金环胡蜂虫草来源真菌的分离鉴定及其代谢产物的抗菌、抗炎活性评价

Isolation and identification of fungi from Vespa mandarinia Smith Cordyceps sensu lato and evaluation of the antibacterial and anti-inflammatory activity of their metabolites

目的探究金环胡蜂虫草的虫生真菌及内生真菌的多样性,筛选出具有抗菌、抗炎活性的真菌,为活性天然药物的开发提供微生物资源。方法通过稀释涂布法从金环胡蜂虫草有性型虫生真菌的子座和子实体、菌核组织以及虫体三部分分离纯化真菌。采用分子生物学方法对子座和子实体部位以及虫生真菌、内生真菌进行ITS rDNA序列分析,并构建系统发育树以确定种属分类情况。以马铃薯葡萄糖培养基(potato dextrose agar,PDA)对所有真菌进行小规模培养。以6株病原细菌为指示菌,通过牛津杯琼脂扩散法检测真菌代谢产物粗提物的抗菌活性。并构建脂多糖(lipopolysaccharide,LPS)诱导的RAW 264.7细胞炎症模型,对粗提物进行抗炎活性筛选。结果有性型的虫生真菌与蜂头线虫草属真菌Ophiocordyceps sphecocephala的同源性最高(>98%)。从子座、子实体和菌核组织分离获得33株虫生真菌,虫体分离获得21株内生真菌。这54株真菌经鉴定隶属于2门,4纲,9目,11科,16属。活性检测结果显示,10株真菌粗提物表现出对不同病原细菌的抑制作用,38株真菌粗提物具有不同程度的抗炎活性。结论金环胡蜂虫草具有丰富的虫生真菌及内生真菌物种,其代谢产物具有抗菌、抗炎活性,这为后续活性先导化合物的发现提供了有价值的微生物资源。

Objective To investigate the diversity of entomogenous and endophytic fungi in Vespa mandarinia Smith Cordyceps sensu lato and the fungi with antibacterial and anti-inflammatory activity were screened out to provide microbial resources for the development of active natural drugs.Methods Fungi were isolated and purified from the stroma,fruiting body,and sclerotia tissue of teleomorph entomogenous fungi from Vespa mandarinia Cordyceps sensu lato,and the insect body using the dilution coating method.The ITS rDNA sequences of stroma,fruiting bodies,entomogenous fungi,and endophytic fungi were analyzed using molecular biological methods.Phylogenetic trees were constructed to determine the taxonomy of these species.All the fungi were cultured on potato dextrose agar medium on a small scale.The antibacterial activity of the crude extract of metabolites from the fungi were detected by an Oxford Cup agar diffusion assay with 6 pathogenic bacteria as an indicator strain.And the RAW 264.7 cells induced by lipopolysaccharide(LPS)were used as an inflammatory model to screen the anti-inflammatory activity of the crude extract of metabolites.Results The teleomorph entomogenous fungi had the highest homology with Ophiocordyceps sphecocephala(>98%).33 strains of entomogenous fungi were isolated from the stroma,fruiting body,and sclerotia tissues.Twenty-one strains of endophytic fungi were isolated from the insect body.The 54 strains of fungi were identified to belong to 2 phyla,4 classes,9 orders,11 families,and 16 genera.The results of the activity test showed that the crude extracts of 10 strains of fungi had different inhibitory effects on pathogenic bacteria.38 strains of fungal crude extracts exhibited varying degrees of anti-inflammatory activity.Conclusion Vespa mandarinia Smith Cordyceps sensu lato were rich in entomogenous and endophytic fungi.The metabolites of these associated fungi exhibited antibacterial and anti-inflammatory activity,offering valuable microbial resources for the discovery of active lead compounds.