| Polyphenols of litchi fruit pericarp,rich in procyanidins A2(PCA2)were shown to ameliorate nonalcoholic fatty liver disease.However,it is poorly absorbable in the gastrointestinal tract.Current evidences indicate that colonic microbiota can degrade procyanidins,producing a large number of metabolites that can be absorbed and exert biological effects in the body.Therefore,the present work aims to analyze the structure of the metabolites of PCA2 with human intestinal microflora incubation using UPLC-Q-TOF-MS,and then explore the effects and underlying mechanism of its metabolites on the inhibitory effects on lipids accumulation in vitro cell model of hepatic steatosis.The main results of this study were presented as follows:(1)Preparation and purification of PCA2 from litchi pericarpIn order to obtain high-purity PCA2 from litchi pericarps,the crude extracts of procyanidins were removed pigments using MCL column,and then further purified by medium-pressure preparative liquid chromatograp with gradient methanol aqueous chromatograph elution.PCA2 was analyzed by HPLC and UPLC-Q-TOF-MS..The purity of PCA2 was 96.38%.(2)Metabolites analysis of PCA2 after incubation with human intestinal microbiotaHPLC results showed that PCA2 was degraded by human intestinal microbiota and produced 14 new components.The structures of the new peaks were analyzed by UPLC-Q-TOF-MS.Peak 2,3,4,5,8,9 and 11 was identified as 3-(3-hydroxyphenyl)propionic acid,epicatechin,benzoic acid,procyanidin B2,catechin,3-(3,4-Dihydroxyphenyl)propionic acid,4-hydroxyphenylacetic acid,respectively.Peak 1,6,7,10,12,13 and 14 needed further confirmation.The antioxidant activities of the metabolites of PCA2 were analysed by the total antioxidant capacity(T-AOC),oxygen radical absorbance capacity(ORAC),DPPH and ABTS radical scavenging ability.The antioxidant capabilities of PCA2 were enhanced after incubation with human intestinal microbiota.Compared with 0 h,T-AOC of PCA2 incubation for 3,6,12,24 h was 1.26,1.40,1.34 and 1.12 times,respectively.The scavenging activities of DPPH· were 1.43,1.45,1.37,and 1.33 times respectively.The scavenging activities of ABTS· were 1.42,2.03,2.15 and 1.58 times,respectively.ORAC of PCA2 incubation for 6 h was 1.70 times compared with the 0 h.(3)The inhibitory effects of metabolites of PCA2 by human intestinal microbiota on hepatocyte steatosis in vitroIn vitro model of hepatic steatosis was induced using 0.25 mM oleic acid incubated with HepG2 cell.Oil red O-hematoxylin staining results showed that lipid droplets were accumulated significantly in model cells after exposing to 0.25 mM oleic acid compared with the control group.The metabolites of PCA2 by human intestinal microbiota significantly reduced the accumulation of lipid droplets in a dose-dependent manner compared with the model cells.25?g/mL metabolites of PCA2 exhibited efficient suppressing effects on TG and TC accumulation with the suppression percentage of 33.28%and 65.80%(p<0.05)respectively.In addition,the metabolites of PCA2 significantly decreased the levels of ROS and MDA equivalent induced by 0.25 mM oleic acid.RT-PCR results showed that compared with the model group,25?g/mL metabolites of PCA2 significantly up-regulated the expression of ABCG1,LXRa and CPTla by 81.32%,83.03%and 103.91%,respectively(p<0.05).While the gene expressions of FAS,miR-122,miR-33a,miR-33b,HMG-CoA reductase and SPRBP-lc were down-regulated by 27.33%,61.99%,42.48%,48.20%,39.46%,42.70%in 25?g/mL metabolites of PCA2 compared with model group.The results suggested that the metabolite of PCA2 may play an important role in the activities of PCA2 improving hepatocyte steatosis. |
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