Based On The Turtle Shell Effect Of The Discovery Of Lead Compounds Research (2)

As a traditional Chinese medicine commonly used in clinical field, Carapax Trionycis,<Shen Nong’s Chinese Materia Medica> included initially, has such activities as replenishing ’yin’, restraining ’yang’, softening and resolving hard masses, reducing fever and removing steam. Clinical experience shows that Carapax Trionycis as the main medicine in the compound has an effective activity of combating liver disease, and modern pharmacological research shows that oligopeptide is onset ingredient for protecting liver. Since oligopeptide separated successively from traditional Chinese medicine showed good activity in anti-tumor, anti-oxidation, anti-fatigue, lowering blood pressure and improving immune activity, more and more attention has been payed to separating peptides for medical scientist. The method of separation and analytical testing is more and more modernized, but there are still some difficulties for peptide separation in fast and effective qualitative and quantitative detection means, and activity guide in vitro model. In addtiton, the current study mainly focuses on oligopeptide substances that naturally present in Carapax Trionycis, meanwhile oligopeptide derived from digestion of Carapax Trionycis have not been reported. There are still such questions to indetify as whether proteins of Carapax Trionycis as an oral medicine through gastrointestinal environment (such as the pH of the solution, pepsin, trypsin) keeps stable, and whether material transformation in vivo occers and play a pharmacodynamic mechanism. In order to solve a series of issues, the thesis under the guidance of the theory of Traditional Chinese Medicine, established qualitative and quantitative methods, as well as detection of pharmacological activity through liver tissue protein binding rate in vitro to guide separation of active oligopeptide compounds. To explore the transformation of aqueous extract of Carapax Trionycis in vivo through investigating digestion in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). And ultimately pure effective parts were got from digestion of aqueous extract of Carapax Trionycis in SGF and SIF based on the methods established.The main results of the subject are summarized on the following pages.1Quantitative determination and on-line detection of oligopeptide from Carapax TrionycisFor the first time study on quantitative determination of oligopeptide by enzyme-linked immunosorbent detector-biuret reaction. The developer was0.02g·ml-1sodium potassium tartrate solution (50ul),5%NaOH(20μl), and1%CuSO4·5H2O(50ul). The wave length of detection was at580nm. The calibration curves were linear at in the ranges of0.00mg·ml-1-5.00mg·ml-1for septa-peptide from Carapax Trionycis.Using this method to compare oligopeptide content in Pre-and-Post-Processed Trionyx Sinensis Carapace. Peptide content in vinegar-processed samples was6.99%,while the same content in crud samples was1.04%.Using this method to detect oligopeptide content of Carapax Trionycis Online in separation process. Baseline is not smooth and the peak is in irregular fluctuations when test solution obtained from reversed phase chromatography was detected at230nm, while the baseline is stable,less affected by the mobile phase and impurities at580nm. The method of enzyme-linked immunosorbent detector colored by biuret is simple and available to observe the separation of oligopeptides.2Exploration on thin layer color of oligopeptide with special a mi no acids by specific chromogenic reagentsFor the first time on establishing the method by chromogenic reagent to specific amino acids(histidine,tyrosine.trytophan,arginine) for the control to ninhydrin to prepare for separation of oligopeptide composed with specific functional groups of amino acids. It shows that color reaction to oligopeptide with histidine is of specificity and high accuracy. And it is simple and feasible to have a good guide for oligopeptide with histidine and itself3Determination of liver tissue protein binding rate of heptapeptide from Carapax Trionycis in miceFor the first time on quantitative determination with heptapeptide derived from Carapax Trionycis and surveying the binding rate of liver tissue protein to have a good guide for separation of oligopeptide, exploring the mode of action of heptapeptide with Liver tissue protein protein in vivo. Equilibrium dialysis was used to imitate the binding process between heptapeptide and Liver tissue protein protein in vivo. The research shows that the binding constants of heptapeptide is0.382mL·mg-1, which indicates this heptapeptide exists largely in free form in vivo with faster transformation between the free and bound drugs, so it is not easy to produce toxic side effects with excess given. And the determination of liver tissue protein binding rate of heptapeptide can provide a reference for other peptide with simple management, high accuracy, good reproducibility and fulfilling the requirement.4Oligopeptide quantitative and liver tissue protein binding rate in mice of aqueous extract of Carapax Trionycis after simulated gastrointestinal digestFor the first time on the transformation exploration of aqueous extract of Carapax Trionycis in vivo through investigating digestion in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), with pepsin enzyme substrate ratio,trypsin enzyme substrate ratio and enzymatic time respectively. Based on the results of Oligopeptide quantitative and liver tissue protein binding rate in mice, it is concluded that a large amount of oligopeptide arises after simulated gastrointestinal digestion with aqueous extract of Carapax Trionycis, with whose activity of liver is significantly stronger than undisposed.5Separation of oligopeptide in aqueous extract of Carapax TrionycisUsing the traditional method of boiling extraction,ethanol precipitation method with different concentrations of oligopeptide compounds of Carapax Trionycis preliminary purification,to get onset composition-W-A-80(precipitation with80%ethanol) with effect on the activity of MAO in the process of the liver fibrosis; with sephadex exclusion chromatography and reversed phase chromatography as the main means of the integrated use of oligopeptides on the turtle shell compounds were isolated and purified, after digestion of aqueous extract W-A-80of Carapax Trionycis in SGF and SIF,while the use of enzyme-linked immunosorbent detector-biuret reaction spectrophotometry, thin layer chromatography, high performance liquid chromatography and other modern means of testing the purity of the obtained compounds. Form a relatively complete Carapax Trionycis oligopeptide compound purification and purity testing method system for oligopeptide compounds, and provide some reference.