Inhibitory Effect Of Blueberry Leaf Polyphenols On α-amylase And α-glucosidase And Its Application In Low GI Rice Cake

With the expansion of blueberry industry in China,a large number of blueberry leaves are treated as waste.Blueberry leaves are rich in polyphenols,protein,VC,mineral elements and other active substances,which have the functions of lowering blood sugar,anti-oxidation,anti-tumor,lowering blood pressure and so on.The purpose of this study was to explore the inhibition effect of blueberry leaf polyphenol extract onα-amylase and α-glucosidase,explore the inhibition mechanism through kinetics,fluorescence,secondary structure,solubility and surface morphology,and develop a low GI rice cake technology,to provide a theoretical basis for the application of blueberry leaves.The main research results are as follows:(1)By screening the extraction solvent of blueberry leaves,the ethanol extract at 1mg/mL had higher α-amylase and α-glucosidase inhibitory activities,the inhibitory rates were 96.70% and 99.03%,respectively.And it is more safe than methanol.The results of composition analysis were mainly composed of polyphenols,in which the content of chlorogenic acid was 42.23%.The results showed that quercetin had the highest inhibitory activity on the two enzymes.The effect of purification on the inhibitory activity of the extract of polyphenols from blueberry leaves was investigated.The results showed that the content and inhibition rate of polyphenols were significantly increased after purification.After purification,the polyphenol content was0.3563±0.0146 mg/mg(g W),α-amylase inhibition rate was 90.49±0.058%,α-glucosidase inhibition rate was 96.76±0.125%,which were 40.61%,138.70% and12.42% higher than that before purification,respectively.The half-inhibitory concentration(IC50)of purified polyphenols from blueberry leaves was calculated.The results showed that the polyphenol extracts of blueberry leaves had higher inhibitory activity against α-glucosidase than α-amylase,with IC50 values of 7.362±0.03 μg/ mL and 12.520±0.65 μg/ mL,respectively.The results of temperature and p H stability of the extract from blueberry leaves were as follows: at 40 ℃,the highest α-amylase inhibition activity of the extract from blueberry leaves was 89.66%.The maximum inhibitory activity of α-glucosidase was 77.03% at 30 ℃.The activity decreased gradually with the increase of temperature,and almost completely lost when the temperature reached90 ℃.Under acidic p H conditions,the extracts of polyphenols from blueberry leaves maintained good inhibitory activity against α-amylase(98%～99%)at p H 2.0～8.0,α-glucosidase(98%～99%)at p H 3.0～6.0,and the highest inhibitory activity was90.01% at p H5.0.Under alkaline conditions,the inhibitory activities of the extract of polyphenols from blueberry leaves decreased,and the inhibitory activities of the extract on α-amylase and α-glucosidase decreased to 46.12% and 5.54% at p H 12.0,respectively.(2)The inhibition mechanism of the extract of polyphenols from blueberry leaves on α-amylase and α-glucosidase was described from five aspects,including kinetics,fluorescence intensity,secondary protein conformation,solubility and surface morphology.The results showed that the competitive inhibition of the extracts onα-amylase and α-glucosidase was higher than the non-competitive reversible inhibition.The competitive inhibition constants Kic of the extracts on α-amylase and α-glucosidase were 1.0415 mg/mL and 0.2229 mg/mL,respectively.The non-competitive inhibition constants Ki U were 0.1504 mg/mL and 0.0041 mg/mL.The effects of the concentration of blueberry leaf polyphenols extract on the fluorescence intensity of α-amylase andα-glucosidase were investigated.The results showed that the blueberry leaf polyphenols extract could quench the fluorescence intensity of enzyme,and the quenching type was static quenching.The binding constants with α-amylase and α-glucosidase were 6.54 and 1265.61,respectively,at 37 ℃.The results showed that the secondary structure ofα-amylase and α-glucosidase were changed by the extract of blueberry leaf polyphenols.The content of α-helix was decreased,and the content of β-folding and irregular coiling were increased.Through the solubility and surface morphology of exploration,blueberry extract leaf polyphenol with enzyme to form a larger aggregate,blueberry leaf extract polyphenols with alpha amylase and alpha glycosidase enzymes were strong hydrophobic interaction,changed the cohesive model,enzyme,enhanced the aggregation of water soluble protein concentration in the supernatant fluid increase.The binding forces of quercetin and chlorogenic acid with α-amylase and α-glucosidase were simulated by molecular docking.The results showed that quercetin and chlorogenic acid had hydrogen bond interaction and hydrophobic interaction with amino acid residues of enzyme protein,and the hydrophobic interaction was greater than the hydrogen bond interaction.(3)The polyphenol extract of blueberry leaves was applied in the development of rice cake with low glycemic index(GI)value,and the effects of different adding concentrations,cooking time,raw materials and raw material ratio on the GI value and quality of rice cake were investigated.The results showed that the optimal formula of low GI rice cake with polyphenols extract from blueberry leaves was as follows: the addition amount of polyphenols extract from blueberry leaves was 0.75%,the cooking time was 15 min,the raw material was tapioca-indica rice flour,and the addition amount of tapioca flour was 50% of rice flour,the GI value of rice cake could be reduced to 40.82,which was in the category of low GI food,and the quality of rice cake was good.