Lipid-lowering Effects Of Averrhoa Carambola Polyphenols And Their Mechanisms Of Ameliorating Liver Steatosis

Lipid metabolism illness is a kind of metabolic syndrome which further increases the prevalence of obesity and other cardiovascular diseases.These diseases affect our health seriously.Accumulating evidence indicates that increased consumption of fruit and vegetables is positively associated with a low incidence of metabolic syndrome.Star fruit,Averrhoa Carambola?A.carambola??belonging to Oxalidaceae family?is a commercial fruit which majorly planted in tropical and subtropical regions.In South China,the fruit is very common.It is worth noting that almost all parts of A.carambola have been used in traditional Chinese medicine,including fruits,roots,leaves and flowers.This study aimed to fill some of the above knowledge gaps and compared the contents of the free,bound and total phenolics and flavonoids,as well as the chemical-based and cell-based antioxidant activities of four A.carambola cultivars.The lipid-lowering effects of four cultivars A.carambola free phenolic extract were determined by cell models,and then the extract which showed the strongest activity in cell models was selected for further study the in vivo activities.High performance liquid chromatography?HPLC?analysis of individual phytochemicals in four cultivars A.carambola free phenolic extracts was also performed.The inhibiting lipogenesis and hepatic lipid accumulation activities of these compounds were tested using cell models,the combinations of active compounds in inhibiting hepatic lipid accumulation effects were also determined.Additionally,the mechanisms of the action of A.carambola fruit phenolics in inhibiting hepatic lipid accumulation were studied.Main results are listed as follows:?1?The free,bound and total phenolic contents of the four cultivars A.carambola were162.5-286.8,6.4-19.7,and 174.5-293.1 mg GAE/100g fresh weight?FW?,respectively.The free,bound and total flavonoid contents were 100.7-234.0,1.1-7.8,and 104.4-235.1 mg CE/100g FW,respectively.The phenolics and flavonoids in the four A.carambola cultivars were mainly in free form,and the contribution of the bound phenolics to total phenolics ranged from 2.17%to 7.69%.Taiguo cultivar A.carambola had the highest total phenolic content.Gallic acid,syringic acid,p-coumaric acid,epicatechin,procyanidin B₂ and isoquercitrin were detected in the four A.carambola cultivars by HPLC.?2?Chemical-or cell-based antioxidant activities of four cultivars A.carambola free phenolic extracts were determined by oxygen radical absorbance capacity?ORAC?,peroxyl radical scavenging capacity?PSC?and cellular antioxidant activity?CAA?assays,respectively.The ORAC values of four cultivars A.carambola free phenolic extracts were23.76-49.84?mol TE/g FW.Taiguo cultivar free phenolic extracts had the highest ORAC value?49.84±3.44?mol TE/g FW?,which was 1.47,1.51 and 2.1 times higher?p<0.05?than those of Xiangmi,Hong and Honglong cultivar's,respectively.The PSC values of four cultivars A.carambola free phenolic extracts were 177.5-457.64?mol Vit.C E/100g FW,and Taiguo cultivar ranked the first.The cellular antioxidant activity?CAA?values with or without phosphate buffered saline wash protocol ranged from 25.80 to 63.29 and from 32.41to 68.96?mol quercetin equivalents/100g FW,respectively.The results showed that four cultivars A.carambola free phenolic extracts all possessed antioxidant activities,and Taiguo cultivar had the highest antioxidant activity among the four tested cultivars.?3?The lipid-lowering effects of four cultivars A.carambola free phenolic extracts.The inhibiting lipogenesis and hepatic lipid accumulation activities of these extracts were tested using cell models.The results suggested that most of the A.carambola free phenolic extracts showed the inhibiting lipogenesis and hepatic lipid accumulation activities in 3T3-L1 cells and L02 cells,respectively.Because Taiguo A.carambola free phenolic extract exhibited the strongest effects on both inhibiting lipogenesis and ameliorating hepatic lipid accumulation.?4?The lipid-lowering effects of Taiguo A.carambola free phenolic extract?ACF?in db/db mice.After administrated with ACF for 8 weeks,the contents of serum TG,NEFA and LDL-C were decreased effectively.The results manifested the hypolipidemic effect of ACF in a dose-dependent manner to some extent.Furthermore,liver TG contents of high-dose group were significantly decreased compared with those in the db/db control group?p<0.05?.The results suggested that ACF ameliorated liver steatosis and effectively decreased blood lipids.?5?The inhibiting lipogenesis and hepatic lipid accumulation activities of these compounds existing in the extracts were tested using cells models,and the combination effects of main active compounds in inhibiting hepatic lipid accumulation were also determined.The results suggested that epicatechin,isoquercitrin,p-coumaric acid and gallic acid showed the inhibiting lipogenesis effects in 3T3-L1 cells.Epicatechin,isoquercitrin,p-coumaric acid,syringic acid and proanthocyanidin B₂ showed the inhibiting hepatic lipid accumulation in L02 cells.When the inhibitory effects against hepatic lipid accumulation in L02 cells were 25%-50%,the combination of epicatechin and proanthocyanidin B₂ showed synergistic effect.?6?The mechanisms of the action of ACF in inhibiting hepatic steatosis.After 8 weeks intervention,the changes of hepatic insulin resistance,antioxidant activities and anti-inflammatory activities were determined.Further assessments by quantitative real-time PCR and Western blot assays were performed to elucidate the lipids synthesis suppression and fat decomposition promotion mechanisms by the protection of ACF.The results suggested that ACF significantly improved the activity of CAT and GSH-Px to suppress oxidative stress in db/db mice.Hepatic insulin resistance was also significantly ameliorated?p<0.05?.However,no significant difference was found in any groups in the contents of inflammatory factors?p>0.05?.Quantitative real-time RT-PCR determined the mRNA expression levels involved in fatty acid synthesis,TC synthesis,fatty acid?-oxidation and cytosolic-lipolysis.Significant differences in fatty acid synthesis genes were found among different groups?p<0.05?.The protein expressions of fatty acid synthesis were carried out to further confirm the evidence for the results of quantitative real-time RT-PCR.The results of quantitative real-time RT-PCR and western blot measurements suggested that the modulation hepatic lipogenesis mechanisms were partly through phosphorylation of AMPK?and down-regulation the expression of SREBP-1c,and further down-regulation of FAS and SCD1?p<0.05?.In addition,the expression levels of mircoRNA-34a and mircoRNA-33 which modulated this signaling pathway were significantly down-regulated by ACF treatment?p<0.05?.