Inhibitory effects of the polyphenol extracts from Chinese bayberry onα-glucosidase in vitro

Abstract: 【Objective】There have been a lot of studies about the inhibitory activities on α-glucosidase ofpolyphenolic compounds such as polyphenol extract from apple,flavonoid-rich extract from leaves of Crataegus cuneata,quercetin and rutin and structure-activity relationships have been analyzed. It has beenshown that these polyphenols have a strong inhibitory effect on α-glucosidase with different types of inhib-itory actions. Chinese bayberry is a unique fruit crop in our country. Previous studies have found that Chinese bayberry has rich polyphenolic compounds,which have a variety of biological activities,such as an-tioxidant,free radical scavenging,anti-inflammatory,visual function improving,blood lipids improv-ing,antibacterial and antitumor etc. However,there has been no report about the inhibitory activity ofthe polyphenol extracts from Chinese bayberry on α-glucosidase and related mechanisms. Therefore,inthis study,we studied the inhibitory effect of the polyphenol extracts from Chinese bayberry on α-glucosi-dase activity,characterizing their kinetic and thermodynamic features,including IC50 value and the typesof inhibition and fluorescence quenching. 【Methods】In this experiment,super versatile microplate,spectrophotometer and fluorescence spectrophotometer were used. The method of folin phenol was used todetermine the content of total phenol in Chinese bayberry; Na NO2-Al(NO3)3-Na OH coloration system wasapplied to determine the content of total flavonoids; and p H differential method was used to measure thetotal anthocyanin content. A commercially available hypoglycemic drug acarbose was used as the positivecontrol,and 4-Nitrophenyl-α-D-glucopyranoside(PNPG) as the substrate of α-glucosidase for testingthe inhibitory effect of the polyphenol extract from Chinese bayberry on the enzyme activity. With a con-stant α-glucosidase concentration,different concentrations(0.5,1.0,1.5,2.0,2.5 mmol·L^-1) of 4-Ni-trophenyl-α-D-glucopyranoside(PNPG) as substrate,added or not added with Chinese bayberry poly-phenol extract,were used to determine α-glucosidase catalyzed reaction rate(V),which was regressedinto Lineweaver Burk double reciprocal plot against substrate concentration to determine the type of inhi-bition. A fluorescence spectrometry was used for thermodynamic studies using 287 nm as an excitationwavelength and 335.6 nm for emission wavelength. The fluorescence emission spectra 15 min after mixingα-glycosidase(0.5 U·mL^-1) with a series of concentrations of Chinese bayberry polyphenol extract(0-40mg·L^-1) were recorded at an optical path length of 10 mm and a range of wavelength from 250 to 500 nmunder three different temperatures(25,30 and 37 ℃).【Results】1. Phenolic compounds in the polyphenolextract from Chinese bayberry were rich,and the total phenol content,total content of flavonoids and con-tent of anthocyanins were 65.52%,36.71% and 13.50%,respectively. The results indicated that the flavo-noids and anthocyanins were the main phenolic compounds in the Chinese bayberry. 2. The polyphenol ex-tract of Chinese bayberry had a strong inhibitory effect on α-glucosidase. The IC50 value was 1.562 mg·L^-1,which was lower than that of acarbose(164.186 mg·L-1),and in a certain range,the inhibition rate in-creased linearly with the concentration of the polyphenol extract from Chinese bayberry. 3. Enzymatic ki-netic analysis showed that the polyphenol extract from Chinese bayberry significantly inhibited α-glucosi-dase activity,and with the increase in the concentration of polyphenol extract,maximum reaction veloci-ty(Vmax) decreased and the Michaelis constant(km) value increased. The result indicated that the inhibi-tion of the polyphenol extract on α-glucosidase was a mixed type of inhibition. 4. Fluorescence spectrumanalysis showed that the quenching constant Ksv decreased with the increase of temperature,indicatingthat the fluorescence quenching activity of the polyphenol extract was mainly due to the static quenchingcaused by the formation of the ground state complex.【Conclusion】The experimental results showed thatthe polyphenol extract from Chinese bayberry had a strong inhibitory effect on α-glucosidase. Chinesebayberry has rich polyphenolic compounds,and the mixed inhibitory effect of Chinese bayberry polyphe-nol extract on α-glucosidase may be due to the different binding sites and binding ways. There are manyactive hydroxyl groups on the polyphenol molecules in the extract of Chinese bayberry. This may be one ofthe important reasons for the strong quenching effect of the polyphenol extract on α-glucosidase-generat-ed fluorescence. However,the specific mechanism of action needs further study. Further research shouldbe done on the structure-activity relationship of the polyphenols in Chinese bayberry in term of α-gluco-sidase inhibition. In the future,we can separate different polyphenols from Chinese bayberry and sepa-rately study their inhibitory activity on α-glucosidase.【Other】 In later studies,we are to isolate flavo-noid monomers from Chinese bayberry and study their inhibitory effects on α-glucosidase activity,andthen do further study on the synergistic effect of different flavonoid monomers on α-glucosidase activity.Based on this study,effect of structure of flavonoid monomers on the inhibitory effect on α-glucosidasewill be unraveled.