Pancreatic α amylase

The α-amylase inhibition assay of the both different seeds extracts (water, methanol, absolute acetone and aqueous acetone) with varying concentrations from 0.16 to 10 mg/ml. was determined to base on the method described by Deguchi et al. [25 (link)] with slight modifications. The reaction mixture contained 500 μl of 1% starch solution, 400 μl of 0.1 M sodium phosphate buffer (pH 7.0), 50 μl of each sample extract dissolved in DMSO and 50 μl of pancreatic α-amylase (Sigma, St. Louis, USA) solution (2 U/ml). After the incubated at 37 °C for 10 min of the reaction medium, 3 ml of 3,5-dinitrosalicylic acid (DNS) color reagent was added. Finally, the solution was diluted with 20 ml of distilled water after its incubation in a boiling water bath for 5 min, and the absorbance was measured at 540 nm. The absorbance of a control sample was prepared accordingly without plant extract and acted as a negative control. The Acarbose was used as positive control. The inhibition capacity of extracts and Acarbose were calculated as following: $$\text{Inhibition Percentage}\left(\%\right)=1-\mathrm{DO}\text{sample}/\mathrm{DO}\text{control}\times 100.$$
The result is expressed as IC50 (mg/ml) which revealed the inhibition concentration of each extract and acarbose of 50% of pancreatic α-amylase. All tests were carried out for three sample replications.

Folin–Ciocalteu, gallic acid, catechin, epicatechin, caffeine, ethyl gallate, rutin, ellagic acid, quercetin, quercitrin, apigenin, chlorogenic acid, DPPH, ABTS, acarbose, pancreatic α‐amylase, and yeast α‐glucosidase were purchased from Sigma‐Aldrich. Maltodextrin (MAX1000® series, Matsutani Chemical Industry Co., Ltd.) was acquired from DKSH Management Ltd., Vietnam. Xilong Scientific Co. Ltd. supplied ethanol, methanol, and acetic acid. All chemicals and reagents were of analytical grade.

Kudzu starch (KS) was purchased from Nanchang Hang Sheng Ecological Agriculture Co, Xinjian, Jiangxi Province, China. Pancreatic α‐amylase (Sigma‐Aldrich Inc., USA), pullulanase (Beijing Solarbio Science & Technology Co., Ltd., China), high‐temperature‐resistant α‐amylase (Shanghai Aladdin Bio‐Chem Technology Co., Ltd., China), glycosylase (Beijing Solarbio Science & Technology Co., Ltd., China), D(+)‐Glucose (Beijing Solarbio Science & Technology Co., Ltd., China). All the other chemicals were of analytical grade.

All solvents and products of analytical grade (99.8%), silica gel, acarbose, apigenin, (−)-catechin, gallic acid, intestinal α-glucosidase, and pancreatic α-amylase were purchased from Sigma Aldrich (Saint-Quentin Fallavier, France).

Rabbit liver glycogen and pancreatic α-amylase were from Sigma. Microcystin-LR was from Enzo Life Sciences. NADH was from Apollo Scientific. Antibodies against pS8 GYS1 and pS8/S11 GYS1 were kindly donated by D. Grahame Hardie (University of Dundee). pS641 GYS1 (#3891) and total GYS1 (#3893) antibodies were from Cell Signaling Technologies. Total GYS1 (sc-81173) and PP1c (sc-7182) antibodies were from Santa Cruz Biotechnology. pS641/645 GYS1 (S486A, 3rd bleed) and GN1 (S197C, 1st bleed) antibodies were generated by the Division of Signal Transduction Therapy (DSTT) at the University of Dundee as previously described [4] (link). Peroxidase conjugated secondary antibodies were from Jackson Immunoresearch. Glutathione Sepharose 4B, NHS-Sepharose FF, Superdex 200 3.2/300, Q-Sepharose HP and Enhanced Chemiluminescent (ECL) reagent were from GE Healthcare. Gel filtration standards were from Bio-Rad (151-1901). All other reagents, unless otherwise indicated, were from Sigma.

Folin‐Ciocalteu reagent, 1,1‐diphenyl‐2‐picraylhydrazyl (DPPH), and pancreatic α‐amylase were products from Sigma‐Aldrich (St. Louis, Mo, USA); 3,5‐dinitrosalicylic acid (DNS), trichloroacetic acid, thiobarbituric acid, sulfuric acid, sodium phosphate, and ammonium molybdate, sodium nitropruside, sodium dodeacyl sulfate were products of BDH Laboratories (Poole, UK), while methanol and ethyl acetate were from JHD (China). All reagents were supplied locally by Shepha Biotech Ventures (Lagos, Nigeria). All other chemicals used were of research and analytical grade.

Folin-Ciocalteu, gallic acid, ascorbic acids, quercetin, butyl hydroxyl toluene (BHT), 1,1-diphenyl-2-picrylhydrazyl (DPPH), bovine serum albumin (BSA), aminoguanidine, captopril, pancreatic α-amylase, yeast α-glucosidase enzyme, p-nitrophenyl-α-D-glucopyranoside, acarbose, furanacroloyl-Phe-Glu-Glu (FAPGG) and trichloroacetic acid (TCA) were purchased from Sigma-Aldrich (Sydney, Australia). All other chemicals used were of analytical or HPLC grade.