广藿香内生真菌Cerrena sp.A593次级代谢产物研究

发布时间：2018-09-04 07:39

【摘要】：广藿香是“十大广药”之一,为我国常用的芳香化湿中药,其主要的药用成分是挥发油,此外,还包括单萜烯、倍半萜烯、酮类、醇类和烷酸类等主要化学成分。经现代药理研究表明广藿香具有抗菌、抗炎、止咳和调节肠道功能等作用。由于植物内生真菌长期生活在植物体内的特殊环境中,产生独特的代谢途径,能产生结构复杂多样和具有丰富生物活性的次级代谢产物。为了发掘广藿香内生真菌的活性代谢产物,本论文从广藿香叶片中分离到1株活性菌株Cerrena sp.A593,通过对其发酵液粗提物进行生物活性测定,发现该真菌的发酵粗提物在100μg·mL-1的浓度下对人肝癌细胞(HepG-2)、人神经癌细胞(SF-268)和人乳腺癌细胞(MCF-7)的抑制率均超过80%。因此,对菌株A593进行了发酵培养、分离提取、结构鉴定和活性测试。本实验通过采用正相硅胶、C18反相硅胶、Sephadex LH-20凝胶以及高效液相色谱等色谱技术从广藿香内生真菌Cerrena sp.A593的发酵粗提物中分离到了13个化合物,经过全面的光谱数据鉴定以及文献比对,鉴定了这13个化合物的结构(图1),包括12个倍半萜类化合物chloriolin B(1),dihydrohypnophilin(2),cucumin F(3),pleurocybellone A(5),chloriolin C(6),antrodin A(7),connatusin A(8),10-hydroxyamorphan-4-en-3-one(9),cerrenain A(10),cerrenain B(11),cerrenain C(12),cerrenain D(13),以及一个对羟基苯乙醇(4),其中化合物10-13为新化合物。本实验中,所有化合物均为首次从Cerrena属真菌中分离得到。采用SRB法测定分离得到的单体化合物的抗肿瘤活性,结果显示,化合物1、5、6和10都具有抑制四种肿瘤细胞SF-268、MCF-7、HepG-2和NCI-H460增殖的活性,其中化合物5、6和10具有良好的细胞毒活性,IC50值分别为32.41、24.96、48.54、32.42μM;30.91、20.22、43.58、28.37μM;41.01、14.43、29.67、44.32μM。以金黄色葡萄球菌(S.Aureus)、枯草芽孢杆菌(B.subtilis)和大肠杆菌(E.coli)为作为供试菌株对所有分离得到的化合物进行体外抗菌活性实验,结果表明化合物5、6和10具有一定的抗菌活性。综上所述,本论文从广藿香内生真菌Cerrena sp.A593中分离鉴定了13个化合物,其中化合物1、5、6和10具有明显生物活性的化合物,化合物10-13为新化合物,研究结果为从内生真菌中筛选具有药用活性的物质提供理论基础。
[Abstract]:Pogostemon patchouli is one of the ten most widely used herbs in China. Its main medicinal component is volatile oil, in addition, it also includes monoterpene, sesquiterpene, ketone, alcohol and alkanoic acid, and other main chemical constituents, such as monoterpene, sesquiterpene, ketone, alcohol, alkanoic acid and so on. Modern pharmacological studies have shown that patchouli has antimicrobial, anti-inflammatory, antitussive and intestinal functions. Because endophytic fungi live in the special environment of plants for a long time, they produce unique metabolic pathways, which can produce secondary metabolites with complex structure and rich biological activity. In order to explore the active metabolites of endophytic fungi of patchouli, a bioactive strain Cerrena sp.A593, was isolated from the leaves of patchouli. It was found that the inhibitory rate of the crude extract of the fungus on human hepatoma cell (HepG-2), human neurocarcinoma cell (SF-268) and human breast cancer cell (MCF-7) was over 80% at the concentration of 100 渭 g mL-1. Therefore, the strain A593 was fermented, isolated, identified and tested for its activity. In this experiment, 13 compounds were isolated from the fermented crude extracts of Cerrena sp.A593, an endophytic fungus of Pogostemon patchouli, by using reverse phase silica gel Sephadex LH-20 gel and high performance liquid chromatography (HPLC). After comprehensive spectral data identification and literature comparison, The structures of the 13 compounds were identified (fig. 1), including 12 sesquiterpenes (chloriolin B (1) dihydrohypnophilin (2) cucumin F (3) chloriolin C (6) antrodin A (7) connatusin A (8) 10-hydroxyamorphan-4-en-3-one (9) cerrenain A (10) cerrenain B (11) cerrenain C (12) cerrenain D (13), and a p-phenylethanol (4), in which 10-13 are new compounds. In this experiment, all compounds were isolated from Cerrena fungi for the first time. The antitumor activity of the isolated monomers was determined by SRB method. The results showed that compounds 1, 5 and 10 had the activity of inhibiting the proliferation of SF-268,MCF-7,HepG-2 and NCI-H460 in four kinds of tumor cells. The IC50 values of compounds 5X6 and 10 were 32.41 ~ 24.96U 48.54N ~ 32.42 渭 m ~ 30.91g ~ 20.222.22 ~ 43.5N ~ 28.37 渭 M ~ (-1) ~ 14.43n ~ (29.67) 渭 m ~ (-1) ~ (29) ~ 44.32 渭 m ~ (-1), respectively. Staphylococcus aureus (S.Aureus), Bacillus subtilis (B.subtilis) and Escherichia coli (E.coli) were used as the tested strains. To sum up, 13 compounds were isolated and identified from the endophytic fungus Cerrena sp.A593 of patchouli. Compounds 1, 5, 6 and 10 have obvious biological activity, and compound 10-13 is a new compound. The results provide a theoretical basis for the screening of substances with medicinal activity from endophytic fungi.
【学位授予单位】：广东药科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R915

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