三株紫苏内生真菌代谢产物抗肿瘤活性的研究

Study on the antitumor activity of metabolites from three endophytic fungi of Perilla frutescens

目的研究3株从紫苏中分离得到的内生真菌次生代谢产物抗肿瘤活性。方法取处于指数生长期的5种人肿瘤细胞(人肝癌细胞株HepG2、人乳腺癌细胞株MCF-7、人肺腺癌细胞株NCI-H1975、人结肠癌细胞株HT-29、人前列腺癌细胞株DU-145),将细胞分为实验组[内生真菌发酵产物乙酸乙酯萃取部分,菌株暹罗刺盘孢菌(ZC-J4)和产黄青霉(BP-Y6)产物给药浓度为25、50、100、300和400μg·mL^(-1),菌株嗜线虫镰刀菌(ZP-G1)产物给药浓度为1.56、3.13、6.25、12.5、25和50μg·mL^(-1))]、阴性对照组、阳性对照组(阳性药物阿霉素,给药浓度为0.06、0.13、0.25、0.50和1.00μg·mL^(-1))。处理48 h后,用噻唑蓝法(MTT)法测定肿瘤细胞的增殖状况,计算内生真菌代谢产物对肿瘤细胞生长的抑制率和半数抑制浓度(IC50);并通过液相色谱-质谱联用技术(LC-MS)分析代谢产物中的主要成分。结果3株紫苏内生真菌发酵产物对肿瘤细胞的增殖有抑制效果,且在一定浓度范围内呈剂量依赖性;不同菌株代谢产物抑制作用差异较大,ZP-G1的次生代谢产物表现出的抑制作用最强,尤其是对HepG2细胞和HT-29细胞,IC50值分别为(6.28±0.09)和(9.03±1.05)μg·mL^(-1);BP-Y6和ZC-J4的次生代谢产物对肿瘤细胞的毒性较弱,对NCI-H1975的IC50值均大于400μg·mL^(-1)。LC-MS结果分析表明,3种内生真菌的代谢产物含有部分相同成分,真菌ZP-G1中含有香紫苏二醇、杨梅素等成分,在其余2株真菌中未发现。结论紫苏内生真菌的部分菌株具有抗肿瘤活性,菌株ZP-G1对肿瘤细胞增殖的抑制效果较好,具有进一步开发抗肿瘤天然活性成分的潜力。

Objective To study the anti-tumor activity of secondary metabolites of three endophytic fungi isolated from Perilla frutescens.Methods Five kinds of human tumor cells(HepG2,MCF-7,NCI-H1975,HT-29,DU-145)in exponential growth stage were cultured.The cells were divided into experimental group[ethyl acetate extract of endophytic fungal fermentation product,with drug concentrations of strains Colletotrichum siamense(ZC-J4)and Penicillium chrysogenum(BP-Y6):25,50,100,300,400μg·mL^(-1),product administration concentration of strain Fusarium nematophilus(ZP-G1):1.56,3.13,6.25,12.5,25,50μg·mL^(-1))],negative control group,positive control group(positive drug doxorubicin,administered at concentrations of 0.06,0.13,0.25,0.50,1.00μg·mL^(-1)).And treated for 48 hours,measure the proliferation status of tumor cells using methyl thiazolyl tetrazolium method(MTT),calculate the inhibitory rate and half inhibitory concentration(IC_(50))of endophytic fungal metabolites on tumor cell growth.The main components in the metabolites were analyzed using liquid chromatography-mass spectrometry(LC-MS).Results The results showed that the fermentation products of three endophytic fungi from Perilla frutescens had an inhibitory effect on the proliferation of tumor cells,and showed a dose-dependent effect within a certain concentration range.The inhibitory effects of metabolites from different strains vary greatly.The secondary metabolites of ZP-G1 exhibit the strongest inhibitory effect,especially on Hep G2 cells and HT-29 cells,with IC_(50)values of(6.28±0.09)and(9.03±1.05)μg·mL^(-1),respectively.The secondary metabolites of BP-Y6 and ZC-J4 are less toxic to tumor cells,and their IC_(50)values to NCI-H1975 are greater than400μg·mL^(-1).LC-MS analysis showed that the metabolites of the three endophytic fungi contained partially identical components.Fungus ZP-G1 contained components such as scareol glycol and myricetin,but was not found in the other two fungi.Conclusion Some strains of endophytic fungi from Perilla frutescens have anti-tumor activity,and strain ZP-G1 has a good inhibitory effect on tumor cell proliferation,which has the potential to further develop natural anti-tumor active ingredients.