中药化学成分抗糠秕马拉色菌活性筛选及协同抗菌作用  

Screening and Synergistic Antibacterial Activity of Chemical Components of Traditional Chinese Medicine Against Malassezia furfur

作　　者：黄燕[1,2] 韦志英[1,2] 张怡婕[1] 李耀华 农清栋 黄冬梅[2] 潘小姣[1] HUANG Yan;WEI Zhiying;ZHANG Yijie;LI Yaohua;NONG Qingdong;HUANG Dongmei;PAN Xiaojiao(Guangxi University of Chinese Medicine,Nanning 530200,China;Ruikang Hospital Affiliated to Guangxi University of Chinese Medicine,Nanning 530011,China)

机构地区：[1]广西中医药大学,广西南宁530200 [2]广西中医药大学附属瑞康医院,广西南宁530011

出　　处：《中国皮肤性病学杂志》2024年第7期731-739,共9页The Chinese Journal of Dermatovenereology

基　　金：广西中医药管理局科研课题(GXZYA20220102);广西中医药大学科研课题(2021MS009)。

摘　　要：目的从30种常见非挥发性中药化学成分中筛选出对糠秕马拉色菌(Malassezia furfur,MF)抑制活性最强的两种化学成分,并对两者的协同抗菌作用及作用机制进行探究。方法采用管碟琼脂扩散法对30种中药单体化合物的MF抑制活性进行初筛;采用96孔板培养联合固体培养基接种法,测定活性最强两种单体化合物、酮康唑(KTZ)及吡啶硫酮锌(ZPT)的最低杀菌浓度(minimum bactericidal concentration,MBC)及其联合应用之后的联合最低杀菌浓度(combined minimum bactericidal concentration,CMBC),并计算联合杀菌指数(fractional bactericidal concentration index,FBCI);采用结晶紫法测定活性成分对MF生物膜的影响;扫描电镜观察活性成分对MF外观形态的影响;CCK-8法检测活性成分对人永生角质形成细胞(HaCaT)的细胞毒性。结果对MF抑制活性最强的两种化学成分分别为肉桂酸(CIN)和盐酸小檗碱(BER),在作用浓度为5.00 mg/mL时,两者抑菌圈直径分别为(15.17±2.37)mm和(11.21±1.03)mm。各样品的MBC大小顺序为:BER(2500.00μg/mL)>CIN(156.25μg/mL)>ZPT(62.50μg/mL)>KTZ(7.81μg/mL)。FBCI试验结果表明:CIN和BER配伍,或者两者分别与KTZ或ZPT配伍,均具有协同杀菌的作用;CIN、BER、KTZ及ZPT相互配伍的CMBC分别为39.06、625.00、1.95、15.62μg/mL,FBCI均为0.5。各样品对MF生物膜抑制率的大小顺序为:CIN(93.08±1.15)%>KTZ(65.10±4.23)%>ZPT(43.00±2.81)%>BER(10.55±1.21)%。扫描电镜结果显示,CIN组MF细胞结构完整,但是大部分菌体细胞壁出现不同程度的凹陷;BER、KTZ及ZPT组MF细胞结构完整,但是大部分菌体整体皱缩,细胞壁表面凹凸不平、粗糙。各样品对HaCaT细胞毒性的大小顺序为:ZPT(IC 50<10.00μg/mL)>BER[IC 50=(27.83±0.66)μg/mL]>KTZ[IC 50=(28.22±0.14)μg/mL]>CIN(IC 50>200.00μg/mL)。结论CIN和BER具有协同抗MF的作用,它们分别与ZPT或KTZ联合应用也有协同抗MF作用;CIN的抗菌作用机制可能与抑制生物膜相关;两者均�Objective To identify the two chemical ingredients with the strongest inhibitory activity against Malassezia furfur(MF)from 30 common non-volatile Chinese medicine chemical ingredients and to explore their synergistic antibacterial effects and mechanisms.Methods The tube-disk agar diffusion method was used to screen the MF inhibitory activity of 30 traditional Chinese medicine monomeric compounds.A 96-well plate culture combined with a solid medium inoculation method was used to determine the minimum bactericidal concentration(MBC)of the two most active identified compounds,and the positive control drug ketoconazole(KTZ)and zinc pyrithione(ZPT),and their combined minimum bactericidal concentration(CMBC),and the fractional bactericidal concentration index(FBCI).The crystal violet method was used to determine the effect of the active ingredients on the MF biofilm.The scanning electron microscope was used to observe the effect of the active ingredients on the appearance of MF.CCK-8 method was used to detect the cytotoxicity of active ingredients on human immortal keratinocytes(HaCaT).Results Cinnamic acid(CIN)and berberine hydrochloride(BER)were the two chemical components with the strongest inhibitory activity against MF.At the concentration of 5.00 mg/mL,the diameter of bacteriostatic zone was(15.17±2.37)mm for CIN and(11.21±1.03)mm for BER.The MBC order was:BER(2500.00μg/mL)>CIN(156.25μg/mL)>ZPT(62.50μg/mL)>KTZ(7.81μg/mL).The FBCI test results showed that the compatibility of CIN,BER,and both compounds with KTZ or ZPT,all had a synergistic bactericidal effect.The CMBC of the compatibility of CIN,BER,KTZ and ZPT were 39.06,625.00,1.95,15.62μg/mL,and the FBCI were all 0.5.The order of the inhibition rate on MF biofilm was:CIN(93.08±1.15)%>KTZ(65.10±4.23)%>ZPT(43.00±2.81)%>BER(10.55±1.21)%.The scanning electron microscope results showed that the structure of MF cells in CIN group was intact,but most of the cell walls were sunken in varying degrees.The structure of MF cells in BER,KTZ and ZPT groups was in

关 键 词：糠秕马拉色菌 肉桂酸 盐酸小檗碱 协同抗菌作用 真菌生物膜 

分 类 号：R284.1[医药卫生—中药学]