Study On The Stability Of Black Bean Seed Coat Anthocyanins In Cell Culture And The Effects On Its Absorption,transport And Antioxidant Activity

Anthocyanins are a class of water-soluble natural pigments that are widely present in plants.They have various effects such as anti-oxidation,anti-inflammatory,liver protection,and cardiovascular disease prevention.Anthocyanins are unstable and are easily affected by factors such as ambient temperature and pH.The low bioavailability of anthocyanins is related to the structure and stability of the compound itself,and to the defense and absorption mechanisms of the body.The phenolic compounds and antioxidant activity of black beans are the highest in edible beans,among which the anthocyanins are the most abundant.In this study,the black bean seed coat anthocyanin cyanidin-3-glucoside was used as the research object to explore its stability under three different cell culture conditions,and to explore its interaction mechanism with fetal bovine serum and its absorption and antioxidant activity,and to study the absorption and transport of cyanidin-3-glucoside in LO2 cells.This study investigated the stability characteristics of anthocyanins of cyanocyanin-cyanidin-3-glucoside under the conditions of in vitro cell experiments,and the aim is to provide scientific basis for anthocyanin concentration,processing time,and medium selection based on in vitro cell experiments.The main findings were as follows:(1)The content of anthocyanins in black bean seed coat was 1.086 mg/g DW.Qualitative analysis of anthocyanins in black bean seed coat by LC-TOF-MS/MS was as follows:the main component of anthocyanins in black bean seed coat were cyanidin and delphinidin glycoside derivatives,specifically including:cy anidin-3,5-diglucoside,delphinidin-3-glucoside,cyanidin-3-glucoside,petunidin-3-galatoside,pelargonidin-3-glucoside,peoni din-3-glucoside,cyanidin-3-arabinoside,malvidin-3-arabinoside,cyanidin-3-(6''-malonoyl)glucoside,apigeninidin-glucoside,delphinidin-3-rutinoside,cyanidin-3,5-diglucoside,delphinidin-3-hexoside,delphinidin-3-hexoside,apigeninidin-3-(6"-malonyl glucoside),pelargonidin-3-(malonoyl)glucoside,petunidin-3-glucoside,malvidin-3-glucoside and delphinidin.Optimize the conditions of AB-8 resin adsorption and desorption of anthocyanins:pH 3.5,sample loading 0.51 mg/g(anthocyanin mass/AB-8 resin mass),80%methanol solution.The anthocyanin extraction solution was initially purified by AB-8 resin column chromatography,and the purity of the anthocyanin extraction solution was significantly improved from 49.85%to 67.34%.The preparative high performance liquid hromatography The preparative liquid phase was further purified to produce anthocyanin monomers with high purity,and 84.67%purity delphinidin-3-glucoside and 93.76%cyanidin-3-glucoside were obtained.(2)Study the stability of black bean seed coat anthocyanin cyanidin-3-glucoside in three commonly used cell culture media:HBSS buffered saline solution,RPMI 1640 medium containing 10%fetal bovine serum(FBS)and RPMI 1640 medium,and explore the mechanism of its interaction with fetal bovine serum and its effect on antioxidant activity.The results show that the stability of cyanidin-3-glucoside in these three solutions was:HBSS>10%FBS+RPMI>RPMI,and the anthocyanin degradation products were protocatechuic acid and phloroglucinaldehyde protocatechuic acid,and the degradation kinetics models were three-order,zero-order,and zero-order kinetic models.At the same time,cyanidin-3-glucoside can quench the endogenous fluorescence of fetal bovine serum,which belongs to static quenching,and its main action was hydrophobic action.Cyanidin-3-glucoside has little effect on the microenvironment of tryptophan and tyrosine in fetal bovine serum,but it will loosen the secondary structure of the protein.The anti-oxidation experiments of DPPH,ABTS,and FRAP have shown that fetal bovine serum significantly reduces the free ABTS radical scavenging ability of cyanidin-3-glucoside,and has little effect on its DPPH free radical scavenging ability and iron atom reducing ability.(3)Fluorescence inverted microscope and HPLC-QQQ-MS were used to study the absorption and transport of cyanidin-3-glucoside in LO2 cells.The results showed that cyanidin-3-glucoside entered LO2 cells as a prototype,and was mainly distributed in the cell membrane.The uptake of cyanidin-3-glucoside by LO2 cells was concentration-and time-dependent.The absorption of C3G was the highest when it was incubated at 80 ?M C3G for 10 h.At the same time,the uptake of cyanidin-3-glucoside by LO2 cells was also temperature-and pH-dependent.LO2 cells were more conducive to the uptake of C3G molecules at 37? and its uptake of C3G at pH 5.0,which was explained that the active transport was taken part in its uptake process.And the uptake of C3G by LO2 cells at 4? indicated that there was a form of passive diffusion participate in the uptake process.The above results indicated that the uptake of C3G by LO2 cells at 37? and pH 7.4 involved active transport and passive diffusion these two processes.WB experiments showed that P-gp,MRP2,GLUT2,and OCT1 transporters exist in LO2 cells,but P-gp and MRP2 transporters do not participate in the efflux process of cyanidin-3-glucoside in LO2 cells.GLUT2 and OCT1 transporters also have no effect on the process of cyanidin-3-glucoside transport in LO2 cells.