| BACKGROUND:Carapax Trionycis is a traditional Chinese medicine which has such ac tivities as replenishing ’yin’,restraining ’yang’and softening the masses.The medicineandi ts compounds could relieve degree of the liver fibrosis,enhance the ability of theimmuni ty and so on. Modern pharmacological research shows its active constituents areoligo-pe ptides which had been proven to show inhibitory effects on the activity of MAO and h ad a certain combinational rate with the liver tissue acorrding to the previous studies an d the precious experiment had found the separation of the oligo-peptides was difficult,w hich was poorly repetitive and time-consuming.The oligopeptide (GAGPHGG) which wa s separated from the medicine had showed well curativeeffect on both acute and chroni c liver damage induced by CCl4and liver fibrosis induced by pig serum,but the experi ment had not studied the action onthe liver injury induced by alcohol which is more co mmon in the daily life. So the theme of the thesis is mainly divided into two aspects:re seach on the method of the separation of the oligo-peptides and the further study of the pharmacologic effects of the oligo-peptides in the Carapax Trionycis.In theaspect of sepa ration, the experiment had investigated the method ofderivationwith DNS-Cl, HPLC-MS/MS and MIAC to go on the work of separation.Inthe aspect of pharmacology,the experi ment focused on the oligo-peptides’effects on the proliferation of the corresponding cell s and the curative effect onthe acute and chronic liver injury induced by alcohol in rats, to elucidate therelated mechanism of Carapax Trionycis on the treatment of liver disease s, providing the basis for further clinicalapplication.The project was sponsered by the na tional natural foundation (Number:81073017) and the creative teamin Beijing University of Chinese Medicine(Number:2011-CXTD-15).METHOD:In the part of separation,the experiment had firstly dealed oligo-peptides with DNS-Cl to go on the separation;as for the part of secondary mass spectrum,the experi ment had determined the primary structure of the oligo-peptides by the method of HPL C-MS/MS;In the part of the complex experiment,the experiment had used the packings of silica gel, polyamide resin,sephadex G10and AB-8macroporous gel resin which wer e all dealed with copper sulfate solution to conduct the separation of the oligo-peptides,t hen optimizing the condition of the complex silica to prepare the complexpackingand co mplex thin layer plate;In the pharmacologic aspect, the cell experiment had used the me thod of MTT to investigate the effect of the oligo-peptides in Carapax Trionycis on the proliferation of human hepatic stellate cells(HSC-T6) and human liver cancer cells (Be17402),human normal liver cell (L02), macrophages(RAW264.7),fibroblasts (L929), basoph ils(rbl-2h3); the protective and therapeutic effect on the injuredL02of peptides in Carapa x Amydae;In the part of the animal experiment,the reseachers used the alcohol tomaket he acute and chronic liver injury model in rats to investigate the curative effect of the oligo-peptide (GAGPHGG) RESULTS:In the aspect of the separation, the experiment had proved the common meth od and derivation method couldn’t get the desired effect;The experiment had identified a n oligo-peptide whose sequence was H-Trp-Pro-Gly-Leu/Ile using the method of HPLC-MS/MS;The results of the complex part indicated the recovery rate of the AB-8absorb macroporous resin was (87.02+2.17)%while the recovery rate of copper sulfate-silica was (86.95±0.87)%; the optimization experiment had produced complex silica gel pac king andcomplex thin-layer plate and the results indicated that the packingcould enrich c ertain amino acids as well as increased the recovery rate significantly; the complex thinl ayer plate could separate the cysteine from the other amino acids; Arginine and histidin e had been separated; glutathione and glycylglycine had also been separated;In the aspec t of pharmacology, the experimentfound that the inhibitory rate of proliferation on the H SC-T6of oligo-peptides in Carapax Trionycis was about10%while the inhibitory rate of the oligopeptides produced by enzymolysis was about40%and the inhibitory rate oft he all oligo-peptides on immune cells were all above-30%;The results in the acute live injury experiment indicated that the serum concentrationsof ALT/AST and content of M DA in liver tissue was significantly decreased in treatment groups after giving the drug (P<0.05);the activity of SOD in the liver tissue in treatment groups were thus significan tly increased (P<0.05).Pathological result showed that the liver contidion in treatment gr oups was significant improved;The results in chronic liver damage experiment indicated the serum concentrations of AST,ALT,MAO,AKP and MDA in treatment groups were al so significantly decreased (P<0.05) while the activity of SOD in the liver tissue increas ed significantly;Pathological result showed thatthe liver contidion damaged by the alcoho1was also significant improved.CONLUSION:In the aspect of separation,the experiment had proved that thereaction of DNS-Cl with the oligopeptides was too complicated to determinethe products and and t he normal method was also not successful;The methodof HPLC-MS/MS could determine the sequence of animo acids in oligo-peptides without getting the monomer;The complex experiment indicated that AB-8macroporous adsorption gel resin could beused to separa te the oligo-peptides initially; the separation performance of silica gel coped with copper sulfate solution had changed greatly and the complex silica thin layerplate couldhave a good separation among certain animo acids like Arg and His;Cys from other amino acid s;In the pharmacologic aspect the experiment of the cellsindicated the active part was th e peptides with low formula weight prodecedby enzymolysis;The results of animal exper iment of acute and chronic liver injury expeiment both indicated that oligo-peptide I-C-F-6had a significantly therapeutic effect on liver injury induecd by alcohol and its mec hanism may be related to increase the vitality of SOD,which could scavenge free radica Is and inhibit lipid peroxidation. |
PDF Full Text Request