,methanol extracts has the strongest antibacterial activity.2.In the common dermatophytes,Trichophyton rubrum is the most sensitive for the methanol extract from Jatropha curcas L.leaves.3.Through animal experiments,further confirmed that methanol extract from Jatropha curcas L.leaves has a strong inhibitory effect for Trichophyton rubrum.