甘肃祁连山冬虫夏草内生真菌的分离及其代谢产物活性评价

Isolation of endophytic fungi from Cordyceps in Qilian mountains of Gansu province and evaluation of their secondary metabolites activity

目的分离甘肃祁连山冬虫夏草内生真菌,并探讨其代谢产物的生物活性。方法采用3种不同培养基,用组织匀浆法和组织切块法对冬虫夏草不同部位内生真菌进行分离纯化。分别对其代谢产物的酶抑制活性、抗菌和抗氧化活性进行评价。利用形态学和分子生物学方法进行高活性菌株分类鉴定,利用现代色谱分离技术对高活性化学成分进行跟踪分离。结果分离得到42株内生真菌,发酵得到84个待测样品,对腺苷脱氨酶(ADA)抑制活性>40%者占比52%。其中高活性菌株Aspergillus sp.C-1乙酸乙酯相样品ADA抑制活性>90%,对5种供试菌均有抑菌活性,二苯基苦基苯肼(DPPH)和羟自由基清除率均>85%,总还原力为(0.039±0.002)mg的L-抗坏血酸(VC)当量,分子鉴定与Aspergillus fumigatus有99%同源性。追踪分离出2个具有ADA抑制活性的化合物,其中C1被鉴定为大黄素甲醚(physcion),IC_(50)为3.58 mmol/L。结论甘肃祁连山冬虫夏草内生真菌资源丰富且次级代谢产物具有良好的生物活性,是活性物质发现的良好来源。

Objective To investigate the bioactivity of Cordyceps'secondary metabolites through the isolation of endophytic fungi from Cordyceps in the Qilian mountains of Gansu province.Methods Three different culture media were used in the study,and the isolation and purification of endophytic fungi from different parts of Cordyceps were carried out by using the tissue homogenization method and tissue sectioning method.The enzyme inhibition activity,antimicrobial activity and antioxidant activity of their secondary metabolites were evaluated.Morphological and molecular biological methods were employed for the classification and identification of highly active strains,and modern chromatographic separation techniques were used to track and isolate highly active chemical components.Results Forty-two strains of endophytic fungi were isolated,and 84 secondary metabolites were obtained through fermentation.Among them,52%had adenosine deaminase(ADA)inhibition activity exceeding 40%.The secondary metabolite of the highly active strain Aspergillus sp.C-1 ethyl acetate phase sample showed ADA inhibition activity was more than 90%.It exhibited antibacterial activity against five tested strains,and its DPPH and hydroxyl radical scavenging rates exceeded 85%.The total reducing power was(0.039±0.002)mg L-ascorbic acid(VC)equivalents,and its molecular identification showed 99%homology with Aspergillus fumigatus.Two compounds with ADA inhibition activity were isolated and tracked,among which C1 was identified as physcion with an IC_(50)value of 3.58 mmol/L.Conclusion The rich resources of endophytic fungi in Cordyceps in the Qilian mountains of Gansu province and the bioactivity of their secondary metabolites make them valuable resources for the discovery of active substances.