The Drug Sensitivity Test Of Dermatophyte To Litsea Cubeba Oil And Clinical Experiment

Objective: clinically, superficial mycosis is a kind ofcommon and frequently-occurring disease which is infected byDermatophyte. It is usually treated with antifungal drug.However the drug resistance of fungi is continuously arising andthe high relapse rate puzzle sufferers and medical workers. Itimpels scientific research workers continuously to look for thenew broad spectrum, high performance and cheap antifungaldrugs. In the past few years,The antifungal drug of thetraditional Chinese medicine were made great progress,andmany antifungal drug of the traditional Chinese medicine wereselected out,of which Litsea cubeba oil is one kind.This test was consulted USA National Committee forClinical Laboratory Standards (NCCLS)Recommend Methodfor broth dilution antifungal susceptibility testing of yeast todetermine the minimum inhibitory concentration (MIC) of Liseacubeaba oil about four kinds of Dermatophyte. We observed theultrastructural changes of Trichophyton mentagrophytes beforeand after test through electron microscope. Moreover, we madeLitsea cubeba oil be cream, and we used the cream to treat tineaof feet and hands,observing curative effect. We used grinders togrind hyphae and counted them by blood cell counting plate inour test. So we made sure of concentration of the fungisuspension and resolved a tough problem that the amount tohave an inoculation is difficult to control. All had made theresults be scientific and believable.Methods: We separated Trichophyton rubrumEpidermophyton floccosum Trichophyton mentagrophytesMicrosporum canis from the lesions. Identify them once more.Then they were cultured in Sabouraud`s dextrose agar(SDA),cultured 10 days at 27℃. At last, we brought out hyphae andgrinded them.We counted hyphae through blood cell counting plate andadjusted the concentration of fungi suspension between 1×106cfu/ml and 5×106 cfu/ml. Litsea cubeba oil was diluted byRPMI-1640 liquid medium to be concentration from80000ug/ml to156ug/ml. Fluconazol was diluted by RPMI-1640liquid medium to be concentration from 128ug/ml to 0.25ug/ml.Get 100ul medicine liquid to the hole of 1~10 file of 96 holeplate in turn. Every concentration medicine liquid was added toin turn. Replace tip after each concentration over. The hole of11st file is the growth comparison,and there was only 100ulRPMI-1640 liquid medium, no antifungus medicines. Get 200ulRPMI-1640 liquid medium to add to the hole of 12nd file asnegative control. Add fungi suspension to the hole of 1~11 filesof 96 hole plate. Be wetting and not wavering, cultured 10 daysat 27℃. Observe the result after 96 hours. In order to determinethe medicine inhibit or kill the fungi, we got liquid mediumcontaining fungi suspension of MIC in test to SDA, culturedthem for 21 days at 27℃, Observed whether the fungi growagain or not. The fungi will grow again, if the medicine is ainhibiter, otherwise it is a fungicide. Trichophytonmentagrophytes were soaked in MIC's concentration Litseacubeba oil for 24 hours. And the control strain was normalTrichophyton mentagrophytes. All of them were fixed byglutaric dialdehyde before sent the electron microscopedepartment. Preparation: add 35ml Litsea cubeba oil to 1000gcream,agitateg for 2 hours,distributed and boxed it. 26 patientswith superficial mycosis: 24 cases suffered from tinea pedis, 2cases suffered from tinea corporis. All cases had representativelesions, and the fungi were examined through microscope.Methods: 3%Litsea cubeba cream,External application, Per day2 times, Re-examine after 14 days,Write down subjectivesymptoms and lesion changes, examination of fungi and sideeffect. Fully recover: Symptom disappear, lesion disappeare,The examinarion of fungi is negative. Improve: Symptomdisappear, lesions partly disappeare,The scales decreases or theblister wither,The examination of fungi is negative. Ineffective:Symptom and lesion continue,and the fungi can be examined.Results: 1 The drug sensitivity test of Dermatophyte toLitsea cubeba oil: Trichophyton rubrum, Epidermophytonfloccosum, Trichophyton mentagrophytes and Microsporumcanis were all susceptible to Litsea cubeba oil, their MIC rangfrom 312.5 ug/ml~156.25ug/ml. Subcultivation experiment...