产抗肿瘤活性物质喜树内生菌分离鉴定

Isolation and identification of Camptotheca acuminata endophytes producing antitumor activity

[目的]从药用植物喜树(Camptotheca acuminate Decne.)中筛选产抗肿瘤活性物质的内生菌。[方法]从喜树不同部位分离纯化喜树内生菌,对其发酵液进行HPLC检测,筛选产生喜树碱(Camptothecin,CPT)和10-羟基喜树碱(Hydroxyamptothecin,10-OH-CPT)的菌株,采用MTT法检测其体外抗肿瘤活性。对抗肿瘤活性强的菌株进行菌落观察、显微镜检及ITS序列分析,鉴定菌种。[结果]从喜树分离出内生菌72株,其中4株真菌代谢过程产生喜树碱或10-羟基喜树碱,以人肝癌细胞(HEP-G2)增殖抑制率为指标,结果发现菌株CS-16代谢产物对HEP-G2细胞增殖抑制率最高,达到83.76%。菌种鉴定结果为泡盛曲霉(Aspergillus awamori Nakaz.)。[结论]菌株CS-16发酵过程中可以产生10-羟基喜树碱396μg/L和喜树碱973μg/L,且对人肝癌细胞HEP-G2的增殖抑制率最高,试验结果对开发产生抗肿瘤药物的药用植物内生菌具有较大的研究价值。

[Objective]To screen the endophytic bacteria producing antitumor active substance from Camptotheca acuminate decne.[Method]Camptothecin and 10-hydroxyamptothecin producing strains were isolated and purified from different parts of Camptotheca acuminata,and their fermentation broth was detected by HPLC,the anti-tumor activity in vitro was measured by MTT assay.The strains with strong anti-tumor activity were identified by colony observation,microscopic examination and ITS sequence analysis.[Result]72 endophytic bacteria were isolated from Camptotheca acuminata,and 4 of them produced camptothecin or 10-hydroxycamptothecin in the process of metabolism,the results showed that the metabolic product of strain CS-16 had the highest inhibitory rate(83.76%)on the proliferation of HEP-G2 cells.Strain CS-16 was identified as Aspergillus awamori Nakaz..[Conclusion]Strain CS-16 could produce 10-hydroxycamptothecin 396μg/L and Camptothecin 973μg/L during fermentation,the results are of great research value for the development of medicinal plant endophytes producing anti-tumor drugs.