| Tibetan Medicine with a long history and a complete theoretical system, one of the most representative in the Chinese traditional ethnic medicine, has been widely used in clinic because of its unique curative effect. Hippophae rhamnoides Linn, is a typical multi bases of the original Tibetan medicine. According to "Jing Zhu Materia Medica'", it is divided into three kinds:large, medium, and small, and the small kind is Hippophae thibetan(sadaer). H.thibetan.with well-developed root system, a widely distributed specie on the Qinghai-Tibet Plateau, has an important ecological value and a momentous function of soil and water conservation in the plateau region. H.thibetan is a major source of Shaji extract, which can be used alone, having a efficacy of "nourishing the liver Yin". It has been recorded in "Standard of Tibetan medicine in Sichuan Province (2014 Edition)"It has been reported that the leaves of H.rhamnoides Linn, also contain a lot of flavonoids and nutrients, with rich resources for collection. It is the raw material of medicine, health food, cosmetics and animal feed and so on, having important economic value. The leaves of H.rhamnoides subsp. sinensis and H.rhamnoides subsp. yunnanensis have been incorporated into "Standard of Tibetan medicine in Sichuan Province (2014 Edition)". At present, there is no related research on leaves of H.thibetana, To take full advantage of H.thibetana resources, this paper investigates the resources distribution and ecological suitability of H.thibetana:contrast with biological determination; researches the components of flavonoids in the leaves, and establishes of quality evaluation method of H.thibetana leaves, ensuring the effectiveness of its clinical application and medicinal resources sustainability, to provide a basis for the resource development and utilization of H.thibetana.Objective:1. Investigate the distribution of resource and ecological suitability of H.thibetana, providing the basis for resource utilization of it.2. Identification the authenticity of H.thibetana.3. Separating and purifying flavonoids from the leaves of H.thibetana, establish methods for TLC to identify and HPLC to determinate the content of H.thibetana, establishing quality standards of H.thibetana leaves preliminarily.Methods:1. Research geographical distribution of H.thibetana in our country, by Maxent ecological models and GIS, and the characteristics of ecological factors that affecting geographical distribution; grades the growth suitability of H.thibetana. potential distribution area.2. Using DNA barcode technology, distinguish H.thibetana and common adulterants.3. Separating and purifying the extraction by column chromatography over macroporous adsorption resin, polyamide and sephadexlh-20, determine the structure of the compound by combining with spectroscopic techniques of 1H-NMR,13C-NMR and others. Establish methods for TLC to identify and HPLC to determinate the content of H.thibetana, with quercetin, kaempferol, isorhamnetin as the control sample, while study the characteristics, inspection, extract and other projects of leaves of H.thibetana.Results:1. Divide suitability distribution area of H.thibetana corresponding to various ecological factor layers, using artificial Manual classification analysis method. The results showed that the distribution of H.thibetana in the Qinghai-Tibet Plateau and the Himalayan alpine grassland regions, was mainly affected by precipitation of July, August, December, and February temperature. In China, suitable area is 477,780 square kilometers, and more appropriate area is 475,500 square kilometers.2. ITS2 sequence can distinguish between H.thibetana and common adulterants.3. Eleven flavonoids isolated and identified from ethanol extract of H.thibetana are as follows:Quercetin-3-O-?-D-glucoside-7-O-?-L-rhamnoside(1),Isorhamnetin-3-O-?-D-gal actoside-7-O-?-L-rhamnoside(2),Isorhamnetin-3-O-?-D-glucoside-7-O-?-L-rhamnosi de(3),Isorhamnetin-3-O-?-L-arabinopyranoside-7-O-?-L-rhamnoside(4), Narcissin(5), Quercitrin(6),Isorhamnetin-3-O-?-D-glucoside(7),Isorhamnetin-7-O-?-L-rhamnoside( 8), Isorhamnetin(9), kaempferol (10), Quercetin (11).4. The establishment of characteristics of H.thibetana leaves, and the method of TLC identification; set the limits of its checks item, the water should not exceed 11.0%, the total ash content not exceeds 5.0%, acid insoluble ash not less than 0.4%, alcohol extract not less than 14.0%; determining the content of H.thibetana leaves:quercetin content in the range of 0.092%-0.282%, with an average of 0.183%; isorhmnetin content in the range of 0.225%-0.666%, with an average of 0.388%; kaempferol content in the range of 0.080%-0.324%, with an average of 0.195%. In dry goods, isorhmnetin not less than 0.310%, quercetin not less than 0.146%, while kaempferol not less than 0.156%.Conclusion:1. H.thibetana resources distribution is mainly in the Qinghai-Tibet Plateau and alpine steppe region of the Himalayas; in ecological factors, altitude and the standard deviation of seasonal temperature changes are the greatest impact factors to suitability of H.thibetana.2.ITS2 sequences can discriminate quickly and accurately of H.thibetana and adulterants, provide new techniques for the identification of Tibetan medicine.3. The analysis of chemical constituents of flavonoids of H.thibetana leaves.4. Initially establish the quality control method of H.thibetana leaves. |
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