Studies On Correlation Factors Of Euodia Ruticarpa Genuine Formation

[Objective]Euodia ruticarpa of Tongren Guizhou is popular at home and abroad because uniform particles, green colour and lustre, strong fragrance, acridity sapor and excellent quality. With high yield and good quality, Tongren Guizhou produces Euodia ruticarpa usually is looked as genuine medicinal materials of Guizhou and one of Guizhou famous medicinal materials, too. The Euodia ruticarpa of Hunan Xinhuang is also genuine medicinal materials for the best quality. In order to establish generic technology system of appraiseing Euodia rutaecarpa of adaptive region, the correlation factors and forming mechanism of Euodia rutaecarpa genuine were researched. Combing with social and economic conditions, the research would not only to provide technical support for producing region composition according to the local conditions and developing advantage varieties, but also to promote the sustainable development of superior medicinal materials of Euodia rutaecarpa.[Methods]Euodia rutaecarpa resource was investigated by network. The main producing areas of Euodia rutaecarpa was investigated via site visits. Plant varieties, introduction, yields, latitudes and longitudes, elevation and soil types were recorded in the main producing areas. Euodia rutaecarpa samples including roots, stems, fruits and soil were collected using five sampling method. The regionalization of Euodia rutaecarpa genuine region was analysed using TCMGIS technology. The genetic diversity of Euodia rutaecarpa with fresh leaves was studied using ITS, SRAP, AFLP molecular markers. Using plate method, the Endophytes in Euodia rutaecarpa were separated and purified and compared in Xinhuang, Liuyang, Xiangxiang, Luodi of Hunan, Tongren Guizhou, Wanzhai Jiangxi and Pingyang, Jiangde Zhejiang. Using HPLC, LC-MS technology, the components about evodiamine, rutaecarpine and structural analogues were analyzed. HPLC method was used to detect the content of Euodiamine and Rutaecarpine in Euodia rutaecarpa. The content of the trace element in Euodia rutaecarpa was determined by atom absorption. Respiration of soil microbe was studied by titration analytical procedures; the microbial physiological groups (decomposing Potassium bacterium, aminate bacteria, Azotobacter, nitrifying bacteria, cellulose decomposition bacteria)in soils were tested by most probable number. Soil enzymatic activities(urease, catalase, sucrase, Proteinase)were determined by colorimetry. The content of the trace element in soil was determined by atom absorption.[Results]1. The result of literature review and investigation showed that the genuineness of Euodia rutaecarpa was associated with the latitude(about27°) and altitude(200～600m). the latitude and altitude were key factors of forming the genuineness of Euodia rutaecarpa. Variety influenced the genuineness of Euodia rutaecarpa distinctly. Using TCMGIS, suitable growth regions was analysed, the resulted showed that82%genuineness regions of E. Benth.var.oficinalis(Dode)Huang located in Guizhou, witch was Completely consistent with records.2. The result of ITS analysis showed that ITS sequence polymorphism of Fructus Euodia was poorer, the sequence was similar, only some samples contain few base mutations.The result of AFLP analysis showed that it was easy to distinguish the genetic diversity of three Medicinal Materials source plants Euodia rutaecarpa, Euodia rutaecarpa (Juss) Benth var. Officinalis (Dode) Huang and Euodia rutaecarpa(Juss) Benth var. bodinieri (Dode) Huang. Plant genetic background was stable in High altitude mountain areas, which are Euodia rutaecarpa authentic region such as Xinhuang of Hunan, Tongren of Guizhou etc. This would be the foundation production quality stable medicinal material. The result of SRAP analysis showed that Euodia rutaecarpa genetic background diversity was obvious. Using cluster analysis of SRAP, we concluded that there were varieties differences in Euodia rutaecarpa genetic background. The genetic differences were obvious between Euodia rutaecarpa (Juss) Benth var. Officinalis (Dode) Huang and Euodia rutaecarpa(Juss) Benth var.bodinieri (Dode) Huang, and there was a trend of same variety to clustering in each region. Obviously, the genetic background of the same variety from different origin, those samples gathered more closely from Euodia rutaecarpa authentic region such as Xinhuang of Hunan, Tongren of Guizhou.3. Content of Euodiamine and Rutaecarpine in different producing areas of the same variety, the same origin of different varieties, the same variety of different harvest time showed that varieties, origin and harvest time influenced on the genuineness of Euodia rutaecarpa distinctly.4. The results of soil factor showed that Zn, sucrase, Cu influenced on the genuineness of Euodia rutaecarpa significantly.5.31endophytic fungi and37endophytic bacteria in Euodia rutaecarpa were separated from8Euodia rutaecarpa region, different specific types of the endophytic fungi and bacteria were separated in different leaves, stems, and fruits, namely with organized specificity. Existing obvious differences in different types of the Endophytes and dominant population, namely with regional specificity.4Euodia rutaecarpa endophytic fungi and1endophytic bacteria were screened which could produce evodiamine, rutaecarpine and structural analogues.6. The result of bacteriostatic experiment on Euodia rutaecarpa volatile oil showed that the bacteriostatic ability of Euodia rutaecarpa volatile oil could be regarded as an important efficacy index of Euodia rutaecarpa genuineness.[Conclusion]Influencing factors of forming genuineness of traditional Chinese medicine Euodia rutaecarpa include internal factors and Extrinsic Factors. Internal factors played decisive role on the formation of genuineness such as the variety difference of Euodia rutaecarpa and the specificity of the genetic background. For example, Euodia rutaecarpa of Xinhuang Hunan was the only variety of Euodia rutaecarpa (Juss) Benth var. Officinalis (Dode) Huang, the genetic background was little difference and stable. Extrinsic Factors included temperature, altitude, the amount and distribution law of soil microorganisms, distribution law of trace elements in soil, witch played key role. The following technology used to research genuineness of Euodia rutaecarpa:AFLP and SRAP molecular markers used for genetic background analysis, TCMGIS-Ⅱ system analysis for genuine origin division, most probable number was applied to the analysis of soil microorganism physiological groups, acid-base titration method was applied to the total content determination of soil microorganisms.