| Nutrients and macromolecular active substances in cereals must be digested,decomposed and absorbed by the gastrointestinal tract before they can participate in human metabolism.The active ingredients of food and their functions are not exactly the same as their effects on the human body.A number of factors may influence the effect of the active ingredient on the body,such as the stability of its macromolecular components and their decomposition products during gastrointestinal digestion,the interaction with other food components,and the ease of passage through small intestinal cells.Therefore,in recent years,more and more attention has been paid to the change mechanism of food raw materials' own system in the process of digestion,absorption and metabolism.This paper focuses on four kinds of common multigrain buckwheat,adlay,red bean and highland barley in China.Buckwheat phenols,the most lipid-lowering single active substance of buckwheat,were screened out by constructing lipid cell model,in vitro digestion,Caco-2 monolayer transwell model and Caco-2/Hep G2 co-culture model to explore the digestive and metabolic mechanisms of phenols in buckwheat,and through animal experiments and the expression of related proteins and genes to explore its regulation mechanism of fat metabolism in high-fat feed induced obesity mice,in order to provide a new idea and theoretical basis for the utilization of polyphenols and related functional food research and development.First of all,we evaluated changes in phytochemical contents of four kinds of coarse cereals(adlay,buckwheat,highland barley and red bean)and their lipid-lowering effect after cooking and in vitro digestion.The results showed that after in vitro digestion the total phenols in buckwheat was 27.78 ± 0.14 mg GAE/100 g,which was highest among all these coarse cereals.The total flavonoids increased after in vitro digestion in red bean,buckwheat and highland,and which increased most in buckwheat by 57.9%.Then the high-lipid model was established using Hep G2 cells.And the concentration of TG and LDL-c was determined with adding of 2.5 mg/m L of polyphenols extract from four coarse cereals.The results showed that the phenolic compounds in buckwheat has good bioavailability and lipid-lowering effect(0.156 ± 0.032 mmol/gprot of TG and 0.025 ± 0.005 mmol/gprot of LDL-c),which shows the best lipid-lowering effect.Twenty main phenolic compounds were identified by UHPLC-Q-Orbitrap mass spectrometry,and the four most abundant hydroxycinnamic acid,quercetin,scoparone and rutin were analyzed.The relative amounts of all phenolic compounds throughout the digestion and intestinal absorption process were determined by UHPLC-Q-Orbitrap MS.The research can provide a reference for the in-depth study of its regulatory mechanism and the development of lipid-lowering related functional food and health products.Then,Caco-2 cell model was constructed to study the changes of polyphenols extracts from digested buckwheat before and after passing through small intestinal epithelial cells.The digestible and easily transported phenolic compounds in buckwheat extract were identified.Herein,four main phenolic compounds and their metabolites were found on both the apical and basolateral sides of the Caco-2 cell model.The transepithelial transport rates in the Caco-2 cell monolayer were scoparone(0.97)> hydroxycinnamic acid(0.40)> rutin(0.23)> quercetin(0.20).The main metabolism of hydroxycinnamic acid,quercetin and scoparone in transepithelial transport was found to be methylation.Thirdly,Hep G2 cells were placed on the BL side of the Caco-2 transport model to construct a Caco-2/Hep G2 hyperlipidemic cell model,so as to study the lipid lowering activity of polyphenols extract from buckwheat digestive fluid on hyperlipidemic cells and its regulation effect on lipid-metabolism-related genes before and after absorption by simulated small intestinal epithelial cells.Results indicated that the triglyceride,low-density lipoprotein cholesterol,total cholesterol,aspartate aminotransferase and alanine aminotransferase levels in Hep G2 cells on the basolateral side of coculture models can be suppressed by 53.64%,23.44%,36.49%,27.98%,and 77.42% compared to the oleic acid-induced group(p<0.05).In addition,the m RNA expression of Fabp4 relative to control was found to be significantly upregulated(85.82 ± 10.64% to 355.18 ± 65.83%)by the easily transported buckwheat polyphenol components in Hep G2 cells(p<0.01).Fourthly,in order to explore the regulating effect of buckwheat on body weight,organ index and blood lipid level in C57BL/6J mice,dietary intervention was carried out with 10%,20% and 40% proportion of buckwheat to replace basic feed and the mixture of buckwheat polyphenol standard substance.The results showed that after 39 d in the diet intervention,compared with high-fat diet group,adding buckwheat feed and buckwheat polyphenol intervention on body weight in mice,the liver index and serum triglyceride(TG),total cholesterol(T-CHO),low-density lipoprotein cholesterol(LDL-c),aspartate aminotransferase(ALT)and third transaminase(AST)increase significantly inhibitory effect,and with the increase of amount of buckwheat add up inhibition increases;The effect of polyphenol intervention was comparable to that of 40% high-dose buckwheat.At the same time,adding buckwheat feed and buckwheat polyphenol could protect and improve the decrease of renal index and HDL-c in mice.It was also the high dose 40% buckwheat and polyphenol group that had the best effect.This indicated that buckwheat intervention could effectively improve the lipid metabolism disorder induced by high-fat diet in obese mice.Last but not least,western-blot and q-PCR were used to investigate the effects of buckwheat feed and polyphenol intervention on the expression of heat-producing proteins and genes(UCP1,PRDM16,PGC1,TCF21 and HOXC8)in white and brown fat in mouse visceral adipose tissue.UCP1,PRDM16,and PGC-1 protein and gene expression levels were significantly decreased in the high-fat diet group compared to the normal diet group,while TCF21 and HOXC8 expression levels were significantly increased(P <0.05).This indicated that the high-fat diet inhibited the expression of heat-producing related proteins,resulting in the decrease of the thermogenic activity of brown fat in mice,while the expression of heat-producing related proteins in white adipose tissue increased,leading to the accumulation of excess energy.After dietary intervention,protein expression levels of UCP1,PRDM16 and PGC-1 in the buckwheat intervention group were significantly up-regulated,while protein expression levels of TCF21 and HOXC8 were significantly decreased(P<0.05).In addition,the 40% buckwheat and polyphenol intervention group significantly increased the expression of UCP1,PRDM16 and pgc-1 genes in mouse visceral adipose tissue(P<0.05),and down-regulated the expression of TCF21 and HOXC8 genes,indicating that buckwheat could significantly improve the thermogenic activity of brown fat,reduce the content of white fat,and regulate the energy metabolism balance.In summary,40% of buckwheat diet intervention or buckwheat polyphenols intervention can activate brown fat and brown white adipose cells in liver tissue to improve obesity and energy metabolism disorders induced by high-fat diet.To sum up,phenolic substances in four common grains(buckwheat,adlay,red bean and highland barley)were investigated.The phenolic substances in buckwheat have the best bioavailability and lipid lowering effect.The main mechanism of its digestion and absorption by the body is the methylation reaction in the process of intestinal epithelial cell transport.The main mechanism of lipid lowering is that buckwheat phenols can enhance the expression of lipid metabolism regulation genes in cells,and improve liver and kidney function of obese mice,effectively regulate lipid metabolism disorder.It provides a new idea and theoretical basis for the utilization of polyphenols in grains and the development of functional foods about weight loss products. |
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