The Inhibition Mechanism Of Flavonoids On α-glucosidase And The Physicochemical Property And Bioactivity Of Flavonoids Binding With Nanoparticles

In recent years, diabetes mellitus, especially typeⅡ diabetes has become one of the major diseases of human life. The activity of α-glycosidase is a key factor in the production of postprandial hyperglycemia, and the clinical use of acarbose has some side effects for different patients, so searching for natural herbal ingredients as newα-glycosidase inhibitors have become the trend of the current study. In this paper by combining fluorescence spectroscopy, synchronous fluorescence spectroscopy,circular dichroism(CD) and molecular simulation technology to study the inhibition of α-glycosidase by kaempferol, myricetin and genistein, and these inhibitors inducedα-glycosidase conformation change, discussing the inhibition mechanism ofα-glycosidase by kaempferol, myricetin and genistein. However, due to the instability of flavonoid compounds and degradation, in this paper, we constructing a kind of high safety, moderate drug-polymer interactions of flavonoids compounds(kaempferol, myricetin and genistein) and BSA/ ?-carrageenan particle system,investigating the stability of BSA/ ?-carrageenan particles coated kaempferol,myricetin and genistein, and the ability of DPPH free radical scavenging, studying the kaempferol, myricetin and genistein in vitro release of gastrointestinal tract and inhibition of α-glycosidase. In this paper, the main research contents and results are as follows:1. The inhibition of α-glycosidase by kaempferol, myricetin and genistein show that kaempferol, myricetin and genistein are reversible α-glycosidase inhibitors, and the inhibition effect of α-glycosidase are greater than the positive control acarbose(IC50 =(2.09 ± 0.03) × 10-4). The inhibition effect of α-glycosidase is myricetin >kaempferol > genistein, their half inhibitory concentration(IC50) values were(0.68 ±0.02) × 10-5,(1.16 ± 0.04) × 10-5 and(1.89 ± 0.01) × 10-5 mol L-1(n = 3),respectively. The experiment proves that kaempferol is a mixed type of α-glycosidase inhibitor, myricetin is a competitive inhibitor while genistein is a non-competitive inhibitor. The kaempferol, myricetin and genistein inhibition constants Ki were(1.31± 0.03) × 10-5,(1.59 ± 0.04) × 10-5 and(1.26 ± 0.02) × 10-5(n = 3), respectively.2. Through spectroscopy method combined with molecular simulation technology to analyze, the results show that kaempferol, myricetin and genistein can quench fluorescence of α-glycosidase. And there are only one binding site(due to the n value is close to 1) between kaempferol, myricetin and genistein and α-glycosidase.Through the radiation energy transfer theory, the binding distance between kaempferol, myricetin and genistein and α-glycosidase were less than 8 nm,indicating that there existed the radiation energy transfer from α-glycosidase to kaempferol, myricetin and genistein in the binding process. And the r value is greater than R0 means that kaempferol, myricetin and genistein may be in a near location ofα-glycosidase, which strongly quenched the fluorescence intensity of enzyme.Molecular simulation results also showed that kaempferol, myricetin and genistein directly into the α-glycosidase activity central, through hydrogen bond and hydrophobic forces combined with the catalytic amino acids, occupy the position of enzyme activity center, hindered substrates pNPG into the enzyme, which have inhibit α-glycosidase activity eventually. There are activity of amino acids such Asp69 around Phe178, Asp215, Glu277, His351, Asp352, Arg315, Phe314 and Val410 in α-glycosidase activity central, these amino acids plays an important role in the catalytic mechanism.3. Based on the synchronous fluorescence spectrum and circular dichroism method, we found kaempferol, myricetin and genistein quenching tryptophan fluorescence intensity of α-glycosidase more obvious than tyrosine, the main fluoresce amino acid of α- glycosidase is tryptophan. And increase the concentration of kaempferol, myricetin and genistein, micro environment of tryptophan and tyrosine of α-glycosidase have changed a little. And the content of α-Helix of α-glycosidase is increased. These results mean kaempferol, myricetin and genistein combined to theα-glycosidase, broken the hydrogen bond network structure of α-glycosidase, then the secondary structure changed, hindered the formation of enzyme activity center and prevent the entry of the substrate, eventually led to the α-glycosidase inactivation.4. BSA/?-carrageenan as the carrier, preparing kaempferol-BSA/?-carrageenan particles, myricetin-BSA/ ?-carrageenan particles and genistein-BSA/ ?-carrageenan particles. The average particle size of BSA/?-carrageenan particles was 110 ± 3 nm,and electromotive force was 35.2 ± 0.2 mV. After added kaempferol, myricetin and genistein, BSA/?-carrageenan particle size changed to 136 ± 4 nm, 145 ± 2 nm and121 ± 2 nm, respectively. Electromotive force was 32.2 ± 0.5 mV, 40.5 ± 0.3 mV and0.3 ± 0.4 mV. Loading rate of kaempferol, myricetin and genistein was 78.41%,89.67% and 71.59% respectively. Kaempferol, myricetin and genistein shown strong combining ability with BSA/?-carrageenan particles, and the combine constant was1.67 × 105,2.60 × 105 and 0.85 × 105 L mol-1. Binding force between flavonoid compounds and BSA/ ?-carrageenan particles may be related to on the number of hydroxyl groups on the B ring. Studies have shown that the more hydroxyl groups on the B ring of flavonoids, the more strong binding between flavonoids and BSA. And,after BSA/?-carrageenan particles coated kaempferol, myricetin and genistein, these flavonoids was more stability than their free form. But the free DPPH radical scavenging ability is relatively weak, this may be kaempferol, myricetin and genistein combined with protein through internal hydrogen bonding and hydrophobic interaction force, these may prevents the flavoniods to provide H+ to eliminate free radicals. After simulated gastrointestinal digestion, kaempferol, myricetin and genistein in gastric(pH 2.0)(0 to 4 h) release a quantity of 9.76%, 11.35% and 8.92%,respectively. Continuing to digest 10 hours after intestinal(pH 7.6), the release rate was 19.54%, 46.74% and 26.54%, respectively. The result may be due to the pH change in digestive environment(pH 2.0 to pH 7.6), enhancing the ionic strength,which directly affects the structure of the BSA, changed combination properties between flavonoids and BSA.