Development Of Plant-Derived Antidandruff Agent And Study On The Mechanism Of Malassezia Inhibition

Dandruff is a large amount of desquamation caused by excessive proliferation of keratinocytes in the scalp.Dandruff has become a common problem and is difficult to eradicate.The main cause of dandruff is the excessive proliferation of Malassezia.Malassezia metabolizes lipids in sebum to produce unsaturated fatty acids,which triggers an inflammatory response.Traditional anti-dandruff agents have problems such as side effects,drug resistance,and environmental pollution,while plant-derived anti-dandruff ingredients have small side effects,wide sources,low prices,and rarely have drug resistance.They are suitable for long-term and preventive applications.So research and development of plant-derived anti-dandruff products have good prospects.In this study,18 plants were selected from the literature,and the extracts were obtained by two extraction methods,water extraction and alcohol extraction,and screening was performed by Oxford Cup Diffusion drug sensitivity test.It was found that clove had the best activity against Malassezia.GC-MS analysis was performed on the clove extract,and the five ingredients with the highest content were tested for drug sensitivity.The minimum inhibitory concentration（MIC）of eugenol was found to be 312.5μg/m L.In order to investigate whether other components would interact with eugenol,eugenol in the extract was quantitatively analyzed using Ultra Performance Liquid Chromatography（UPLC）,and the same eugenol concentration was guaranteed to compare the antifungal ability of eugenol and the extract.It was found that eugenol did not interact with other components.We conducted further research on eugenol,it was found that the eugenol’s fungicidal activity against Malassezia was dose-time dependent,and the inhibition rate of eugenol with MIC concentration against Malassezia was higher than Ketoconazole at MIC concentration.The interaction between eugenol and traditional antidandruff agents was studied.It was found that the combination of eugenol and ketoconazole showed a synergistic effect,and the combination with ZPT,OCT,and climbazole showed an additive effect.When combined with ketoconazole（0.25×MIC eugenol+0.25×MIC ketoconazole）,the fungicidal effect is better than eugenol or ketoconazole used alone.The thiazole blue（MTT）method was used to evaluate the toxicity of each drug to keratinocytes.It was found that the half-inhibitory concentration(IC₅₀)of eugenol was higher than that of traditional antidandruff agents.In human patch test,eugenol at a concentration of 2%（w%）did not cause adverse reactions,indicating that eugenol has higher safety.Scanning electron microscopy and transmission electron microscopy were used to observe eugenol-treated Malassezia cells at MIC concentrations.It was found that compared with the control group,biofilm synthesis was significantly reduced,cell walls appeared sunken and shrunk,cell membranes sunken,and plasma wall separation occurred.Membrane and mitochondrial membranes dissolve.In the sorbitol binding experiment,the MIC of eugenol was doubled,which further proves that eugenol targets the cell wall of Malassezia.Ergosterol binding experiments showed that eugenol did not bind to ergosterol in cell membranes,but the results of nucleic acid leak test and quantitative ergosterol experiments showed that eugenol can greatly reduce the synthesis of ergosterol in cell membranes,thereby destroying the integrity of cell membranes and permeability,causing the leakage of cell contents.This study screened plant extracts for antifungal activity and safety evaluation,and preliminary studied the antifungal mechanism of eugenol,providing a theoretical basis for the application of eugenol in antidandruff products.