【作者】 刘川；

【导师】 张艺；

【作者基本信息】 成都中医药大学 ， 药物化学， 2016， 硕士

【摘要】 近年来,随着市场需求和自然环境变化的影响,藏药资源遭到破坏。因此,摸清藏药资源分布状况、开展野生资源的保护显得尤为紧迫。另一方面,藏药长期存在的“同名异物”、“同物异名”等多基原现象,导致藏药品种鉴定困难,严重影响了藏药质量控制和评价研究。本文分为两个部分：第一,以复杂多基原藏药“蒂达”((?))为研究对象,重点采用DNA条形码开展其品种鉴定研究；第二,以常用藏药中国沙棘Hippophae rhamnoides L. subsp. sinensis Rousi为研究对象,开展其资源调查、规范化种植、生态适宜区划、化学成分积累与生态因子相关性研究。以期为藏药品种鉴定、野生资源保护和适宜区推广人工栽培种植提供科学依据。目的：建立藏药“蒂达”的DNA条形码鉴定方法；对中国沙棘进行科学的潜在分布预测和适生等级划分；确定影响中国沙棘叶黄酮类成分积累的生态因子及其最佳范围。方法：1.采用试剂盒法对藏药“蒂达”提取总DNA,采用通用引物ITS2(2F/3R)进行PCR扩增,并对扩增成功的PCR产物进行测序。同时对序列进行拼接、比对,计算种内种间K2P距离,构建系统进化NJ树,评价其barcoding gap,从而实现对藏药“蒂达”的DNA条形码分子鉴定；2.从全球气候数据库中提取生态因子,基于Maxent模型设置ROC评价曲线,同时运用ArcGIS软件,对中国沙棘进行适宜区区划研究；3.采用TLC法对中国沙棘叶黄酮类成分进行定性鉴别；采用HPLC法对不同产地中国沙棘叶黄酮类成分进行含量测定；对17批次不同产地中国沙棘叶进行HPLC特征指纹图谱研究,构建准确可靠的中国沙棘叶色谱指纹图谱。4.应用Pearson相关分析、逐步回归分析和偏最小二乘回归法(PLSR),分析不同产地中国沙棘叶化学成分与其生态因子间的相关性。结果：1.ITS2序列可准确鉴定龙胆科藏药“蒂达”类药材：样品共151条序列,比对后序列长度达231 bp；平均GC含量为61.40%,种内变异大,共31个单倍型。其中四数獐牙菜Swertia tetraptera、祁连獐牙菜S. przewalskii、镰萼喉毛花Comastoma falcatum、辐状肋柱花Lomatogonium rotatum各只有1个单倍型。13个“蒂达”物种中,除二叶獐牙菜S. bifolia和华北獐牙菜S. wolfangiana2个种外,11个物种均能准确鉴定。2.课题组建立了产业化治沙科技试验示范基地、红原县藏医院沙棘规范化栽培示范基地和松潘县川主寺贝母基地(沙棘规范化栽培示范基地)等3个沙棘防沙漠化规范化栽培示范基地,共计6亩。基于Maxent模型区划结果表明：中国沙棘在我国分布的适宜指数为0-0.708。适宜区面积大59.05万km~2,占全国总面积的6.13%,分布范围涉及山西、陕西、宁夏、四川、甘肃、青海、四川、西藏、内蒙古、辽宁等10个省(自治区)；较适宜区面积达55.25万km~2,占全国总面积的5.73%,分布范围包括河北、辽宁、内蒙古省(自治区)。3.以槲皮素、山柰素和异鼠李素为对照品建立了中国沙棘叶的薄层色谱鉴别方法；17批次中国沙棘叶HPLC含量测定结果表明：槲皮素含量在0.1929%-0.5236%之间、异鼠李素含量在0.2242%-0.6236%之间、山柰素含量在0.0772%-0.1775%之间。可见,不同产地间中国沙棘叶中槲皮素、山柰素和异鼠李素含量差异明显；不同批次的中国沙棘叶HPLC指纹图谱研究结果显示,17批次中国沙棘叶共有14个共有峰,与标准品对照,可确定山柰素-3-O-β-D-槐二糖-7-O-a-L-鼠李糖苷、异鼠李素-3-O-p-D-槐二糖-7-O-a-L-鼠李糖苷、异鼠李素-3-0-p-D-葡萄糖-7-O-a-L-鼠李糖苷、芦丁、异鼠李素-3-O-p-D-葡萄糖苷等5个色谱峰。对17批中国沙棘叶的特征指纹图谱进行相似度计算,结果表明17批样品中,相似度均在0.80以上,来自四川省阿坝州红原县、西藏拉萨市达孜县、西藏拉萨市曲水县、陕西省晋中市4批次相似度小于0.9,其余13批次相似度达0.9以上。4.确定了影响中国沙棘叶中槲皮素、山柰素和异鼠李素含量积累的主导生态因子及其最佳范围,在最佳范围内,相应化合物含量最高,具体如下：影响槲皮素积累的主导因子为海拔高度、1月月均降水量和8月月均降水量,其最佳范围分别为3009-3320 m、4.0-6.0mm和118-152mm；影响山柰素含量积累的主导因子为海拔高度、最冷季度降水量、12月月均降水量和3月平均气温,其最佳范围分别为2999-3315 m、9.0-12.0mm、1.0-2.0 mm和-1.9-0.1℃；影响异鼠李素含量积累的主导因子为8月月均降水量、1月月均降水量、最冷季度降水量和海拔高度；其最佳范围分别为：118-122mm、4.0-5.0 mm、13.0-22.0 mm和3158-3568 m。结论：1.DNA条形码能够快速、准确鉴定龙胆科藏药“蒂达”类药材。在鉴定工作中,应综合运用不同鉴定手段,以达到鉴定目的。2.中国沙棘适宜区分布在我国10省(自治区),其适宜区域和较适宜区面积广,资源数量大,适合可持续开发利用。更多还原

【Abstract】 In recent years, with the increasing of the market demand and changes in the environment, Tibetan medicine resources was sevciously damaged. Finding out the distribution of resources and protect the wild resources is particularly urgent. On the other hand, the phenomenon of synonymies and homonymies in Tibetan medicine, resulting in varieties identification difficult, seriously impact the quality control and evaluation. Therefore this paper aims at the complex multi original Tibetan medicine "Dida" as the research object, focused on its species identification by DNA barcode; Besides, then carry out Hippophae rhamnoides L.subsp. sinensis Rousi’resources investigation, normalized planting, ecological suitability zoning, accumulation of chemical components and ecological factors,to provide scientific basis for variety identification, the protection of wild resources and the promotion of artificial cultivation in the suitable habitat.Objectives:1. To identify Tibetan medicine "Dida" by DNA barcoding; To select the suitable habitat Hippophae rhamnoides L. subsp. sinensis Rousi. and to investigate the dominant ecological factors whith affect the accumulation of chemical components.Methods:1. ITS2 sequence was Used to identify different of "Dida", to provide the basis of varieties identification from the molecular level.2. Based on the Maxent model, the suitable area is studied;3. TLC was used to identify, the contents of flavonoids were determined by HPLC:4. Pearson correlation analysis, stepwise regression analysis and partial least squares (PLS) were used to analyze the correlation between the chemical components and their ecological factors.Results:1.ITS2 sequence can be used for "Dida" identification. ITS2 sequence length were 231 bp, GC content was an average of 61.40%, Phylogenetic analysis demonstrated that every species clustered into their own clade, except Swertia bifolia and Swertia wolfangiana clade2.Widely distributed in the provinces of China, wide distribution area, large number of resources. Like the sun, for the water has certain requirements. Often with shrubs, grass shrub vines formed structure.Established 3 standardized cultivation.demonstration base, a total of 6 acres.3.China’s regional distribution of the suitable index for 0-0.708. suitable area 59.05 ten thousand km~2, accounting for 6.13% of the total area. More suitable area 55.25 ten thousand km", accounting for 5.73% of the total area.With quercetin, kaempferol and isorhamnetin as controls, Determination of the content of 17 batches by HPLC:quercetin 0.1929%-0.5236%, isorhamnetin 0.2242%-0.6236%, kaempferol 0.0772%-0.1775%. Results show that the content difference among different producing areas.4. Determine the dominant ecological factor and it’s optimal range that effect the accumulation of quercetin,kaempferol and isorhamnetin.Effect of quercetin content accumulation:altitude is 3009-3320m; the average monthly precipitation in January and August are 4.0-6.0mm and 118-152mm respectively.Effect of kaempferol accumulation:altitude is 2999-3315m;the aerage monthly precipitation in the coldest quarter and December are -9.0-0.1℃. Effect of isorhamnetin accumulation:the average monthly precipitation in August,January and the coldest quarter are118-122mm,4.0-5.0mm and 13.0-22.0mm;altitude is 3158-3568m.Conclusions:1. ITS2 sequence as the Gentianaceae Tibetan medicine Dida’s DNA barcode with good accuracy.2. Hippophae rhamnoides L. subsp. sinensis Rousi is widely distributed in china.with a wide area and a large number of resources, which is suitable for sustainable development and utilization.更多还原