珙桐产黄酮内生真菌的分离及鉴定
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摘要
珙桐(Davidia involucrate Baill.)为我国特有的第三纪古热带植物的孑遗树种,被列为我国一级保护植物。研究表明,珙桐叶提取物中含有槲皮素、山奈酚等黄酮类物质。本研究从内生真菌的角度出发,对珙桐中的内生真菌进行研究,结果如下:
1、采用组织分离法从珙桐组织中分离了126株内生真菌。经对比实验,内生真菌分离时,植物组织的表面消毒以75%酒精和0.1%升汞结合的方法较好;对珙桐茎部的升汞消毒时间以4～5min为宜,珙桐叶子的升汞消毒时间应控制在2min左右。珙桐中分离到的126株内生真菌主要分布在树叶和茎中。
2、以金黄色葡萄球菌(Staphylococcus aureus)、枯草芽孢杆菌(Bacillus subtilis)、大肠杆菌(Escherichia coil)为测试菌种,对初选的126株珙桐内生真菌进行代谢产物活性筛选。其产物对三种细菌的抑菌率分别为65%、71%、25%,其中D37菌株对金黄色葡萄球菌的抑制作用最强。
3、对具抑菌活性的菌株D37进行液体发酵。经高效液相色谱(High PerformanceLiquid Chromatography,HPLC)分析证明菌株D37的次生代谢产物中含有槲皮素、山奈酚二种黄酮类物质,每100g干菌丝可产黄酮31.9mg/100g。
4、根据菌株D37的形态学特征,结合核糖体内部转录序列(intemal transcribedspacer,ITS)的测定结果,确定菌株D37为曲霉属,烟曲霉种(Aspergillus fumigatus)。
本文从珙桐分离的产黄酮内生真菌D37即可作为生产黄酮类药物的候选菌株,此外,分离到的126株内生真菌是进一步进行分离其它生物活性物质、进行抗菌及药物等研究的资源库。
Objective To isolate endophytic fungi producing active substances from Davidia involucrate Baill.
Methods Endophytic fungi strains were isolated by issue - culture method from Davidia involucrate Baill.The result showed that,the endophytic fungi in Davidia involucrate Baill is isolated by the surface sterilization method with 70%of ethyl alcohol and 0.1%of HgCl_2.The best sterilization time of the stem is 4～5min to Davidia involucrate Baill.And The best sterilization time of the leaf should be controlled about 2min.
Results To isolate endophytic fungi producing active substances from Davidia involucrate Baill.The inhibitory activity of endophytic fungi was detected taking Staphylococcus aureus,Bacillus subtilis and Escherichia coli as indicative bacteria.The active substances in the products of endophytic fungi was confirmed by High Performance Liquid Chromatography(HPLC).
Conclusion One hundred and twenty six endophytic fungi strains were isolated from the leaves and branches of Davidia involucrate Baill,and their metabolic products had inhibitory activity,the inhibition rates to the three bacteria were 65%,71%,25% respectively.The endophytic fungi strain D37 was selected to screen its active substance as its activity of inhibitory effects on Staphylococcus aureus was the highest.The strain D37 produced quercetin and kaempferol about 31.9 mg per 100g of dry mycelium confirmed by HPLC.The endophytic fungi D37 was identificated as Aspergillus fumigatus according to the morphology of fungal and the similarity of nucleotide sequence of internal transcribed spacer(ITS) between rDNAs.
Significance The endophytic fungi D37,isolated from Davidia involucrate Baill, could produce flavonoids.The endophytic fungi strain seperated from Davidia involucrate would be a candidator for flavonoids preparation.These endophytic fungi strains is resources for further research other bioactive compound,antibiosis experiment and so on.
1、采用组织分离法从珙桐组织中分离了126株内生真菌。经对比实验,内生真菌分离时,植物组织的表面消毒以75%酒精和0.1%升汞结合的方法较好;对珙桐茎部的升汞消毒时间以4～5min为宜,珙桐叶子的升汞消毒时间应控制在2min左右。珙桐中分离到的126株内生真菌主要分布在树叶和茎中。
2、以金黄色葡萄球菌(Staphylococcus aureus)、枯草芽孢杆菌(Bacillus subtilis)、大肠杆菌(Escherichia coil)为测试菌种,对初选的126株珙桐内生真菌进行代谢产物活性筛选。其产物对三种细菌的抑菌率分别为65%、71%、25%,其中D37菌株对金黄色葡萄球菌的抑制作用最强。
3、对具抑菌活性的菌株D37进行液体发酵。经高效液相色谱(High PerformanceLiquid Chromatography,HPLC)分析证明菌株D37的次生代谢产物中含有槲皮素、山奈酚二种黄酮类物质,每100g干菌丝可产黄酮31.9mg/100g。
4、根据菌株D37的形态学特征,结合核糖体内部转录序列(intemal transcribedspacer,ITS)的测定结果,确定菌株D37为曲霉属,烟曲霉种(Aspergillus fumigatus)。
本文从珙桐分离的产黄酮内生真菌D37即可作为生产黄酮类药物的候选菌株,此外,分离到的126株内生真菌是进一步进行分离其它生物活性物质、进行抗菌及药物等研究的资源库。
Objective To isolate endophytic fungi producing active substances from Davidia involucrate Baill.
Methods Endophytic fungi strains were isolated by issue - culture method from Davidia involucrate Baill.The result showed that,the endophytic fungi in Davidia involucrate Baill is isolated by the surface sterilization method with 70%of ethyl alcohol and 0.1%of HgCl_2.The best sterilization time of the stem is 4～5min to Davidia involucrate Baill.And The best sterilization time of the leaf should be controlled about 2min.
Results To isolate endophytic fungi producing active substances from Davidia involucrate Baill.The inhibitory activity of endophytic fungi was detected taking Staphylococcus aureus,Bacillus subtilis and Escherichia coli as indicative bacteria.The active substances in the products of endophytic fungi was confirmed by High Performance Liquid Chromatography(HPLC).
Conclusion One hundred and twenty six endophytic fungi strains were isolated from the leaves and branches of Davidia involucrate Baill,and their metabolic products had inhibitory activity,the inhibition rates to the three bacteria were 65%,71%,25% respectively.The endophytic fungi strain D37 was selected to screen its active substance as its activity of inhibitory effects on Staphylococcus aureus was the highest.The strain D37 produced quercetin and kaempferol about 31.9 mg per 100g of dry mycelium confirmed by HPLC.The endophytic fungi D37 was identificated as Aspergillus fumigatus according to the morphology of fungal and the similarity of nucleotide sequence of internal transcribed spacer(ITS) between rDNAs.
Significance The endophytic fungi D37,isolated from Davidia involucrate Baill, could produce flavonoids.The endophytic fungi strain seperated from Davidia involucrate would be a candidator for flavonoids preparation.These endophytic fungi strains is resources for further research other bioactive compound,antibiosis experiment and so on.
引文
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3.史央,戴传超,陆玲等.大戟科4种药用植物内生真菌3种胞外酶活性的比较.植物资源与环境学报.2002,11(2):17-20。
4.刘芸,殷红,彭辉等.植物内生真菌一潜在的天然药物新来源.中国现代中西医杂志,2005年第三卷第五期
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6.宋云龙,张万年,季海涛等.喜树碱类抗肿瘤药物的量子化学与定量构效关系研究.计算机与应用化学.Vol 19.No.2002
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