Study On The Active Anti-hepatic Fibrosis Principles And Fingerprint Of Carapax Trionycis

Carapax Trionycis,as a traditional chinese medicine,originated from the shell of Trionyx sinensis Wiegmann.It has such activities as replenishing "yin",restraining "yang",softening and resolving hard masses,reducing fever and removing steam;It is used for anti-fever,abdominal mass,windy syndrome agents.Clinical experience indicated that traditional chinese medicine compound with Carapax Trionycis showed curative effect when used for the therapy of liver fibrosis;Modern pharmacological researches revealed that extracts of Carapax Trionycis showed beneficial effects on animal model of liver fibrosis.Under the guidances of traditional chinese medicine theory,this treatise reported the active anti-hepatic fibrosis principles of Carapax Trionycis,and its chromatographic fingerprint by applying modern technology.Portions with different molecular weight were separated from the extracts of Carapax Trionycis using the method of ultrasonic extraction and membrane dialysis;the active anti-hepatic fibrosis principle was determined to be portion cantaining compounds molecular weight lower than 6000,by using such pharmacological assays as HSC proliferation assay,HSC activation assay and collagen synthesis assay. We determinated the mass range of free and hydrolyzed amino acids in the active anti-hepatic fibrosis principle,using Hitachi L8800 High Speed Acid Analyzer.The results showed that the content of free amino acid is 1.32%, while the content of hydrolyzed amino acid is up to 58.0%,suggesting that main active principle was peptides.Under guidance of Pharmacological assay,nine fractions were isolated from the active principle of Carapax Trionycis is using Sephadex G-25, among which four sections showed anti-fibrosis activity,the most active fraction was divided into seven subfractions by high performance liquid chromatography.the subfraction 7,showing better activity,has purity of 90%. We analyzed its structure by using 4700 Tandem Time-Of-Flight mass spectromenter,which producing following results:Peptide 572,with sequence:H G R F G(His-Gly-Arg-Phe-Gly );Peptide 542 with sequence: N P N P T(Asn-Pro-Asn-Pro-Thr);Peptide 526 with sequence:N D D Y(Asn-Asp-Asp-Tvr);Searching from NCBI,The three peptides are obtained from natural world for the fist time.It is the first that formulate the fingerprint of the active principle of Carapax Trionycis by HPCE.The analytical method has good credibility and stability.Ten batches of samples from Hubei,Zhejiang,Jiangsu province were analyzed by HPCE and 16 shared peaks were found in the fingerprints.The identified peptides were also determined in the fingerprint ofsamples.However,the fingerprints of Carapax Trionycis from different areas showedsignificant differences.So it provided good scientific foundation for searching active anti-fibrosis principles and evaluating the quality of Carapax Trionycis.