I. Oligopeptides In Cam Model Vinegar Turtle-based Active Ingredients

Biejia for turtle species (Trionyx sinensis Wiegmann)carapace, is one of clinical commonly used animal medicine , alias snapping turtle, Vinegar snapping turtle. Vinegar turtle, a processed product using the stir-baking in sand with hot vinegar quenched.Tenth Five-Year Plan period, 50 kinds of Chinese medicine such as Biejia Pieces of processing, are listed as key national key project with a view to adopting systematic research, and the use of modern medical theories connected by means of science and technology to Chinese medicine theory and methods pursuiting truth, to reform and improve the methods of traditional Chinese medicine processing purposes.The team of researchers based on it aiming turtle chemical composition, processing technology and quality standards, carried out a series of research work. Confirmed in further studies, turtle also contain Oligopeptide constituents, and the oligopeptide separation, purification and identification of vinegar turtle shell powder in the water extract , which has a seven-peptide compounds. In order to obtain more oligopeptide compounds, we do depth studies in vinegar turtle on the water extraction with Alcohol sinking, including: vinegar Biejia Screening of active ingredients, separation and purification of the active site research, and to explore structural modification applied separation and purification of vinegar turtle activity oligopeptideMainly focusing on the following studies:Literature Review1 A comprehensive evaluation of the chemical composition of vinegar turtle, pharmacological effects and clinical research applications , Focused on the chemical composition and application of vinegar Biejia. For the latter part of the development of viable research programme to identify and study the starting point indicators and research work has laid a solid foundation, a total of 17 references.2 Review the oligopeptide pharmacological compounds on the progress, purification and structure determination of the level of a total of 51 references.3 Review the chicken chorioallantoic membrane (CAM) model in drug inhibiting angiogenesis research the application time of the drug into the choice of CAM microvascular changes in the quantitative study were introduced. CAM angiogenesis future research will focus on finding CAM angiogenesis inhibition method to control tumor growth and metastasis, a total of 24 references.Experimental study1 Preliminary studies using macroporous resin - HPLC, isolated and identified a compound AAHGPCG in vinegar turtle, for the first time from the compound isolated from the natural world.2 Applied chicken chorioallantoic membrane (CAM) model, vinegar Biejia condensed water extract powder dry paste, a screening study in 20% alcohol precipitation, 40% alcohol precipitation, 60% alcohol precipitation, and 80% alcohol and 95% alcohol precipitation. The results suggested that: vinegar turtle powder extracts of the six have inhibited angiogenesis effect (p <0.05) and, in particular 80% alcohol Shen stronger activity;Further activity screening results in three parts peptide from 80% of alcohol using sephedaxLH-20 suggest that: three sites have inhibited angiogenesis effect (p <0.05), especially F2 stronger activity; parts of the three peptides were purified from the three samples of chicken embryo experiments also suggested that inhibit angiogenesis effect (p <0.05), F3-22 activity stronger.3 We used gel chromatography (Sephadex LH-20), the combination of biuret reagent tracking, mainly on the F2 and F3 activity strong position of the 80% alcohol-Parts for isolation and purification. F2 water purification liquid acetone precipitation, the RP-C18 column repeated purification. F2-20% of which methanol elution part of the F2-105, and the amount was white solids. F3 water purification liquid acetone precipitation, the RP-C18 column color, and is divided into five parts, and 20% methanol elution part of the F3-22, and the amount was white solids. Three pure F1-107, F2-105, F3-22 chromatography of the blank control, it is clear that F2-105, the highest purity of the samples. We do F2-105 of the 1H-NMR spectrum, 13CNMR spectrum, H-H COSY, QC and BC spectrum, and then through the amino acid sequence was obtained for a structure: G / A - E / P - A / G -G--A/P--Q/I-G.4 Using DNS-Cl structural modification , so peptide compounds reduce its polarity at the same time, the introduction of fluorescent groups. And the establishment of the first polyamide polyamide thin layer chromatography, separation peptide derivatives are four parts white solid barⅠ,Ⅱ,ⅢandⅣ,Ⅲthrough polyamide repeatedly purification obtaining the white solid barⅢ- 1 andⅢ-2.Ⅳthe structure identified as DNS-OH; Results suggest thatⅣwe have not derived separated fromⅠ,Ⅱ,Ⅲ, andⅠ,Ⅱ,Ⅲcan be clearly observed yellow fluorescent peptide in the polyamide, HPLC also has a good separation.So the polyamide can be successfully applied vinegar Biejia crude extract of the peptide derivatives purified by the method is easy to operate, with good reproducibility, polyamide can be activated to re-use, less consumption It is a purified peptide derivatives good way.