Study On The Quality Of Alliin And The Intracorporal Process By Intravenous Administration In Mice

Objective:To determin chemical constituents in garlic by pharmacopoeia means and study on the correlation between them;To optimize the separation and purification technology of alliin;To establish the HPLC fingerprint of alliin（50%and 90%）and study the related substances;To study the correlation between alliin and H₂S by intravenous administration in mice.Methods:1.The content of the components in the garlic was determined by the United States Pharmacopoeia,the India Pharmacopoeia,the European Pharmacopoeia,the British Pharmacopoeia,the Chinese Pharmacopoeia and the team method.2.On the basis of the original technology,the effects of resin type,concentration and pH of sample solution,eluent concentration on alliin separation were investigated,and recrystallization by ethanol,the purified product was determined by UV,IR,MS and NMR,and the purity was determined by HPLC.3.The HPLC fingerprint of alliin（50%and90%）was established by derivatization,and the contents of amino acids were analyzed.4.The UPLC-MS/MS method for simultaneous determination of alliin and H₂S alliin in biological samples,To study the pharmacokinetics and tissue distribution of alliin and its correlation with H₂S by determining the content of alliin and H₂S in plasma and tissues at each time point.Results:1.The content of alliin was measured in the garlic by USP,India Pharmacopoeia,team method were 2.97%,1.47%and3.10%,the content of potential allicin in garlic was 1.29%by EP,the content of in garlic allitricin was 0.273%by ChP.2.Using cation exchange resin as filler,the garlic extract was concentrated and adjusted pH,then eluted with water and ammonia water,and then collected pH<7 part of the eluent,the yield was 74%.The results showed that the purified products obtained by recrystallization were in good agreement with the molecular structure of Alliin.The results of melting point and optical rotation were consistent with those reported,the purity of alliin was more than 99%by HPLC,and the total yield was 2.8%.3.The HPLC fingerprint of alliin（50%）was established,14 common peaks were found and identified 9 peaks,the similarity>0.97;and the HPLC fingerprint of alliin（90%）was established,12 common peaks were found and identified 8 peaks,the similarity>0.99;The contents of amino acids in alliin（50%and 90%）were significantly different.4.Simultaneous determination of alliin and H₂S in blood and tissues in mice by UPLC-MS/MS,the main pharmacokinetics parameters of alliin were as fllows,K_e:4.79×10^-3min^-1,T_1/2:148.03min,C_max:266.61μmol/L,AUC_{（0-120min）}:9217.71 min·μmol/L,AUC_（0-）∞:13365.49 min·μmol/L,V_d:6.99L/kg,CL:0.03L/min/kg.The main pharmacokinetics parameters of metabolites H₂S were as fllows,K_e:4.59×10^-33 min^-1,T_1/2:154.34 min,C_max:0.452μmol/L,T_max:20min,AUC_{（0-120min）}:34.79 min·μmol/L,AUC_（0-∞）:79.33 min·μmol/L,V_d:1208.22 L/kg,CL:5.48 L/min/kg.Alliin could be determined and the contents of H₂S were increased in different degree in the plasma and tissues of mice after administration.The contents of alliin in tissues reached the highest value was 10²0min after administration,the contents of alliin in tissues were:kidney>heart>lung>spleen>brain>liver.The contents of H₂S in plasma and tissues reached peak was 20min and 15³5min after administration,the amount of H₂S increased at the peak of each site were kidney>liver>brain>spleen>heart>lung>plasma.Conclusion:1.Alliin is the index components of garlic,there is no significant difference between the results the contents of alliin by team method and USP,enzyme inactivation is not complete in pretreatment process,the result of alliin was lower by IP,There was a correlation between the results of EP/BP and USP,the results of allicin could indirect reflect alliin in garlic,and there was no correlation between the content of alliin and allitricin by ChP,and the results of allitricin could not reflect of alliin in garlic.So it is more reasonable to choose the appropriate method to control the quality of garlic with alliin and allicin.2.This study determined the best type of resin,the concentration and pH of sample solution,the concentration of eluent,clarify that it is necessary to adjust pH of sample solution,improve the yield of alliin by ethanol,purified alliin by recrystallization and reduced the process from 3 times to 2 times,and improved the yield and reduced the cost.It is to lay the foundation for the industrialization of alliin to apply for new drug.3.The HPLC fingerprints of alliin（50%and 90%）were in accordance with the technical requirements of the fingerprint of the extract,they could be used for process control and quality evaluation of alliin（50%and 90%）,and the related substances in the 10batch of alliin（50%and 90%）were amino acids and sugars,and the content of amino acids related substances was different.4.In this study,UPLC-MS/MS method was established for simultaneous determination the contents of alliin and H₂S in blood and tissues in mice,and the results showed that alliin could metabolize H₂S in mice,and laid the foundation for further study on the pharmacodynamics of alliin.