中草药及其单体抗马拉色菌作用的研究
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摘要
为了得到有效抑制马拉色菌的中草药及其单体，本研究采用体内外实验相结合的方法，即中草药及其单体抗马拉色菌体外药敏试验结合中草药单体治疗豚鼠皮肤马拉色菌感染的动物实验，筛选有效抑制马拉色菌的中草药及其单体。
首先应用单一基因区PCR－限制性内切酶分析法（单一基因区PCR-REA）对马拉色菌7种参考菌株及28株临床分离株进行鉴定分型，即设计特异引物对马拉色菌属大亚基核糖体RNA(LSU rRNA)基因区进行扩增，并以3种限制性内切酶（BanI、 HaeII and MspI）对PCR产物进行酶切。结果7种参考菌株均得到了各自的条带格局，快速、清晰地分辨出马拉色菌的7个不同种。28株临床分离株共得到5种马拉色菌，分别为M.furfur 12株(42.9%),占首位，其余4型依次为M.globosa 6株(21.4%)、M.restricta 6株(21.4%)、M.sympodialis 3株(10.7%)、M.slooffiae1株 (3.6%)。其中15例花斑癣标本，分离到M.furfur 8株(53.3%)、M.globosa 4株(26.7%)、M.sympodialis 2株(13.3%)、M.restricta 1株(6.7%)。9例健康头屑标本，分离到M.restricta 5株(55.6%)、M.globosa 2株(22.2%)、M.furfur 1株(11.1%)、M.slooffiae 1株(11.1%)。由此得到结论，单一基因区PCR-REA法可快速、准确地对马拉色菌属7个种进行分型。
中草药及其单体抗马拉色菌体外药敏试验参照美国国家临床实验室标准化委员会（NCCLS）制订的M27-A方案中酵母菌微量稀释法，选择23种中草药及14种单体对马拉色菌进行体外抑菌初筛试验，结果显示23种中草药中，香茅、蛇床子对马拉色菌有较强的抑制作用（MIC31.25～62.5mg/L）；14种中草药单体中，苦参碱、氧化苦参碱、柠檬醛、丁香酚有强的抗马拉色菌作用（MIC0.98～7.81mg/L）。5味中草药混合液及5种单体混合液的MIC值均不低于其单味药的MIC值。在此基础上，进一步选择苦参碱、柠檬醛、丁香酚3种中草药单体对7种马拉色菌参考菌株及5种临床分离株进行体外抑菌、杀菌试验，并在培养基中加入10％人血清，观察高蛋白环境下3种单体的抑菌情况，计算其抑菌活性降低分数。结果显示，苦参碱对M.globosa、M.slooffiae、M.furfur具强抑菌作用（MIC0.39～0.78 mg/L）, 柠檬醛对M.globosa、M.slooffiae有强抑菌作用（MIC0.195～0.78 mg/L），丁香酚对M.globosa、M.furfur抑菌作用最强（MIC0.195～0.78 mg/L）。其中 M.globosa对3种单体均最敏感，尤其是对柠檬醛、丁香酚，其MIC值为0.195mg/L。苦参碱对M.restricta的MFC值及丁香酚对M.globosa的MFC值均较其MIC值高几个浓度梯度，其余的MFC与MIC值相同或高1个浓度梯度。3种单体中，柠檬醛与蛋白亲和力最高，抑菌活性降低分
数为1.3。
豚鼠皮肤感染马拉色菌的动物模型采用3种方法（涂菌1次法、涂菌1次并皮下注射甲基强的松龙法、连续7天涂菌法）进行诱导，分别于末次涂菌后4、7、12天观察其皮损情况、真菌培养阳性率及皮损区菌落数。发现连续7天涂菌法适合于此模型的建立，得到了满意的皮损及菌落数。此外，皮肤菌落计数更能直观、客观地反映药物的短期疗效，是评价皮肤马拉色菌感染动物模型较科学的指标。
将苦参碱、柠檬醛、丁香酚3种中草药单体制成2％霜剂，采用自身对照法，并且设立阳性、阴性、空白对照组治疗豚鼠皮肤马拉色菌感染。采用连续涂菌7天法建立动物模型，并于末次涂菌后1天对皮损区进行局部治疗，连续用药4天或7天后观察各组皮损区菌落数。结果显示，连续用药4天，柠檬醛、丁香酚组菌落数明显少于阴性及空白对照组，虽高于酮康唑组，但无统计学意义。苦参碱组菌落数虽明显低于阴性及空白对照组，却明显高于酮康唑和其它两种单体组；连续用药7天后，苦参碱的疗效则明显提高，该3种单体组的菌落数均明显低于阴性对照组及空白对照组而与酮康唑组接近，无显著差异。
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 MFCs
of 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
首先应用单一基因区PCR－限制性内切酶分析法（单一基因区PCR-REA）对马拉色菌7种参考菌株及28株临床分离株进行鉴定分型，即设计特异引物对马拉色菌属大亚基核糖体RNA(LSU rRNA)基因区进行扩增，并以3种限制性内切酶（BanI、 HaeII and MspI）对PCR产物进行酶切。结果7种参考菌株均得到了各自的条带格局，快速、清晰地分辨出马拉色菌的7个不同种。28株临床分离株共得到5种马拉色菌，分别为M.furfur 12株(42.9%),占首位，其余4型依次为M.globosa 6株(21.4%)、M.restricta 6株(21.4%)、M.sympodialis 3株(10.7%)、M.slooffiae1株 (3.6%)。其中15例花斑癣标本，分离到M.furfur 8株(53.3%)、M.globosa 4株(26.7%)、M.sympodialis 2株(13.3%)、M.restricta 1株(6.7%)。9例健康头屑标本，分离到M.restricta 5株(55.6%)、M.globosa 2株(22.2%)、M.furfur 1株(11.1%)、M.slooffiae 1株(11.1%)。由此得到结论，单一基因区PCR-REA法可快速、准确地对马拉色菌属7个种进行分型。
中草药及其单体抗马拉色菌体外药敏试验参照美国国家临床实验室标准化委员会（NCCLS）制订的M27-A方案中酵母菌微量稀释法，选择23种中草药及14种单体对马拉色菌进行体外抑菌初筛试验，结果显示23种中草药中，香茅、蛇床子对马拉色菌有较强的抑制作用（MIC31.25～62.5mg/L）；14种中草药单体中，苦参碱、氧化苦参碱、柠檬醛、丁香酚有强的抗马拉色菌作用（MIC0.98～7.81mg/L）。5味中草药混合液及5种单体混合液的MIC值均不低于其单味药的MIC值。在此基础上，进一步选择苦参碱、柠檬醛、丁香酚3种中草药单体对7种马拉色菌参考菌株及5种临床分离株进行体外抑菌、杀菌试验，并在培养基中加入10％人血清，观察高蛋白环境下3种单体的抑菌情况，计算其抑菌活性降低分数。结果显示，苦参碱对M.globosa、M.slooffiae、M.furfur具强抑菌作用（MIC0.39～0.78 mg/L）, 柠檬醛对M.globosa、M.slooffiae有强抑菌作用（MIC0.195～0.78 mg/L），丁香酚对M.globosa、M.furfur抑菌作用最强（MIC0.195～0.78 mg/L）。其中 M.globosa对3种单体均最敏感，尤其是对柠檬醛、丁香酚，其MIC值为0.195mg/L。苦参碱对M.restricta的MFC值及丁香酚对M.globosa的MFC值均较其MIC值高几个浓度梯度，其余的MFC与MIC值相同或高1个浓度梯度。3种单体中，柠檬醛与蛋白亲和力最高，抑菌活性降低分
数为1.3。
豚鼠皮肤感染马拉色菌的动物模型采用3种方法（涂菌1次法、涂菌1次并皮下注射甲基强的松龙法、连续7天涂菌法）进行诱导，分别于末次涂菌后4、7、12天观察其皮损情况、真菌培养阳性率及皮损区菌落数。发现连续7天涂菌法适合于此模型的建立，得到了满意的皮损及菌落数。此外，皮肤菌落计数更能直观、客观地反映药物的短期疗效，是评价皮肤马拉色菌感染动物模型较科学的指标。
将苦参碱、柠檬醛、丁香酚3种中草药单体制成2％霜剂，采用自身对照法，并且设立阳性、阴性、空白对照组治疗豚鼠皮肤马拉色菌感染。采用连续涂菌7天法建立动物模型，并于末次涂菌后1天对皮损区进行局部治疗，连续用药4天或7天后观察各组皮损区菌落数。结果显示，连续用药4天，柠檬醛、丁香酚组菌落数明显少于阴性及空白对照组，虽高于酮康唑组，但无统计学意义。苦参碱组菌落数虽明显低于阴性及空白对照组，却明显高于酮康唑和其它两种单体组；连续用药7天后，苦参碱的疗效则明显提高，该3种单体组的菌落数均明显低于阴性对照组及空白对照组而与酮康唑组接近，无显著差异。
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 MFCs
of 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
引文
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40 Chai FC, Auret K, Christiansen K, et al. Malignant otitis externa caused by Malassezia sympodialis. Head Neck 2000,22:87-89.
41 Guillot J, Bond R. Malassezia pachydermatis; a review. Med Mycol 1999, 37:295-306.
42 Crespo MJ, Abarca ML, Caba?es FJ. Atypical lipid-dependent Malassezia species isolated from dogs with otitis externa. J Clin Microbiol 2000, 38:2383-2385.
43 Crespo MJ, Abarca ML, Caba?es FJ. Isolation of Malassezia furfur from a cat. J Clin Microbiol 1999,37(5):1573-1574.
44 Raabe P, Mayser P, Weiss R. Demonstration of Malassezia furfur and M.sympodialis together with M.pachydermatis in veterinary specimens. Mycoses 1999,42(11-12):673-674.
45 Crespo MJ, Abarca ML, Cabanes FJ. Otitis externa associated with Malassezia sympodialis in two cats. J Clin Microbiol 2000,38:1263-1266.
46 Crozier WJ, Wise KA. Onychomycosis due to Pityrosporum. Aust J Dermatol 1993,34:109-112.
47 Silva V, Moreno GA, Zaror L, et al. Isolation of Malassezia furfur from patients with onychomycosis. J Med Vet Mycol 1997,35:73-74.
48 Escobar ML, Carmona-Fonseca J, Santamaria L. Onychomycosis due to Malassezia. Rev Iberoam Micol 1999,16:225-229.
49 Gueho E, Oster C, Improvisi L, et al. Antifungal in vitro susceptibility of Malassezia yeasts. In: Proceedings of the Journees de Mycologie Médicale; 28-29 November 1997; Paris. Paris: Société Fran?aise de Mycologie Médicale; 1997. p. 6.
50 Hammer KA, Carson CF, Riley TV. In vitro activities of ketoconazole, econazole, miconazole and Melaleuca alternifolia (tea tree) oil against Malassezia species. Antimicrob Agents Chemother 2000,44:467-469.
51 Nakamura Y, Kano R, Murai T, et al. Susceptibility testing of Malassezia species using the urea broth microdilution method. Antimicrob Agents Chemother 2000,44:2185-2186.
52 Gupta AK, Kohli Y, Li A, et al. In vitro susceptibility of the seven Malassezia species to ketoconazole, voriconazole, itraconazole and terbinafine. Br J Dermatol 2000,142:758-765.
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37 Aractingi S, Cdranel S, Reygagne P, et al . Neonatal pustulosis caused by Malassezia furfur. Ann Dermatol Venereol 1991,118:856-858.
38 Pont V, Grau C, Del Pino M, et al. Neonatal Malassezia furfur pustulosis. Actas Dermosifiliogr 1998,89:199-205.
39 Rapelanoro R, Mortureux P, Couprie B, et al. Neonatal Malassezia furfur pustulosis. Arch Dermatol 1996,132:190-193.
40 Chai FC, Auret K, Christiansen K, et al. Malignant otitis externa caused by Malassezia sympodialis. Head Neck 2000,22:87-89.
41 Guillot J, Bond R. Malassezia pachydermatis; a review. Med Mycol 1999, 37:295-306.
42 Crespo MJ, Abarca ML, Caba?es FJ. Atypical lipid-dependent Malassezia species isolated from dogs with otitis externa. J Clin Microbiol 2000, 38:2383-2385.
43 Crespo MJ, Abarca ML, Caba?es FJ. Isolation of Malassezia furfur from a cat. J Clin Microbiol 1999,37(5):1573-1574.
44 Raabe P, Mayser P, Weiss R. Demonstration of Malassezia furfur and M.sympodialis together with M.pachydermatis in veterinary specimens. Mycoses 1999,42(11-12):673-674.
45 Crespo MJ, Abarca ML, Cabanes FJ. Otitis externa associated with Malassezia sympodialis in two cats. J Clin Microbiol 2000,38:1263-1266.
46 Crozier WJ, Wise KA. Onychomycosis due to Pityrosporum. Aust J Dermatol 1993,34:109-112.
47 Silva V, Moreno GA, Zaror L, et al. Isolation of Malassezia furfur from patients with onychomycosis. J Med Vet Mycol 1997,35:73-74.
48 Escobar ML, Carmona-Fonseca J, Santamaria L. Onychomycosis due to Malassezia. Rev Iberoam Micol 1999,16:225-229.
49 Gueho E, Oster C, Improvisi L, et al. Antifungal in vitro susceptibility of Malassezia yeasts. In: Proceedings of the Journees de Mycologie Médicale; 28-29 November 1997; Paris. Paris: Société Fran?aise de Mycologie Médicale; 1997. p. 6.
50 Hammer KA, Carson CF, Riley TV. In vitro activities of ketoconazole, econazole, miconazole and Melaleuca alternifolia (tea tree) oil against Malassezia species. Antimicrob Agents Chemother 2000,44:467-469.
51 Nakamura Y, Kano R, Murai T, et al. Susceptibility testing of Malassezia species using the urea broth microdilution method. Antimicrob Agents Chemother 2000,44:2185-2186.
52 Gupta AK, Kohli Y, Li A, et al. In vitro susceptibility of the seven Malassezia species to ketoconazole, voriconazole, itraconazole and terbinafine. Br J Dermatol 2000,142:758-765.