| Gasirodiae Rhizoma is a perennial parasitic plant of Orchidaceae gastrodia, the tuber of which is medicine. The effect of it is income wind Zhijing, stabilize liver Yang, dispelling wind and removing obstruction in the meridians. And it can cure insomnia, headache, tinnitus,numbness, hypertension etc. Moreover, gastrodia have a large demand in and out of China, whch can be used in the area of food, heatth care products, cosmetic etc. At present, many areas raise gastrodia,including Shanxi, Yunnan, Guizhou, Sichuan, Hubei etc. Pingwu in Sichuan Province is the wild real estate areas, and grow it by manual work from1980s, which have a long history of30years. In order to coordinate enterprise and local government’s standardization cultivate of gastrodia SOP optimizing promotion and authentication of GAP. Together with modern chemistry analysis and molecule biology technology, we adopt traditional crude drug research method to do systematic evaluation research of gastrodia’s quality. The main content and result is as follows:1Morphological and histological study on Gastrodia elata Bl.f.glauca, Gastrodia elata Bl.f.viridis, Gastrodia elata Bl.f.elata has been.did.As a result, find out their main identification characteristics of original plant, medicinal materials characters and microscopic characteristics. We observe their polliniums with the aid of scanning electron microscope for the first time,which provides a reference for its varietal indentification.1.1The plant height, the length of the inflorescence.the color of stem and flower are the main identifications of the original plant morphology.1.2In terms of medicinal materials characters, there are not significant difference among the three different varieties. However, they have slightly difference in shape,size and surface links.1.3The main identification of microscopic characteristics are the columns,shape and degree of bolt of hypodermis cell,the columns of cortex cell and the shape of outer cells of tubers cross-section. 1.4Polliniums of the three different varieties under the scanning electron microscope reveals that although the overall is similar, they are different in the size of pollen and network ridge. The characters of pollen of Gastrodia elata Bl.f.viridis and Gastrodia elata Bl.f.elata. are more similar in comparision with Gastrodia elata Bl.f.glauca.2Established a more abundant of information on TLC identification method,on the base of the current edition of "Chinese Pharmacopoeia".We compared different areas,different species of Gastrodia with this methods and found their similarities and differences. This method provides a referencer for the analysis of the differences in chemical composition.It can establish TLC identification method of rich information for Gastrodiae Rhizoma by improving the method of test solution preparation and investigating development system n the amount of spotting and the expanded distance, which can be better isolate n had the clear spots and suitable RF value with chloroform-methanol-water-formic acid (3:1:0.1:0.1) as developing solvent.Comparison of TLC identification of20batches of samples indicated:Gastrodia elata Bl.f.glauca、Gastrodia elata Bl.f.elata and Gastrodia elata Bl.f.viridis in Pingwu Sichuan had the same of Chromatography spot position and color, but the individual spot shade of color was different.Gastrodia elata Bl.f.viridis (sixteenth sample) the spot light color when RF=0.8,which had slight difference with the same origin of Gastrodia elata Bl.f.glauca and Gastrodia elata f.elata.The different origin of Gastrodia elata Bl.f.elata had different chromatographic spots and difference in shade of color with same chromatographic spots in location,so origin was a factor that not only leaded to the difference of the amount of the same chemical composition,but also resulted in the generation of different types of chemical ingredients.3Builded the analysis method of HPLC fingerprint of Gastrodiae Rhizoma,Comparison of fingerprints of the different varieties of the same origin and the same species of different origin of Gastrodiae Rhizoma,laid the foundation for further study 3.1By selection of chromatographic conditions, the choice of method of preparation of the test solution and the study of methodology, Gastrodiae Rhizoma’s HPLC fingerprint analysis method has been proposed.By Analysised of20batches of samples of Gastrodiae Rhizoma’s fingerprint chromatogram, to determine the13peaks as the reference fingerprint characteristic peaks. The method was stable and reliable, good repeatability, and it could be used for the overall comparative study of the the Gastrodiae Rhizoma’s chemical composition information.3.2Similarity analysis results showed that20samples excepted NO.1,NO.7,NO.18,No.20, the similarity was more than0.9.illustrating that16batch of gastrodin medicinal chemical composition is substantially the same.3.3The result of cluster analysis showed that20batches of samples to divide into two classes-A and B by a distance of20.The No.4. No.10samples were clustered into one class, other samples were clustered the other class. Clustering analysis diagram showed that No.5,No.13,No.20were clustered into one class. NO.1,NO.7were clustered into another class.So it can speculated that in the same place were similar in chemical composition.3.4HPLC fingerprint of Gastrodia elata Bl.f.glauca of Pingwu and Gastrodia elata Bl.f.elata of Pingwu had higher similarity. It Shows that the convergence of their chemical components. Gastrodia elata f.viridis and other two varieties of fingerprint had differences, but because of its less samples.it needs further testing. Gastrodia elata Bl.f.glauca and Gastrodia elata Bl.f.elata in different origins had significant difference in the number of peaks and peak area size.It indicated that the origin factors influenced their chemical composition significantly.4Established the ultraviolet spectrophotometry determination method of Gastrodiae Rhizoma polysaccharides content, determined the content of Gastrodia elata extract,Gastrodine and polysaccharide. In addition, we used the weighted scoring method on the determination results of comprehensive evaluation, in order to provides reference basis to make clear the quality situation of cultivated Gastrodia elata in pingwu. 4.1According to the2010edition of "Chinese Pharmacopoeia" alcohol soluble extract assay hot-dip method,we determined20batches of Gastrodiae rhizoma samples. The results manifest that these samples contents about6.78%～17.79%extract.6batchs of these samples does not conform to the regulation of current edition of "Chinese pharmacopoeia"(shall not be less than10.0%).4.2According to the determination method of gastrodine in2010edition of "Chinese pharmacopoeia".we determined gastrodine content of20batches of samples.The results manifest that in these samples the highest gastrodine contents is0.75%, and the lowest is0.22%. All samples are comply with the current edition of "Chinese pharmacopoeia"(must not be less than0.2%)4.3through the preparation of test sample solution and methodology, rhizoma gastrodiae polysaccharides content was established method of uv spectrophotometry, and the samples of20batches of rhizoma gastrodiae polysaccharides content determination.Polysaccharides of Gastrodia elata Bl.f.glauca content is high, polysaccharides of Gastrodia elata Bl.f.virid is content up to32.5%. Same breed of gastrodia elata its polysaccharide content in different regions have differences,4.4the weighted scoring method is adopted to extract of20batches of samples, carries on the comprehensive score of gastrodine and polysaccharide content, results showed that Gastrodia elata Bl.f.glauca with every town of pingwu of sichuan higher scores; Gastrodia elata Bl.f.elata in hubei yichang, pingwu of sichuan higher scores; The same origin of different varieties of gastrodia elata, Gastrodia elata Bl.f.glauca is superior to the Gastrodia elata Bl.f.elata.5. Different varieties is studied by using theory of SRAP molecular markers for the first time gastrodia elata genetic relationship, by means of gastrodia elata DNA extraction, PCR, electrophoresis conditions, etc, to establish gastrodia elata SRAP analysis method, comparison of different varieties of gastrodia elata, provide theoretical basis to further explore the screening of fine varieties. The research results show that the similarity coefficient of0.55as the threshold, the11samples of gastrodia elata blume can be divided into A, B two groups, including gastrodia tuber gathered for A class,Gastrodia elata Bl.f.elata and Gastrodia elata Bl.f.virid gathered for A class, suggests thatGastrodia elata Bl.f.virid with Gastrodia elata Bl.f.elata close genetic relationship. Methods established in this paper is stable, reliable, high repeatability, can be used for analysis of genetic relationship between samples with different varieties, for gastrodia elata screening of excellent germplasm resources provides a scientific basis. |
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