| To get Chinese herbs and components which inhibit Malassezia species effectively, the studies on in vitro anti- Malassezia activity and 3 components efficacy against experimental cutaneous Malassezia infection in guinea pigs were determined.Firstly,in order to identify the seven currently recognized Malassezia species,a system based on PCR and restriction endonuclease analysis(PCR-REA) were used. 7 kinds of Malassezia reference strains and 28 isolates were examined. A single primer pair was designed to amplify the large subunit ribosomal RNA(LSU rRNA) gene of the 7 Malassezia species,and identification was achieved by digestion of the PCR products with 3 restriction endonucleases: BanI, HaeII and MspI. A specific restriction endonuclease analysis pattern was determined for each species investigated. We have got 5 Malassezia species from 28 isolates that were 12 M.furfur(42.9%), 6 M.globosa(21.4%), 6 M.restricta(21.4%), 3 M.sympodialis (10.7%) and 1 M.slooffiae (3.6%), respectively. Among 15 isolates of pityriasis versicolor, 8M.furfur(53.3%), 4M.globosa(26.7%), 2 M.sympodialis (13.3%) and 1 M.restricta(6.7%) were identified. 5M.restricta(55.6%), 2M.globosa(22.2%), 1M.furfur(11.1%), 1M.slooffiae (11.1%) were found from 9 isolates of dandruff. It concluded that PCR-REA of only the LSU rRNA gene is a reliable and rapid method to identify all of Malassezia species.The susceptibility testings of Malassezia species in vitro were studied by NCCLS M27-A microdilution method. Cymbopogon citratus, Cnidium monnieri have strong anti- Malassezia activity(MIC31.25～62.5mg/L)from 23 kinds of Chinese herbs. Matrine, oxymatrine, citral, eugenol have stronger anti-Malassezia activity from 14 kinds of Chinese herbal components(MIC0.98～7.81mg/L). Matrine ,citral and eugenol were selected in the further fungistatic and fungicidal activity testings.It has shown that M.globosa was the species most susceptible to 3 components as mentioned above,especially to citral and eugenol(MIC 0.195mg/L ).The MFCsof matrine and eugenol were several gradients higher for M.restricta and M.globosa , respectively, while others were equivalent or 1 gradient apart between MFC and MIC. To predict the anti-Malassezia activity of the 3 components in infected skin tissues,we investigated the effect of serum as a keratinic substance on the in vitro activity of them. 10% human serum-containing Malassezia broth medium was used. The genomic MIC of 3 components to Malassezia species referance strains were taken. The reduced activity of citral was 1.3 which was lower than other 2 components and had a highest affinity to keratin.In an attempt to develop an animal model of cutaneous Malassezia infection useful for the pre-clinical evaluation of anti-Malassezia drugs,experimental cutaneous Malassezia infection were achieved in guinea pigs by 3 methods(inoculum applied once time,inoculum applied once time with 3 subcutaneous injections of methyl-prednisolone,inoculum applied daily for 7 consecutive days). Skin lesions,positive percentage of fungal culturing and CFU number were evaluated at 4th,7th,12th days after the final inoculum. It demonstrated that the 3rd method appling test(infection induced daily for 7 consecutive days) was suitable for the animal model which got higher lesion scores and CFU number. CFU number was useful for studies on the animal model of cutaneous Malassezia infection.The testing of 3 components efficacy against experimental cutaneous Malassezia infection in guinea pigs was performed by self-controlled method.It comprised of 3 testing groups (3 components), 1 positive group (ketoconazole) and 2 negative groups(vehicle and untreated control groups). The animal model was produced by appling inoculum daily for 7 consecutive days. Matrine, citral, eugenol were made into 2% cream which were applied topically once daily for 4 or 7 consecutive days from 24 hours after stopping infective testing. The therapeutic efficacy was evaluated microbiologically as described above by calculating CFU number of skin lesions at 24 hours aft...
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