| Kai-Xin-San(KXS)is a classic traditional Chinese medicine compound which has been used clinically for the treatment of neurasthenia and Alzheimer’s disease.The study took KaiXin-San as the research object based on the high resolution mass spectrometry technology.Through the association analysis of the in vitro and in vivo chemical constituents and pharmacological effects in different groups,the rules of formula compatibility and the pharmacodynamic material basis of KXS were explored,and the pharmacokinetics comparison of the main components in normal and AD rats was also studied,which provided the basis for the comprehensive development and utilization of KXS.An ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry was established to identify the chemical components of compound KXS in vitro.Initially,the fragmentation patterns,diagnostic product ions and neutral loss of each category of compounds were summarized by collision-induced dissociation analysis of representative standards.Combined with multiple product ions filtering technique,neutral loss filtering technique and other data processing methods,108 constituents were identified including 47 ginsenosides,9 polygala saponins,17 oligosaccharide esters,8 xanthones,8 triterpene acids and 19 other compounds.The compounds database in vitro was established and the main chemical material basis of KXS was elucidated.An ultra high performance liquid chromatography quadrupole time-of-flight mass spectrometry was developed to analyze the plasma and brain samples after administration of compound KXS.The prototype compounds were extracted based on the established database,and multiple data processing methods combined with the drug metabolism reaction rules was employed to identify metabolites.47 prototype constituents including 16 ginsenosides,3 polygala saponins,8 oligosaccharide esters,4 xanthones,5 triterpene acids,11 other compounds and 22 metabolites were characterized in rat plasma.The compounds database in plasma was established.9 constituents were firstly characterized in rat brain including triterpene acids and phenylpropanoids.The main effective composition in vivo was preliminarily elucidated.The AD model was induced by intraperitoneal injection of D-gal and bilateral hippocampal injection of Aβ25-35 to investigate pharmacodynamics in AD rats after oral administration of different combinations of KXS,the Morris Water Maze behavioral test and brain tissue pathological biopsy were carried out at the seventh week.The behavioral test showed that the spatial learning and memory abilities of KXS group were significantly better than model rats.Brain tissue pathological biopsy showed that KXS could significantly improve the morphology of nerve cells in the hippocampus CA1 section of AD rats.The results indicated that KXS has preventative and therapeutic effect on AD.Different groups also affected the the spatial learning and memory abilities of AD rats in different degrees,and the disassembled prescriptions which had significant differences compared with the model group all contain Panax ginseng or Polygala tenuifolia.The two drugs may play a major role in KXS compound,which might be the monarch and minister medicine.At the same time,contrasted between each drug group,the increase of the drug flavor could significantly improve the spatial learning and memory ability of AD rats,and indirectly proved the importance of Panax ginseng,Polygala tenuifolia,Poria cocos,and Acorus tatarinowii Schott to KXS efficacy,also explained the rationality of the compatibility combination in classic TCM compound KXS.In vitro samples and plasma samples were analyzed with the established qualitative analysis method.Combined with the established in vitro and in plasma compounds database,the qualitative and semi-quantitative analysis of each prescription chromatogram was carried out.After attribution and addition processing of the obtained chemical data,the grey correlation analysis between chemical data and pharmacological data was carried out to explore the rules of formula compatibility.The results of in vitro and plasma samples were basically consistent,and the grey correlation degree between pharmacological data and chemical data of each drug namely the contribution of each drug to pharmacological action:Panax ginseng>Polygala tenuifolia>Poria cocos,Acorus tatarinowii Schott.The results also supported the pharmacological experimental results,which is Panax ginseng is monarch herb,Polygala tenuifolia is minister herb,Poria cocos and Acorus tatarinowii Schott are adjuvant and guide herbs.However,the composition of TCM compound is complex,based on the principles of formula compatibility,the pharmacodynamic material basis of KXS was studied,and the three TCM medicines were combined with the minister herb ginseng.The grey correlation analysis between chemical data and corresponding pharmacological data was carried out to explore the pharmacodynamic material basis.The results showed that protopanaxadiol type compounds ginsenoside Rc and ginsenoside Rb1;protopanaxatriol type compounds ginsenoside Rgl and ginsenoside Re;polygala saponins compounds reinioside A and tenuifolin;polygala oligosaccharide esters compounds sibiricose Al,sibiricose A6,tenuifoliside A and arillanin A;polygala xanthones compound sibiricaxanthone B;polygala oxanthrone compound telephenone B and corresponding metabolites made up the pharmacodynamic material basis of KXS.The chemical composition from different sources composed the pharmacodynamic material basis of the compound KXS,suggested that different kinds of chemical composition may exert efficacy through different action pathways.It also showed the advantages of KXS in preventing and treating Alzheimer’s disease through multiple targets and multiple pathways.A method of ultra-fast liquid chromatography with tandem mass spectrometry was developed and validated for the simultaneous quantitation of polygalaxanthone Ⅲ,sibiricaxanthone B,tenuifolin,sibiricose A5,sibiricose A6,tenuifoliside A,ginsenoside Re and ginsenoside Rbl in rat plasma after oral administration of KXS.The plasma samples were extracted by liquid-liquid extraction using digoxin as internal standard.Chromatographic separation was performed on a Venusil MP C18 column with methanol and 0.05%acetic acid in water as mobile phase.The tandem mass spectrometric detection was performed in the multiple reaction monitoring with turbo ion spray source in the negative ionization.Validation parameters were within acceptable ranges.The established method has been successfully applied to comparing the pharmacokinetic profiles of the analytes between normal and Alzheimer’s disease rats.The results indicated that there were significant differences in pharmacokinetic parameters of polygalaxanthone Ⅲ,tenuifolin,sibiricose A6,ginsenoside Re and ginsenoside Rb1 between two groups while the others had no significant differences,which may be due to the mechanisms of Alzheimer’s disease and pharmacological effects of the analytes. |
