【作者】 刘丽；

【导师】 郭巧生；

【作者基本信息】 南京农业大学 ， 药用植物学， 2012， 博士

【摘要】 活血丹Glechoma longituba (Nakai) Kupr.为唇形科多年生草本植物。其干燥地上部分入药,药材名为连钱草(《中国药典》(2010年版)收录),主产于江苏、浙江一带,有清热解毒、利尿通淋、散瘀消肿的功能,主治胆囊炎、胆结石、肾结石等症。其种质资源系统收集评价、种质资源库的构建和药材品质评价未见报道。本文综合运用药用植物学、药用植物资源学、食品营养学、药用植物生态学知识,结合现代分析技术和分子生物学手段,在系统收集我国20省市活血丹种质基础上,从DNA分子水平揭示了不同种质的亲缘关系；并对不同活血丹种质从植物形态、营养品质和药材品质等方面进行了对比分析,此外对南京紫金山活血丹的生长发育和营养、药材品质的年积累动态进行了分析,主要结果如下：1从全国20个省市收集到58份活血丹种质,其分布北起黑龙江哈尔滨、南至云南麻栗坡(中越边境),东起上海、西至云南昆明。调查发现活血丹自然群落以无性繁殖为主,在条件适宜的小环境中有性繁殖可以成功。不同活血丹种质植株形态有一定差异,其叶片直径与叶柄长度、茎节直径显著相关,茎节数与叶柄长度显著相关,茎节长度与和芽数显著相关。2首次利用ISSR分子标记技术对分布于我国的54个活血丹居群进行了遗传多样性研究。从47条ISSR引物中筛选出18条,共检测到189个位点,其中多态性位点148个,DNA片段大小范围在200～500bp之间,总多态位点比率为78.31%,表明活血丹具有丰富的遗传多样性。3首次利用AFLP分子标记技术对对分布于我国的55个活血丹居群进行了遗传多样性研究。从64对引物组合中筛选出8对引物组合,共检测到1199个位点,其中多态性位点1184个,DNA片段大小范围在73～450bp之间,总多态位点比率为99.75%,表明活血丹具有丰富的遗传多样性。4以药材水分、总灰分、酸不溶性灰分、醇溶性浸出物、总黄酮、熊果酸、齐墩果酸为药材活性成分,兼顾Cu、Pb、Cd、As、Cr等5种重金属,对分布于我国的41个活血丹居群从药材有效性和安全性两方面进行了品质评价。不同活血丹种质活性成分含量差异明显,江南江淮丘陵山地一带和云贵高原两个药材区划区域种质资源浸出物、总黄酮和熊果酸三种活性成分平均含量均显著高于全国其它药材区划区域种植资源,为优良种质资源区；不同活血丹种质中重金属元素含量差异明显,Pb含量0.025～22.300mg·kg-1；Cu平均含量15.562mg·kg-1,符合国家绿色行业标准；Cd含量0.013～1.013mg·kg-1；Cr含量0～17.75mg·kg-1；As仅在北京、广东广州、辽宁丹东和南京栖霞山、珍珠泉产地的5个居群检出,余均未检出。综合5种重金属元素含量的检测结果可见,西南地区、江淮地区活血丹种质安全性较高。5以3种基本营养成分(可溶性蛋白、可溶性糖、脂肪)、10种营养元素(4种常量元素P、K、Ca、Mg),6种微量元素(Zn、Cu、Fe、Al、Mn和B)为指标,对分布于我国的41个活血丹居群从营养品质角度进行了评价。基本营养成分南方高于北方,东部高于西部；Ca、Mg和K3种常量元素在不同活血丹居群间的差异特征基本一致；微量元素在不同活血丹居群中的表现为：zn和Cu含量变化特征一致、Fe和Al元素含量变化特征一致、Mn和B元素含量变化特征基本一致。6对南京紫金山活血丹自然分布地野生群体进行连续三年的动态观测发现：紫金山活血丹2月上旬恢复生长,3月直立生长阶段后开花、4月盛花期后种子成熟,5月底进入匍匐生长阶段,地上部分可安全越冬；一年内可分为恢复生长期、营养生长期、开花期、果实种子成熟期、匍匐生长期和枯黄期几个阶段。7同一年内,不同采收期的紫金山活血丹药材水分、浸出物含量均符合药典标准规定,总黄酮、熊果酸和齐墩果酸含量年动态整体变化趋势均呈现“升—降—升—降”的双峰型曲线,但灰分含量仅4-8月达标,因此可选择4-8月作为活血丹的最佳采收期。8同一年内,不同采收期的紫金山活血丹基本营养成分(可溶性蛋白、可溶性糖、脂肪)含量冬春季高于夏秋季；Zn、Mn、B、Fe含量冬季相对较高；Ca和Mg含量在1-2月较高、P和K含量从3月份开始到次年1月份逐渐下降。更多还原

【Abstract】 Glechoma longituba (Nakai) Kupr.(Labiatae) is an herbaceous medicinal plant. Its dried aerial part, Glechomae herba, is a standard medicinal material in the Chinese Pharmacopoeia (2010edition) and is used for the treatment of cholecystitis, gall-stone, kidney stone, etc. So far, there are few reports on the collection, establishing of germplasm bank and quality evaluation of the G. longituba germplasm resource. In present doctoral dissertation, the theory and method of Medicinal Botany, Science of Medicinal Plant Resource, Food Nutriology, Medicinal Plant Ecology as well as modern inorganic element analysis and Molecular Biology method were synthetically applied. Based on the multidiscipline theory and method, we collected different G. longituba germplasm resources from20provinces in China and compared the biological characteristics, nutrient quality and pharmaceutical quality. And we explored the genetic relationship among germplasm resources based on ISSR and AFLP markers as well as ITS sequence analysis. In addition, dynamic change of growth characteristic, contents of nutrient elements, heavy metals, and bioactive components in G. longituba from Zijinshan Mountain was investigated. The main results are as follows.1. Fifty-eight G. longituba populations were collected from main contribution regions in China. The main reproductive mode of wild G. longituba is vegetable propagation, and sex reproduction is possible under appropriate microclimate conditions. There are marked difference in morphological characteristics among G. longituba populations. The correlation analysis showed that leaf diameter was linearly correlated with petiole length and internodes diameter, and internode number was linearly correlated with petiole length. Additionally, internodes length and bud number showed linear correlation.2. Present study was conducted to assess molecular genetic diversity among54G. longituba populations in China by ISSR markers for the first time. Eighteen ISSR primers which were screened from47ISSR primers generated a total of189bands and varied in size from200to500bp. Of the189bands obtained,148bands were polymorphic (78.31%). The results of ISSR marker showed that G. longituba populations from different areas of China exhibited abundant genetic variation.3. Simultaneously, molecular genetic diversity among54G. longituba populations was evaluated based on AFLP markers. Eight primers which were screened from64ISSR primers generated a total of1199bands and varied in size from581to734bp. Of the1199bands obtained,1184bands were polymorphic (99.75%). The results of AFLP marker showed that G. longituba populations from different areas of China exhibited abundant genetic variation.4. In terms of pharmaceutical quality and safety, quality evaluation of41G. longituba populations was carried out based on contents of water, total ash, acid-insoluble ash, ethanol-soluble extractive, total flavonoids, ursolic acid (UA) and oleanolic acid (OA), and contents of five heavy metals, viz:Cu、Pb、Cd、As、 Cr. The results showed that different G. longituba. populations showed significant variation in contents of ethanol-soluble extractive, total flavonoids, UA and OA. In generally, the high-quality G. longituba populations that are rich in ethanol-soluble extractive, total flavonoids, ursolic acid, such as the population from Jianghuai Hilly region and Yunnan-Guizhou Plateau, should be interesting to candidates for future research aiming to elevate major bioactive compounds levels in cultivated G. longituba material. Different G. longituba populations showed significant variation in contents of Cu、Pb、Cd、As、Cr in aerial parts. Lead content ranged from0.025to22.300mg·kg-1. The copper content in different G. longituba populations met Chinese standard with the average value of15.562mg·kg-1. The contents of cadmium and chromium ranged from0.013～1.013mg·kg-1, and0～17.75mg·kg-1, respectively. Arsenic content in G. longituba populations met Chinese standard except for samples from few locations. In generally, G. longituba populations from Southwest China and Huaihe region contained lower heavy metals contents.5. Nutritional quality evaluation of41G. longituba populations was carried out based on contents of soluble protein, soluble sugar and fat as well as contents of ten nutrient elements, viz:P, K, Ca, Mg, Zn, Cu, Fe, Al, Mn, and B. The overall trend was that the contents of soluble protein, soluble sugar and fat in samples from South China was higher than those from North China, while the fat content in ones from East china were higher than those from West China. The variation patterns of Ca, Mg, and K were similar. In the case of microelement contents, Zn content changed following the similar pattern of Cu content. The similar variation tendency was found in Fe content and Al content, Mn content and B conten. 6. The growth of wild G. longituba in Zijinshan Mountain was carried out in continuous three years. G longituba emerged in early February; blossomed after rapid growth in March; seeds matured in April; and crept in May. The aerial part could live safely through the winter. Its growth stages could be divided into six stages, viz:emerging, vegetable growth, florescence, seed maturity, creeping, and wilt.7. The contents of water and ethanol-soluble extractive in G herba from Zijinshan Mountain met Chinese pharmacopeia standard irrespective of harvesting time. And the general dynamic change of total flavonoids, ursolic acid and oleanolic acid showed double-peak curve. The optimum harvesting time should be April-August.8. The dynamic change of contents of soluble protein, soluble sugar and fat as well as contents of8nutrient elements, viz:P, K, Ca, Mg, Zn, Fe, Mn, and B. in wild G. longituba from Zijinshan Mountain was measured. The resulted showed that the contents of soluble protein, soluble sugar and fat in G longituba collected in winter and spring were higher than these collected in summer and autumn.In addition, the highest value of calcium and magnesium contents were found in January and February, and the relatively higher contents of Zinc, manganese, boron and iron were found on winter. The contents of phosphorus and potassium decreased from March to January of the nest year.更多还原