| Traditional Chinese medicine (TCM) is the valuable treasure of Chinesepharmacology. It is very important to explore its action mechanism systematically anddevelop it drastically, then make it win the world recognition. Although it has beenused clically for thousands of years, it can not march towards the world with activeposture. The major reason lies in three key points:(1) the real bioactive componentsare not clear;(2) exact action mechanism is unambiguous;(3) there is insufficienteffective method to assess the safty of TCM and the theory of TCM uptill now.While steroid glycosides have many health benefits, the potential development ofthem for the prevention/treatment of neurological disorders is largely hindered bytheir complexity as well as by limited knowledge regarding their bioavailability,metabolism, and bioactivity, especially in the brain. This study integrated multiplemodern methodologies and techniques in the discipline of Pharmaceutical Analysisincluding multiple-dimensional liquid chromatography tandem mass, microdialysissampling, metabolomics and pattern recognition methods. The aim of this paper wasto systematically discuss the chemical substances and mechanism of TSA, and tooffer the reference of technology for related investigation of other herbal medicine.1. In the work presented here a novel approach to comprehensive two-dimensionalliquid chromatography is evaluated. Reversed-phase liquid chromatography wasemployed for the frst-dimension separation and polyamine chromatography waschosen for the second-dimension separation mode. The comprehensive RP×polyamine II-MS system proposed here not only simplifed complex plant samples,but also enlarged peak capacity and enhanced selectivity. The two dimensions arehighly orthogonal and the separation efficacy of the developed octadecylsilica×polyamine was tested by separating an extract from Anemarrhena asphodeloides. Thisallowed for the successful separation of co-eluted and minor compounds. As a result,a total of31compounds, including5compounds of low amount and4steroid isomerswere unambiguously identifed with this approach. In contrast only22compounds, allof which were highly abundant in their samples, could be identifed usingconventional1D-LC. This method could be considered as an important demonstrationfor “nonseparation analysis” of complex system.2. The present work aims to elucidate the multi-component metabolic characteristics of herbal medicine by the combination of plasma pharmacochemistry andmicrodialysis sampling. Anemarrhena asphodeloides, a well-known traditionalChinese medicine, was chosen as a model. After oral administration of A.asphodeloides saponin extract to rats, microdialysis samples were collectedcontinuously in the jugular vein and analyzed by ultrahigh-performance liquidchromatography/quadrupole-time-of-fight mass spectrometry. The identification ofcompounds in bio-samples was achieved by accurate mass measurement and detailedfragmentation pathway analysis. The results showed that unbound constituents inblood circulation of the rat included seven parent saponins and six metabolites, whichmight be the potential active components in vivo. Among which, three metaboliteshave not been previously reported and were identifed in this study. Compared withconventional biological sampling techniques, microdialysis offers direct in vivosampling, low invasiveness, real-time monitoring and simple sample preparation. Asdemonstrated in our study, microdialysis sampling assisted QTOFMS effectively forsimultaneous analysis of multiple minor drug metabolites with potentially importantfunctions in vivo. Some important advantages of this approach are: the short analysistime, the absence of any unlabelled compounds, and also the ease of use. In summary,this developed method is generally applicable to include more active componentsscreening of TCM in mammalian body and to better clarify its pharmacological actionmechanism.3. We established a dynamic microdialysis sampling method coupled withultrahigh-performance liquid chromatography/quadrupole-time-of-fight massspectrometry (UHPLC/QTOFMS) based metabolomics. By using this approach, tensaponins/metabolites have been identified in rat hippocampus microdialysates after asingle oral administration of total saponins of Anemarrhena asphodeloides (TSA), andthe pharmacokinetic behaviors of them were real-time monitored. Comparing thebody dynamics of each constituent, timosaponin A-III and dehydrogenatedsarsasapogenin, with faster absorption, slower metabolism, higher peak concentrationand larger AUC values were considered to be candidate hit. Furthermore, behavioralassessment was consistent with pharmacokinetic data, which suggested these twocomponents were bioactive markers in TSA for its neuroprotective effects. This is thefirst attempt to systematically reveal the in vivo process of steroid glycoside in thebrain. Our results indicated that pharmacokinetics screening using UHPLC/QTOFMSbased metabolomics can not only be used to explore potential active components contributing to central nervous system (CNS) pharmacological effects of herbalmedicine, but also to get a promising perspective of new brain-targeted leads in drugdiscovery from natural agents.4. Despite recent advances in understanding the pathophysiology of dementia and themechanisms of anti-dementia drug action, the development of biomarkers fordiagnosis and therapeutic monitoring in dementia remains challenging. Metabolomicsprovides a powerful approach to discover diagnostic and therapeutic biomarkers byanalyzing global changes in an individual’s metabolic profle in response topathophysiological stimuli or drug therapy. In this study, we performed combined gaschromatography-mass spectrometry (GC-MS), reversed-phase iquidchromatography mass spectrometry (RPLC-MS) and a hydrophilic interaction liquidchromatography-mass spectrometry (HILIC-MS)-based metabonomics in serum ofscopolamine-induced demented rat models before and after2-week total saponins ofAnemarrhena asphodeloides (TSA) treatment, to detect potential biomarkersassociated with dementia and intervention effects of TSA. Twenty-one markermetabolites contributing to the complete separation of demented rats from matchedhealthy controls were identifed, with myo-inositol, uric acid, and tryptophan showingthe maximum combined classifcation performance. Metabolic pathways includingenergy metabolism, antioxidant defense systems, neurotransmitter metabolism, fattyacid biosynthesis, and phospholipid metabolism were found to be disturbed indemented rats and partially normalized following TSA treatment. Further study ofthese metabolites may facilitate the development of new potential biomarkers andmore efficient therapeutic strategies for dementia. |
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