In Vitro Metabolism And Biological Activity Of The Steviol Glycosides

The sweet components of steviol glycosides have the same molecular skeleton?ent-kaurenoic acid?.Steviol glycosides have a wide range of physiological activities due to its special terpene structure.In recent years,because of the naturalness of steviol glycosides and its constantly discovered physiological effects,as well as the characteristics of high sweetness and low calories,it has been widely used in many fields instead of the traditional edulcorant of sucrose.At present,the safety and bioactivity study of stevioside,rebaudioside A and its metabolite steviol dgradually increased but with a certain amount of controversy.In vitro metabolic mechanism of steviol glycosides and the effect of edulcorant on detoxification based on phase ? metabolic enzymes are still unclear.The cytotoxicity of steviol glycosides needs to be recognized,explored and utilized.There are few studies on their inhibition on human cancer cells.In this research,the phase ? metabolites and kinetics of stevioside and UDPGA reactions catalyzed by human liver microparticles and UGT single enzyme were analyzed in depth,to explore the effect of exogenous substances on steviol glucuronidation,and to clarify the in vitro metabolic mechanism of steviol glycosides.To explore the hypoglycemic activity of steviol glycosides,and focus on the effect on the growth of cancer cells in the human digestive system,and to analyze the inhibitory effect of steviol glycosides on human tumor cells and the possible signal pathway.Through the research,to further understand the metabolic safety of steviol glycosides,provide support for the deep utilization of steviol glycosides as a kind of bioactive sweetener,and also provide guidance for the use of other bioactive food additives.The main research contents of this paper are as follows:1.The in vitro metabolic mechanism study found that HLM induced higher steviol glucuronide formation rates than UGT2B7 did.5-Fu,BSA,4-MU and glucose showed weak inhibition on steviol glucuronidation.Although high concentration of glucose would weaken the aldehyde acidification of steviol,the effects of Stevioside?St?and Rebaudioside-A?RA?on the aldehyde acidification of steviol were not significant.Several chemicals tested showed that they were friendly to use with steviol.In vivo and in vitro extrapolation?IVIVE?was adopted and found that human liver microspheres?HLM?showed stronger in vivo clearance than UGT2B7.AutoDock software was used for assistant analysis of the binding sites of steviol and UGT2B7.It was found that the active pockets of UGT2B7 was consisted of Arg352,Leu 347,Lys 343,Phe 339,Tyr 354,Lys 355 and Leu 353 amino acids,of which only Lys 343 formed hydrogen bonds with steviol.2.The metabolic intermediates of stevioside such as steviolbioside,rubusoside,steviol monoglucosyl ester and steviolmonoside have low cytotoxicity to human normal cells,and the cytotoxicity level is grade 1.While the end product of metabolism,steviol,has a broad spectrum of inhibitory effect on human cells.The two-dimensional topological similarity and three-dimensional shape similarity methods based on ligand similarity method in Drug Target Prediction System were selected to predict the network diseases pharmacology of steviol.It was found that the possible targets of steviol were tyrosine phosphatase 1B,11-?-hydroxysteroid dehydrogenase 1,microtubule-associated protein tau,DNA polymerase beta,cyclooxygenase-1,matrix metalloproteinase-2,cytochrome P450 2C9,etc.which were treated through network diseases.It was predicted that steviol may have the potential to treat diseases such as diabetes and cancer through network diseases pharmacological charts.3.The hypoglycemic experiment in mice showed that steviol could significantly reduce the blood sugar level of diabetic mice,promote insulin release,reduce the level of glycosylated hemoglobin?HbA1c?,improve the glucose tolerance of diabetic mice,and improve the oxidative stress index at the same time.The hypolipidemic experiments showed that steviol could reduce the weight of mice,reduce the level of triglyceride,total cholesterol and low density lipoprotein cholesterol,and increase the level of high density lipoprotein cholesterol.The effects of stevioside and steviol on glucose metabolism in liver,brain,myocardium and kidney of ¹⁸F-FDG labeled mice were observed by micro-PET dynamic imaging.It was found that steviol could increase the uptake of glucose in myocardium and brain of diabetic mice within 60 minutes,and decrease the accumulation of glucose in the liver and kidney.4.Steviol was found to possess intensive anticancer activity on the human gastrointestinal cancer cells and human osteosarcoma U2OS cell.They inhibit the proliferation of cancer cells through cycle arrest and apoptosis,and increase of Bax/Bcl-2 ratio,activation of p21 and p53;and caspase 3-independent mechanism was also involved.But different cancer cells have different arrest periods..Based on Sanger miRBase Release 21 miRNA microarray analysis,steviol-treated cells showed different miRNA regulation,miR-203a-3p?log2=1.32?and miR-6088?log2=-2.54?,MKN in HCT-116 1268b?log2=19.85?and miR-23c?log2=-2.05?in-45,miR-146a-5p?log2=1.88?and miR-615-5p?log2=-2.87?in HepG2,and miR-486-3p in U2OS?log2=4.53?.