Soybean Soluble Polysaccharides Regulate The Absorption, Metabolism And Nutritional Effects Of Genistein

Soybean originated in China and has been cultivated for more than five thousand years.As one of the most important food crops in the world,soybean and its products are rich in nutrients.SSPS(Soluble soybean polysaccharides)and genistein are the main components of functional sugar and isoflavone,respectively,have a wide range of physiological activities,such as lipid lowering and,blood circulation and gastrointestinal health promoting activities.At present,the research on this topic at local and international level is limited to the separation,purification,and nutritional function evaluation of single component.But those results could not accurately reflect the nutritional characteristics of the coexistence and interaction of food components,and ignored the fact that genistein had low bioavailability in oral administration.In this study,the liver injury induced by high L-carnitine and the obesity induced by HFD(high fat diet)were used as the animal models to systematically analyze the liver protection and weight loss effects of the combined intervention of SSPS and genistein.Furthermore,the potential mechanism of SSPS promoting the bioavailability of genistein was explored.The main research contents are as follows:(1)The total polysaccharides content of SSPS determined by phenol-sulfuric acid method was 84.86%.The results of HPLC-UV analysis showed that the main identified monosaccharide compositions of SSPS were galacturonic acid,glucose,galactose,and arabinose,indicating that SSPS was acidic heteropolysaccharides.Mice were administered intragastrically with SSPS and/or genistein against high L-carnitine induced hepatic injury for 12 consecutive weeks.Analysis results of UPLC-qTOP/MS showed that compared with genistein treatment alone,the content of genistein aglycone and the total content of genistein in urine of mice were increased by 26.81%and 30.42%,respectively.Further,L-carnitine-fed mice developed severe hepatic lipid metabolism disorders,oxidative stress,and inflammatory responses,leading to the liver damage.Interestingly,the hepatic TC(Total cholesterol),LDL(Low-density lipoprotein)and MDA(Malonaldehyde)concentrations in mice were reduced,the levels of inflammatory cytokines IL-1(Interleukin-1),IL-6(Interleukin-6)and TNF-?(Tumor necrosis factor-?)were inhibited,and the activities of SOD(Superoxide dimutase)and GSH-Px(Glutathione peroxidase)were enhanced,and finally the important biomarkers AST(Aspartate aminotransferase),ALT(Alanine transaminase)and ALP(Alkaline phosphatase)of liver injury were down-regulated by intragastric administration of SSPS and genistein.Importantly,combined treatment was more efficient than the individual treatment of SSPS or genistein in regulating these imbalances in mice.Moreover,the liver H?E(hematoxylin-eosin)and oil red O staining results further verified the liver protective effect of combined administration is better than that of single treatment.(2)To prevent obesity in mice,2.5%SSPS and 0.5%genistein were added to HFD for 12 consecutive weeks.The UPLC-qTOF/MS results showed that urine concentrations of genistein were increased from 259.33 ?g/mL of the mice treated with genistein alone to 332.61 ?g/mL of the mice treated with SSPS and genistein,which was consistent with the results of the first chapter.The results also showed that SSPS combined with genistein was more effective than genistein alone in reducing the abnormal increases in body weight,fat and fat index induced by HFD in mice,and the results were also confirmed by adipose and liver histopathological biopsies.At the same time,the combined supplementation of SSPS and genistein also more effectively down-regulated the expressions of FAS(Fatty acid synthase),ACC(Acetyl CoA carboxylase)and SREBP-lc(Sterol regulatory element binding protein-1c),resulting in reduced abnormal fat accumulation than that of SSPS and genistein alone.In addition,the combined intervention was more effective than the single intervention in enhancing the oxidative defense system and inhibiting the release of inflammatory factors IL-1 and TNF-? by down-regulating the protein expression of the NF-?B(Nuclear factor-?-gene binding)pathway.Cluster analysis based on obesity-related biochemical indicators showed that mice treated with SSPS combined with genistein were clustered together with mice fed normal diet,which also confirmed that the combined addition of SSPS and genistein had a better weight loss effects than those treated with SSPS and genistein alone.(3)This study was designed to investigate the mechanism of SSPS combined with genistein to prevent obesity by regulating energy metabolism and intestinal bacteria in mice.The HFD led to severe energy metabolism imbalance,manifested as the hyperglycemia and the insulin resistance.The OGTT(Oral glucose tolerance test)and the ITT(Insulin tolerance test)results showed that the combined SSPS and genistein intervention significantly reduced the area under the curve for blood glucose,thereby improving the abnormal glucose metabolism in HFD mice.In addition,joint intervention significantly reduced the fasting glucose and insulin levels by 29.22%and 28.63%than that in HFD mice,thereby reducing the HOMA-IR(Insulin resistance index).It is important that SSPS and genistein effectively inhibited the expressions of the key protein AMPK(Adenosine monophosphate activated protein kinase),PPAR-?(Peroxisome proliferators-activated receptor-?)and PPAR-?(Peroxisome proliferators-activated receptor-y),thereby balancing the energy metabolic disorder caused in obese mice.In addition,SSPS combined with genistein could produce more SCFAs(Short chain fatty acids)than that of SSPS or genistein alone.Moreover,the combined intervention of SSPS and genistein was more effective than the single intervention in maintaining intestinal microbiota homeostasis by reducing the abundance ratio of F/B(Firmicutes to Bacteroidetes),promoting the growth of beneficial bacteria(Norank-F_Bacteridales_S24-7,Lachnospiraceae NK4A136_group,Allobaculum,Lactobacillus and Alloprevotella),and inhibiting the growth of harmful bacteria(Helicobacter)in mice.(4)The molecular mechanism of SSPS regulating genistein absorption and metabolism in mice was investigated by intragastric administration of SSPS and genistein in healthy mice.The concentrations of genistein and its metabolites in blood,urine and feces of mice were evaluated by UPLC-qTOF/MS.It was found that SSPS could dose-dependently enhance the bioavailability of genistein.Specially,SSPS can significantly improve thelevel of genistein in the feces of mice by 167.54 ?g/g,while the concentrations of DH-GEN(Dihydrogen)in feces;a characteristic metabolite of genistein produced by gutmicroorganism,were reduced by 27.21%and 57.42%at lower and higher doses,respectively.In addition,the experimental results showed that SSPS could inhibit the expressions of the phase ? metabolic enzymes UGT(UDP-glucuronosyltransferase)and SULT(Sulfotransferase)in small intestine,thereby reducing the transformation of genistein to the low bioactive metabolites.Furthermore,it was found that SSPS could reduce the expression levels of the efflux transporters P-gp(P-glycoprotein),MRP1(Multidrug resistance-associated protein 1)and MRP2(Multidrug resistance-associated protein 2)in the small intestine of mice,thus reduce the phase ? metabolites of genistein excreting from intestinal epithelial cells to enteric cavity.At the same time,the expression levels of phase ? metabolic enzymes and efflux transporters in the colon and liver was also detected.Results indicate that the small intestine was the target organ of first-pass metabolism of genistein,not the large intestine and the liver.It is revealed that SSPS inhibit the degradation of genistein and reduce the activities of UGT,SULT,P-gp,MRP1 and MRP2,which is the exhibit potential mechanism of SSPS improving the bioavailability of the genistein in mice.In this study,it was found that SSPS could promote the absorption of genistein and enhance its nutritional effects in mice.In addition,the potential mechanism of enhancing bioavailability of genistein by SSPS has also be explored.These findings will provide a solid scientific basis for the development and the utilization of soybean-based nutrient food.