br Materials and methods br Results br Discussion Here we

Materials and methods
Results
Discussion Here, we investigated the inhibitory effects and associated mechanisms of phloroglucinol on α-glucosidase. Our results showed that phloroglucinol reversibly inhibited α-glucosidase in a dose-dependent manner (Fig. 1), with the double-reciprocal plot showing that increased phloroglucinol concentrations changed the Vmax, but not the Km value associated with the reaction and suggesting that phloroglucinol inhibition is non-competitive (Fig. 3). Reversible inhibition was confirmed by plots involving [E] (Fig. 2) that passed through the origin. Additionally, the inactivation reaction occurred quickly, with time-course kinetics observed only under low concentrations of phloroglucinol (2.5–10mM) (Fig. 4). Compared to the previously reported bicyclo[2.2.0]hexane-2,3,5-triol that has a similar structure of phloroglucinol [41], the IC50 value of phloroglucinol (7.80±0.56mM) was higher than bicyclo[2.2.0]hexane-2,3,5-triol (0.558±0.079mM). Moreover, the Ki value for phloroglucinol was 2.07±0.16mM, which is larger than that of cobalt (0.78±0.08mM) [30] or hydroxysafflor yellow A (1.04±0.23mM) [29], suggesting a relatively low-affinity binding event on the part of phloroglucinol as compared with these molecules. To identify structural and conformational changes in the enzyme during the inhibition, we evaluated protein aggregation and fluorescence emission. Interestingly, although intrinsic fluorescence intensity was reduced along with elevated phloroglucinol concentration, the maximum peak wavelength did not change greatly (~330nm) throughout the assay (Fig. 6). Additionally, the ANS-binding fluorescence remained unchanged, indicating that the MG 149 australia resides inside of the tertiary structure were not exposed to solvent (Fig. 7) and suggesting a relatively stable structure maintained during inhibitor binding, which agreed with results showing a lack of phloroglucinol-induced protein aggregation (Fig. 6).
Acknowledgments This work was supported by Zhejiang Province Public Technology Application Research (No. 2017C32053) and Zhejiang Provincial Top Key Discipline (ZS2017010). Dr. Yong-Doo Park was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP; Ministry of Science, ICT & Future Planning) (No. 2017R1A6A3A11033277).
Introduction Diabetic mellitus (DM) is a metabolic disease characterized by hyperglycemia, caused by abnormal metabolism of fats, proteins and carbohydrates in terms of insulin resistance or insulin deficiency (Teng & Chen, 2017). α-Glucosidase (EC 3.2.1.20), located in the brush-border of small intestine, is an exo-type carbohydrase that acts upon α-(1 → 4) glycosidic bonds of key disaccharides and oligosaccharides in the final step of digestive process (Zeng, Ding, Hu, Zhang, & Gong, 2019). Retarding its activity is one of the effective treatment of DM, and acarbose, voglibose, miglitol, etc. are common α-glucosidase inhibitors (Choi et al., 2010). However, these drugs were reported be accompanied with some side-effects. Therefore, new potential natural inhibitors remain developed in view of safety. In general, considering that α-glucosidase participates the metabolic process of polysaccharides (Gong et al., 2017), understanding of the effect and mechanism of biomolecule inhibitors on regulating α-glucosidase is of great significance. Proanthcynidins (PAs) are a class of natural phenolic compounds characterized by oligomers or polymers of flavan-3-ols (Haihua Yang et al., 2011). According to the different constitutive units, PAs could be classified into three categories: propelargonidins, procyanidins and prodelphinidins, corresponding to units of (epi)afzelechin, (epi)catechin, and (epi)gallocatechin or their esterification products (Hellström et al., 2007, Monagas et al., 2010). PAs were mainly distributed in some leaves, seeds, fruits of plants and its average intake in daily diet (U.S. adults) was about 95 mg/day (Li et al., 2018). Chinese bayberry (Myrica rubra Sieb. et Zucc.) is a fruit originated in Eastern China with more than 2000-year cultivation history, and its leaves are a new source of abundant PAs. PAs in Chinese bayberry leaves (BLPs) were discovered and developed for structural characteristic and activity analysis. Our previous research revealed that BLPs belong to prodelphinidins family and its terminal units and most extension units are epigallocatechin-3-O-gallate (EGCG) (Fu et al., 2014). And some related reports showed that BLPs had positive effect on antioxidant, antiproliferation, anti-obesity, anti-cancer, neuroprotective and melatonin functions (Zhang et al., 2016, Zhang et al., 2018, Zhou et al., 2017). However, the study on hypoglycemic effect of BLPs was rarely till now.