發(fā)布時(shí)間：2018-09-04 07:39

【摘要】：廣藿香是“十大廣藥”之一,為我國常用的芳香化濕中藥,其主要的藥用成分是揮發(fā)油,此外,還包括單萜烯、倍半萜烯、酮類、醇類和烷酸類等主要化學(xué)成分。經(jīng)現(xiàn)代藥理研究表明廣藿香具有抗菌、抗炎、止咳和調(diào)節(jié)腸道功能等作用。由于植物內(nèi)生真菌長期生活在植物體內(nèi)的特殊環(huán)境中,產(chǎn)生獨(dú)特的代謝途徑,能產(chǎn)生結(jié)構(gòu)復(fù)雜多樣和具有豐富生物活性的次級(jí)代謝產(chǎn)物。為了發(fā)掘廣藿香內(nèi)生真菌的活性代謝產(chǎn)物,本論文從廣藿香葉片中分離到1株活性菌株Cerrena sp.A593,通過對(duì)其發(fā)酵液粗提物進(jìn)行生物活性測定,發(fā)現(xiàn)該真菌的發(fā)酵粗提物在100μg·mL-1的濃度下對(duì)人肝癌細(xì)胞(HepG-2)、人神經(jīng)癌細(xì)胞(SF-268)和人乳腺癌細(xì)胞(MCF-7)的抑制率均超過80%。因此,對(duì)菌株A593進(jìn)行了發(fā)酵培養(yǎng)、分離提取、結(jié)構(gòu)鑒定和活性測試。本實(shí)驗(yàn)通過采用正相硅膠、C18反相硅膠、Sephadex LH-20凝膠以及高效液相色譜等色譜技術(shù)從廣藿香內(nèi)生真菌Cerrena sp.A593的發(fā)酵粗提物中分離到了13個(gè)化合物,經(jīng)過全面的光譜數(shù)據(jù)鑒定以及文獻(xiàn)比對(duì),鑒定了這13個(gè)化合物的結(jié)構(gòu)(圖1),包括12個(gè)倍半萜類化合物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),以及一個(gè)對(duì)羥基苯乙醇(4),其中化合物10-13為新化合物。本實(shí)驗(yàn)中,所有化合物均為首次從Cerrena屬真菌中分離得到。采用SRB法測定分離得到的單體化合物的抗腫瘤活性,結(jié)果顯示,化合物1、5、6和10都具有抑制四種腫瘤細(xì)胞SF-268、MCF-7、HepG-2和NCI-H460增殖的活性,其中化合物5、6和10具有良好的細(xì)胞毒活性,IC50值分別為32.41、24.96、48.54、32.42μM;30.91、20.22、43.58、28.37μM;41.01、14.43、29.67、44.32μM。以金黃色葡萄球菌(S.Aureus)、枯草芽孢桿菌(B.subtilis)和大腸桿菌(E.coli)為作為供試菌株對(duì)所有分離得到的化合物進(jìn)行體外抗菌活性實(shí)驗(yàn),結(jié)果表明化合物5、6和10具有一定的抗菌活性。綜上所述,本論文從廣藿香內(nèi)生真菌Cerrena sp.A593中分離鑒定了13個(gè)化合物,其中化合物1、5、6和10具有明顯生物活性的化合物,化合物10-13為新化合物,研究結(jié)果為從內(nèi)生真菌中篩選具有藥用活性的物質(zhì)提供理論基礎(chǔ)。
[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.
【學(xué)位授予單位】：廣東藥科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R915

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