Analysis Of Chemical Components And Material Basis For Neuroprotective Effect Of Ershiwuwei Shanhu Pill

’Ershiwuwei Shanhu’ pill (ESP) is famous prescription of traditional Tibetan medicine, which was recorded in a Tibetan medical classics called ’The Four Medical Tantras’(AD773-783), and now it has been officially recorded in the Chinese Pharmacopoeia (2010Edition). It has a long history of empirical use by traditional Tibetan physicians for the treatment of Bai-Mai disease, a traditional Tibetan medicine term referring to central nervous system diseases such as Alzheimer’s disease, epilepsy, migraine and so on. Nowadays, it is in clinical use for the treatment of cerebrovascular and neurologic diseases, and proved effective clinically in migraine and epilepsy. Though ESP has been widely used clinically for a long time, the bioactive ingredients, their metabolites and the mechanism of action of ESP are still not well understood, which restricts its clinical application and further development. In addition, in Chinese Pharmacopoeia (2010Edition), there only have simple requirements of chemical reactions, qualitative identification of Carthamus tinctorius L. and Crocus sativus L. by TLC as well as quantitative limitation of aconitine for ESP, which refers to few herbs, without enough representation and lack of prescription quality control indicators and quantitative criteria, it is difficult to ensure the stability of product quality as well as safety and efficacy of medication, therefore, the quality standards of ESP should to be improved and enhanced.In this study, multiple analytical methods, including UPLC-DAD/Q-TOF-MS, HPLC-ELSD, ICP-MS and GC-MS, were used to systemic analysis of the main components of ESP. The aqueous fingerprint of ESP was established by UPLC-DAD/Q-TOF-MS, and61main compounds belonging to6components were identified, which were attributed to11herbal medicines. The amino acids of ESP were analyzed by HPLC with a pre-column derivatization method, totally15amino acids were determined which mainly came from Pericarpium Lagenariae. Twenty two mineral elements, including4macroelements and18microelements at conjugated, ionized and solid forms, were determined by ICP-MS, which were attributed to10mineral medicines. The fructose and glucose in ESP before and after acid hydrolysis were determined by HPLC-ELSD. The volatile compounds of ESP were analyzed by GC-MS, and18main compounds belonging to3components were identified, which were attributed to5herbal medicines. In the aspect of the single medicine research of ESP, a rapid near-infrared reflection (NIR) spectroscopy analysis method was developed for discrimination of the dried rhizome part of Acorus calamus L. and Acorus tatarinowii Schott, two kinds of traditional Chinese herbs that are sometimes mixed or used interchangeably, and the simultaneous determination of their main components. LC-MS method was used to comparative analysis of the fingerprints of Aconitum kusnezoffii Reichb. and Aconitum pendulum busch to verify the rationality of using Aconitum kusnezoffii Reichb. instead of Aconitum pendulum busch; the rationality of compatibility of Aconitum kusnezoffii Reichb. with Glycyrrhiza uralensis Fisch and Terminalia chebula Retz. was also tested by comparing the contents of diester acdnitines in fingerprint of Aconitum kusnezoffii Reichb. before and after co-extracted with Glycyrrhiza uralensis Fisch and Terminalia chebula Retz..In the aspect of the bioactive material basis of ESP, both in vitro and in vitro methods were used to screen the bioactive compounds in ESP on the OGD-R induced apoptosis of SH-SY5Y cell. In vitro study, the compounds information of different extracts of ESP obtained by extraction and UPLC-MS analysis coupled with the bioactive test results of different ESP extracts with several concentrations, virtual screen of the potential bioactive compounds from ESP extracts was conducted by artificial neural network (ANN); in vitro study, the methodology of plasma pharmacoehemistry was adopted to analyze the potential bioactive compounds in rat plasma, heart, liver, kidney and brain after administration of ESP extracts, and36absorbed components, including20prototype compounds and16metabolites, were discovered. The results of virtual screen and absorbed components analysis were combined to obtain the main bioactive compounds which were further validated by monomer. The inverse docking method was used to predict the target protein of bioactive compounds of ESP and relative pathways. In this study, the compound composition of ESP was basically clarified. The main compounds of ESP, especially the characteristic compounds of each medicine, were determined which help to improve the quality standard of ESP. The bioactive material basis of ESP were confirmed by virtual screen in vitro and absorbed components analysis in vitro, which provide useful chemical information for further study of the pharmacology and mechanism of action of ESP on molecular level and also lay the foundation for the secondary development of ESP product. Finally, make it modern preparation with clear material composition and mechanism of action as well as more safety, stability and effectiveness.