【作者】 杨舒婷；

【导师】 傅承新；

【作者基本信息】 浙江大学 ， 植物学， 2011， 博士

【摘要】 玄参为玄参科植物玄参(Scrophularia ningpoensis Hemsl.)的干燥根,为传统的“浙八味”中的一种,其性微寒,味甘、苦、咸,具有凉血滋阴,泻火解毒等功效,其中主要的活性化学成分为环烯醚萜类和苯丙苷素类。作为传统中药,玄参在中国有着1000多年的栽培历史,然而对于玄参次生代谢的研究并不多,尤其是综合研究其与遗传变异因素和环境因子关系尚无人报道。基于此,本研究从遗传和环境两个方面入手,对玄参的化学多样性开展了相关研究,分析各因素对药材化学多样性的贡献率,探讨玄参品质的成因,为玄参质量评价和品种选育提出科学的建议,为玄参药材的可持续发展奠定研究基础。主要研究结果如下：(1)利用HPLC-DAD分析技术和多变量数据处理方法构建了玄参化学指纹图谱与四种主要活性成分(即毛蕊花糖苷、安格洛苷C、哈巴俄苷和肉桂酸)定量分析相结合的化学多样性评价体系,为本文后续研究及玄参药材质量评价提供了技术手段。(2)基于ISSR标记的玄参的种质遗传分析揭示了玄参栽培群体和野生群体之间已经出现了明显的遗传分化,栽培群体的遗传多样性远低于野生群体,而且栽培群体内的遗传分化较大。认为造成该现象的原因在于栽培群体长期的人工选育和克隆繁殖,群体间缺乏基因流。同质园实验揭示了玄参的栽培群体和野生群体之间在营养生长、生物量分配和化学多样性上均出现了明显的分化,野生群体普遍在地上部分的生长上要优于栽培群体,在地下部分根的生物量分配上要小于栽培群体,在花果器官上的生物量分配较大,认为与其在自然环境中的繁殖方式有一定的关系。野生玄参的化学多样性受遗传因素的影响较大,而栽培玄参则受环境的影响较大,作者认为这是野生和栽培群体的遗传多样性不同造成的。江西冷水(WJX)、湖南平江(WHN)和浙江磐安(WPA)的野生玄参群体在同质园下表现出茎秆粗壮,抗倒伏性好,地下部分产量较高,与栽培群体相对比较接近,因此从生产的角度来看,可作为品种选育和杂交的优选野生种质。(3)对于栽培玄参来说,温度和海拔是影响其化学多样性最主要的原因,其中环烯迷萜苷类成分哈巴俄苷的含量与温度因子呈正相关。对浙玄参四种活性成分季节变化的研究也证实了安格洛苷C、哈巴俄苷对温度存在一定的响应。建议在规范化栽培中选择较低的海拔或温度适宜的地区作为玄参种植地区有利于高含量活性成分的玄参的生产。(4)对来自五个主产地的栽培玄参药材样品进行了化学多样性评价后发现浙玄参已经在化学组成上形成了一个比较特殊的群体,并且相似度分析也表明了浙江磐安产区的玄参药材在化学指纹相似度基本上大于95%,揭示其化学成分较稳定。并且相对于其他四个栽培群体来讲,浙玄参在哈巴俄苷和安格洛苷C的平均含量上也是最高的,支持传统认为浙产玄参为道地玄参的观点。结合种质遗传分析和同质园实验结果,作者认为在长期的人工栽培过程中,浙玄参已经适应了浙江地区的气候环境特点,形成了一个具有一定遗传基础的化学型。但浙玄参药材道地性的形成与浙江产地特殊的地理气候环境(如较低的海拔高度、较高的月均和年均温度、较长的日照时数等)有着更为密切的关系。(5)通过对浙玄参的生长、生物量分配规律及活性成分的季节变化研究,确定了11月中下旬为浙玄参的最佳采收期。发现去花去果农艺操作对浙玄参的生长、生物量分配以及药用部位的化学多样性影响不大,而不同的加工处理方法则会极大地影响玄参的化学成分组成和含量,认为在传统药材加工的过程中,玄参根部本底水平的次生代谢物可能经历了一些降解和转化,而加工温度则是一个关键的因素。建议以毛蕊花糖苷、安格洛苷C、哈巴俄苷和肉桂酸四种成分为主要活性目标物的情况下,推荐优先采用鲜材切片烘干的加工处理方法。更多还原

【Abstract】 Radix Scrophulariae, the dry root of Scrophularia ningpoensis, is one of the famous traditional herb medicine named as "Zhe ba wei". It is widely used in China for cooling the blood, invigorating yin, purging fire and removing toxin. The iridoids and phenylpropanoid glycosides are considered to be the main bioactive components. As a traditional Chinese medicine, it has a long cultivation history of more than one thousand years. However, the studies on its secondary metabolites remain fairly limited, especially the comprehensive study on the effects of both genetic variation and environmental factors on the secondary metabolites of S. ningpoensis. In the present research, the chemical diversity of S. ningpoensis was evaluated under diverse genetic background and environmental conditions, to determine which parameter(s) contribute more to its secondary metabolic variation, and clarify the causes for good-quality of Radix Scrophulariae, so that scientific suggestions can be proposed for breeding and quality assessment of S. ningpoensis, as well as its sustainable development. The main results are listed as following:1. Applying HPLC-DAD technique and multivariate statistic analysis, both chemical fingerprints and the four bioactive components (i.e. acteoside, angroside C, harpagoside and cinnamic acid) quantitative analysis were combined to construct the chemical diversity evaluation system of S. ningpoensis. This evaluation system provides technical means for the present study and quality evaluation of Radix Scrophulariae.2. Genetic analysis based on ISSR data revealed significant genetic differentiation between cultivated and wild groups. Much lower genetic diversity and stronger differentiation were observed among cultivated populations than wild populations. This could be explained by the long-term artificial selection, the mode of clonal propagation, and limited among-population exchange of genetic materials in cultivated populations of S. ningpoensis. Common garden experiments demonstrated evident differences of growth characters, biomass allocation and chemical diversity between cultivated and wild groups. The over-ground part of wild populations basically grew better and bigger than cultivated populations, and the wild populations partitioned more on the flowers and fruits biomass but less on the root biomass. This phenomenon can be attributed to their different reproductive system. In addition, as a result of the discrimination of genetic diversity between the two groups, genetic background contributed more to wild populations’chemical diversity, while environment played a more important role in chemical diversity of cultivated populations. Under the consistent condition of common garden in Zhejiang Province, the wild populations introduced from Jiangxi Province (WJX), Hunan Province (WHN) and Zhejiang Province (WPA), grew vigorously, possessed a good ability of lodging resistance and obtained large root biomass. These characters are very similar to cultivated populations, so we proposed that these wild germplasm resources can be applied to agricultural practice for breeding and hybridation.3. As for cultivated S. ningpoensis, temperature and altitude are the main environmental factors that influence its chemical diversity. Our present study indicated the content of the most abundant iridoid, harpagoside, in Radix Scrophulariae is positively correlated with temperature. Investigation on seasonal variation of the four bioactive components in S. ningpoensis also confirmed the contents of harpagoside and angroside C were related to the temperature variation. Relatively higher average temperature and lower altitude were recommended for the cultivation of S. ningpoensis for GAP.4. Chemical diversity evaluation of cultivated populations from the five main production regions revealed that S. ningpoensis cultivated in Zhejiang Province (ZJ) had became a special group, all the samples from ZJ population shared high chemical fingerprints similarity (>95%) indicating stable chemical profiles in this cultivated population. Compare to other four cultivated populations, ZJ also had the highest average contents of harpagoside and angroside C. All these evidences support the traditional popular believing that Radix Scrophulariae from Zhejiang Province is geo-authentic. Based on the results of genetic analysis and common garden experiments, we concluded that the cultivated S. ningpoensis from Zhejiang Province had adapted to the special local environment during the long-term cultivation, and shaped as a typical chemotype that possesses a certain genetic background. However, the formation of geo-authenticity of Radix Scrophularea from Zhejiang province is more related to the typical environment in Zhejiang Province, such as lower altitude, higher annual or monthly temperature, and longer sunshine duration.5. Observations on the growth characters, biomass allocation and the four bioactive components accumulation of S. ningpiensis cultivated in Zhejiang province during its growth period support that mid-to-late November is the best time for harvest. Furthermore, no differences of growth, biomass allocation and chemical diversity were observed after flower and fruit removal treatment comparing to the control group. While different processing methods dramatically affect the chemical composition and content. It is supposed that the basal level of secondary metabolites in the root of S. ningpoensis may suffer some kind of degradation and transformation during traditional processing, and the processing temperature is probably the key factor. With the four components (i.e. acteoside, angroside C, harpagoside and cinnamic acid) as the main targeting compounds, a processing of slicing and oven-drying the fresh root of S. ningpoensis was recommended.更多还原