Absorption Of Se-enriched G.frondosa Polysaccharide In Caco-2 Cell Model And Metabolic Kinetics In Rats

Grifola frondosa is rich in protein,dietary fiber,polysaccharides,micronutrients and other nutrients,which polysaccharides are its main active substances and have biological activities such as immunomodulatory,anti-oxidation,anti-tumor,hypoglycemic and hypoglycemic.Se-enriched Grifola frondosa polysaccharide is a kind of organic selenium compound with dual biological activities of polysaccharide and selenium.Previous studies of this research group have found that Se-GFP extracted and purified from Se-enriched Grifola frondosa fruit body has good anti-tumor activity in vivo,which can enhance the immune activity and antioxidant activity of immunosuppressed mice.However,studies on the absorption of Se-GFP in vitro and metabolic kinetics in vivo have not been reported.Therefore,the monolayer culture model of Caco-2 cells was established to study the transport and uptake of Se-GFP in vitro.Se-GFP was fluorescently labeled by fluorescein isothiocyanate?FITC?through amide reaction to investigate the metabolic kinetics in rats.This study can provide a basis for the research and application of absorption and metabolic kinetics of polysaccharide compounds,and has important theoretical and practical significance.The main research contents are as follows:?1?The transport and uptake of Se-enriched Grifola frondosa polysaccharide?Se-GFP?were investigated by Caco-2 monolayer cell culture model from two aspects-the polysaccharides and selenium of Se-enriched Grifola frondosa.The results revealed that the transport of Se-GFP in Caco-2 cells was time-and concentration-dependent,which was more easily transported on the apical?AP?side than on the basolateral?BL?side.The determination results of the apparent permeability coefficients?Papp?and transport rates indicated that Se-GFP was a moderately absorbed biological macromolecule,mainly penetrating Caco-2 cells through the same pathway as endocytosis.The uptake of Se-GFP in Caco-2 cells was time-and concentration-dependent,and the uptake rate on the AP side was significantly higher than that on the BL side.The uptake of Se-GFP may be a macrocytopathic pathway,which was an accumulation from cytoplasm to nucleus process.Determination of selenium content showed that organic selenium Se-GFP was more easily absorbed than that in the inorganic selenium control group?sodium selenite?.?2?Se-enriched Grifola frondosa polysaccharide?Se-GFP?was fluorescently labeled with fluorescein isothiocyanate to study the stability of the labeled Se-enriched Grifola frondosa polysaccharide?Se-GFP-FITC?in biological samples.The experimental results showed that the Se-enriched Grifola frondosa polysaccharide was connected to the amino group through reductive amination reaction,and the polysaccharide-tyramine compound?Se-GFP-Tyr?was obtained.Fluorescein isothiocyanate was used to fluorescently label the aminated polysaccharide-tyramine compound?Se-GFP-Tyr?to obtain Se-enriched Grifola frondosa polysaccharide?Se-GFP-FITC?,and the substitution degree of FITC was0.96%.The purified components of Se-GFP-FITC were relatively uniform,the peak shape was symmetrical,the molecular weight was nearly unchanged,and there was no free fluorescein,indicating that the Se-enriched Grifola frondosa polysaccharide was successfully labeled by FITC fluorescence.The results of stability tests in vitro showed that Se-GFP-FITC had good stability in phosphate buffered solution?PBS?,blank plasma and urine of rats and other media in vitro.It can be seen that fluorescently labeled Se-enriched Grifola frondosa polysaccharides can be used in subsequent experimental studies.?3?The metabolic kinetics of Se-GFP-FITC in rats were studied by fluorescence quantitative detection methods in vivo.The non-compartmental model fitting Se-GFP-FITC plasma concentration-time data in rats showed that the half-life(t_1/2)of Se-GFP-FITC in rats was 16.92 h,the peak was delayed,and the values of reaching the time required for maximum blood concentration(T_max),mean residence time?MRT?,apparent volume of distribution?V?,clearance rate?CL?and t_1/2 were independent of the dose administered,of which the maximum blood concentration(C_max)and the drug-time curve area AUC?0-??were positively correlated with the composition of each dose,indicating that Se-GFP-FITC in rats presented linear metabolic kinetic characteristics in vivo.Tissue distribution experiments showed that Se-GFP-FITC could be distributed to most tissues after intragastric administration for1 h,with the highest concentration in the intestine,followed by the stomach and liver.The concentration of Se-GFP-FITC was the highest in liver tissues after 24 h of intragastric administration,followed by kidney,muscle,intestine,stomach and brain.The results of the excretion experiments showed that 83.87%of Se-GFP-FITC was excreted in feces and urine after 72 h of single gavage,and most of it was excreted in feces.It can be seen that the method was suitable for the study of the metabolic kinetics of Se-GFP-FITC in rats.In summary,the study was conducted on the absorption of Se-enriched Grifola frondosa polysaccharide?Se-GFP?in vitro based on Caco-2 cells.Isothiocyanate fluorescence labeling was carried out on the Se-enriched Grifola frondosa polysaccharide after reduction amination to study the metabolic kinetics of Se-enriched Grifola frondosa polysaccharide after fluorescent labeling?Se-GFP-FITC?in rats,which can provide a basis for the absorption of polysaccharide compounds and the research and application of metabolic kinetics.