| The research based on the preparation for abstinence BinChaShuan, a kind of Chinese Traditional Medicine created by Professor Wang Zeshi of Zhejiang TCM College. Toxin of Agkistrodon halys pallas is main part of the preparation, which can significantly control the withdrawal symptoms. It is necessary to find the role of toxin of Agkistrodon halys pallas in analgesia in order to reveal the mechanism of BinChaShuan. Because of complexity in parts of the toxin with different pharmacologic function, it is essential to purify a kind of analgesic protein from the toxin and clarify its properties, then begin mechanism studies. On the purpose, we separated a basic protein from the toxin, revealed its properties, role passage and modulation on neurotransmitters, and founded the theory for the new medicine development. Part I Studies on target gene and target drug in BinChaShuanAfter the discovery of effective control on monkey's heroin withdrawal symptoms by BinChaShuan, we try to find differential expression of genes by the way of screening in striatum of monkey brain.First formed the model of heroin addicted monkeys by injection heroin in increasing dosage through scoring withdrawal symptoms to determine the grades of dependence. Then killed the monkeys in the groups of Normal, Negative and BinChaShuan treatment by injecting air into heart during withdrawal. Immediately cut the striatum and stored it in liquid nitrogen. Through the extract of total RNA, pre-hybridization, probe labeling of Cy3 or Cy5 to the control or treatment group, judging the genes of different expression by comparing the intensity and ratio of fluorescence.Results showed the score of BinChaShuan group was significantly lower than that of negative group, but not different with that of positive clonidine group. During withdrawal of heroin addicted monkeys, gene expression changed in striatum after addiction and treatment by BinChaShuan. They were preproenkephalin gene and HLA-SB(DP) alphagene. Preproenkephalin gene had lower expression after heroin addiction, and higher after treated by BinChaShuan, but HLA-SB(DP) alpha gene expression decreased in treatment. The result showed BinChaShuan promoted expression of preproenkephalin gene on one hand, inhibited expression of HLA-SB(DP) alpha gene on the other hand, then controlled withdrawal symptoms. The separating recipe study on BinChaShuan by even designing showed toxin of Agkistrodon halys pallas was the key part in analgesic function of BinChaShuan.Part II Separation, Screening of analgesic C4 protein in toxin of Agkistrodon Halys Pallas and researches on its propertiesWe tried to find relevant proteins in toxin of Agkistrodon halys pallas, which was active analgesic matter in BinChaShuan. On the platform of AKTA-Explorer100 purifying system, combining experiments of analgesic effect and toxicity, we purified the various proteins in toxin of Agkistrodon halys pallas, and screened the best analgesic component, then determined its molecular weight, isoelectric point, purity by SDS-PAGE, Isoelectric focusing and HPLC, further clarified its properties by UV spectrum, amino acid sequence, etc.In the separating experiments we inspected the effectiveness of different columns among cation exchange, anion exchange and size exclusion, applied orthogonal design L9 (34) to inspect the effect of buffer pH, flow rate, salt concentration on separation by method of scouting, and decided to use ion exchange first, two kind of size exclusion latter to purify and get proteins in purity 97.5%.We got 14 proteins from toxin of Agkistrodon halys pallas after separation. They were 5 acid proteins, 5 basic proteins and 4 neutral proteins. Since flow rate had great influence on separating effect with size exclusion, slower flow rate should be chosen.Using mice hot plate method and acetic acid twisting body method to determine ED50 and LD50 of different proteins, we discovered a basic protein had stronger analgesic function and less toxicity. Its LD50 was 13.3mg/kg ip.,EDso was 0.63mg/Kg ip., it app...
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