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题名: 铁线莲和滑桃树及其共生菌的抗真菌成分研究
作者: 杜芝芝
学位类别: 博士
答辩日期: 2005
授予单位: 中国科学院昆明植物研究所
授予地点: 中国科学院昆明植物研究所
导师: 郝小江
关键词: 铁线莲 ; 清桃树 ; 抗真菌 ; 美登木素类化合物 ; 内生菌 ; 化学防御
学位专业: 植物学
中文摘要: 近年来由于真菌感染呈上升趋势,以及真菌对目前使用药物产生耐药性等问题,所以寻找新的抗真菌剂刻不容缓。本论文以从植物和植物共生微生物天然产物中寻找具有抗真菌活性的化合物为主线,在初步活性筛选的基础上,选定以藏药甘青铁线莲(Clematis tangutica)以及热带植物滑桃树(Trewia nudiflora)作为研究对象,从植物体、植物愈伤组织培养、植物共生微生物三个方面开展了相关研究,取得了以下研究结果:从甘青铁线莲地上部分的乙醇提取物中,通过活性追踪分离得到两个新的抗真菌三菇皂昔。根据光谱数据结合化学反应,将它们的结构分别鉴定为3-0-a-L-arabinoPyranosyl hederagenin28-O-a-L-rhamnopyranosyl ester(1)和3-0-β-D-glucopyranosyl-(1-4)-a-L-arabinopyranosyl hederagenin28-0-α-L-rhamnopyranosyl ester②。这两个化合物对7株真菌的抗菌活性试验表明,它们对Saccharomyces cerevisiae较好的抑制活性(MIA2.5μg/disc),与阳性对照两性霉素B的活性相当;对Penicillium avellaneumUC-4376,Cadidan glabrata, Trichosporon beigelii的抑菌活性一般(MIA10μg/disc)。对用于筛选的大多数菌株,化合物2比化合物1的活性更强。关于该属植物的三菇皂昔已有较多的研究和文献报道,但这是首次报道从该属植物发现具有抗真菌活性的三菇皂昔。活性追踪分离结合传统的植物化学成分分离方法,对滑桃树果皮不同材料 (新鲜、风干、发霉、机械损伤等)的抗真菌等化学成分进行了对照研究,从中共分离并鉴定了25个不同化合物,其中有3个为新的阿替烷型二菇(BP3,BP7,AC2),1个为新的闭花烷型二菇(TC7)。闭花烷型二枯与分离到的其它二菇有显著差异,对其来源进行了初步的探讨,实验结果表明只有发霉的滑桃树果皮中含有该化合物,因此推断Tc7可能为滑桃树果皮受真菌胁迫所产生的应激代谢产物(stress metabolite)。活性追踪分离实验结果表明,滑桃树果皮中抗真菌活性最强的成分为美登木素类化合物,并且以特利回生(trewiasine)为主,因此认为美登木素类化合物是滑桃树植物的组成型抗真菌次生代谢产物,它可能在植物对外界有害微生物的化学防御中起重要作用。从滑桃树种子萌发的无菌苗成功地诱导出愈伤组织,并进行了继代培养;对愈伤组织的化学成分进行了研究,分离鉴定了巧个化合物,包括5个新的阿替烷型二菇(T ncl,10,12,13,14)(其中2个同时在滑桃树果皮中分离得到);实验结果表明:滑桃树愈伤组织在目前使用的培养条件下不能产生美登木素类化合物。从温室栽培滑桃树的不同部位、滑桃树无菌苗、愈伤组织进行内生菌的分离,共分离纯化了未鉴定的内生真菌57株(i solates)、内生放线菌10株、内生细菌2株。采用共培养拮抗实验(antagonistic test)与小规模发酵产物的活性测试相结合,对分离到的菌株进行抗真菌活性筛选,发现具有抗真菌活性的菌株10株,其中真菌8株、放线菌2株。对活性较好的4株真菌进行了分类学鉴定,从中挑选了2株(ITnRZ-4和ZTnPI-2)进行了化学成分研究。活性追踪从菌株ITnRu-4(lindrocarPon sP.)琼脂固体发酵(7L)产物中,分离到抗真菌活性成分-化合物CSEI(brefeldinA)及其衍生物CSE3(7,acetylbre免ldinA)、cSE4(7-dehydrobrefeldinA),其中CSE3为一新天然产物。初步的抗癌活性测试表明:化合物CSEI及CSE3具有抗癌活性。这是首次从柱抱属(。lindrocarPon)真菌中分离到这类化合物。从中分离到的其它次生代谢产物还包括鞘糖脂(cSES)、街体和脂肪酸等。活性追踪结合常规的化学成分分离方法,对菌株ZTnPI-2(Fusarium sP.)的次生代谢产物进行了研究,从ZL琼脂固体发酵的产物中分离并鉴定了3个化合物FSI、FsZ、Fs3,其中FS3为一新化合物。初步的抗癌活性测试结果表明:Fsl具有抗癌活性。最后在论文的综述部分,介绍了植物利用次生代谢产物作为防御外界病原菌、植食性动物等侵害的武器,这些化学防御涉及3种不同类型:成型化学防御、诱导型化学防御以及共生化学防御。
英文摘要: In the past years, the increasing prevalence of fungal infection and inefficiency of antifungal therapy because of drug resistance have stimulated the search for new antifungal agents. The initiatives of this dissertation were to investigate antifungal compounds from the natural products of plants and plant commensal microbes. Based on traditional knowledge and bioassay, two plant species, Clematis tangutica and Trewia nudiflora were selected for further investigation. Research works were carried out on three aspects including antifungal chemical constituents from the two plants, plant cell cultures, and the isolation and antifungal secondary metabolites of endophytes from T. nudiflora. The main findings of this dissertation are summarized as follow: From bioassay-guided fractionation of the ethanol extract of aerial parts of C. tangutica, two new antifungal triterpene saponins were isolated and identified. Their structures were determined to 3-O-a-L-arabinopyranosyl hederagenin 28-Q-a-L-rhamnopyranosyl ester (1) and 3-O-(3-D-glucopyranosyl-(l->4)-a-L-arabinopyranosyl hederagenin 28-0- a-L-rhamnopyranosyl ester (2) on the basis of spectral data and chemical evidence. Inhibitory activities of the two saponins against seven fungal strains were evaluated. Compounds 1 and 2 showed evident antifungal activity (Minimum inhibition amount ~ 2.5u.g/disc) against Saccharomyces cerevisiae, similar as the positive control amphotericin B. The antifungal activity of compound 2 is stronger than compound 1. Furthermore, even though the triterpene saponins from Clematis plants were described in many references it is the first time to identify new antifungal saponins from genus Clematis The investigation of antifungal constituents and other components from different pericarp samples (fresh, dry, mold-infected, damaged) of T. nudiflora resulted in the isolation of 25 different compounds, including three new atisine type diterpenes (Bp3, Bp7, AC2) and one cleistanthin diterpene (TC7). The striking difference of TC7 from other diterpenes isolated from the pericarp of T. nudiflora, which provided us the inspiration to investigate the origin of this compound. The relevant results showed that only the pericarp which damaged by fungal-infection could be the host of TC7. Therefore TC7 was deduced as a stress metabolite of pericarp infected by fungi. Trace amounts of maytansinoids were isolated by antifungal-activity-guided fractionation of the ethanol extracts of pericarp of T. nudiflora and determined by LC-ESI-MS analysis; they were prominent antifungal constituents in the pericarps of T. nudiflora. The calli were successfully induced from germ free seedlings of T. nudiflora and remained successive transfer culture for more than one year. Moreover, studies on the chemical constituents of callus culture yield 15 compounds, including 5 new atisine type diterpenes (among them 2 diterpenes were isolated from pericarp siraultaneouly). But no maytansinoids were isolated or detected by LC-ESI-MS in the extracts of the calli, which means the callus culture can not produce maytansinoids on such culture conditions. 57 fungi isolates, 10 actinomycete isolates and 2 bacteria isolates were isolated from different plant parts and calli of T. nudiflora, Antifungal bioassays were carried out by antagonistic test as well as activity detection of small scale fermentation metabolites. 10 isolates showed antifungal activity including 8 fungi strains and 2 actinomycete strains, among which 4 strains of fungi were determined with prominent antifungal activities. Antifungal secondary metabolites of strains lTnR2-4 and 2TnPl-2 were studied accordingly. Bioassay-guided fractionation of solid agar fermentation (7L) extracts of strain lTnR2-4 lead to the isolation of a macrolide compound CSEl (brefeldin A) and its derivates CSE3 (7-acetylbrefeldin A)> CSE4 (7-dehydrobrefeldin A), including one new natural product CSE3. This is the first report of isolation brefeldin A and its derivates from Cylindrocarpon, Antitumor bioassay showed that CSE3 and CSEl have activity. Other chemical constituents isolated from strain lTnR2-4 involve steroid, fatty acid and cerebroside. Bioassay-guided fractionation of solid agar fermentation (2L) extracts of strain 2TnPl-2 affored 3 compounds (FS1, FS2, FS3), including one new compound Fs3. The test of antitumor activity showed that compound Fsl had prominent bioactivities. In the last part of this dissertation, a review which described how plants use secondary metabolites to defend the attack or invasion by phytopathogens and herbivor was presented. The chemical defenses are divided into 3 different types, e.g. constituentive chemical defense, induced chemical defense and symbionts for chemical host defense.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/778
Appears in Collections:昆明植物所硕博研究生毕业学位论文_学位论文

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铁线莲和滑桃树及其共生菌的抗真菌成分研究.杜芝芝[d].中国科学院昆明植物研究所,2005.20-25
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