【作者】 杨倩；

【导师】 王沫；

【作者基本信息】 华中农业大学 ， 药用植物学， 2018， 硕士

【摘要】 本课题从蕲春蕲艾（Artemisia argyi）根茎部分离纯化得到一株内生真菌命名为HCH285,之后进行菌种鉴定、bostrycin方法学构建,同时对该HCH285内生菌代谢产物和bostrycin进行安全性和活性分析,主要结果如下:1.HCH285菌种鉴定。利用微生物学与分子生物学两种方法对HCH285内生真菌进行鉴定。微生物形态学观察发现菌落呈圆形,生长初期为白色绒状毛且生长蓬松,后期逐渐成红色且菌落背面的红色产物也清晰可见。HCH285菌丝体为无色有隔菌丝。它产单个、球形或椭圆形、黑色光滑的分生孢子。此外通过分子生物学技术对其ITS序列测定确定该菌株属于黑孢霉属球黑孢霉菌（Nigrospora oryzae）。2.HCH285内生菌产bostrycin分离制备方法建立。利用多种化学试剂对HCH285内生菌代谢产物提取,确定乙酸乙酯为最佳萃取试剂,以半制备液相进行化合物分离,优化卷线孢菌素bostrycin的HPLC制备方法,使用高效分析型液相检测分离纯度,其分离制备方法为:柱温:30℃,进样体积:700μL,流速:3 mL/min,流动相比例:甲醇:0.3%甲酸=50:50（v/v）,检测时间:30 min,紫外检测:302 nm;采用400MH_Z核磁共振波谱仪进行结构鉴定确定为卷线孢菌素bostrycin。3.Bostrycin活性分析。Bostrycin抗氧化活性分析证明,对三种自由基抗性顺序由大到小为O₂^–>DPPH^–>OH^–。它的抗菌活性表明有较好的抗菌作用,对革兰氏阳性菌比革兰氏阴性菌较好。CCK-8法体外抗癌活性研究表明对U251,B10F16癌细胞的半致死浓度分别为4.30μM和6.05μM。4.Bostrycin及HCH285菌代谢色素的安全性分析。引入秀丽隐杆线虫（C. elegans）为生测材料,以食品级红曲红色素为对照,研究对C.elegans各生理指标的影响。试验表明HCH285菌产色素对C.elegans的急性与慢性毒性、摄食与运动能力、生长发育状态、卵块孵化率,均没有明显抑制作用且与食品级红曲红色素作用效果一致,综合各指标显示500μg/mL内为安全剂量。以相同方法和指标,分析bostrycin对C.elegans作用,确定剂量在10μg/mL内无毒副作用。更多还原

【Abstract】 In this study,an endophytic fungus HCH285 was isolated and purified from the rhizome of Artemisia argyi in the laboratory.The HCH285 strain was identified,and its metabolites were separated and identified,and their toxicity and activity were analyzed.Furthermore,the endophytic metabolites were analyzed for the toxicity.The active compounds were isolated for the activity analysis as follows:1 Identification of HCH285 strain.HCH285 Endophytic fungi have been traditionally studied and described based on both morphological characteristics and molecular methods.In the present study,the results show that the colonies were rounded,with white fleece and fluffy growth in the early growth stage.The mycelia gradually became red in the late stage,and the red metabolism was observed on the back of the colonies.The mycelium was produced segmented hyphae and sporeless round conidia.Also,internal transcribed spacer（ITS）sequence was determined by using molecular techniques to find whether this strain belonged to Nigrospora oryzae.2 Compound preparation method for the separation of HCH285 endophyte metabolic pigment.Different chemical reagents were used to extract the metabolites,so ethyl acetate was selected as the best extraction reagent.The compounds were separated in semi-preparative liquid phase,and the HPLC method was optimized for the separation of monomer substances.The separation purity and method using 400MR structural analysis for bostrycin:the column temperature was 30℃;injection volume:700μL;flow rate:3 mL/min;mobile phase:methanol:0.3%formic acid=50:50.3 Bostrycin bioassays.Antioxidant,antibacterial and the anticancer analysis of the bostrycin showed significant activity and provedas a good antioxidant,antibacterial and the anticancer compound.The order of the resistance to the three free radicals were O₂^–>DPPH^–>OH^–.CCK-8 in vitro showed that at the median lethal concentration U251and B10F16 cancer cells were about 4.30μM and 6.05μM.4 HCH285 Pigments and Toxicity Analysis of bostrycin.C.elegansalong withfood grade Monascus red was applied as a control treatment to assess the various characteristics of C.elegans.Results show that the acute and chronic toxicity of pigments produced from HCH285 strains and C.elegans were exhibited no significant effects on feeding ability,life history traits,and egg hatching rate.While food-grade monascus red at500μg/mL showed a consistent effect.Results also show that the toxicity analysis of bostrycin on C.elegansat the rate of 10μg/mL are not toxic for C.elegans.更多还原