title: Modeling of cooked starch digestion process using recombinant human pancreatic α-amylase and maltase-glucoamylase for in vitro evaluation of α-glucosidase inhibitors.
abstract: In human, digestion of cooked starch mainly involves breaking down of α-amylase to α-limit dextrins and small linear malto-oligosaccharides, which are in turn hydrolyzed to glucose by the gut mucosal maltase-glucoamylase (MGAM). Human pancreatic α-amylase (HPA), amino- and carboxyl-terminal portions of MGAM (ntMGAM and ctMGAM) catalyze the hydrolysis of α-D-(1,4) glycosidic linkages in starch, playing a crucial role in the production of glucose in the human lumen. Accordingly, these enzymes are effective drug targets for the treatments of type 2 diabetes and obesity. In this study, a Plackett-Burman based statistical screening procedure was adopted to determine the most critical factors affecting cooked starch digestion by the combination of HPA, ctMGAM and ntMGAM. Six factors were tested and experimental results showed that pH and temperature were the major influencing factors, with optimal pH and temperature at 6.0 and 50 °C, respectively. Surprisingly, ntMGAM had no significant contribution to the glucose production from starch digestion compared to the HPA and ctMGAM. The optimal proportion of HPA and ctMGAM in a starch digestion system was further determined by response surface methodology. Results showed a maximum starch digestion (88.05%) within 0.5 h when used HPA:ctMGAM=1:9 (U). The inhibitory effects of various inhibitors on the cooked starch digestion by HPA1/ctMGAM9 were evaluated by determining their half maximal inhibitory concentration (IC50) values. Acarviostatin II03 showed the highest inhibitory activity, with 67 times higher potency than acarbose. Moreover, acarviostatin II03 could significantly depress postprandial blood glucose levels in mice, better than that by acarbose. These findings suggest that our in vitro enzymatic system can simulate in vivo starch digestion process, and thus can be used to screen and evaluate α-glucosidase inhibitors.
authors.affiliation: College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, People's Republic of China.
authors.forename: Xiaofang
authors.initials: X
authors.lastname: Cao
authors.affiliation: College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, People's Republic of China.
authors.forename: Chen
authors.initials: C
authors.lastname: Zhang
authors.affiliation: College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, People's Republic of China.
authors.forename: Yangyang
authors.initials: Y
authors.lastname: Dong
authors.affiliation: Basic Medical College, Tianjin Medical University, Tianjin 300070, People's Republic of China.
authors.forename: Peng
authors.initials: P
authors.lastname: Geng
authors.affiliation: College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, People's Republic of China. Electronic address: baifang1122@nankai.edu.cn.
authors.forename: Fang
authors.initials: F
authors.lastname: Bai
authors.affiliation: College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, People's Republic of China.
authors.forename: Gang
authors.initials: G
authors.lastname: Bai
mesh_terms: D020909:Acarbose / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D065089:Glycoside Hydrolase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008297:Male; D051379:Mice; D015394:Molecular Structure; D055292:Pancreatic alpha-Amylases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D013213:Starch / Q000378:metabolism*; D013696:Temperature; D000520:alpha-Glucosidases / Q000235:genetics* / Q000378:metabolism
chemical_list: D065089:Glycoside Hydrolase Inhibitors; D011994:Recombinant Proteins; D013213:Starch; D055292:Pancreatic alpha-Amylases; D000520:alpha-Glucosidases; D020909:Acarbose
keywords: Human pancreatic α-amylase; Maltase-glucoamylase; Response surface methodology; Starch digestion; α-Glucosidase inhibitors