In Vitro Catabolism Of Procyanidin A2 By Rat Intestinal Microbiota And Its Inhibitory Effects On The Formation Of Macrophage-derived Foam Cells

Polyphenols of litchi fruit pericarp,rich in procyanidins A2(PCA2)were shown to prevent atherosclerosis(AS),and thebioavailability of PCA2 is very low.Previous researches showed thatmetabolites of phenolics by intestinal microflora play important role in the physiological functions of phenolics.Therefore,this study aims to analyze the structure of the metabolites of PCA2 by rat intestinal microflora using UPLCQ-TOF-MS,and then explore the effects and underlying mechanism of its metabolites on the formation of macrophage foam cell.The main results are as follows:(1)Preparation and purification of PCA2 from litchi pericarpIn order to obtainhigh-purityPCA2 from litchi pericarps,the crude extracts of procyanidins were obtained by ethanol extraction,and t hen were purified by AB-8 macroporous resins and preparativeHPLC.PCA2 was analyzed by ultraviolet and infrared spectroscopy,HPLC and UPLC-Q-TOF-MS.The purtity and yields of PCA2 was 94.08%.(2)Metabolites and changes in antioxidant activity of PCA2 after incubation with rat intestinal microbiotaThe total antioxidant capacity(T-AOC),oxygen radical absorbance capacity(ORAC),DPPH and ABTS radical scavenging ability were used to compare the antioxidant activity of PCA2 and its metabolites by rat intestinal microflora.The antioxidant capabilities of PCA2 were enhanced after incubation for 6 h.Compared with 0 h,T-AOC,DPPH,ABTS radical scavenging ability of PCA2 incubation for 6 h were significantly increased by 2.01,2.16 and 1.34,respectively.HPLC results showed that PC A2 wasdegradedby intestinal microbiotaand produced 8 new components.The structures of the new peaks were analyzed by UPLC-Q-TOF-MS.Peak 1,2,3,4 and 6 was identified as 4-hydroxyphenylacetic acid(4-HPAA),epicatechin,3-(4-hydroxyphenyl)propionic acid(HPPA),B type proanthocyanidins,5,7,2'-Trihydroxyflavanone,respectively.Peak 5,7 and 8 needed further confirmation.(3)Effects ofmetabolites of PCA2 by intestinal microbiotaon the formation of macrophage foam cellFoam cell model was induced using 80 ?g/mL ox-LDL incubated with RAW264.7 macrophages at 24 h.O il red O-hematoxylin staining results showed thatlipid droplets was accumulated largely in model cells after exposing to 80 ?g/mL ox-LDL for 24 h.HPPA and 4-HPAA,the metabolitesof PCA2 by intestinal microbiota significantly reduced the accumulation of lipid droplets in a dose-dependent manner compared with the model cells.12.5 ?g/mLHPPA and 4-HPAA significantly decreased the levels of intracellular total cholesterol(TC),free cholesterol(FC),cholesterol esters(C E).CE/TC in 12.5 ?g/mL HPPA and 4-HPAA group significantly was decreased by21.63% and 27.64%,respectively.RT-PCR results showed that compared with the model group,HPPA significantly up-regulated the expression of ABCA1 and SR-B1 by 2.13 and 2.10 times,respectively(P< 0.05).4-HPAA significantly up-regulated PPAR? and ABCG1 expression level by 2.02 and 2.44 times,respectively(P< 0.05).The results showed that the PCA2 were degraded by the intestinalmicrobiota and produced 4-HPAA,epicatechin,HPPA,B type proanthocya nidins,5.7.2 '-trihydroxyflavanone and several unknown compounds.HPPA and 4-HPAA significantly restrainedthe foam cell formation by ox-LDLthrough upregulation of PPAR?,ABCA1,SRB1,ABC G1 expression,which suggested that the anti-atherosclerosis activity of PCA2 can be partially attributed to its metabolites by intestinal microbiota.