黄芩苷发酵转化成黄芩素和千层纸素A的黄芩内生真菌的筛选及鉴定
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摘要
目的:采用现代微生物发酵转化技术开发传统中药——黄芩,从黄芩植株体内筛选可发酵转化黄芩苷的内生真菌,以期替代传统化学方法获得天然药效成分,同时缓解该中药资源紧张的现状。方法:以新鲜黄芩植株为材料,采用马铃薯葡萄糖琼脂(PDA)平板培养消毒组织块,分离单菌落,黄芩药粉选择性培养法初筛,黄芩苷发酵转化实验复筛,HPLC分析转化产物。结果:从新鲜黄芩植株根部分离得到一株发酵转化黄芩苷的内生真菌R3,该菌在添加0.1%黄芩苷的马铃薯葡萄糖培养液(PDB)中能转化黄芩苷,同时产生黄芩素和千层纸素A共2种产物,于28℃,150 r·min~(-1)培养5 d转化率61.09%,其中黄芩素和千层纸素A的摩尔比3∶5。利用形态分析及显微鉴定并结合分子生物学技术手段,鉴定该菌为青霉菌,定名为Penicillium sp. R3。结论:筛选得到黄芩内生真菌Penicillium sp. R3,该菌能够转化黄芩苷得到活性更强、更易吸收的黄芩素,以及近年来因更多药效不断被发现而备受关注的千层纸素A,具有一定的研究价值与应用价值。
Objective: In order to explore natural active ingredients of Scutellariae Radix and alleviate the pressure of traditional medicinal resources, this research aimmed to screen endophytic fungus strains from Scutellariae Radix which could transform baicalin into some active ingredients. Method: Taking fresh plants of Scutellariae Radix as strain resource,endophytic fungus strains were isolated by potato dextrose agar( PDA) plate separation,Scutellariae Radix powder selective culture and HPLC detection. Result: An endophytic fungus strain R3 was isolated and it can transform baicalin to baicalein and oroxylin A simultaneously in potato dextrose broth( PDB) contained 0. 1% baicalin and the conversion rate reached 61. 09% in 5 d cultured at 28 ℃ and150 r·min~(-1),molar ratio of baicalein and oroxylin A was 3 ∶ 5. With the method of morphological analysis,microscopic identification and 18 Sr DNA sequence analysis,the bacterium was identified as Penicillium sp. R3.Conclusion: Penicillium sp. R3 can transform baicalin to baicalein and oroxylin A with certain research value and application value.
Objective: In order to explore natural active ingredients of Scutellariae Radix and alleviate the pressure of traditional medicinal resources, this research aimmed to screen endophytic fungus strains from Scutellariae Radix which could transform baicalin into some active ingredients. Method: Taking fresh plants of Scutellariae Radix as strain resource,endophytic fungus strains were isolated by potato dextrose agar( PDA) plate separation,Scutellariae Radix powder selective culture and HPLC detection. Result: An endophytic fungus strain R3 was isolated and it can transform baicalin to baicalein and oroxylin A simultaneously in potato dextrose broth( PDB) contained 0. 1% baicalin and the conversion rate reached 61. 09% in 5 d cultured at 28 ℃ and150 r·min~(-1),molar ratio of baicalein and oroxylin A was 3 ∶ 5. With the method of morphological analysis,microscopic identification and 18 Sr DNA sequence analysis,the bacterium was identified as Penicillium sp. R3.Conclusion: Penicillium sp. R3 can transform baicalin to baicalein and oroxylin A with certain research value and application value.
引文
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[14]姚磊,张敏,王鹏娇,等.黄芩中黄芩苷生物转化工艺优化[J].中国实验方剂学杂志,2015,21(9):22-24.
[15]贺美,邱德全,柏仕杰,等.米曲霉两步活化法对黄芩苷的生物转化及转化产物的纯化与鉴定[J].广东海洋大学学报,2007,27(6):41-44.
[16]管悦,孙佰贺,边淑玲,等.固定化酵母菌和米曲霉生物转化黄芩的实验研究[J].哈尔滨医科大学学报,2017,51(3):206-210.
[17]苏龙,梁广波,龙文英,等.黄芩苷微生物转化菌的筛选鉴定及发酵条件优化[J].湖北农业科学,2017,56(8):1483-1488.
[18]Stierle A,Strobel G,Stierle D.Taxol and taxane production by Taxomyces andreana,an endophytic fungus of pacific yew[J].Science,1993,260(5105):214-216.
[19]宝艳儒,刘悦,解生旭,等.内生真菌对药用植物活性成分影响研究进展[J].人参研究,2017,29(5):40-45.
[20]张黎光.黄芩内生真菌的初步研究[D].西安:陕西师范大学,2011.
[21]国家药典委员会.中华人民共和国药典.一部[M].北京:中国医药科技出版社,2015:301-302.
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[23]魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1979:501-512.
[2]Muto R,Motozuka T,Nakano M,et al.The chemical structure of new substance as the metabolite of baicalin and time profiles for the plasma concentration after oral administration of sho-saiko-to in human[J].Yakugaku Zasshi,1998,118(3):79-87.
[3]Yim J S,Kim Y S,Moon S K,et al.Metabolic activities of ginsenoside Rb1,baicalin,glycyrrhizin and genposide to their bioactive compounds by human intestinal microflora[J].Biol Pharm Bull,2004,27(10):1580-1583.
[4]周锡钦,梁鸿,路新华,等.中药黄芩主要黄酮类成分及其生物活性研究[J].北京大学学报:医学版,2009,41(5):578-584.
[5]陈卫卫,刘灵杰,张自力,等.千层纸素A的药理作用及制剂开发的最新进展[J].中华中医药学刊,2018,36(2):412-416.
[6]Dordick J S,Clark D S.Biocatalysis and biotransformation[J].Curr Opin Chem Bio,2002,6(2):121-122.
[7]郑丽娜,张明春.中药有效成分的酶法生物转化[J].时珍国医国药,2010,21(10):2556-2558.
[8]LI M X,ZHU H Y,YANG D H,et al.Production of secoisolariciresinol from defatted flaxseed by bacterial biotransformation[J].J Appl Mirobiol,2012,113(6):1352-1361.
[9]马宗敏,段绪红,秦梦,等.微生物发酵技术在中药苷类生物转化中的应用进展[J].世界科学技术—中医药现代化,2017,19(5):858-864.
[10]刘伶文,司磊,任树勇,等.人肠道菌对黄芩苷的生物转化[J].天然产物研究与开发,2012,24(10):1437-1440,1392.
[11]汪红,高陪,廖勇,等.微生物发酵转化黄芩苷生成黄芩素的研究[J].四川大学学报:自然科学版,2009,46(3):795-798.
[12]陈丽艳,张迎,金爽,等.黄芩经侧耳菌和黑曲霉发酵后黄酮类成分的变化[J].中国实验方剂学杂志,2011,17(5):63-65.
[13]杨希,刘伶文,王璐倩,等.白腐真菌转化黄芩苷生成黄芩素的研究[J].西安工程大学学报,2015,29(1):57-61.
[14]姚磊,张敏,王鹏娇,等.黄芩中黄芩苷生物转化工艺优化[J].中国实验方剂学杂志,2015,21(9):22-24.
[15]贺美,邱德全,柏仕杰,等.米曲霉两步活化法对黄芩苷的生物转化及转化产物的纯化与鉴定[J].广东海洋大学学报,2007,27(6):41-44.
[16]管悦,孙佰贺,边淑玲,等.固定化酵母菌和米曲霉生物转化黄芩的实验研究[J].哈尔滨医科大学学报,2017,51(3):206-210.
[17]苏龙,梁广波,龙文英,等.黄芩苷微生物转化菌的筛选鉴定及发酵条件优化[J].湖北农业科学,2017,56(8):1483-1488.
[18]Stierle A,Strobel G,Stierle D.Taxol and taxane production by Taxomyces andreana,an endophytic fungus of pacific yew[J].Science,1993,260(5105):214-216.
[19]宝艳儒,刘悦,解生旭,等.内生真菌对药用植物活性成分影响研究进展[J].人参研究,2017,29(5):40-45.
[20]张黎光.黄芩内生真菌的初步研究[D].西安:陕西师范大学,2011.
[21]国家药典委员会.中华人民共和国药典.一部[M].北京:中国医药科技出版社,2015:301-302.
[22]钱存柔,黄仪秀.微生物学实验教程[M].北京:北京大学出版社,2006:29-31.
[23]魏景超.真菌鉴定手册[M].上海:上海科学技术出版社,1979:501-512.