两种药用植物内生真菌次生代谢产物及其生物活性的研究.doc

两种药用植物内生真菌次生代谢产物及其生物活性的研究 论文分类号： 2 学校代码： 10708 学 号： BS0908005 博 士 学 位 论 文 Dissertation for Doctors Degree 两种药用植物内生真菌次生代谢产物 及其生物活性的研究 SECONDARY METABOLITES OF ENDOPHYTIC FUNGI FROM TWO MEDICINAL PLANTS AND THEIR BIOACTIVITIES 张弘弛 指导教师姓名：马养民 学 科 名 称：应用化学 论文提交日期： 论文答辩日期： 学位授予单位： 答辩委员会主席： 评阅人： 申请 工学博士 学位论文 论文题目： 两种药用植物内生真菌次生代谢产物 及其生物活性的研究 学科门类：工学 一级学科：化学工程与技术 培养单位：化学与化工学院 博士生：张弘弛 导 师：马养民 教授 2012 年10 月 SECONDARY METABOLITES OF ENDOPHYTIC FUNGI FROM TWO MEDICINAL PLANTS AND THEIR BIOACTIVITIES A Dissertation Submitted to Shaanxi University of Science and Technology in Partial Fulfillment of the Requirement for the Degree of Doctor of Engineering Science By Zhang Hongchi Dissertation Supervisor: Professor Ma Yangmin October, 2012 两种药用植物内生真菌次生代谢产物及其生物活性的研究 摘 要 植物内生真菌是具有高度多样性的微生物资源，代谢产物多种多样，目 前被认为是抗菌、抗病毒、抗肿瘤、抗氧化、杀虫等天然活性物质的重要资 源。为了寻找更多的生物活性物质，本论文采用活性追踪的方法，研究了4 株来源于药用植物的内生真菌的活性代谢产物。内容包括：药用植物（杜仲 和无花果）内生真菌的分离的初步鉴定；抗菌活性菌株的分级组合筛选；活 性菌株的鉴定和发酵条件考察；活性成分的追踪分离；单体化合物的结构解 析；单体化合物生物活性的初步评价。概括如下: （1）以杜仲和无花果为研究对象，共获得了 119 株内生真菌。从杜仲 根、茎、叶中分离得到62 株内生真菌，初步形态学鉴定，这些内生真菌分 属于7 目，9 科，15 属，其中曲霉属Aspergillus sp.，镰孢霉属Fusrium sp.， 链格孢属 Alternaria sp.是杜仲内生真菌的优势种群。从无花果根、茎、叶中 分离得到57 株内生真菌，初步形态学鉴定，分属于7 目，10 科，21 属。其 中镰孢霉属Fusrium sp.，青霉属Penicillium sp.，曲霉属Aspergillus sp.是无 花果内生真菌的优势种属。不同部位分离内生真菌的数量、种类和分布不尽 相同，这就表明这两种药用植物的不同部位内生真菌的数量、分布和种群存 在差异。 （2 ）采用抗菌活性筛选为主、代谢产物化学成分筛选为辅的二级组合 筛选模式，得到如下结果：抗菌活性初筛确定活性菌株共有20 株（杜仲内 生真菌11 株，无花果内生真菌9 株），筛选率为16.8 %；抗菌活性复筛筛 选到12 株高抗菌活性的内生真菌，筛选率为10.1 %；结合化学成分复筛最 后确定了ER12 、EL09 、FR02 、FL10 这4 株具有高抗菌活性，并且代谢产 物种类丰富的内生真菌作为本论文的研究对象。 （3 ）根据菌株的形态、培养特征、18S rDNA 以及ITS 序列分析结果， 对活性菌株ER12 、EL09 、FR02 和FL10 进行菌种鉴定，分别鉴定为刀孢蜡 蚧菌、黑曲霉、溜曲霉以及三线镰孢。以菌丝体生物量、发酵产物重量、抗 菌活性、薄层色谱分析以及高效液相色谱分析为评价依据，对4 株活性内生 真菌进行了发酵产物稳定性试验，培养基选择试验，发酵时间试验，萃取实 验，确定了4 株活性内生真菌的扩大培养条件以及提取条件。 （4 ）经过扩大培养，采用常规的硅胶柱层析、反相硅胶柱层析，凝胶 I Sephadex LH-20 柱层析、制备薄层层析以及重结晶等分离手段，从4 株活性 内生真菌分离到 90 个单体化合物。利用各种现代波谱技术（EI-MS 、 1 13 1 1 HR-ESI-MS 、H-NMR 、 C-NMR 、DEPT 、H- HCOSY 、HSQC 、HMBC 等） 并结合化学方法鉴定了这90 个化合物的结构，其中新化合物3 个，分别是 malformin E （19）、cyclo- N-methyl-Trp-Leu （62 ）和helovlic acid ester （83）。 从ER12 中分离到34 个化合物，其代谢产物的结构类型是12 个环二肽 类化合物（3-10，13-16），1 个新的环五肽化合物（19），4 个fumiquinazoline 生物碱类化合物（17，18，22 ，23 ），2 个二苯酮类化合物（20 ，21 ），1 个 咪唑类化合物（12），2 个甾体化合物（1，2 ），8 个苯的衍生物（11，24-28 ， 31，32 ）及4 个核酸苷（29 ，30，33，34 ）。从EL09 中分离到18 个化合物， 其代谢产物的结构类型是 9 个甾体类化合物（38-44, 1, 2 ），3 个脂肪酸 （35-37 ），2 个呋喃类化合物（45 ，46 ），2 个多羟基醇化合物（47 ，48 ）及 2 个吲哚类化合物（49 ，50 ）。从FR02 中分离到22 个化合物，其代谢产物 的结构类型是 13 个吲哚二酮哌嗪类化合物（53-65，其中62 为新化合物， 65 为新天然产物），2 个萜类化合物（51，52 ），2 个甾体化合物（1，39 ）， 2 个多元羧酸（67，68 ）和1 个多元醇（66 ）。从FL10 中分离到33 个化合 物，其代谢产物的结构类型是7 个gliotoxin 类化合物（74-80），3 个烟曲霉 酸的衍生物（81-83 ），3 个环二肽化合物（13，14，84），5 个脂肪酸（69-71， 36，37 ），4 个甾体化合物（1，39，72，73 ），3 个苯的衍生物（87-89 ），4 个碱基或核酸苷（29 ，30，85，86）及4 个多元羧酸或多元醇（47 ，48 ，68， 90 ）。 （5 ）采用MIC 法，以11 种指示菌（4 个细菌和7 个植物病原真菌） 为模型，对4 株活性内生真菌产生的90 个单体化合物进行抑菌活性评价， 结果表明有 26 个化合物对指示菌显示了较强的抑制作用。新化合物 19 （malformin E ）和83 （helovlic acid ester ）对多种指示菌表现了很强的抗菌 作用。综合分析抗菌活性测试的结果，明确了4 株活性内生真菌生物活性物 质的类别，ER12 Lecanicillium psalliotae 的活性物质主要是fumiquinazoline 类生物碱和环肽类化合物；EL09Aspergillus niger 的活性物质主要是甾醇类 和呋喃类化合物；FR02 Aspergillus tamarii 的活性物质主要是吲哚二酮哌嗪 生物碱类化合物；FL10 Fusarium tricinctumi 的活性物质主要是gliotoxin 类 生物碱和烟曲霉酸类化合物。 采用MTT 法，以3 个肿瘤细胞株（MCF-7 人乳腺癌细胞、HepG2 人肝 癌细胞、A549 人肺癌细胞）为模型，对分离得到的新化合物和生物碱类代 II 谢产物的抗肿瘤活性进行了初步评价，结果表明这些化合物对不同肿瘤细胞 系表现出不同程度的增殖抑制活性。其中新化合物malformin E （19）MCF-7 和 HepG2 系表现出很好的细胞毒活性，IC50 分别是 0.65 和 2.42 M ； fumiquinazoline C （18）和fumiquinazoline J （23 ）对HepG2 和A549 肿瘤 细胞的增殖有抑制作用，verruculogen （56 ）对HepG2 和A549 肿瘤细胞表 现出很强的细胞毒性，IC50 分别是1.95 和2.56 M 。 总之，本文经过筛选获得 4 株活性内生真菌，通过分离鉴定阐明了 4 株内生真菌中90 个化合物的结构，其中包括3 个新化合物。评价了这些单 体化合物的抗菌活性和抗肿瘤活性，发现了26 个单体化合物具有较强抗菌 活性和4 个单体化合物具有较强抗肿瘤活性。上述研究结果证明植物内生真 菌作为植物生态一个重要的组成部分，是新结构和新活性物质的主要来源， 是寻找药物先导化合物的重要资源。 关键词： 药用植物；内生真菌；活性筛选；次级代谢产物；生物活性 III SECONDARY METABOLITES OF ENDOPHYTIC FUNGI FROM TWO MEDICINAL PLANTS AND THEIR BIOACTIVITIES ABSTRACT Endophytic fungi are a special and important group of microorganisms with taxonomic diversity, which show high potential as sources of antimicrobial, antiviral, anticancer, antioxidant, and insecticidal compounds. In order to investigate the potential bioactive compounds derived from endophytic fungi, this dissertation described the discovery of several antimicrobial compounds isolated from four endophytic fungi of medicinal plants by bioassay-guided fractionation. Studies include isolating of endophytic fungi, seleeting aimed strains, fermentation studies, bioassay-guided fractionation, structural elucidation, preliminary evaluation of antimicrobial and anti-tumor activities of pure compounds. The results are concluded as follows: Sixty-two strains isolated from roots, stems and leaves of Eucommia ulmoides, were classified, identified, and belonged to fifteen genera, nine families, and seven orders according to their morphological and micro-structure characters. Alternaria sp., Fusrium sp. and Alternaria sp. are dominant endophytic fungi population in Eucommia ulmoides . Fifty-seven strains from Ficus carice were classified, identified, and belonged to twenty-one genera, ten families, and seven orders while Fusrium sp., Penicillium sp. and Alternaria sp. are dominant endophytic fungi population. It showed that the quantities, species, and distribution of the endophytic fungus varied in different parts of Eucommia ulmoides and Ficus carice . The endophytic fungi isolated from Eucommia ulmoides and Ficus carice , were first screened by antimicrobial assay to obtain 20 strains which showed strong antimicrobial activities, and possessed 16.8 % of total 119 strains tested. The 20 strains were subjected to the flow antimicrobial secondary screening and 10 strains were found to have more strong activities, which possessed 10.1 % of total 119 strains tested. On the basis of antimicrobial activities, the results of IV TLC and HPLC analysis, four ested strains, ER12, EL09, FR02 and FL10, were chosen as the aimed strains to investigate their bioactive metabolites in this study. According to their morphology, cultural characteristics, 18S rDNA and ITS sequences, the strains ER12, EL09, FR02 and FL10 were indentified as Lecanicillium psalliotae, Aspergillus niger , Aspergillus tamari and Fusarium tricinctum, respectively. On the basis of mycelia biomass, weight of fermentation extracts, antimicrobial activities, the results of TLC and HPLC analysis, stability-tests for the bioactive components and fermentation studies perfomred on the aimed strains. The time course experiments for the fermentation of four strains were then carried out, followed by solvent extraction tests for the active components. Then, large-scale fermentation and preparation of the active fractions were perfomred to obtain the active fractions of the four strains. Ninety compounds were isolated from the four active strains by repeated column chromatography on silica gel, Sephadex LH-20, preparative thin layer chromatography PTLC and recrystallization. The structures of these compounds were elucidated by means of spectroscopic methods including 1 13 ESI-MS, HR-ESI-MS, 1D-NMR H NMR, C NMR, DEPT and 2D-NMR 1 1 H- H COSY, HSQC, HMBC as well as chemical method. Among them there were three new compounds, malformin E 19 , cyclo- N-methyl-Trp-Leu 62 and helovlic acid ester 83 . Thirty-four compounds were isolated from fungus ER12, and the chemical structure types of compounds were involved in cyclic dipeptides 3-10, 13-16 , malformin E 19 , fumiquinazolines 17, 18, 22, 23 , diphenyl ketones 20, 21 , imidazoles 12 , sterols 1, 2 , benzene derivatives 11, 24-28, 31, 32 and nucleosides 29, 30, 33, 34 . Eighteen compounds were isolated from fungus EL09, and the structure types of compounds were involved in sterols 38-44, 1, 2 , fatty acids 35-37 , furan derivatives 45, 46 , polyhydric alcohols 47, 48 and indole derivatives 49, 50 . Twenty-two compounds were isolated from fungus FR02, and the structure types of compounds were involved in indolyl diketopiperazine analogs 53-65 , terpenes 51, 52 , sterols 1, 39 , polycarboxylic acids 67, 68 and polyhydric alcohols 66 . Thirty-three compounds were isolated from fungus Fl10, and the structure types of V compounds were involved in gliotoxins 74-80 , helvolic acid derivatives 81-83 , cyclic dipeptides 13, 14, 84 , fatty acids 69-71, 36, 37 , sterols 1, 39, 72, 73 , benzene derivatives 87-89 , nucleosides 29, 30, 85, 86 , polyhydric alcohols and polycarboxylic acids 47, 48, 68, 90 . Ninety compounds were evaluated for their antimicrobial activities agains eleven tested strains. The results indicated that twenty-six compounds showed strong antimicrobial activities and the structure types of bioactive compounds were involved in indolyl diketopiperazines, gliotoxins, quinazoline alkaloids, cyclic dipeptides and sterols. Remarkably, malformin E and helovlic acid ester show strong antimicrobial activities against different tested strains. The results of antibacterial activity test indicated bioactive substances of four active fungi. Active substances of ER12 were mainly fumiquinazoline alkaloids and cyclic peptide compounds. Active substances of EL09 were sterols and furans. Active substances of FR02 were indolyl diketopiperazines. Active substances of FL10 were gliotoxins and helvolic acid derivatives. Three new compounds and isolated alkaloids were evaluated for their cytotoxicities against three cancer cell lines, MCF-7, HepG2 and A549, by the MTT method. The results indicated that four of them showed significant cytotoxicities against different cancer cell lines and others were weak. Among them, new compond, malformin E, showed significant inhibitory activities against MCF-7 and HepG2 cell lines with IC50 0.65, 2.42 M respectively. Fumiquinazoline C and fumiquinazoline J exhibited moderate activities against HepG2 and A549. Verruculogen showed significant inhibitory activities against HepG2 and A549 with IC50 1.95, 2.56 M. Summarily, this work obtained four active fungi from two medicinal plants and ninety compounds, including three new compounds and a new natural product, were isolated from the fungi. The bioactivities results indicated that twenty-six compounds showed significant antimicrobial activities and four compounds showed significant cytotoxicities against different cancer cell lines. This thesis indieated that endophytic fungi, as an important part of plant system, which contain novel and bioactive compounds in its secondary metabolisms, is an important source for searching lead compounds for drugs. KEY WORDS: medicinal plant, endophytic fungi, active screening, secondary metabolites, bioactivity VI 缩略词语 HPLC high performance liquid chromotography ITS Internal Transcribed Spacer PDB Potato Dextrose Broth SDB Sabourauds Dextrose Broth CDB Czapek-Dox Broth PYG Peptone Yeast extract Glucose medium MeOH methanol CHCl3 chloroform Pet. petroleum DMSO demethylsulfoxide EtOAc ethyl acetate TLC thin-layer chromatography Recrys. Recrystallize NMR nuclear magnetic resonance DEPT distortionless enhancement by polarization transfer HMBC 1H-detected heteronuclear multiple bond connectivity HMQC 1H-detected heteron