The Effects And Mechanism Of Lotus Seeds Bound Phenolics On Lipid Metabolism In 3T3-L1 Preadiposes And Obese Mice

Lotus seeds have positive effects on clearing heat,lowering blood pressure and promoting sleep.The modern epidemiological research showed intake of foods riched in phenolic compounds play an important role in preventing chronic diseases such as cardiovascular diseases,hyperlipidemia,atherosclerosis and diabetes.Therefore,in this study,the lotus seeds bound phenolics(LBP)was extracted by alkali method,and the extract conditions were optimized by Box-Behnken central composite design.And the bound phenolic compouds and their antioxidant activity in vitro were determined.In addition,to explore the molecular mechanisms of LBP in regulating lipid metabolism,the lipid metabolism-related genes and protein expression in 3T3-L1 adipocytes and high-fat diet fed mice,as well as the diversities of gut microflora were analysed.This research provides great significance for revealing the mechanisms of health effects and guiding the dietary consumption of lotus seeds.The main research contents are as follows:(1)The extraction of bound phenolics from Jianning lotus seeds was optimized using a central composite design.Optimization experiments were carried out by investigating the extraction efficiency from the factors including solvent pH,alkali concentration,solid-tosolvent ratio and extraction time.The results showed that the optimum extraction conditions were determined as follows: NaOH concentration,2.8 mol/L;solid-to-solvent ratio,1:25(g/mL);and extraction time,3.7 h.Under these conditions the yield of bound phenolics was(8.483 ± 0.063)mg/g of dry matter.(2)The extract was fractionated with solvents of different polarities to obtain different fractions which were comparatively from their bound phenolic contents and antioxidant activity.Additionally,the bound phenolic compounds were analyzed by high performance liquid chromatography(HPLC).The results showed the n-butanol fraction contained the higher bound phenolics content,and ethyl acetate and n-butanol fractions possessed higher total antioxidant capacity,1,1-diphenyl-2-picrylhydrazyl(DPPH),and hydroxyl radical scavenging capacity in a dose-dependent manner.Additionally,the n-butanol fraction was rich in protocatechuic acid(0.42±0.12mg/g),caffeic acid(0.23± 0.08mg/g)and ellagic acid(0.15±0.06mg/g).Collectively,lotus seeds n-butanol fraction contained abundant bound phenolics and exhibited higher antioxidant capacity.(3)The 3T3-L1 preadipocytes were used to investigate the potential anti-obese action of lotus seeds bound phenolics(LBP).Our results showed that LBP below 100?g/mL significantly decreased intracellular lipid accumulation in a dosedependent manner without toxicity toward 3T3-L1.Moreover,it showed that LBP increased phosphorylation of adenosine monophosphate-activated protein kinase(AMPK)accompanied by down-regulation of lipogenesis-related genes including PPAR?,SREBP-1c,aP2,LPL,C/EBP?,FAS and up-regulation of adiponectin gene.Taken together,these observations suggested lotus seeds bound phenolics may exert anti-adipogenic and anti-lipogenic activities,in part by modulating the AMPK signaling pathway.(4)HFD-fed obeity mice were used as a research mobel to explore the antiobesity effects of lotus seeds bound phenolics in vivo.All mice were acclimated for one week before randomly separated into four groups(n= 6 in each group): normal diet(ND)group,high-fat diet(HFD)group,LBP group received HFD with 400 mg/kg bw of LBP,and CGA group(the positive group)received HFD with 150 mg/kg bw of chlorogenic acid.After 6-week administration,the serum,liver and epididymal fat in mice were separated for analysis.The results showed that LBP could improve plasma lipid profiles,inhibit adipocyte hypertrophy and reduce liver lipid accumulation in HFD-induced obesity mice.In addition,LBP treatment could activate AMPK accompanied with down-regulation of lipogenesis related genes(PPAR?,C/EBP?,SREBP-1c,LPL,FAS,aP2,GPR43)and up-regulation of lipolysis genes(adiponectin).Collectively,our data demonstrated LBP possesses anti-obesity actions which are,at least partially,mediated by the activation of AMPK signaling pathways.(5)To further study the anti-obesity action of lotus seed bound polyphenols(LBP),the intestinal microbiota composition of mice in different groups were investigated by high-throughput sequencing.The results showed that compared with HFD group,ND group had significantly decreased Firmicutes/Bacteroidetes,and LBP treatment could modestly reduce the phylum of Firmicutes and Bacteroides,prevalenting the growth of beneficial family Verrucomicrobiaceae and its genus Akkermansia,as well as the inhibition of obesity-related family Desulfovibrionaceae.In conclusion,LBP may be beneficial in regulating the composition of intestinal microflora in obese mice fed with HFD.