雷公藤内生真菌的分离鉴定及活性研究

Isolation and bioactivity studies of endophytic fungi from Tripterygium wilfordii Hook.f.

摘要:

【背景】雷公藤是一种传统中药，具有多种药理活性和较高的应用价值。但由于产量少、提取过程复杂而限制了其在临床上的应用。植物内生真菌的次级代谢产物能够产生相似的药理活性，有望解决植物资源匮乏的难题，成为新药筛选的潜在资源。【目的】从卫矛科植物雷公藤中分离内生真菌，对其产物的体外抗菌活性及抗肿瘤活性进行测定，并利用活性菌株对雷公藤粗提物进行转化，以达到增效减毒的作用。【方法】采用组织块法从雷公藤茎中分离内生真菌；打孔法测定发酵产物对金黄色葡萄球菌和大肠埃希氏菌的抑菌活性；MTT (噻唑蓝)比色法测定发酵产物及转化产物的抗肿瘤活性；ITS (Internal transcribed spacer)序列测序分析确定活性菌株的种属。【结果】从雷公藤茎中分离得到43株内生真菌，其中5株对金黄色葡萄球菌有抑菌作用，LGT7几乎与1 μg/mL青霉素钠的作用效果相当；6株对MCF-7、SKOV3细胞有抗肿瘤活性，LGT7、LGT41对肿瘤细胞生长抑制率达90%以上；4株对雷公藤粗提物具有转化活性。活性菌株分别被鉴定为拟茎点霉和腐皮壳属。【结论】雷公藤内生真菌具有抗菌抗肿瘤作用，并可以通过转化产生增效减毒作用。

Abstract:

[Background] Tripterygium is a traditional Chinese medicine with great value of clinical application for its diversity pharmacological activity. However, its lack of output and complicated extraction process limit the clinical application. Secondary metabolites of plant endophytic fungi can produce similar pharmacological activity, which is expected to solve the problem of lack of plant and become a potential resource for screening new drugs. [Objective] To isolate endophytic fungi from tripterygium wilfordii Hook.f., and screen their anti-microbial and anti-tumor activities in vitro. To transform the crude extracts of tripterygium wilfordii Hook.f. with active strains to achive increasing efficiency and reducing toxicity. [Methods] Endophytic fungi were isolated from tripterygium wilfordii Hook.f. with tissue block method; antibacterial activity against Staphylococcus aureus and Escherichia coli was tested with disk diffusion method; anti-tumor activity to two cell lines (MCF-7/SKOV3) was tested with MTT (Thiazolyl blue) assay. The ITS (Internal transcribed spacer) sequence sequencing analysis was used to identify the active strains species. [Results] 43 strains of endophytic fungi were isolated from Tripterygium wilfordii Hook.f. Five of them showed bacteriostasis to Staphylococcus aureus and the activity of LGT7 was similar to that of 1 μg/mL of penicillin sodium; 6 of them showed cytotoxicity to MCF-7 as well as SKOV3 and the growth inhibition ratio of LGT7 and LGT41 was above 90%; 4 of them showed transforming activity to crude extracts from Tripterygium wilfordii. The active strains were identified as Phomopsis sp. and Diaporthe sp.. [Conclusion] Endophytic fungi isolated from Tripterygium wilfordii Hook.f. have anti-microbial and anti-tumor activity in vitro. They can enhance the activity and reduce the toxicity by transformation.