| Huangqi-Danshen is one of the herb-pairs commonly used for invigorating qi and activating blood circulation.In clinic,this herb pair was widely used in the treatment of coronary heart disease,ischemic brain injury,diabetic nephropathy.The literatures mainly focused on its pharmacology and clinical application.Up to now,few researches were reported on its chemical components in vitro and in vivo.Based on the main idea of similar components having the similar fragmentation behavior,the diagnostic ion technology could rapidly screen and identify the chemical constitutes of traditional Chinese medicine(TCM)coupled the application of highresolution mass spectrometry.This study intends to construct the analysis method based on the diagnostic ion technology,to systematically analysis the chemical constituents of Huangqi-Danshen in vivo and in vitro,which provided the scientific basis for its clinical applications.Objective:This work aimed to globally identify the constitutes of Huangqi –Danshen in vivo and in vitro based on the preferential multi-diagnostic ions of the chemical constituents of Huangqi and Danshen.Methods:1.Establishment of the strategy for the analysis of Huangqi compounds based on the diagnostic ion technique.First,Huangqi constitutes were collected and established its in house library,where the parent nucleus and substituents types of flavonoids and saponins were summarized and classified.The potential chemical components were predicted through the analysis of the potential link between the new substituents to its existed parent nucleus,which were then collected in a predictive compound database.Secondly,the diagnostic ions and auxiliary diagnostic ions were selected by UHPLC-Q Exactive MS by analysis of the cleavage of Huangqi standard and representative components at different collision energies.The expected and unexpected constitutes of Huangqi were discriminated and confirmed based on the diagnostic ions.Meanwhile,the chemical components of Huangqi were untargetedly searched and identified through Compound Discoverer software and other network databases.Finally,the chemical composition of Huangqi was systematically characterized.2.Establishment of the strategy for chemical constituents of Danshen based on diagnostic ion technique,which was same as Huangqi.3.UHPLC-Q Exactive MS technology was used to obtain the chemical profiles of Huangqi-Danshen herb pair,which were assigned and identified by the above method of diagnostic ion technique.4.The plasma samples were collected after multiple administration of high-dose Huangqi-Danshen herb pair in 15 min,30 min,1 h,1.5 h,2 h,4 h and 8 h,respectively.The chemical constituents of Huangqi-Danshen herb pair in vivo were tested by UPLC-Q-TOF-MS technology coupled the above approach.Results:1.Based on the established approach,a total of 121 compounds,including 60 flavonoids,41 saponins,17 amino acids,2 coumarins and 2fatty acids were identified in Huangqi.2.A total of 136 compounds consisting of 92 phenolic acids,36 tanshinones and 8 amino acids were detected in Danshen.3.The composition of Huangqi-Danshen herb pair were comprehensive analyzed by diagnostic ion technology,which identify the 190 ingredients,including 39 flavonoids,24 saponins,78 salvianolic acids,30 tanshinones,16 amino acids,2 coumarins and 2 fatty acids.4.Using UPLC/Q-TOF-MS,a total of 57 commands of were identified in plasma samples after oral administration of Huangqi-Danshen herb pair,which included 14 prototype components and 43 metabolites.And flavonoids,tanshinone,salvianolic acids were the main composition of plasma samples.Meanwhile,the results also suggest the oxidation,reduction,esterification,glucuronidation and sulfate methylation were their major metabolic reactions in vivo.Conclusion:This research established the analytical methods of chemical composition of Huangqi and Danshen by the diagnosis ions,which was carried out to research the chemical profile of Huangqi-Danshen herb pair in vitro and in vivo.This study provided a scientific basis for the quality standard for the TCM preparations containing Huangqi-Danshen herb pair,and laid a foundation for their development and research. |
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