RESUMO
A practical approach to control glycemia in diabetes is to use plant natural products that delay hydrolysis of complex sugars and promote the diminution of the release of glucosyl units into the blood plasma. Polyphenolics have been described as being effective in inhibiting amylases and α-glucosidases. Grape pomace is an important sub product of the wine industry, still rich in many compounds such as polyphenolics. In this context, the purpose of this study was to search for possible effects of a grape pomace extract on salivary and pancreatic α-amylases and α-glucosidase, as well as on intestinal glucose absorption. The Merlot grape pomace extract (MGPE) was prepared using a hydroalcoholic mixture (40% ethanol + 60% water). In vitro inhibition was quantified using potato starch (for amylases) and maltose (for α-glucosidase) as substrates. In vivo inhibition was evaluated by running starch and maltose tolerance tests in rats with or without administration of MGPE. Ranking of the extract compounds for its affinity to the α-amylases was accomplished by computer simulations using three different programs. Both α-amylases, pancreatic and salivary, were inhibited by the MGPE. No inhibition on α-glucosidase, however, was detected. The IC50 values were 90 ± 10 µg/mL and 143 ± 15 µg/mL for salivary and pancreatic amylases, respectively. Kinetically this inhibition showed a complex pattern, with multiple binding of the extract constituents to the enzymes. Furthermore, the in silico docking simulations indicated that several phenolic substances, e.g., peonidin-3-O-acetylglucoside, quercetin-3-O-glucuronide and isorhamnetin-3-O-glucoside, besides catechin, were the most likely polyphenols responsible for the α-amylase inhibition caused by MGPE. The hyperglycemic burst, an usual phenomenon that follows starch administration, was substantially inhibited by the MGPE. Our results suggest that the MGPE can be adequate for maintaining normal blood levels after food ingestion.
Assuntos
Diabetes Mellitus , Vitis , Animais , Simulação por Computador , Inibidores de Glicosídeo Hidrolases/farmacologia , Extratos Vegetais/farmacologia , Ratos , alfa-Amilases , alfa-GlucosidasesRESUMO
Amylases are interesting targets for antidiabetic drugs because their inhibition is able to lower glycaemia without the need of hormonal control, as promoted by insulin or glibenclamide. In this context, the comparison between the binding features of α-amylases with their substrate and known inhibitors may provide insights aiming at the discovery of new antidiabetic drugs. In this work, the structure of the porcine pancreatic α-amylase was modelled with the acarbose pentasaccharide inhibitor, and used in structure-based virtual screening simulations based on a library containing the structures of amylose (AMY), acarbose (ACA) and the more representative structures of condensed tannin (CTN) and hydrolysable tannin (HTN). After validation of the methodology by redocking (mean rmsd ~ 0.8 Å), the scores provided by programs AutoDock/Molegro were contradictory (- 1.5/- 23.3; - 3.5/- 24.6; - 4.3/- 14.6; -/- 19.5 for AMY, ACA, CTN and HTN respectively), indicating that a more sensitive methodology was necessary. The ΔGbinding was calculated by the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method, which indicated that the HTN, ACA and CTN had higher affinities for the enzyme regarding the AMY substrate, with values of - 350.0, - 346.2, - 320.5 and - 209.2 kJ mol-1, respectively. The predicted relative affinities of HTN and CTN are in agreement with those obtained experimentally. The results provided useful information for the characterization of tannin binding to α-amylase, which can be applied in future studies aiming at finding new hypoglycaemic molecules among natural products.