采用OSMAC法对Streptomyces roseofulvus M63次级代谢产物及其抗肿瘤成分的研究

Isolation of Secondary Metabolites from Streptomyces roseofulvus M63 by OSMAC Methods and Their Anti-tumor Activity

本文采用OSMAC方法通过4种发酵方式从玫瑰黄链霉菌Streptomyces roseofulvus M63中获得其次级代谢产物,并研究它们的抗肿瘤活性。首先应用正相硅胶柱色谱、薄层色谱、羟丙基葡聚糖凝胶Sephadex LH-20、反向硅胶、制备色谱等多种方法进行分离纯化,其次用NMR和MS等波谱技术解析化合物的结构,最后采用MTT法筛选具有抗肿瘤活性的化合物。从玫瑰黄链霉菌M63中共分离鉴定出14个化合物,分别为对甲基苯酚(1)、4-羟基苯乙醇(2)、2,3,4,5-四羟基苯甲基酯(3)、3,4-二羟基苯甲酸(4)、4-羟基苯甲酸(5)、1H-吡咯-2-羧酸(6)、(22E,24R)-麦角甾-7,22-二烯-3β-醇(7)、4-羟基苯甲醛(8)、3β-羟基-5α,8α-过氧化麦角甾-6,22-二烯(9)、4-羟基苯乙酸(10)、5-(2-甲基苯基)-4-戊烯酸(11)、3β-羟基-麦角甾-5,7,22-三烯(12)、(5-羟基-2-氧代-2H-吡喃-4-基)乙酸甲酯(13)、(4S,5S,9AR-5-(E)-4-羧基,甲基-8-烯)-10,13-二甲基-6-萘甲酸(14)。上述14个化合物均为首次从玫瑰黄链霉菌M63中分离得到,其中化合物11、13对子宫颈癌细胞(He La)、卵巢癌细胞(SKOV3)均有不同程度的抑制作用;化合物11在24h时的半抑制浓度值(IC_(50))低于阳性对照物顺铂,为38.26、14.25μmol/m L;化合物13对He La细胞、Skov3细胞在24、48、72 h时半抑制浓度值(IC_(50))分别为25.62、11.34、6.524μmol/m L;7.892、10.32、5.671μmol/m L。

In this study, secondary metabolites of Streptomyces roseofulvus M63 were obtained under four fermentation ways using OSMAC methods, and their anti-tumor activity was examined, Compounds were firstly isolated and purified by column chromatography on Silica gel, Silica gel thin layer chromatography, Sephadex LH-20, reverse phase silica gel and preparative chromatography. The structures of purified compounds were identified by NMR and MS. Their anti-tumor activity was eventually evaluated using MTT method. The results showed that, fourteen compounds were purified and identi- fied as p- cresol （ 1 ） ,4 -hydroxyphenyl ethanol （ 2 ）, 2,3,4,5-tetra hydroxy, methyl ester （ 3 ）, 3,4 -dihydroxy benzoic acid （4） ,4-hydroxy benzoic acid （ 5 ）, 1H-Pyrrole-2-carboxylic acid （ 6 ）, （ 22E, 24R ） -Ergosta-7,22-dien-3β-ol （ 7 ）, 4-hy- droxy benzaldehyde （8） ,3β-hydroxy-5α,3β-hydroxy-5α,8α-ergosterol peroxide-6,22-diene（9） ,4-hydroxy phenylacetic （ 10 ）, 5- （2-Methylphenyl） -4-penten oic acid （ 11 ）, 3β-hydroxy-ergosterol-5 ,7 ,22 -triene （ 12 ）, （ 5-hydroxy-2 -oxo-2H-py- ran-4-yl） acetate （ 13）, （4S,5S,9αR） -5-（E） -4-carboxy methyl-8-en） -10,13-dimethyl-6- deca hydrronaphthalene-carboxylic acid（14）. It was the first time to obtainthese compounds from Streptomyces roseofulvus M63, cervical cancer （He-La） cells and ovarian cancer（ Skov3 ）were inhibited by compound 11,13 for different degrees. The IC50 values of compound 11 was 38.26 and 14.25 μmol/mL that less thanpositive control cisplatin only at 24h. The IC50 values of compound 13 on HeLa cells and Skov3 cells was 25.62,11.34,6. 524 and 7. 892,10.32,5. 671, at 24 h,48 h and 72 h,respectively.