Study On The Chemical Constituents Of The Traditional Chinese Medicine Cistanche And The Effective Substances In The Body Against Vascular Dementia

Cistanches Herba(CH),one of the most valuable herbal drugs in traditional Chinese medicine,is of high medicinal and edible value and has been honored as "Ginseng of the desert".In recent years,chemical constituents of CH,especially phenylethanoid glycosides(PhGs),has been intensively studied.However,scarcely any research of simultaneous quantitative analysis focused on both hydrophilic and hydrophobic components.The structure of PhGs,the main active components in CH,possesses a number of unstable groups,which are easy to degrade during the analysis process,affecting the accuracy of the analytical results.Therefore,it is necessary to establish a reliable analytical method for the accurate quality control of CH.CH total glycosides capsules,derived from the total glycosides of Cistanche,are widely used in clinical practice for mild to moderate vascular dementia(VD)caused by deficiency of marrow.However,pharmacodynamic substances and effective mechanism of CH are not clear.Therefore,in view of above research shortcomings,this thesis systematically studied in vitro and in vivo chemical components of CH,and explored the pharmacodynamic substances and mechanism of CH to treat VD based on pharmaco-metabolomics.The findings obtained in this thesis are expected to improve the quality analysis method of CH and provide powerful evidence for the clinical application of CH to treat VD and anti-dementia drug discovery.The detailed research contents and results are as follows:1.Comprehensive qualitative and quantitative analysis of chemical constituents of CHPressurized solvent extraction module(PLE)was established to achieve micro-extraction of CH.Extraction efficiency of PLE module was demonstrated to be comparable with conventional ultrasonication using LC-IT-TOF-MS,although limited volume of water,a bit of crude materials,and only three minutes were consumed.The mass spectrometric patterns and diagnostic cracking pathways of PhGs,iridoids,and lignans on IT-TOF-MS platform were summarized,and 79 components from PLE collection of CH were putatively identified.Meanwhile,PLE module was online hyphenated with high performance liquid chromatography(HPLC),and online PLE-TFC-HPLC system was configured for simultaneous quantitation of eight PhGs in CH.The method validation results demonstrated that the proposed online PLE-TFC-HPLC system can be precise,accurate,reproducible,and reliable for quantitative analysis of CH.All findings demonstrated that the home-made online PLE-TFC-HPLC analytical platform is advantageous at direct and green analysis in terms of time-,labor-,solvent-,and material-savings,and reduces the possibility of unstable components degradation of CH during extraction.The versatile and automated platform offers a promising green choice for direct chemical analysis of solid complex matrices such as traditional Chinese medicine(TCM)and biological samples.The chemical composition of CH was holistically characterized for further study the metabolites of CH.Multiple scan modes of Qtrap-MS,such as predefined multiple reaction monitoring mode(pMRM),neutral loss scan(NL),precusor ion scan(Prec),enhanced product ion scan(EPI),and enhanced MS(EMS),etc.,were employed.NL scans and Prec scans based on characteristic fragments were developed to excavate chemical constituents of PhGs,iridoids and lignans.The pMRM scan based on databases such as HMDB and literatures was established to detect primary metabolites.Overall,a total of 513 chemical components in CH were detected,and the chemical metabolome of CH was basically clarified.Reverse phase liquid chromatography hyphenated with hydrophilic interaction chromatography was coupled to mass spectrometry(RPLC-HILIC-MS/MS).And a total of 27 abundant and minor components covering most chemical families in CH,i.e.amino acids,nucleosides,organic acids,PhGs,sugars,etc.,were simultaneously determined using RPLC-HILIC-MS/MS platform.Inferior parameters acquired by online optimization strategy of mass parameters rather than the optimal ones were applied for those abundant ingredients to purposely suppress product ion generation and advance the upper limits of quantitation,such as echinacoside,acteoside and mannitol.And scheduled MRM(sMRM)was adopted to monitor trace components in CH,The method validation demonstrated that the proposed RPLC-HILIC-MS/MS could extend the linearity range and achieve satisfactory retention for hydrophilic analytes.It could be a feasible and robust tool to practice holistic quality evaluation for TCM.2.Analysis of in vivo metabolites of CHBased on structural decomposition and simplification strategies,LC-MS was employed for systematic investigation on in vivo metabolism of tyrosol,hydroxytyrosol,caffeic acid,salidroside,cistanoside F,decarboxy rosmarinic acid,acteoside,echinacoside and CH.It was found that ester bond and glycosidic bond of compounds,such as PhGs and phenylpropanoid glycosides,were easily hydrolyzed after oral administration.This means that in vivo metabolites of CH were degradation products of PhGs,such as HT,CA,HVA and other phenolic acids,which were hydrolyzed in the gastrointestinal tract.After being absorbed into blood,these degradation metabolites further underwent sulfation and glucuronidation in the liver and excreted in the urine.Moreover,time to peak levels of in vivo metabolites were different among single compounds and CH.This goes to show that single compounds cannot represent the real process of gastrointestinal metabolism of multiple components from herbal medicines.The systematic study on the metabolism of single compounds and total extracts explained in vivo metabolome of CH,and provides a new strategy for the study of TCM metabolism.3.Study on anti-vascular dementia of CH based on pharmaco-metabolomicsThe VD model was established by the ligature of bilateral common carotid artery(2VO).Morris water maze test and pathological section results demonstrated that CH can reduce the hippocampus neuron damage caused by 2VO and significantly promote learning and memory in VD rats.The pharmaco-metabolomics study based on LC-MS was carried out to characterize plasma metabolome changes of VD rats induced by CH treatment.Multivariate statistical analysis indicated that endogenous metabolite levels in plasma showed significant differences between the sham-operated group vs the VD group and the VD group vs the CH-treated group.Meanwhile,39 metabolites that contributed to the VD model were identified,and CH significantly regulated 26 of those metabolites.Combined with analysis of metabolic pathways associated with those markers,major in vivo metabolites of CH,phenolic acids such as HVA and 3-HPP,were involved in the metabolism of aromatic amino acids,such as phenylalanine,tyrosine and tryptophan,and also affected the biosynthesis of neurotransmitter.Besides,glycerophospholipid metabolism and arachidonic acid metabolism were also regulated after CH treatment.Therefore,CH may participate in the regulation of neurotransmitter metabolism via its in vivo metabolites,regulate lipid metabolism disorder,alleviate oxidative damage and inflammatory response to exert anti-vascular dementia.Potential pharmacodynamic substances,such as HT,HVA and so on,showed inhibitory activity of OGD/R-induced HUVEC cell damage,exhibiting protective activity on cerebral vascular endothelial cells.The results indicated that in vivo metabolites of CH were pharmacodynamic substances against VD.Above all,these work provided abundant methods for holistic qualitative and quantitative analysis of CH,which clarified in vitro and in vivo metabolome of CH.The study elucidated that metabolites of CH were pharmacodynamic substances and involved in neurotransmitter metabolism to exert pharmacological effects of CH to treat VD.