Relevance Between The Holistic Quality And Efficacy Of Rehmanniae Radix Praeparata Processed By Steaming And Wine Stewing

Rehmanniae Radix Praeparata(RRP)is the processed product of the fresh or dried root of Rehjnannia glutinosa Libosch.(Scrophulariaceae),which is commonly used for nourishing in clinical.RRP processed by steaming(RRP-S)and wine-stewing(RRP-W)have been documented in the 2020 edition of China Pharmacopeia.However,no systematic comparative study has been reported on the differences of quality and efficacy between RRP-S and RRP-W.Based on the "multi-component,multi-target" therapeutics of traditional Chinese medicines(TCM),the traditional efficacy of RRP,and the advancement of the holistic quality/efficacy evaluation methods of TCM,a method for holistic quality-efficacy evaluation of RRP was established and applied to compare the quality and efficacy between RRP-S and RRP-W.The relevance between the holistic quality and efficacy of RRP-S and RRP-W was preliminarily discussed as well.Firstly,to evaluate the intrinsic quality of RRP-S and RRP-W,glycomics and metabolomics were integrated to compare the glycome(polysaccharides,oligosaccharides and free monosaccharides)and metabolome(non-sugar small molecules)respectively in RRP-S and RRP-W.Secondly,to compare the Yin nourishing effects of RRP-S and RRP-W,traditional pharmacological approaches,metabolomics and gene sequencing were used for comparison of the effects of RRP-S and RRP-W on classical physiological and pathological indexes,endogenous small molecules and gut microbiota in a Yin deficiency mouse model.Finally,facilitated by multivariate statistical analysis,the relevance between the quality and efficacy of RRP-S and RRP-W was evaluated.1.Comparison between holistic chemical profiles of RRP-S and RRP-WSamples of RRP-S and RRP-W were prepared according to the 2020 edition of Chinese Pharmacopoeia.Polysaccharides and small molecular components of RRP-S and RRP-W were respectively prepared by water extraction and alcohol precipitation.The molecular weight range of polysaccharides was determined using HPGPC-ELSD.Compared with RRP-S,the molecular weight range of polysaccharides in RRP-W(1.01×103～6.98×106 vs 1.02×103～5.54×106 Da)was broader,and the proportion of larger molecular weight polysaccharides was higher.The sugar composition of polysaccharides was analyzed using a pre-column derivatization HPLC-DAD method.Both the polysaccharides in RRP-S and RRP-W were composed of mannose,galacturonic acid,glucose,galactose and arabinose,but with different molar ratios(2.57:0.05:1.00:0.87:0.10 vs 2.07:0.07:1.00:1.05:0.17).Oligosaccharides were determined using HPLC-ELSD.Stachyose,manninotriose and melibiose were detectable in both RRP-S and RRP-W with the content of stachyose significantly higher in RRP-W than that in RRP-S(P<0.01).Free monosaccharides were also analyzed using the above-mentioned pre-column derivatization HPLC-DAD method.Glucose,fructose,rhamnose,mannose,galactose and arabinose were detected in both RRP-S and RRP-W,with the contents of glucose(P<0.01),rhamnose(P<0.05)and mannose(P<0.05)significantly lower,whereas galactose higher(P<0.05)respectively,in RRP-W than that in RRP-S.A total of 29 non-sugar small molecules with significant abundance difference between RRP-W and RRP-S were identified or tentatively assigned using a UPLC-QTOF-MS/MS-based metabolomics approach,including nine iridoid glycosides,ten phenylethanoid glycosides and one furfural derivative.The quantitative determination of the representative non-sugar small molecules showed that the contents of catalpol and leonuride were 19.9 and 19.7 times higher in RRP-W respectively.The content of rehmannioside D was also higher in RRP-W(P<0.01),whereas melittoside(P<0.05)and 5-hydroxymethylfurfural(5-HMF,P<0.01)lower in RRP-W,than that in RRP-S.2.Comparison of the Yin nourishing effects of RRP-S and RRP-WThe Yin nourishing effects of RRP-S and RRP-W were compared in mice with L-thyroxine-induced Yin deficiency.Compared with the model group,RRP-S and RRP-W both significantly improved the appearance and the survival rate of mice,alleviated the thyroid and kidney injuries,and regulated the secretion of thyroid related hormones and oxidation-related factors.A UPLC-QTOF-MS/MS-based metabolomics approach was used for comprehensively analyzing serum metabolites.Twenty-two metabolites responsible for group discrimination between model and control groups were identified.Metabolic pathways of thyroid hormone synthesis,glycine metabolism,serine metabolism,threonine metabolism and tyrosine metabolism were found to be associated with the 22 identified potential biomarkers.Both of RRP-S and RRP-W significantly regulated six potential biomarkers,including thyroxine,13-L-hydroperoxylinoleic acid,isobutyryl-L-carnitine,9-hexadecenoylcamitine,LysoPC(20:4(8Z,11Z,14Z,1 7Z))and LysoPC(22:6(4Z,7Z,10Z,13Z,16Z,19Z)),which were associated with metabolic pathways of thyroid hormone synthesis,linoleic acid metabolism and tyrosine metabolism.Besides,RRP-S significantly regulated etoposide glucuronide and L-palmitoylcamitine,while RRP-W significantly regulated N-acetylaspartylglutamic acid,4-hydroxy-estazolam,coprocholic acid and trans-2-tetradecenoylcarnitine.3.Comparison of the effects of RRP-S and RRP-W on gut microbiotaThe 16S rRNA gene sequencing was used to evaluate the effects of RRP-S and RRP-W on gut microbiota of mice with Yin deficiency.Compared with normal mice,the relative abundance of Firmicutes was higher,and the relative abundance of Bacteroidota was lower in Yin deficiency mice.Both of RRP-S and RRP-W decreased the relative abundance of Firmicutes and increased the relative abundance of Bacteroidota.Besides,RRP-S significantly promoted the relative abundance of Actinobacteriota,while RRP-W significantly increased the relative abundance of Actinobacteriota.In the top 10 most abundant genus,RRP-S significantly promoted the relative abundance of Blautia and Bifidobacterium,while RRP-W significantly increased the relative abundance of Bacteroides,Prevotellaceae_UCG-001 and Akkermansia.4.Relevance evaluation between the holistic quality and efficacy of RRP-S and RRP-WTo explore the holistic quality-efficacy relevance of RRP-S and RRP-W,Spearman correlation analysis was used to analyze the correlation between pathological indexes,endogenous metabolites and gut microbiota.The results showed that thyroxine,13-L-hydroperoxylinoleic acid and LysoPC(20:4(8Z,11Z,14Z,17Z))were significantly correlated with Bifidobacterium.It is probable that RRP-S could regulate thyroid hormone synthesis(hypothalamic-pituitary-adrenal axis)and linoleic acid metabolism by promoting Bifidobacterium.The contents of stachyose,galactose,catalpol and leonuride were significantly higher in RRP-W,which might contribute to the proliferative effects of RRP-W on Akkermansia and Bacteroides.Isobutyryl-L-carnitine was significantly correlated with Akkermansia and Bacteroides.It is probable that RRP-W could regulate the lipid metabolism pathway and exert the Yin nourishing effects.The above results indicated the significant difference in intrinsic quality between RRP-S and RRP-W.The quality variations could obviously affect the Yin nourishing effects of RRP-S and RRP-W,and there was relevance between the quality and efficacy.This study provided a reference for improving the quality control and the clinical precision application of RRP-S and RRP-W.