【作者】 吴令上；

【导师】 秦路平； 韩婷；

【作者基本信息】 第二军医大学 ， 生药学， 2012， 博士

【摘要】 本课题以我国珍稀保护药用植物南方红豆杉内生真菌为研究对象，对其生物多样性及代谢产物和其与宿主的相关性进行系统地研究，以期为开发新的天然药物资源和提高药材品质提供新的思路。与已报道的同属植物相比，南方红豆杉具有更加丰富的内生真菌种类。从3个产地（江西井冈山、浙江景宁、重庆缙云山）南方红豆杉3个部位（叶片、枝条、树皮中）共分离得到2442株内生真菌，根据菌株群落的培养特征及5.8S和ITS区片段序列相似性比较和系统发育分析，共鉴定为145个分类单元（分子鉴定143个分类单元，2个未能进行分子鉴定的形态型），归属到真菌界3个门6个纲20个目66个属，其中125个分类单元（占总数的86.2%）属于子囊菌门，14个（占9.8%）属于担子菌门，5个（3.4%）属于接合菌门，1个（0.7%）未确定分类地位。南方红豆杉内生真菌生物多样性及丰度受组织类型影响显著，枝条、树皮中内生真菌多样性及丰度为叶片的1.2-3倍。地理位置、组织类型对南方红豆杉内生真菌组成及分布具有显著的影响，优势类群（目、属、种水平上）存在产地间、部位间明显差异。鉴定的145个分类单元中，仅10个分类单元为3个地点共有，20个分类单元为2个地点共有，其他分类单元只在1个地点出现。Hypoxylon、Pezicula、Pestalotiopsis为不同产地特有的优势属；最优势目Magnaporthales、Botryosphaeriales、Glomerellales、Diaporthales、Xylariales、Helotiales、Hypocreales，最优势属Phyllosticta/Guignardia、Colletotrichum/Glomerella、Phomopsis、Hypocrea/Trichoderma、Pezicula的分布具有显著的组织特异性。大多数优势种都表现出明显的地理特异性或组织特异性，许多分离频率低的菌还表现出地理专一性或组织专一性。由于产地和部位的作用，更多种类的内生真菌从南方红豆杉中获得。南方红豆杉丰富多样的内生真菌种类是极其重要的资源宝库，为内生真菌后续研究工作提供了重要素材。利用高效液相色谱法测定有效成分（Taxol、10-DABⅢ）的含量，并结合南方红豆杉相关化学成分的HPLC-TOF/MS数据分析，比较3个产地南方红豆杉3个部位化学成分包括有效成分的差异。结果表明南方红豆杉Taxol与10-DABⅢ含量存在部位间、产地间显著差异。3个产地的南方红豆杉的Taxol含量均以树皮部位明显高于枝条、叶片部分，且以江西产地树皮含量最高；3个产地的南方红豆杉10-DABⅢ含量则均以叶片明显高于枝条、树皮部分，且以江西产地叶片中含量最高。从化学成分质谱数据初步鉴别结果来看，南方红豆杉植物中的化学成分极为复杂，包含紫杉烷类化合物，部分松香烷类、木脂素类等化合物。南方红豆杉叶片、枝条、树皮3个部位化学成分的差异显著，这与内生真菌菌群部位间的差异结果相符。通过对飞行时间质谱数据的PLSDA分析筛选出18个在部位间分布有明显差异的化合物，比对确立的南方红豆杉化学成分数据库初步鉴别出11个化合物，8个紫杉烷二萜类（P2、P5-P7、N1、N2、N7-N9）、1个松香烷二萜类（N6）、和2个木脂素类（N3、N5）。进一步考察南方红豆杉化学成分包括有效成分与内生真菌之间的关系，发现18个部位间差异化合物的量与一些具有组织特异性的内生真菌有关，它们之间存在正相关关系和负相关关系，表明内生真菌部位间的差异与宿主植物部位间成分差异相关。有效成分（Taxol与10-DABⅢ）含量与一些有组织特异性的优势类群的分布存在显著的相关性。与Taxol含量显著相关的有：在目水平上，Helotiales、Hypocreales、Xylariales目；在属水平上，Hypocrea，Pezicula属；在种水平上，Hypocrealixii。与10-DABⅢ的含量显著相关的有：在目水平上，Botryosphaeriales、Magnaporthales目；在属水平上，Fusarium/Gibberella、Phyllosticta/Guignardia属；在种水平上，Colletotrichumgloeosporioides,Glomerella lindemuthiana,Magnaporthales sp.2、Xylariales sp.1。Taxol或10-DABⅢ的含量和与其相关的不同内生真菌类群的相对频率之间的关系均最符合立方模型（CubicModel）。研究阐明内生真菌对药材品质（有效成分的含量）的影响，对提高药材品质、筛选优质药材具有重要的理论价值和现实意义。通过稻瘟霉模型筛选和与宿主化学成分比较分析相结合的方法，对鉴定的145个内生真菌分类单元发酵提取物进行筛选，以Perenniporiatephropora（Z41）的抗稻瘟霉活性最好，并且与宿主共有的分子特征离子数目最多，因此对该菌发酵物乙酸乙酯提取部位进行分离，利用波谱方法共鉴定了5个化合物，1个新型的倍半萜类化合物PerenniporinA和4个已知化合物2-5依次为ergosterol、rel-(+)-(2aR,5R,5aR,8S,8aS,8bR)-decahydro-2,2,5,8-tetramethyl-2H-naphtho[1,8-bc]furan-5-ol、albicanol、para-methoxybenzoicacid，这些化合物都是首次从Perenniporia属真菌分离得到。Perenniporia tephropora发酵物的乙酸乙酯提取物（EPT）及5个化合物对HeLa细胞，SMMC-7721和PANC-1细胞系具有较好的细胞毒活性。倍半萜类化合物1、3、4都是潜在的肿瘤抑制剂。以生物活性和化学成分分析相结合的方法筛选得到的担子菌门内生真菌Perenniporia tephropora在筛选活性或新型化合物展现出较好的应用前景。更多还原

【Abstract】 In order to find new resources for novel drugs and new way to improve the quality of herbal materials, plant chemistry, endophytic fungi and their secondary metabolites in the bark, branches and leaves of Taxus chinensis var. mairei from the Jiangxi, Zhejiang and Chongqing regions of China were mainly studied. The results were showed as follows.T. chinensis var. mairei from three localities (Jiangxi, Zhejiang and Chongqing in China) harbours high and novel diverse fungi. A total of2442strains were isolated from bark, branches and leaves of T. chinensisvar. mairei at three localities. A total of145fungal taxa were identified according to the morphology and ITS sequences, of these125taxa (86.2%) belonged to Ascomycota,14(9.7%) to Basidiomycota,5(3.4%) to Zygomycota and1(0.7%) to undefined fungus. The species richness and diversity of endophytic fungi were significantly affected by tissue, and were1.2-3fold higher in the branches and bark when compared to the leaves.The shape of the endophyte assemblages were strongly influenced by locality and tissue. In addition, the distributions of dominant fungi at different taxonomic levels including orders, genera and species levels were greatly influenced by locality and tissue. Hypoxylon was the dominant genus in Zhejiang, and Pezicula and Pestalotiopsis were the dominant genera in Jiangxi and Chongqing. Among the145fungi,10taxa shared among the three localities,20taxa occurred at two localities, and the others were special to one locality. The most dominant orders i.e. Magnaporthales, Botryosphaeriales, Glomerellales, Diaporthales, Xylariales, Helotiales, Hypocreales and the most dominant genera Phyllostictal Guignardia, Colletotrichuml Glomerella, Phomopsis, Hypocreal Trichoderma, Pezicula showed obvious tissue-specificity. Most of the dominant fungi showed spatial heterogeneity and tissue specificity or preference and many fungal taxa with low frequency were special to one locality or one tissue. More fungal taxa could be obtained in T. chinensis var. mairei as functions of locality and tissue. These endophytic fungi are important material sources and are currently being investigated by our laboratory for bioactive substances which can be screened for novel drugs.HPLC for determining the contents of taxol and10-DAB Ⅲ combined with HPLC-TOF/MS data analysis on the chemical compositions were applied to compare the differences in the chemical compositions of yew trees among the localities or tissues. The results showed that taxol and10-DAB Ⅲ were both found to be significantly different in quality between tissues and across the localities. The levels of taxol and10-DAB III in yew trees from Jiangxi both were the highest. At each locality, the leaf contained the highest concentration of10-DAB Ⅲ while the bark contained the highest concentration of taxol.The chemical compositions in plants of T. chinensis var. mairei were extremely complex,148compounds were preliminary identified including the taxanes, the abietane, lignan and so on. The chemical compositions among tissues were significantly different. This result was agreed with the difference in the distributions of domiant fungi among tissues. According to PLSDA analysis on TOF mass data,18compounds with great difference among tissues were selected. Of them,11compounds including8taxane diterpenoid (P2, P5-P7, N1, N2, N7-N9),2lignans (N3, N5), and abietane diterpenes (N6) were preliminary identified by comparing to chemical composition database of T. chinensis var. mairel.The relationships between the18selected compounds and endophytes assembleges were further studied. The result that these compounds were related to the distribution of some endophytic fungi with tissue-specificity indicated that endophytic fungal compositions and chemical compositions were related. There was a significant correlation between the levels of active ingredients (taxol and10-DAB Ⅲ) and the distribution of some endophytic fungi especially the dominant groups. Several dominant fungi at different taxonomic levels were significantly related to the content of taxol, e.g. Helotiales, Hypocreales and Xylariales at order level, Hypocrea and Pezicula at the genus level, and Hypocrea lixii at the species level. Also, several dominant fungi at different taxonomic levels were significantly related to the content of10-DAB III, e.g. Botryosphaeriales, Magnaporthales at order level, Fusarium/Gibberella and Phyllosticta/Guignardia at genus level and Colletotrichum gloeosporioides, Glomerella lindemuthiana, Magnaporthales sp.2and Xylariales sp.l at species level. The relationships between RF of these mentioned dominant fungi and the content of taxol or10-DAB III were all fit to the Cubic Model. Clarified the influences of endophytic fungi on the quality of medicinal plants (the levels of active ingredients) has important theoretical value and practical significance in improving the quality of medicinal plants and screening high-quality medicinal plants. Based on bioactivity and comparative analysis on chemical composition, the EtOAc extract of a culture broth of the endophytic fungus Perenniporia tephropora Z41, with strongest anti-Pyricularia oryzae activity and largest number of similar compositions with the host, afforded a new sesquiterpenoid, perenniporin A (1), together with four known compounds, ergosterol (2), rel-(+)-(2aR,5R,5aR,8S,8aS,8bR)-decahydro-2,2,5,8-tetramethyl-2H-naphtho[1,8-bc]furan-5-ol (3), albicanol (4) and para-methoxybenzoic acid (5). Their structures were elucidated by means of spectroscopic methods. All the isolated compounds and the EtOAc extract of Perenniporia tephropora Z41(EPT) were evaluated for their cytotoxic activity against three human cancer cell lines (HeLa, SMMC-7721and PANC-1). EPT demonstrated significant cytotoxicity with IC50values ranging from2to15μg/mL. Compound2was the most cytotoxic constituent against the tested cell lines with IC50values of1.16,11.63and11.80μg/mL, respectively, while compounds1,3,4and5exhibited moderate cytotoxicity with IC50values ranging from6to58μg/mL. Our results indicate that sesquiterpenoids1,3, and4could be valuable candidates as potent tumor inhibitors and be beneficial in the therapy of cancer diseases. Our study also underscores that endophyte P. tephropora is a promising sources of natural bioactive and novel sesquiterpenoids metabolites, though without diterpenoids like taxols.更多还原