Holistic Evaluation On Quality And Efficacy Of Rehmanniae Radix Praeparata In "Nine Cycles Of Steaming And Drying" Processing

Rehmanniae Radix(RR),derived from the dried root of Rehmannia glutinosa Libosch,is one of the most important Chinese medicinal herbs that was firstly documented in "Sheng-Nong-Ben-Cao-Jing".Two types of RR,raw and processed RR(RRR and PRR),are commonly used in traditional Chinese medicine(TCM)practice for different medical purposes.According to the theory of TCM,RRR is used to "reduce heat in blood,nourish yin and promote the production of body fluids",while PRR can "nourish yin and replenish blood,reinforce essence and marrow".RRR is traditionally processed by nine cycles of repeated steaming and drying to generate PRR.However,the scientific basis involved in the traditional processing approach still remains largely elusive.Herbal medicines are characterized by multiple components and exemplify the principle of "multi-components against multi-targets" in therapeutic and prophylactic applications.Therefore,guided by the view of holism,this study first integrated chromatographic technique based chemomics,including targeted glycomics and untargeted secondary metabolomics strategy to reveal the variations of glycome and metabolome of nine cycles of repeated steaming and drying-processed RR.Then,in order to investigate the efficacy change of the processed RR,chromatography-tandem mass spectrometry-based effcicacy metabolomics combined with classical pharmacological indexes were conducted.By these experiments,the holistic variation rules of quality and efficacy in "nine cycles of steaming and drying" processing of PRR were elucidated,so that the scientific connotation of the processing would be clarified,and innovative processing method would be objectively optimized.Moreover,the research outcomes can also offer new ideals and approaches for the inheritence and innovative research on traditional processing methods of other Chinese medicinal herbs.The dissertation consists of five chapters as follows:Chapter 1 Advances in related research fieldsThe current research achievements regarding effects of processing on chemicals and pharmacological activities as well as quality control methods of RR in the near decade were summarized.Previous related studies were generally based on the determination of few chemical markers and pharmacological index,which were unable to reflect the well-accepted " multi-components against multi-targets"therapeutic feature of TCMs,thus are not objective to reveal the scientific connotation of the traditional "nine cycles of steaming and sun-drying" processing for PRR.Given the fact we proposed the integrated omics(chemomics and pharmaco-metabolomics)strategy so as to systemically investigate the holistic variations of chemical components and pharmacological activities of processed RR.Chapter 2 Integrating secondary metabolomics and glycomics to investigate processing chemistry of RRHyphenated chromatographic technique-based chemomics including untargeted secondary metabolomics and targeted glycomics strategy were integrated for qualitatively and quantitatively characterizing overall chemical components(secondary metabolites and carbohydrates)of RR,and then the obtained data were further processed by multivariate statistical analysis to reveal the variations of the chemome of RR during the processing.UPLC-PDA-QTOF-MS/MS-based untargeted secondary metabolomics was employed to characterize the secondary metabolites in RR.A total of fourty-one chemicals,including iridoid glycosides,phenethylalcohol glycosides and furfural derivatives were identified or tentatively assigned.Selected ion monitoring(SIM)mode of UPLC-QTOF-MS/MS was employed for quantitative determination of seven typical secondary metabolites.Meanwhile,targeted glycomics by HPGPC-ELSD,HPLC(Ci8)-UV and HPLC(NH2)-ELSD were applied to qualitatively and quantitatively characterize polysaccharides,oligosaccahrides and monosaccahrides in RR.It was demonstrated that the integrated strategy holistically illuminated the variations in glycome and metabolome of RR during processing,and futher discussed the processing-induced chemical transformation mechanisms of secondary metabolited and carbohydrates.The two PCA results derived from glycome and metabolome analysis unequivocally manifested that the processing inconsistently changed the carbohydrates and secondary metabolites in the RR sample.This situation suggested that unreliable conclusions might be drawn if only carbohydrates or secondary metabolites were individually investigated.By integrating glycomics and metabolomics,we could conclude that nine cycles of steaming and drying might be unnecessary for RR processing since neither the glycome nor metabolome changed significantly after the 6th processing cycle.Chapter 3 Combining chemomics-based marker compounds mining and mimetic processing to elucidate chemical mechanisms invoved in RR processingThen,a novel strategy by combining chemomics-based marker compounds mining and mimetic processing is proposed for further exploring the chemical mechanisms involved in RR processing.First,the differential marker compounds between raw and processed herb were rapidly discovered by untargeted chemomics-based mining approach through multivariate statistical analysis of chemome(secondary metabolome and glycome)data obtained by metabolomics and glycomics analysis.Second,the marker compounds were mimetically processed under the simulated physicochemical conditions as in herb processing,and the final reaction products were chemically characterized by targeted chemomics-based mining approach.Third,the main chemical transformation mechanisms involved were clarified by linking up the original marker compounds and their mimetic processing products.Using this strategy,a set of differential marker compounds including saccharides,glycosides and furfurals in raw and processed RR were rapidly found,and the major chemical mechanisms involved in RR processing were elucidated as stepwise transformations of saccharides(polysaccharides,oligosaccharides and monosaccharides)and glycosides(iridoid glycosides and phenethylalcohol glycosides)into furfurals(glycosylated/non-glycosylated hydroxymethylfurfurals)by deglycosylation and/or dehydration.Chapter 4 Employing pharmaco-metabolomics to compare the hematopoietic effects of RRR and PRRAfter the chemical investigation,UPLC-QTOF-MS/MS-based pharmaco-metabolomics was established to portray the plasma and urine metabolic characteristics of model and RR-treated rats.The obtained data were further processed by multivariate statistical analysis for systemically investigating the variations of hematopoietic effects of RR by the processing.Blood deficiency model was induced by the combination of acetyl phenylhydrazine(APH)and cyclophosphamide(CP).Then,on the model rats,the hematopoietic effects of RRR0,PRR6 and PRR9 were evaluated and compared based on the pharmaco-metabolomics as well as classical pharmacological indexes(mortality,weight,peripheral blood indexes and organ indexes).The results indicated that the effects of RR treatment were ranked as PRR6>PRR9>RRR0.In the statistical analysis of pharmaco-metabolomics,41 potential biomarkers contributing to the PCA separation of model group and control group were tentatively identified from the plasma and urine,and 6 metabolic pathways involved in the blood deficiency modeling were explored(impact>0.10).The treatments by RRRO,PRR6 and PRR9 regulated the perturbed pathways.To be specific,PRR9 and RRRO significantly improved the hemeostasis of biomarkers(P<0.05)mainly related to pentose and glucuronate interconversions and sphingolipid metabolism,while PRR6 involved glyoxylate and dicarboxylate metabolism.Chapter 5 Employing pharmaco-metabolomics to compare the effects on nourishing kidney-yin of RRR and PRR UPLC-QTOF-MS/MS-based pharmaco-metabolomics was established to portraythe plasma and urine metabolic characteristics of model and RR-treated rats.The obtained data were further processed by multivariate statistical analysis for systemically investigating the variations of effects on nourishing kidney-yin of RR by the processing.Kidney-yin deficiency model was induced by levothyroxine sodium.Then,on the model rats,the effects on nourishing kidney-yin of RRRO,PRR6 and PRR9 were evaluated and compared based on the pharmaco-metabolomics as well as classical pharmacological indexes(body weight and pathological photographs).The results indicated that the effects of RR treatment were ranked as PRR9>(or≈)PRR6>RRR0.In the statistical analysis of pharmaco-metabolomics,46 potential biomarkers contributing to the PCA separation of model group and control group were tentatively identified from the plasma and urine,and 7 metabolic pathways involved in the kindey-yin deficiency modeling were explored(impact>0.10).The treatments by RRRO,PRR6 and PRR9 regulated the perturbed pathways.To be specific,PRR6 significantly improved the hemeostasis of biomarkers(P<0.05)mainly related to sphingolipid metabolism,RRRO imediated the metabolism of pentose and glucuronate interconversions,while PRR9 involved the metabolism of tryptophan metabolism.