【作者】 陈自宏；

【导师】 虞泓；

【作者基本信息】 云南大学 ， 植物学， 2013， 博士

【摘要】 兰坪虫草Ophiocordyceps lanpingensis H. Yu&Z. H. Chen主要分布于滇西北地区,是名贵中药冬虫夏草的近缘种。本论文从兰坪虫草的分类地位、生态调查、活性成分、代谢产物的分离提取、药用价值等多个方面开展兰坪虫草的生物学及药用价值研究,为兰坪虫草的开发利用奠定基础,对缓解冬虫夏草资源紧缺具有重要意义。1从形态特征和分子序列对兰坪虫草系统分类地位进行研究兰坪虫草属于线虫草属Ophiocordyceps,其形态特征与该属其它物种有明显的区别：子座细长,纤维质；子囊壳细小；子囊孢子细长,不断裂成次生子囊孢子,隔细胞细小。兰坪虫草与线虫草属近缘种的ITS系统发育分析表明,兰坪虫草与罗伯茨虫草、冬虫夏草、锈壳虫草、珊瑚虫草的亲缘关系较近。进一步分析了兰坪虫草与其近缘种的5基因(nrSSU,nrLSU, tef-1α, rpb1和rpb2)和交配型基因(MAT1-2-1)的系统发育关系,结果表明兰坪虫草与罗伯茨虫草、冬虫夏草亲缘关系更近,但它们也有着明显的进化距离。对这3个近缘种的tef-1α, rpb1, rpb2和MAT1-2-1基因编码的氨基酸序列进行比对分析表明,它们的tef-1α和rpb1序列具有较高的相似性(98%-99%),而rpb2和MAT1-2-1序列差异较大,异质性都大于5%。形态特征比较和分子序列分析都充分支持兰坪虫草是线虫草属中的一个新种,它是冬虫夏草的一个姊妹种。根据柯赫法则证明兰坪虫草无性型是兰坪被毛孢。在云南钩蝠蛾幼虫上接种兰坪被毛孢菌,50多天的人工诱导培养,成功培养出兰坪虫草子实体,与野生兰坪虫草形态一致。2兰坪虫草生态环境调查其生境的海拔、植被、昆虫寄主、虫生真菌、土壤、温度、湿度、坡向等多个方面的因素构成了兰坪虫草物种形成的基础。兰坪虫草主要分布于横断山区,是横断山造山运动过程中分化出来的新种,分布在海拔2600-3400m的高山草丛。其生境植被中主要以牛膝菊、艾蒿、酸模、西南鸢尾等草本植物为优势种。昆虫寄主云南钩蝠蛾和剑川钩蝠蛾的食物多样,主要以草本植物如艾蒿、西南鸢尾、血满草、白藻、大蓟等的幼嫩根茎为食,偶以枯枝落叶层的腐殖质为食。兰坪虫草的生境中虫生真菌有多种,包括细脚棒束孢、蝙蝠蛾棒束孢、白僵菌和粉棒束孢等。生境土壤类型为棕壤或黑褐土,微酸性,含水量在34%-42%左右。空气湿度在53%-67%,年平均气温10.7-11.7℃,虫草生长旺季的气温10-21℃；随温度、湿度及海拔差异,坡向有阴坡与阳坡的选择。3优化兰坪被毛孢培养工艺对4个样地的兰坪被毛孢菌的活性成分进行研究,筛选出产活性成分性能最佳的优良菌株OLLJ14。对该菌株的培养工艺进行优化,产腺苷最佳培养条件为：以OLM-3培养基为基质,以14-16℃的温度为培养温度,恒温培养30天。野生兰坪虫草宜在6月中下旬采收,活性成分积累达到高峰,质量最好。野生兰坪虫草、兰坪被毛孢发酵培养物与质量最好的西藏冬虫夏草的15种活性成分的含量非常相似,多种活性成分的综合搭配较均衡。4兰坪被毛孢发酵培养物代谢产物分离提取根据溶剂种类将提取物分为不同部分：石油醚部分、乙酸乙酯部分、无水乙醇部分和水提部分。分离纯化得到7个单体化合物,皆为已知成分,其中β-谷甾醇、乌苏酸、D-甘露醇和尿苷具有重要的生物学功能,乌苏酸为首次在广义虫草属中分离到。5药理实验兰坪被毛孢发酵培养物的粗多糖对果蝇幼虫和成虫都具有显著延缓衰老的效果。0.3%兰坪被毛孢发酵培养物的粗多糖浓度能修复因丙酸致衰老的果蝇幼虫,使果蝇幼虫完成整个生活史,与对照无统计学差异。0.2%兰坪被毛孢发酵培养物的粗多糖浓度能明显延缓雌、雄果蝇的平均寿命和半数死亡时间,提高其最高寿命,达到统计学极显著水平,且显示出性别差异,雌性的平均延寿率高于雄性。兰坪虫草及兰坪被毛孢发酵培养物的镇痛效果明显。与生理盐水比较,兰坪被毛孢发酵培养物的粗提物的石油醚部分、乙酸乙酯部分、无水乙醇部分和水提部分对外周性疼痛都具有显著的镇痛效果。兰坪被毛孢发酵培养物中起镇痛效果的不同成分需要组合起来才能更好的发挥外周镇痛效果。综上所述,兰坪虫草系统分类地位与冬虫夏草非常接近,是冬虫夏草的姊妹种,野生兰坪虫草和兰坪被毛孢发酵培养物的活性成分也都与冬虫夏草接近,兰坪被毛孢发酵培养物有较明显的延缓衰老效果和镇痛效果,故兰坪虫草具有较高的开发潜能。更多还原

【Abstract】 Ophiocordyceps lanpingensis H. Yu&Z. H. Chen was mainly distributed in northwest Yunnan and was a close phylogenetic affinity species of Ophiocordyceps sinensis, a famous and precious Chinese traditional medicine. Biological studies of O. lanpingensis were performed from several aspects, including taxonomic status, ecological investigation, functional component, the separation and extraction of metabolite, and medicinal value. The attempt was to lay the foundation for the development and utilization of O. lanpingensis and it had an important significance for remitting the resources lack of O. sinensis.1The classification of O. lanpingensis was identified based on morphology characters and the molecular data analyses. This species was belonged to Ophiocordyceps genus and characterized by thinner stroma, smaller perithecium, thinner ascospore (multiseptate with short septation). The phylogenetic analyses based on the ITS sequences of O. lanpingensis and its related species in Ophiocordyceps showed that O. lanpingensis had closer evolution relationship with Ophiocordyceps robertsii, Ophiocordyceps rubiginosoperitheciata, Ophiocordyceps stylophora and O. sinensis. The phylogenetic analyses based on5-gene dataset and the mating type gene showed that O. lanpingensis had the closest evolution relationship with O. robertsii and O. sinensis, but still had obvious distances to them. The amino acids sequences of the genes tef-la, rpb1, rpb2and MAT1-2-1were respectively aligned among the3related species. Their sequences of tef-la and rpbl exhibited high identities (98%-99%). However, obvious variations occurred at the rpb2and MAT1-2-1sequences and the heterogeneities were above5%. Both morphological characteristics and systematic analysis supported that O. lanpingensis was a new species of Ophiocordyceps and it was a sister species of O. sinensis.According to Koch’s postulate, the anamorph of O. lanpingensis was Hirsutella lanpingensis H. Yu&Z. H. Chen. Thitarodes yunnanensis larva was inoculated by H. lanpingensis. After the artificial culturation for more than50d, the fruitbody of O. lanpingensis was successfully cultured and the morphology of its stroma was similar to natureal O. lanpingensis.2The ecological environment of O. lanpingensis was investigated. Such as the elevation, vegetation, insect host, entomogenous fungi, soil, air temperature, air humidity, exposure and so on, were surveyed in the present study.O. lanpingensis was mainly spreaded over Hengduan Mountains Region and was a new species generated during the process of orogenic movements. It was distributed in mountain grass of2,600-3,400m elevation. The dominant species of vegetation were herbages such as Galinsoga parviflora, Artemisia argyi, Rumex dentatus and Iris bulleyana. The host of O. lanpingensis was T. yunnanensis and Thitarodes jianchuanensis, whose food was various, mainly the young roots of herbaceous plant, such as Artemisia argyi, Iris bulleyana, Sambucus adnata, Fragaria nilgerrensis, Cirsim chlorolepis, occasionally the humus of the litter layer. Several entomogenous fungi existed in the habitat of O. lanpingensis, such as Isaria tenuipes, Isaria hepiali, Beauveria bassiana and Isaria farinosa. The soil types of its habitat were brown or black, being subacidity, and the moisture content was within34%-42%. The air humidity was from53%to67%. The annual average temperature was10.7-11.7℃, while the temperature in the growth season of O. lanpingensis was10-21℃. O. lanpingensis existed exposure selection of shady slopes and sunny slope with the difference of temperature, air humidity and elevation.3The culture process of H. lanpingensis was optimized. The strains of H. lanpingensis from4sample plots had been conducted active component analysis and the quality isolate OLLJ14was screened to have the optimum functional component. The culture process of the isolate was optimized, and the best culture condition for producing adenosine was selected as the OLM-3medium, culturing at14-16℃constantly for30days. Natural samples of O. lanpingensis should be harvested in mid to late June, when their functional component accumulated to the peak and their quality was the best. The active ingredients of O. lanpingensis and H. lanpingensis were similar to O. sinensis of Tibet.4The metabolic products of H. lanpingensis were separated and extracted with traditional solvent extraction method. The extracts were divided into different parts according to the solvents: petroleum ether, ethyl acetate, absolute ethyl alcohol and water. Seven monomeric compounds were separated and purified. All the seven compounds were known composition, such as β-Sitosterol, ursolic acid and D-mannitol and uridine had important biological function. Ursolic acid was separated from Cordyceps s.1. for the first time.5The results of pharmacology experiment suggested that the crude polysaccharide of H. lanpingensis culture had the effect of delaying senescence. The0.3%crude polysaccharide of H. lanpingensis culture could pull through the decrepit Drosophila melanogaster larva caused by propionic acid, completing the larva their whole life history, no statistical differences comparing with the control. The0.2%crude polysaccharide could delay the average lifetime and the time of half lethal, improve the maximum life-span of male and female D. melanogaster, reaching statistically significant level. It was showed that the sex differences was obvious, and the delay rate of lifetime was much higher in female insect than male insect.The crude extract of H. lanpingensis culture, including petroleum ether part, ethyl acetate part, absolute ethyl alcohol part and water part, all had very significant difference comparing with normal saline on reducing the writhing times in mice induced by HAc. It was suggested that all the polar parts of H, lanpingensis culture had prominent analgesic effects to the peripheral neurogenic pains. The combination of all the polar parts of H. lanpingensis culture was better to restrain the peripheral neurogenic pains.In conclusion, the systematic position of O. lanpingensis was closer to O. sinensis, being as the sibling species of O. sinensis. The active ingredients of O. lanpingensis teleomorph and anamorph culture were both similar to O. sinensis. H. lanpingensis culture had the effect of delaying senescence and analgesia. Therefore, O. lanpingensis was well potential to substitution of O. sinensis.更多还原