【作者】 夏静；

【导师】 潘超美；

【作者基本信息】 广州中医药大学 ， 生药学， 2012， 硕士

【摘要】 前人研究表明,很多传统中药的内生菌能产生与宿主相同或相近的有效成分。为了更好的保护野生中药资源,近年来许多学者开始从药用植物内生菌中筛选活性菌株或活性代谢产物、利用微生物发酵技术实施工业化生产天然药物。鸦胆子是常用的清热解毒,截疟止痢的良药,来源于苦木科鸦胆子属植物Brucea javanica(L.)Merr.的干燥成熟果实,其味苦、性寒,具有较好的抗菌、杀虫功效。现代药理研究显示,鸦胆子油乳因为能有效抑制幽门螺杆菌在临床上用于抗消化道溃疡而鸦胆子油的乙醇提取物对金黄色葡萄球菌等人类致病菌较强的抑制作用。本研究选择传统中药鸦胆子为研究对象,对其内生菌的种群结构特征、次生代谢产物的抗菌活性进行考察,开发鸦胆子内生菌的药用价值,对解决鸦胆子原料紧缺的状态、保护现有的野生资源和传统中药的二次开发具有重要的意义。本文的主要研究内容如下：1国内外研究综述通过查阅大量相关的国内外文献,对其进行归纳整理。综述中主要分析了鸦胆子在化学成分、药理作用、制剂研究、临床应用、资源等方面的研究进展,重点讨论了药用植物内生菌的生物多样性、活性代谢产物的筛选尤其是抑菌作用和拮抗致病菌方面的研究近况。2鸦胆子内生菌的初步研究鸦胆子内生菌的多样性研究——对鸦胆子不同部位的内生菌进行分离、纯化、鉴定,发现鸦胆子内生菌的组成具有多样性。本实验共分离得到151株鸦胆子内生菌,其中内生真菌92株,内生细菌59株。其中从叶中分离的内生真菌数目最多,茎中的内生细菌最多,果实中仅有1株内生真菌和5株内生细菌,且无专属性。鸦胆子内生真菌的多样性指数H=2.65,表明其多样性相对较高。内生真菌在鸦胆子植株内的分布既有广泛性又有组织专属性,在不同的组织内的内生菌种类差别明显,同属真菌在不同组织的分布情况差别也较大。92株内生真菌,分别归属于20个属。59株内生细菌包含球菌53株(有荚膜类14株,无荚膜类39株)、杆菌4株、弧菌2株。鸦胆子内生菌的抑菌活性——以4种人类致病菌为靶标菌,用滤纸片法考察鸦胆子内生菌发酵液的抑菌活性,共筛选出45株有活性的菌株,其中12株有活性较强,余者对4种靶标菌的抑菌圈均在8mm以下。其中,来自镰孢霉属的7株内生真菌对金黄色葡萄球菌和枯草芽孢杆菌、2株内生细菌对金黄色葡萄球菌、4株对枯草芽孢杆菌的抑制作用较明显。鸦胆子的组织培养研究——在采用组织块法分离鸦胆子内生菌的过程中,同时以鸦胆子茎段为外植体,进行了鸦胆子离体培养试验。探讨了基本培养探讨基本培养基和外源激素对其离体再生的初代培养、继代增殖、生根培养的影响并进行移栽试验,完善了前人对鸦胆子组织培养的研究。鸦胆子的不定芽增殖、壮苗、生根的最佳培养基分别为MS+6-BA1.0mg·L-1+IBA0.1mg·L11,1/2MS+香蕉汁20g·L-1;1/2MS+IBA0.1mg·L-1+NAA0.1mg·L-1。炼苗基质为细沙时,成活率最高。更多还原

【Abstract】 It’s shown in the relevant literature that endophytes in many traditional Chinese medicine can produce the same or similar active ingredients with their hosts. In order to better protect and develop the limited wild Chinese medicine resources, many scholars screened many effective strains and the active metabolite to bmicroorganism in special ecological environment has close relations with special secondary metabolites, which can be applied in the microbial fermentation technology to the implementation of the industrialized production of natural drugs.Brucea javanica is the seeds of the plant Brucea javanica(L.)Merr. which belongs to Brucea of Simaroubaceae. It’s a common tradional Qingrejiedu drugs, and it also showed good antibacterial, insecticides efficacy. Pharmacological studies have shown that Brucea javanica oil emulsion was significantly inhibited Helicobacter pylori,therefore it was often used as clinical anti-petic ulcer drugs. The the ethanol extracta of its oil also showed strong inhibition of Staphylococcus aureus and other human pathogens.Based on the theories above, searching the population structure of endophytes in Brucea javanica and their antibacterial activity of secondary metabolites is of great significance to explore new drug source and the secondary development of traditional Chinese medicine.The paper includes several parts:1. The review of domestic and foreign literatureAfter reviewing lots of relevant literature, authors summarized the general research situation of Brucea javanica on the chemical composition, pharmacological effects, clinical application of fromulations and resources. Then we turned to the recent progress of medicinal endophyte,including the biological diversity of endophytes in medicinal plant, screening of the active metabolite,especially antibacterial and antagonism of pathogens.2.The preliminary studies on endophytes of Brucea javanica(L.)Merr.Studies on the biodiversity of endophytes from Brucea javanica:By separating and purifying endophytes from different organs of Brucea javanica again and again,151strains of endophytes were isolated, including92strains of fungi and59strains of endophytic bacteria. The biodiversity is obvious both in the composition of the endophytes species and the viarious distribution in different ogans. The largest number of endophytic fungi,bacteria and actinomycetes were respectively from leaves,sterm and inflorescence. The species of endophyte in fruit is simple and without specificity.The diversity index of endophytic fungi H reaches2.65, indicating a high variety of strains relatively. The distribution of endophytic fungi showed both extensity and the specificity in different tissues from Brucea javanica.Simultaneously, the types of endophytes varied in different plant organs, and the same endogenous strain also had clear differences in distribution within different organizations.92endophytic fungi were morphologically identified belonging to20genera.59endophytic bacteria containing53cocci (14with capsule,39without capsule)4bacillus and2vibrio.Antibacterial experiment of endophyte from Brucea javanica:To investigate the antibacterial activity of the endophytes’fermentation broth, we chosed4kinds of human pathogens as target bacteria, using the filter paper method, screened a total of45active strains, including12showed strong activity.Tissue culture and rapid propapagation research:When using tissue explant method to isolate the endophyte with the sagments of Brucea javanica as explants, like sterm,leaves and inflorescence,we also studied the effects of minimal medium and plant growth regulators on the tissue culture and rapid propapagation. The result showed that the best formula of proliferation, robust seedling and rhizogenesis were MS+6-BA1.0mg-L-1+IBA0.1mg-L-1,1/2MS+banana juice20g-L-1,1/2MS+IBA0.1mg-L-1+NAA0.1mg-L-1, respectively. The survival rate reached the top when using sands as culture medium.更多还原