Study On In-vitro And In-vivo Metabolism Of The Pivotal Phenolic Compound Of Lychee(litchi Chinensis Sonn.)pulp By Intestinal Microbiota

Lychee pulp is abundant in phenolic compounds,including a large number of flavonoids and trace amounts of phenolic acids.Phenolic compounds were generally considered as the key source of the bioactivities of lychee pulp.The bioactivities of phenolic compounds are closely related to the gut microbial flora.However,the metabolic mechanism of the pivotal phenolics in lychee pulp by gut microbiota and its regulatory effects on the microbial flora structure were still unclear.In the present work,the metabolic fate of lychee pulp phenolics(LPP),its regulatory functions in metabolic pathway and the specific microbial species participating in LPP metabolism were investigated,based on fecal metabolomics of mice and individual bacterial fermentation.Furthermore,the pivotal phenolic in lychee,quercetin-3-Orutinose-7-O-?-L-rhamnoside(QRR)was subjected to fecal fermentation to investigate the interaction between phenolics and gut microbiota.(1)Study on the fecal metabolomics of LPP-fed mice: UHPLC-ESI-QTOF-MS/MS was applied to analyse the distinction of colonic metabolites between normal-fed and LPP-fed mice after 21 days of intervention.According to the Orthogonal partial least square discriminant analysis(OPLS-DA),the metabolic profile of LPP-fed mice was different from that of normal-fed mice.compared with normal-fed mice,a total of 124 differentially expressed metabolites were found in LPP-fed mice in positive ion mode while only 69 differentially expressed metabolites in negative ion mode.Among them,the contents of hydroxybutyric acid,mevalonic acid and phenylethylamine were significantly(p < 0.05)increased,but no flavonoids and phenolic acids were detected.Based on the KEGG database,annotation and enrichment analysis were conducted to search for metabolic pathways significantly affected by LPP,including linoleic acid metabolism,biotin metabolism and nicotinate and nicotinamide metabolism.It was preliminarily confirmed that LPP was completely degraded in the colon of body,and suggested that the existence of amino products in the intestinal metabolism of polyphenols and their health effects.(2)Reciprocal interactions between lychee pulp phenolics and specific gut microbial species: Based on the preliminary analysis results of intestinal flora composition of LPP-fed mice,five selected gut microbial strains(Bacteroides uniformis,B.thetaiotaomicron,Lactobacillus rhamnosus,L.plantarum and L.acidophilus)were separately incubated with LPP in an anaerobic fermentation mode,and changes in the viable cell counts,p H,total phenolic contents and antioxidant activites during the whole fermentation period were monitored.High performance liquid chromatography(HPLC)analysis confirmed that B.uniformis,L.rhamnosus and L.plantarum were involved in LPP utilization,contributing to52.37%,28.33% and 45.11% of LPP degradation after 48 h fermentation,respectively.The qualitative analysis based on ESI-MS/MS suggested that the metabolic pathway of the pivotal phenolic compound quercetin-3-O-rutinose-7-O-?-L-rhamnoside by L.plantarum appeared to be the direct fission of C-ring.Meanwhile,it was fully confirmed that B.uniformis catalyzed the cleavage of glycosidic bonds of flavonoids glycoside,such as QRR,isorhamnetin rutinose and isorhamnetin rhamnosylrutinoside,as well as the cleavage of C-C bonds on procyanidin A2,resulting in the increased contents of quercetin and(+)-catechin.Besides,the results of viable cell counts showed that LPP exerted a promoting effect on the proliferation of Bacteroides and Lactobacillus.spp.(3)Investigation of the metabolic characteristics of QRR and quercetin-3-Orutinose(QR)by gut microbial flora: After clarifying the gut microbes involved in LPP metabolism,the unclear metabolic gut metabolic characteristics of QRR,the pivotal phenolic compound in litchi pulp,were further investigated,and the structural analogues quercetin-3-O-rutinose(QR)was used as the control.The QRR and QR were subjected to colonic fermentation individually,and their metabolic characteristics were systematically investigated from the perspectives of metabolites and intestinal flora.HPLC analysis showed that QR was not found in the gut microbial metabolites of QRR,and the contents of aglycone from QRR were significantly lower than that from QR.It was observed that the synthesis of acetic,propionic and butyric acid was significantly up-regulated after QRR supplementation,while only acetic acid was promoted after QR treatment.Based on these results,it could be deduced that the metabolic fate of QRR was different from that of QR.LC-MS identification confirmed that QRR deconjugated to form quercetin-3-O-glucoside-7-O-rhamnoside by gut microbial flora,instead of QR.Unprecedentedly,hydroxymethoxyl-myricetin and hydroxyethoxyl-isorhamnetin were found and identified in the QRR metabolites,indicating that glycosyl moiety of flavonoid was directly utilized by gut microbiota without the breakage of glycosidic bond and the existence of amination during phenolic metabolism.Through methylation,deconjugated glycosylation and C-ring cleavage,QR was catabolized into isorhamnetin,quercetin and hydroxyphloridin.However,it was observed that fewer phenolic acids were found in QRR metabolites,while a variety of chromogen ketones were produced,indicating the cleavage of C-C bond between C-and B-rings.Based on taxonomic composition analysis,it was found that QRR supplementation significantly increased(p <0.05)the relative abundances of Akkermansia,Bacteriodes,Lactobacillus and Faecalibacterium in gut microbial flora,while Lactobacillus and Weissella were significantly(p < 0.05)enriched by QR.Similarly,both of them effectively inhibited the proliferation of Shigella.Therefore,it could be confirmed that glycosylated quercetin exerted a structureactivity relationship in gut microbial regulation.The intestinal metabolic mechanism of litchi pulp phenolics was clarified systematically from the perspectives of gut microbes and metabolites in this study.The results of this study provide a scientific basis regarding the nutritional value of lychee pulp phenolics and also present better insights into the interaction between gut microbes and bioactive components of fruits.