Study On Effective Components And Mechanism Of Cistanche Deserticola In Relieving Physical Fatigue

In the early stage of the research,the "spectrum-effect" method was conducted to study the effective components,anti-oxidation effect in vitro and relieving physical fatigue effect in vivo of Cistanche deserticola.Two kinds of effective components,phenylethanoid glycosides and polysaccharides,which are different in pharmacodynamic effect,but same in poor oral absorption and poor intestinal absorption in the earlier studies and related reports published by the research group.Therefore,the anti-fatigue components and mechanism also need to be further explored.In this paper,different methods,including the qualitative and quantitative characterization methods of chemical composition,components metabolism and pharmacodynamic experiments in vitro and in vivo,metabonomics and high-throughput intestinal flora sequencing technology,are used to systematically study the effective components which relieved physical fatigue and mechanism of Cistanche deserticola.A method for studying the correlation between the chemical constituents of the traditional Chinese medicine Cistanche deserticola and the anti-physical fatigue effect has been established.These components regulating physical fatigue and their function in regulating abnormal biomarkers related with physical fatigue and metabolic pathway disorders are abtained to provide scientific basis for indicating its mechanism and its clinical application and development.1.Study on the chemical composition and quality control of Cistanche deserticola in vitro and vivoOn-line rapid identification of 70%ethanol extract of Cistanche deserticola by UHPLC-Q-tof HRMSE technique is used to identify 44 compounds,including phenylethanoid glycosides,iridoid glycosides and lignin glycosides.We apply high-performance gel chromatography-multi-angle laser scattering equipment,combined with ion chromatograph and high-resolution liquid chromatography-mass spectrometer for the first time to identify the molecular weight distribution of polysaccharides from Cistanche deserticola,monosaccharide composition,the degree of polymerization and sequence structure of oligosaccharide fragments to establish a characterization system for the macromolecular polysaccharides to provide the foundation for the subsequent research on the quality evaluation and pharmacodynamic mechanism of Cistanche deserticola.We use UPLC-Q/TOF MSE and MDF identification method to analyze the metabolism of 70%ethanol extract of Cistanche deserticola in vivo to figure out 20 prototype components,32 metabolites in plasma,urine and feces.There are 13 prototype components and 16 metabolites in plasma,18 prototype components and 24 metabolites in urine,17 prototype components and 17 metabolites in feces,indicating that it has undergone extensive metabolism in vivo.The metabolism transformations exist in the form of hydrolysis,methylation,sulfonation,and glucuronidation.In vivo metabolic characteristics analysis finds that most of the prototypic compounds of phenylethanoid glycosides and the metabolites such as reduction,methylation and sulfate conjugation including the parent compound and hydrolyzation are detected in the feces,which further confirm that the phenylethanoid glycosides have low oral absorption and are difficult to be absorbed into the blood,but can be metabolized by the intestinal flora and also cause low intestinal bioavailability.The main components of Cistanche deserticola,phenylethanoid glycosides,act as a prodrug to be metabolized in vivo to be activate.The secondary components,iridoid glycoside compounds,are also more easily absorbed into the blood after being metabolized into aglycone,which preliminarily clarifies the metabolic and material basis of Cistanche deserticola,providing a basis for screening its pharmacodynamic substances and clinical mechanism.Based on UHPLC-Q-Exactive Orbitrap HRMS and in vitro intestinal flora culture,the in vitro metabolism of four representative phenylethanoid glycosides in Cistanche deserticola were studied.It was found that these compounds were easily hydrolyzed in glycosidic bonds and ester bonds,and finally hydrolyzed into their aglycones,hydroxytyrosol and caffeic acid.The degraded metabolites will continue to undergo phase I and phase II metabolic reactions to form new metabolites,and the metabolic sites are mainly on the phenolic hydroxyl structural unit,confirmed the role of the intestinal flora in the metabolism of phenylethanoid glycosides.In addition,the activity of 9 prototype glycosides and 12 metabolites was evaluated by DPPH antioxidant experiments in vitro,figuring out that the antioxidant activity was related to o-diphenol hydroxyl groups.Caffeic acid and hydroxytyrosol have the similar antioxidant activity as the phenylethanoid glycoside.It is speculated that the phenylethanoids exist mainly in the form of prodrugs,which are metabolized by the intestinal flora to the same active phenolic acids after oral administration,such as hydroxytyrosol and caffeic acid to offer pharmacological effects after being absorbed into blood.The results of metabolism studies in vitro and in vivo assays indicate that the main chemical constituents,phenylethanoid glycosides,are metabolized by the gastrointestinal tract,producing the hydroxytyrosol and caffeic acid and their derivatives,which have the same action of the prototype components.In addition,using the multi-reaction detection(MRM)mode of UPLC-Q/Trap-MS/MS technology,the internal determination method was used to establish the simultaneous determination method of 10 compounds in Cistanche deserticola.The results showed that under the optimized experimental conditions,the methodology both meet the requirements of the pharmacopoeia,indicating that the established quantitative method is sensitive,fast and reliable.In addition,combined with the determination of polysaccharide content,it can be more comprehensively used for the study of quality standards of Cistanche deserticola.2.Study on phannacodynamics and metabolomics mechanism of Cistanche deserticola in relieving physical fatigue and anti-oxidationFor the first time,a variety of pharmacodynamic evaluation assays and HPLC-Q-Exactive Orbitrap HRMS-based metabolomics techniques were used to study serum metabolism of mice with long-term weight-loaded Forced swimming-induced fatigue models,as well as those treated with total glycosides,polysaccharides,and glycosides-polysaccharides combination.Different pretreatment methods and chromatographic-mass spectrometry conditions were used to comprehensively analyze the endogenous metabolites of different polarities to obtain 27 potential biomarkers most relevant to physical fatigue diseases,mainly involving in vivo amino acid metabolism,lipid metabolism and disorder of purine and pyrimidine nucleotide metabolism.The study on the effect of total glycosides,polysaccharides and their combination on the metabolomics of body fatigue finds that the combination can adjust the most metabolites,followed by the total glycosides and polysaccharide components alone,and improve the disorder of related metabolic pathways,and provide a basis for studying the anti-fatigue effective components and mechanism of action based on the pharmacodynamic indicators related to anti-fatigue.3.Regulation of intestinal flora disturbance and total effective components in mice with physical fatigue16S rRNA gene sequencing was used to analyze the intestinal flora structure of mice during physical fatigue.The results showed that the diversity of intestinal microorganisms and the composition of phylum and genus in mice with physical fatigue were different,suggesting that these intestinal microflora may play an important role in the occurrence and development of physical fatigue diseases.Firmicutes and Bacteroides and their related genuses are closely related to physical fatigue.Then the effects of total effective components on intestinal flora disturbance induced by physical fatigue in mice were investigated.The results showed that it could regulate the disorders of the composition of phylum and genus in mice with physical fatigue.The total effective components of Cistanche deserticola can regulate the intestinal flora disorder of physical fatigue mice and play the pharmacodynamic role.However,the preliminary results based on the current single method of bacterial flora sequencing need to be further validated with other methods.To sum up,this paper applies UPLC-Q-TOF/MS technology to study the non-targeted metabolomics and intestinal flora,and explains the mechanism of the active ingredients from different levels and perspectives in combination with pharmacodynamics,metabolism in vivo and in vitro,and the regulation of intestinal flora.Especially,the ingredients with low bioavailability,poor absorption but significant effect after oral administration may be activated through the transformation of intestinal flora,then act on the flora,regulate the intestinal micro-ecological environment to achieve the regulatory effect on the body.