The Inhibition Mechanism Of Different Flavonoids On α-amylase And The Development Of Dandelion Jelly

Type 2 diabetes is a global chronic disease that accounts for more than 90%of people with diabetes^[1].In recent years,some studies have shown that hydrolysis of dietary carbohydrates in vivo is a major factor for the postprandial glycemic response.A low-carbohydrate diet is also widely recognized for the treatment of diabetes^[2,3].Pancreaticα-amylase is one of the major digestive enzymes catalyzing starch hydrolys in the small intestine,which can catalyzing starch hydrolys into maltotriose,maltose,α-dextrin and a small amount of glucose.Then these disaccharides and polysaccharides,which affects the postprandial blood glucose,were further decomposes into glucose byα-glucosidase.When the glucose were too much that can’t be metabolize by the body,they will accumulate in the blood,increase the postprandial blood glucose,eventually cause diabetes.Recent studies indicated that someα-amylase andα-glucosidase inhibitors used clinically would accompanied with serious gastrointestinal side effects such as diarrhea,gastrointestinal bloating,abdominal distention,and intestinal cystic emphysema^[4].Thus,find some new types ofα-amylase inhibitors with little or no side effects is important for the prevention and control of diabetes.Plant components of natural origin have good physiological functional activity and become research hotspots due to their minimal toxic side effects.Flavonoids are widely found in a variety of vegetables and fruits.They have been extensive attention for their broad biological activities,such as anti-inflammatory,anti-bacterial,antioxidant,anti-cancer,antiviral,and anticardiovascular and cerebrovascular diseases.In this study,the inhibition activity and structure relationship of nine structure similar flavonoids on porcine pancreaticα-amylase（PPA）were investigated by enzyme inhibition dynamics,multiple spectroscopy（fluorescence,circular dichroism（CD）spectroscopy,UV–visible absorption spectra）and molecular simulation.Two representative flavonoids,luteolin and quercetin,were selected to study the mechanism of their interaction with porcine pancreasα-amylase.And dandelion jelly was made with dandelion as the raw material.The main results are as follows:1.The inhibition studies showed that all the nine flavonoids can inhibit PPA activity,among which luteolin,Apigenin,Quercetin and Diosmetin displayed the best inhibitory effect on PPA,the values of IC50 of them were 0.35±0.023,0.56±0.014,0.78±0.019 mg/m L,respectively.And the inhibitory activity of Myricetin and Isorhamnetin were the weakest,the values of IC50 were 6.1±0.390 and 7.89±0.135mg/m L,respectively.The inhibitory activity ranged from strong to weak were Luteolin>Apigenin>Quercetin>Diosmetin>Chrysin>Kaempferide>Baicalein>Myricetin>Isorhamnetin。The results of inhibition kinetics indicated that all the nine flavonoids were of mixed inhibition type and preferred competitive inhibition.They have only one or one class of binding sites when interacting with PPA.2.The results of fluorescent spectroscopy showed that all the nine chosen flavonoids could effectively quench the endogenous fluorescence of PPA,and the quenching mechanism was static quenching.These nine flavonoids bind to the PPA with only one class or one binding site（n≈1）.3.The results of molecular docking suggested that hydrogen bonding may be an important force for the interaction of flavonoids with PPA,and the force ofπ-πstacking also can’t be ignored.All the nine flavonoids could enter into the active center of the PPA and bind to the amino acid residues surround with.In the meantime,they could also bind to the amino acid residues around the entrance of the PPA active center which impede the substrate enter into the active site,and inhibited the catalytic activity of the PPA ultimately.4.Structure-efficiency analysis illustrated that hydroxylation at C6,C3’and C4’favored the inhibitory activity,hydroxylation at C3 and C5’disfavored the inhibitory activity of flavonoids,and methoxylation of C4’diminished the inhibitory activity of flavonoids on PPA.5.The results of Fluorescence spectrum and fluorescence thermodynamic indicated that the binding process of representative flavanols Luteolin or Quercetin and PPA is an entropy-driven endothermic reaction,and the main interaction force between Luteolin and Quercetin was hydrophobic interactions,and the role of hydrogen bonding cannot be ignored.According to the results of calculation about the binding distance of Luteolin and Quercetin to PPA,it could be indicated that the high possibility of nonradiative energy transfer between PPA and luteolin or Quercetin.6.The results of synchronous fluorescence spectrum,3D fluorescence spectrum,circular dichroism and UV spectroscopy showed that the binding of luteolin and quercetin to PPA,mainly affected hydrophobic microenvironment of tryptophan residues.These conclusion indicated that the binding sites of flavonoids on PPA were closer to the tryptophan residues.The addition of both Luteolin and Quercetin led to the decrease of the contents ofα-helix and the random coil,and the increase of the contents ofβ-Sheet andβ-Turn.The secondary structure of PPA becomes loose and the hydrogen bonds break,eventually resulting in a reduced catalytic activity of PPA.7.The best formula of dandelion jelly:dandelion tea extract added 60%,citric acid added 0.04%,arhat fruit sweet side added 0.01%.