| Black soybean(Glycine max(L.)Merr.)has been widely consumed as medicinal food in Asia for hundreds of years and its seed coat contains abundant anthocyanins that possess various bioactivities,such as antioxidative,anticancer,antidiabetic,antimicrobial,and neuroprotective effects,etc.However,the present studies on bioactive mechanisms of anthocyanins are not very comprehensive.Besides,the unstable properties of anthocyanins usually affect their performance and biological activities,while,the commonly used stabilization methods of anthocyanins have some disadvantages and the related mechanisms are not very clear.In addition to the beneficial nutritional anthocyanins,black soybean contains several antinutritional factors,like trypsin inhibitors(BBTI),which can affect digestion and absorption of proteins.Recently,most inactivation strategies of BBTI focus on physical and chemical methods that have negative effects on BBTI,there are few studies on the mechanism of using natural active constituents to inactivate BBTI.Tea(Camellia sinensis L.),the second most consumed beverage in the world next to water,has a long drinking history in China.Tea and black soybean are often consumed in the daily diet,and the tea polyphenols are natural active constituents with safety property and various beneficial bioactivities.While,little is known about using tea polyphenols to inactivate BBTI.Based on these problems,the present study investigated antioxidant,antidiabetic and neuroprotective activities of black soybean anthocyanins(BSSCE)and their related mechanisms.The stabilization mechanism of BSSCE,nanoparticle system for proanthocyanidin delivery and the inactivation mechanism of BBTI were also studied,which could provide a theoretical basis for black soybean anthocyanins as a functional food or medicine and facilitate the extensive application of black soybean.The main contents are as follows:1.Black soybean anthocyanins(BSSCE)were extracted from its seed coat,and the compositions of which were identified as cyanidin-3-O-glucoside(90.2%),delphinidin-3-O-glucoside(5.3%),and peonidin-3-O-glucoside(2.3%)by HPLC-MS.BSSCE exhibited an effective protection effect on H2O2-induced oxidative damage in Hep G2 cells through reducing intracellular ROS accumulation and enhancing endogenous antioxidant enzyme system.In fact,BSSCE(120?g/m L)significantly increased catalase(CAT)by 48.9%,superoxide dismutase(SOD)by 77.1%,and glutathione peroxidase(GPX)by 95.2%(P<0.05).The malondialdehyde(MDA)level in BSSCE treatment group was also significantly decreased by 55.7%(P<0.05).BSSCE also exhibited strong neuroprotective activity against Human amyloid-?1-42protein(A?1-42)-mediated toxicity in PC-12 cells(100?g/m L BSSCE increased PC-12cell viability reduced by A?1-42 from 59.5%to almost 89.0%).This finding suggested that the neuroprotective effect of BSSCE might be associated with the protection of antioxidant defense system.The molecular docking studies revealed that both hydrophobic effects and hydrogen bonds between Cy3G and target proteins might be responsible for the antioxidant activity and neuroprotective effect of BSSCE.2.In order to illustrate the hypoglycemic mechanism of black soybean anthocyanins(BSSCE),the effects of BSSCE and cyanidin-3-O-glucoside(Cy3G,the major anthocyanins of BSSCE)on the regulation of Protein Kinase B(Akt)and Glucose transporter protein 4(GLUT4)in L6 rat skeletal muscle cells were studied.The results showed that BSSCE and Cy3G significantly enhanced the glucose uptake in L6myotubes,compared with the negative control(P<0.05).Especially,150?g/m L BSSCE and 40?M Cy3G remarkably increased the 2-NBDG uptake level by 1.8-fold and 1.9-fold compared with that of negative control,respectively.Furthermore,the expressions of Phosphorylated-Protein Kinase B(p-Akt)and GLUT4 were increased and the GLUT4 translocation was promoted by BSSCE and Cy3G,which evidenced the involvement of the Akt/GLUT4 signal pathway in the hypoglycemic mechanism of BSSCE.These findings revealed that Cy3G-enriched BSSCE might be a promising functional food or medicine for the treatment of T2DM and the related disorders.3.The interaction of soy protein isolate(SPI)with cyanidin-3-O-glucoside(Cy3G)was investigated to study the stabilization effect of preheated protein on black soybean anthocyanins by UV-Vis spectrophotometry,Fourier transform infrared spectroscopy(FTIR),circular dichroism(CD)and fluorescence spectroscopy.Preheat treatment and binding of Cy3G effectively changed the secondary structures of SPI,with a decrease in?-helix,random coil structure and an increase in?-sheet and?-turn.Among 40,60,80,100 and 121?,SPI preheated at 121? exhibited the strongest binding affinity towards Cy3G with strong Ks of 147.40×104 M-1 and effectively increased the thermal stability of black soybean anthocyanins(BSSCE)via decreasing its degradation rate by67%and improved the oxidation stability of BSSCE by decreasing its degradation rate by 23%.SPI interacted with Cy3G mainly through hydrophobic interactions and static quenching process.These results suggested that preheated SPI-Cy3G interaction system(formed by hydrophobic interactions and static quenching process)could effectively protect anthocyanins'stability.4.A novel and effective multifunctional PEG modified Ce O2@Si O2 nanoparticle(CSP-NPs)system that could deliver hydrophilic and hydrophobic molecules was successfully fabricated.Proanthocyanidin(PAC)and curcumin(Cur)were loaded onto CSP-NPs and formed as PAC-NPs and Cur-NPs to demonstrate the potential application of CSP-NPs.FTIR,X-ray diffraction(XRD),scanning electron microscopy(SEM),transmission electron microscopy(TEM),and dynamic light scattering(DLS)were used to characterize the prepared NPs.The bioactivities including antioxidative,neuroprotective and anticancer effects were studied.FTIR and XRD analysis confirmed the layer-by-layer deposition of Ce O2,PEG,PAC or Cur onto the Si O2 core.CSP-NPs,PAC-NPs and Cur-NPs displayed spherical shape with about 35-45 nm size and showed a noticeable sustainable release performance.Confocal fluorescence microscope analysis revealed that the empty CSP-NPs system could effectively load PAC and Cur to exhibit potent protection effect on H2O2-induced oxidative damage.PAC-NPs and Cur-NPs at the dose of 1.0 mg/m L,exhibited strong acetylcholinesterase(Ach E)inhibitory activity with 81.4±0.8%,78.8±2.0%inhibition rate,respectively,and potent neuroprotective activity against A?1-42-mediated toxicity in PC-12 cells by an increased cell viability compromised by A?1-42 from 57.5%to 81.0%at the dose of 25?g/m L.In addition,PAC-NPs and Cur-NPs showed effective antiproliferative effects on Hep G2and Hela cells(P<0.05).These findings indicated that CSP-NPs might be a promising delivery system for hydrophilic and hydrophobic drugs against the oxidative damage,neurodegenerative diseases and cancer.Moreover,these results may provide a potential delivery system for hydrophilic drug PAC or hydrophobic drug Cur,which could promote their wide applications.5.The inactivation mechanism of BBTI induced by tea polyphenols(TPs),EGCG and EGC was firstly investigated using fluorescence,FTIR,CD spectroscopy,isothermal titration calorimetry(ITC)and molecular docking technologies.The BBTI inhibition test revealed that TPs,EGCG and EGC could effectively inactivate BBTI with a maximal inhibition rate of 35%,51%and 21%,respectively.The higher inhibition effect of EGCG on BBTI may result from its molecular structure that contains more hydroxyl groups than EGC.The mechanism studies indicated that EGCG and EGC interacted with BBTI via static quenching process and hydrophobic interactions.TPs,EGCG and EGC induced a transition of BBTI conformation from disorder to order.ITC analysis and molecular docking study revealed the interactions of EGCG-BBTI and EGC-BBTI were spontaneous,and hydrophobic interactions and hydrogen bonds were the predominant forces.Together,these findings clearly suggested that EGCG could be a promising inactivated agent for BBTI,which had the potential value for improving the safety and nutritional properties of black soybean products.In conclusion,the present studies comprehensively investigated the bioactivities mechanisms,stabilization mechanism of nutritional factors(BSSCE and Cy3G)and the inactivation mechanism of antinutritional factor(BBTI)of black soybean as well as the nanoparticle delivery system for proanthocyanidin and curcumin delivery.These findings may establish a foundation for the research and development of black soybean as functional food or medicine candidate,and provide theoretical basis for the extensive utilization of black soybean resources. |
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