Proceedings of International Congress on Bioactive Compounds and International Workshop on Bioactive Compounds
Proceedings of 1st International Congress on Bioactive Compounds and 2nd International Workshop on Bioactive Compounds - Vol. 1 - 2018

Introduction: Non-extractable polyphenols (NEPPs) are bound to the food matrix or in polymeric form, hence making them unable to be extracted in the conventional form with which extractable polyphenols (EPP) are obtained. Recently, a growing number of studies has been showing the NEPPs potential for beneficial effects, especially related to gastrointestinal health. α-glucosidase is an enzyme excreted in the small intestine that catalyzes the break of carbohydrates to generate glucose. α-glucosidase inhibitors have a major role in the control of diseases such as type-2 diabetes since they can lower the blood glucose levels by slowing down carbohydrate digestion. Objective: The present study has the objective of evaluating and comparing the inhibition of α-glucosidase by EPP and NEPP fractions of guarana (Paullinia cupana) powder. Methods: The EPP fraction was obtained by extraction with water:acetone:methanol (20:56:24 v:v:v), and the NEPP fraction was obtained by alkaline hydrolysis with sodium hydroxide, followed by extraction with acetone:water:acetic acid (70:29.5:0.5 v:v:v). The NEPP fraction was further purified using solid-phase extraction (SEP-PAK cartridge). Both fractions were evaporated to eliminate organic solvents and resuspended in water. The Folin-Ciocalteou method was used to determine total reducing capacity (TRC), the enzymatic assay was carried out using steady state assumptions, and the Lineweaver-Burk plot was used to determine the mode of inhibition and appropriate constants. The phenolic profile was obtained by HPLC-ECD and LC-MS/TOF for EPP, and MALDI-TOF/TOF for NEPP. Results and discussion: The TRC was 473.01 µg GAE/mL for EPP and 19.20 µg GAE/mL for NEPP. These values were used as an estimative of phenolic content for the enzymatic assay. IC50 values were 9.504 µg GAE/mL for EPP and 1.624 µg GAE/mL for NEPP, a difference of approximately 6-fold. Both fractions exhibited a mixed mode of inhibition, with values of apparent Km of 0.489 and 0.292 mM and values of apparent Vmax of 0.003 and 0,007 OD/min for EPP and NEPP, respectively. When compared with the values obtained for the control, the value of Km was increased, and the value of Vmax was decreased in both cases, which is consistent with the effect of mixed-type inhibitors. The dissociation constants, Ki and K’i, were 0.403 and 1.735 µg/mL for EPP and 0.287 and 0.847 µg/mL for NEPP. Even for the lower relative amount of phenolics in the non-extractable fraction, the lower dissociation constants suggest that the equilibrium is dislocated in favor of the inhibitor-enzyme (or inhibitor-enzyme-substrate) complex formation. The phenolic profile of the EPP fraction consists of catechin, epicatechin, proanthocyanidin B1 and B2, epigallocatechin gallate and type-A trimer. The phenolic profile of the NEPP fraction shows a complex array of compounds, including esterified and glycosylated monomers, flavylium and flavan units with different degrees of substitution, and polymeric structures up to pentamers. The variability, especially of monomers, may be partially explained by the degradation effects of the extraction process itself and the laser treatment of the MALDI technique. Conclusion: The NEPP fraction of guarana powder has a promising potential for studies with α-glucosidase inhibitors and, subsequently, diabetes control.