【作者】 王玲；

【导师】 孙令；

【作者基本信息】 大连医科大学 ， 皮肤病与性病学， 2007， 硕士

【摘要】 目的:本研究采用中药提取、体外药物敏感试验和扫描电镜的方法,观察黄连、黄柏、土槿皮、白鲜皮、蛇床子、地肤子等六味常用中药的水提取成分和乙醇提取成分,对7种浅部真菌的体外抗真菌活性,研究不同中药单体、不同提取成分对不同浅部真菌的抑菌作用和杀菌作用,拟寻找来源广泛,价格低廉,副作用小的有效抗真菌中药。方法:将黄连、黄柏、土槿皮、白鲜皮、蛇床子、地肤子六味中药分别用蒸馏水和95%乙醇提取得中药水提取和乙醇提取粉末或浸膏。采用药基琼脂稀释法,检测对红色毛癣菌、须癣毛癣菌、石膏样小孢子菌、犬小孢子菌、絮状表皮癣菌、白念珠菌、糠秕马拉色菌等7种浅部真菌的体外抗真菌活性。白念珠菌组置于37℃温箱孵育24～48小时,糠秕马拉色菌组置于37℃温箱孵育3～4天;皮肤癣菌组置于26℃温箱孵育7天判读最小抑菌浓度(MIC)。将新鲜的沙氏液体培养基(SDA)倒入没有真菌生长的含药培养基中,接种环轻刮培养基表面,沙氏液体培养基震荡充分后倒出,白念珠菌组置于37℃温箱孵育3天、皮肤癣菌组置于26℃温箱孵育7天判读最小杀菌浓度(MFC);糠秕马拉色菌组,将倒出的沙氏液基离心,弃上清液,沉淀物接种于含4%橄榄油的SDA培养基,置于37℃温箱孵育7天判读MFC。将浓度为2.5mg/ml水提黄连溶液和浓度为0.3125mg/ml乙醇提蛇床子溶液加入已转种活化7天的红色毛癣菌标准株T.1a试管中,分别于作用2小时、8小时、24小时,各取出一块菌块,采用扫描电镜观察水提黄连、醇提蛇床子对红色毛癣菌形态影响的连续性变化。结果:1. 5种10株皮肤癣菌对水提黄连、黄柏、土槿皮、白鲜皮、蛇床子、地肤子、乙醇提黄连、黄柏、土槿皮、白鲜皮、蛇床子、地肤子的MIC值范围分别在0.3125~2.5mg/ml,0.625~1.25mg/ml,1.25~≥5.0mg/ml,>5.0mg/ml,≥5.0mg/ml,>5.0mg/ml,0.156~1.25mg/ml,0.156~1.25mg/ml,0.156~2.5mg/ml , 0.039~0.156mg/ml , 0.0195~0.156 mg/ml ,0.625~2.5mg/ml;3株白念珠菌MIC值范围分别在0.625~1.25mg/ml,≥5.0mg/ml , 2.5~5.0mg/ml , > 5.0mg/ml , > 5.0mg/ml , > 5.0mg/ml ,1.25mg/ml , 0.625~1.25mg/ml , 0.3125~0.625mg/ml ,≥2.5mg/ml ,≥2.5mg/ml ,≥2.5mg/ml ; 3株糠秕马拉色菌MIC值范围分别在0.625~1.25mg/ml,>5.0mg/ml,>5.0mg/ml,>5.0mg/ml,>5.0mg/ml,> 5.0mg/ml, 1.25mg/ml, > 2.5mg/ml, > 2.5mg/ml, > 2.5mg/ml,≥2.5mg/ml,>2.5mg/ml。2. 5种10株皮肤癣菌对水提黄连、黄柏、土槿皮、白鲜皮、蛇床子、地肤子、乙醇提黄连、黄柏、土槿皮、白鲜皮、蛇床子、地肤子的MFC值范围分别在2.5~≥5.0mg/ml,>5.0mg/ml,>5.0mg/ml,>5.0mg/ml,>5.0mg/ml,>5.0mg/ml,1.25~≥2.5 mg/ml,>2.5mg/ml,>2.5mg/ml,>2.5mg/ml,0.078~0.3125 mg/ml, >2.5mg/ml;3株白念珠菌MFC值范围分别在>5.0mg/ml,>5.0mg/ml,5.0mg/ml,>5.0mg/ml,>5.0mg/ml,>5.0mg/ml,>2.5mg/ml,>2.5mg/ml,0.625~2.5mg/ml,>2.5mg/ml,>2.5mg/ml,>2.5mg/ml;3株糠秕马拉色菌对水提中药成分MFC值范围均为>5.0mg/ml;对乙醇提中药成分MFC值范围均为>2.5mg/ml。3.扫描电镜观察结果:分别用2.5mg/ml水提黄连溶液和0.3125mg/ml乙醇提蛇床子溶液与红色毛癣菌作用2小时、8小时、24小时表现出菌丝与孢子的连续性破坏过程。结论:1.水提黄连、黄柏、土槿皮、蛇床子对皮肤癣菌有抑菌作用、水提黄连、黄柏、土槿皮对白念珠菌有抑菌作用、水提黄连对糠秕马拉色菌有抑菌作用;乙醇提黄连、黄柏、土槿皮、白鲜皮、蛇床子、地肤子对皮肤癣菌均有抑菌作用、乙醇提黄连、黄柏、土槿皮、蛇床子对白念珠菌有抑菌作用、乙醇提黄连对糠秕马拉色菌有抑菌作用。2.水提黄连、乙醇提黄连和蛇床子对皮肤癣菌有杀菌作用;土槿皮水提和乙醇提取成分对白念珠菌均有杀菌作用。更多还原

【Abstract】 Objective: The studies dealt with in vitro antifungal activity against seven pathogenic superficial fungi by means of aqueous and ethanol extracts from six kinds of traditional Chinese medicines (TCMs). The experiments were evaluated on the basis of the following outcome Variables : minimal inhibitory concentration(MIC), minimal fungicidal concentration(MFC) and the results of scanning electron micrograph (SEM). In order to screen the efficacy of antifungal extracts and influence of different strains and pathways.Methods : The aqueous and ethanol extracts from 6 TCMs were obtained , including Rhizoma Coptidis, cortex Phellodendri, Cortex pseudolaricis tincture, Dictamnus dasycarpus Turcz., Fructus cnidii, Kochia scoparia Schrad.. The test organisms were Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum gypseum, Microsporum canis, Epidermophyton floccosum, Candida albicans, Malassezia furfur. The 16 strains were inoculated to obtain young, actively growing cultures. The resulting suspensions were adjusted to a concentration of 1×106～6×106 CFU /ml using blood cell counting plate. The aqueous and ethanol extracts were dissolved in sterile distilled water or dehydrated alcohol, and ten twofold serial dilutions were made. The solutions mixed with SDA slants and SDA slants containing 4% olive oil , and then inoculated the prepared suspensions. C. albicans slants were incubated at 37℃for 24～48 hours,M. furfur. slants were incubated at 37℃for 3～4 days,and dermatophytes slants were incubated at 26℃for 7 days. The MICs were defined as the lowest drug concentrations that inhibited visible growth of the fungi. The MFCs for C. albicans and dermatophytes were determined by subculturingfrom each slant with no visible growth onto liquid SDA mediums. C. albicans mediums were incubated at 37℃for 3 days and dermatophytes mediums were incubated at 26℃for 7 days. Every M. furfur. slant with no visible growth was subcultured onto SDA slants containing 4% olive oil, and then incubated at 37℃for 7 days. The MFC was defined as the lowest concentration which yielded a negative subculture. Trichophyton rubrum morphology with SEM was observed after infused with 2.5mg/ml aqueous extract from Rhizoma Coptidis and 0.3125mg/ml ethanol extract from Fructus cnidii.Results:1. The MIC range of aqueous extracts from Rhizoma Coptidis, cortex Phellodendri, Cortex pseudolaricis tincture, Dictamnus dasycarpus Turcz., Fructus cnidii, Kochia scoparia Schrad. and ethanol extracts from above 6 TCMs against 10 dermatophyte strains are 0.3125~2.5mg/ml, 0.625~1.25mg/ml, 1.25~≥5.0mg/ml, >5.0mg/ml,≥5.0mg/ml,>5.0mg/ml, 0.156~1.25mg/ml, 0.156~1.25mg/ml,0.156~2.5mg/ml, 0.039~0.156mg/ml, 0.0195~0.156mg/ml, 0.625~2.5mg/ml; against 3C. albicans strains are 0.625~1.25mg/ml,≥5.0mg/ml, 2.5~5.0mg/ml, > 5.0mg/ml, > 5.0mg/ml, >5.0mg/ml,1.25mg/ml, 0.625~1.25mg/ml, 0.3125~0.625mg/ml,≥2.5mg/ml,≥2.5mg/ml,≥2.5mg/ml; against 3 M. furfur. strains are 0.625~1.25 mg/ml,>5.0mg/ml,>5.0mg/ml,>5.0mg/ml,>5.0mg/ml,>5.0mg/ml,1.25mg/ml , > 2.5mg/ml , > 2.5mg/ml , > 2.5mg/ml ,≥2.5mg/ml , >2.5mg/ml.2. The MFC range of aqueous and ethanol extracts from above 6 TCMs against 10 dermatophyte strains are 2.5~≥5.0mg/ml, > 5.0mg/ml,>5.0mg/ml,>5.0mg/ml,>5.0mg/ml,>5.0mg/ml,1.25~≥2.5 mg/ml,>2.5mg/ml,>2.5mg/ml,>2.5mg/ml,0.078~0.3125 mg/ml,>2.5mg/ml;against 3C. albicans strains are >5.0mg/ml,>5.0mg/ml,5.0mg/ml,>5.0mg/ml , > 5.0mg/ml , > 5.0mg/ml , > 2.5mg/ml , > 2.5mg/ml ,0.625~2.5mg/ml,>2.5mg/ml,>2.5mg/ml,>2.5mg/ml;The MFC range of aqueous extracts from above 6 TCMs against 3 M. furfur. strains are>5.0mg/ml all,and the MFC range of ethanol extracts from above 6 TCMs against 3 M. furfur. strains are>2.5mg/ml all.3. The results of SEM exhibited that the serial variation ofTrichophyton rubrum morphology was closely related to the infusion time by 2.5mg/ml aqueous extract from Rhizoma Coptidis and 0.3125mg/ml ethanol extract from Fructus cnidii.Conclusions:1. The results showed that the 6 aqueous extracts and 6 ethanol extracts demonstrated different antifungal activity against different superficial fungi.2. The aqueous extracts from Rhizoma Coptidis and the enthanol extracts from Rhizoma Coptidis, Fructus cnidii against dermatophytes dexhibited fungicidal activity. The aqueous and ethanol extracts from Cortex pseudolaricis tincture against C. albicans exhibited fungicidal activity.更多还原