| 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.
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