Interaction Effects Of Stachyose Regulating Absorption,Metabolism And Hepatoprotection Of Flavonoids

Human health is seriously threaten by liver injury and obesity induced by chronic fructose sweeteners ingestion.Recently,dietary flavonoids,including flavanols,isoflavones,flavones and flavanone et al.,have attracted much attention in the field of prevention and control of hepatic injury.However,long-term intake of dietary flavonoid alone to prevent sub-health can trigger and even accelerate the intestinal first-pass metabolism of flavonoids,thus lead to decrease bioavailability of flavonoids.Previous studies indicated that some non-digestible saccharides could effectively enhance bioavailability of dietary flavonoids and thus strengthen its physiological function.Nevertheless,there is no evidence showing that stachyose,a non-digestible saccharide rich in diet,can strengthen absorption and hepatoprotective activity of dietary flavonoids with different molecular structures,and its releated mechanism is not understood.In the present work,soybean stachyose,tea flavanols(TF)and genistein were used as main nutritional factors in dietary intervention study,and high fructose induced liver injury in mice was used as animal model,and conventional biochemical and metabonomics analysis technologies were employed to study the interaction effects of stachyose in regulating absorption,metabolism and hepatoprotective activity of dietary flavonoids.The main research contents and results are as follows:(1)Mice were administrated by feeding stachyose and TF for 8 weeks continuously.The GC-MS analysis revealed that the ingestion of stachyose combined with TF elevated the concentrations of EC(Epicatechin),EGC(Epigallocatechin),ECG(Epicatechin-3-gallate),EGCG(Epigallocatechin-3-gallate)and total flavanols in the serum of mice fed with high fructose diet,especially the serum levels of catechins in gallate-esterified form(ECG and EGCG)were increased by 4.40%and 10.08%,respectively.The biochemical analysis showed that co-ingestion of stachyose and TF effectively prevented the increase of body weight induced by HF,the decrease of serum TC(Total cholesterol),TG(Total triglyceride)and LDL(Low-density lipoprotein-cholesterol)levels,and elevation of serum HDL(High-density lipoprotein-cholesterol)level in mice when compared to ingestion of TF or stachyose alone.Furthermore,intervention of TF combined with stachyose was more effective in inhibiting HF-caused an increase in hepatic MDA(Malonaldehyde)level,the decrease of hepatic SOD(Superoxide dimutase)and GSH-Px(Glutathione peroxidase)activities,and elevation of the serum hepatic injury biomarker ALT(Alanine aminotransferase),AST(Aspartate aminotransferase)and CRP(C-reactive protein)levels in mice,compared to that of TF or stachyose alone.The metabonomic analysis showed that ?-hydroxybutyrate,oleic acid(C18:1)and elaidic acid(C18:1t),involved in butanoate and fatty acid metabolism,were the potential biomarkers of HF-induced liver injury.The disorganized metabolism of ?-hydroxybutyrate,oleic acid and elaidic acid were ameliorated effectively by ingestion of TF together with stachyose than that of TF or stachyose alone.All these results revealed stachyose can enhance hepatoprotective activity of the tested flavonoids to prevent the HF-induced injury via promoting absorption of TF.(2)Mice were pre-treated with stachyose at different dose and duration,and were subsequently treated with TF at a same dose.The results showed that serum levels of EC,EGC,ECG and EGCG were time-and dose-dependently increased with stachyose pre-treatment in mice.In addition,pre-treatment with stachyose elevated the total flavanol concentration at least 152.46 ng·mL-1 in serum of mice.The results of ELISA assay revealed that pre-treatment of stachyose time-and dose-dependently inhibited the expressions of phase II metabolic enzymes SULT(Sulfotransferase)and UGT(UDP-glucuronosyltransferase)and efflux transporters P-gp(P-glycoprotein)and MRP1(Multidrug resistance-associated protein 1)in the small intestine.Furthermore,correlation analysis showed that there were significant negative correlations between serum catechins levels and phase II metabolic enzymes or efflux transporters levels in small intestine.These results indicated that stachyose increased the absorption of TF through inhibiting the expressions of phase II metabolic enzymes and efflux transporters in the intestine.(3)Genistein,a sensitive flavonoid for microbial metabolism in gut,was used to study the effects of stachyose on degradation in gut,absorption and hepatoprotection of deity flavonoid aglycones and their interrelationships.The present results exhibited that stachyose dramatically increased total genistein concentrations in serum and urine of mice.Besides,the total genistein concentration in faeces of mice treated with genistein and stachyose increased by 66.03%compared to solely genistein treated mice.The biochemical analysis revealed that the HF-induced abnormal weight gain,increasing fasting serum glucose levels,serum insulin concentrations and HOMA-IR(Homeostasis model assessment for insulin resistance)in mice were effectively prevented by ingestion of genistein combined with stachyose than that of genistein or stachyose alone.Meanwhile,co-treatment of genistein and stachyose effectively inhibited the increase of FAS(Fatty acid synthase),TC,TG,LDL and Apo-B(Apoprotein B)levels,and the decrease of HDL and Apo-A1(Apoprotein A1)levels in HF-fed mice,compared with that of treatment with genistein or stachyose alone.Moreover,the HF-induced increases of hepatic IL-1(Interleukin-1),IL-6(Interleukin-6)and TNF-?(Tumor necrosis factor-a)levels and serum AST,ALT and ALP activities were effectively decreased by administration of genistein together with stachyose relative to the administration of genistein or stachyose alone.Additionally,metabonomic profiling analysis of fatty acids revealed that the inhibition of the HF-induced metabolic disorders of serum esterified C16:1(palmitoleic acid),serum free C20:3(mead acid)and hepatic free C18:lt(elaidic acid)were more effective by combined intervention of genistein and stachyose than intervention of genistein or stachyose alone.These results preliminarily indicated that stachyose increased the absorption of genistein through inhibiting degradation of genistein in gut to strengthen liver protection.(4)Mice were treated with stachyose together with genistein for continuous 4 weeks.The UPLC-qTOF/MS analysis confirmed that stachyose dose-dependently increased the levels of genistein,GEN-4'-G(Genistein-4'-?-D-glucuronide),GEN-7-G(Genistein-4'-?-D-glucuronide)and GEN-7-S(Genistein-7-sulfate)in serum and urine of the mice co-treated with stachyose and genistein chronically.Particularly,stachyose significantly increased the genistein level in faeces of mice by 88.58 ?g·g-1,while the levels of DH-GEN,a characteristic metabolite of genistein by gut microorganism,were dose-dependently decreased by 10.34%?26.37%.Additionally,elevation levels of phase ?metabolic enzymes(SULT and UGT)and efflux transporters(P-gp and MRP1)in the intestine,caused by chronic genistein intake,was decreased with the increase of co-treatment dose of stachyose.These results suggested that stachyose increased bioavailability of genistein through inhibiting degradation of genistein in gut and down-regulating expression of first-pass metabolism related proteins during the process of chronic genistein ingestion.To sum up,the present work reveals that stachyose can enhance bioavailability and hepatoprotection of dietary flavonoids,and illuminate the related mechanism for the first time.These results facilitate the further studies on the mechanism of interaction between non-digestible saccharides and dietary flavonoids.Furthermore,all the results above will provide scientific basis for the exploitation of compound functional food.