CAMILA GABRIEL KATO

Título da Tese: INIBIÇÃO DAS AMILASES SALIVAR E PANCREÁTICA POR PRODUTOS NATURAIS: CINÉTICA, MECANISMOS E EFEITOS FISIOLÓGICOS

Orientadora: Prof.ª Dr.ª Rosane Marina Peralta

 Data da Defesa: 22/02/2019

RESUMO GERAL

INTRODUCTION AND AIMS – Important enzymes of carbohydrate metabolism, the salivary and pancreatic amylases and the intestinal α-glucosidase, β-galactosidase e βfrutofuronosidase (invertase), they are regulated by the action of inhibitors with consequent improvement in disturbances such as diabetes and obesity. These enzymes are related to metabolic disorders such as diabetes and obesity. In consequence, a series of inhibitors of these enzymes, such as acarbose, miglitol and voglibose, are available in the market. Tannins are one of the most extensively studied molecules able to inhibit amylases. Hydrolysable tannins such as pentagaloyl glucose (PGG) can be found in several plants and other tannins have also been identified in a P. pluviosa stem bark extract (PPSB extract), a tree also known as "sibipiruna". It is generally believed that the discovery of new materials rich in tannins with enzyme inhibitory properties can contribute for the discovery of new drugs useful in the control and treatment of diabetes and obesity. One of the most extensively studied condensed tannin (proanthocyanidin) is that one extracted from the bark of the black wattle tree (Acacia mearnsii). It is rich in the catechin-like flavan-3-ols monomers robinetinidol and fisetinidol. One of the most simple and common hydrolysable tannin is the gallotannin with up to 12 esterified galloyl groups and a glucose core. This structure is particularly abundant in the gallotannin from the chinese natural gallnuts. The aim of article 1 was to examine in detail the kinetics of the inhibition of both salivary and the pancreatic αamylases using starch as the substrate. Furthermore, molecular dynamics simulations were done in order to get some insight into the molecular events determining binding of PGG to the enzymes. Finally, in vivo experiments were done in rats aiming at finding out if inhibition of the α-amylases by PGG is effective or not in diminishing the hyperglycemic excursion that usually follows starch ingestion. The aim of article 2 was to quantify the antioxidant activity of the hydroalcoholic extract of the stem bark of P. pluviosa and to investigate its possible effects on a set of carbohydrate hydrolysing enzymes, namely α-amylase, α-glucosidase, βgalactosidase, and β-fructofuranosidase. Efforts were also undertaken in order to assert the safety of the extract, especially after oral administration, in order to obtain a more or less complete picture of its potentialities as a future alternative therapeutic agent. METHODS – Porcine pancreatic α-amylase (Type VI-B), human salivary α-amylase (HAS) and acarbose, a well known commercial amylase inhibitor (empirical formula C25H43NO18, molecular weight 645), potato starch and diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich Co. Barks of P. pluviosa were collected at the campus of the University of Maringá (UEM), Maringá, Paraná, Brazil (lat: -23.4253005981445 long: - 51.9385986328125 err: ±19250 WGS84). A voucher specimen was deposited at the herbarium of the Universidade Estadual de Maringá under number HUEM-12492. The kinetic experiments with the HAS and pancreatic -amylase were carried out at 37 oC in 20 mM phosphate buffer pH 6.9 containing 6.7 mM NaCl. Potato starch (Sigma-Aldrich) was used as substrate. The substrate and one of the three inhibitors, acarbose, PGG e extract PPSB were mixed and the reaction was initiated by adding the enzyme. The reaction was allowed to proceed for 10 min. The produced reducing sugars were assayed by the dinitrosalicylic acid method, using maltose as standard. The other enzymes responsible for carbohydrate hydrolysis were withdrawn from the small intestine of rats and the inhibition experiment was performed. Statistical analysis of the data was done by means of the Statistica program (Statsoft, Inc., Tulsa, OK). Fitting of the rate equations to the experimental initial rates was done by means of an iterative non-linear least-squares procedure using the Scientist software from MicroMath Scientific Software (Salt Lake City, UT). The decision as to the most adequate model (equation) was based on the model selection criterion (MSC) and on the standard deviations of the optimized parameters. All docking calculations and procedures were performed using DOCK 6 and standard flexible ligand protocols and all molecular dynamics simulations were done using the Amber16 simulation software. Male healthy Wistar rats weighing 200–250 g were used in all in vivo experiments. Rats were divided into 5 groups (n = 4 rats per group). To group I (positive control) commercial corn starch (1 g per kg body weight) was administered intragastrically. Group II (negative control) received only 11 tap water. Group III received intragastrically commercial corn starch plus acarbose (50 mg/kg). In the PGG article, group IV received intragastrically commercial maize starch plus PGG (50 mg/kg) and group V received commercial maize starch and PGG (100 mg/kg). In the PPSB extract article, group IV received intragastrically commercial maize starch plus PPSB extract (50 mg/kg), group V received commercial maize starch and PPSB extract (250 mg/kg). Finally, group VI received commercial maize starch and PPSB extract (500 mg kg). Fasting blood glucose levels were determined before the administration of starch and amylase inhibitors (0 time). Later evaluations of blood glucose levels took place at 15, 30, 45 and 60 min. Blood glucose from cut tail tips was determined using aAccu-Chek® Active Glucose Meter. MAIN RESULTS, DISCUSSION AND CONCLUSION – In article 1, PGG inhibited both the pancreatic and the salivary α-amylases. With starch as the substrate, complexation with the free enzyme form either predominates (pancreatic enzyme) or is the only complexation that is effectively allowed (salivary enzyme; competitive inhibition). Inhibition was of the parabolic type, i.e., binding of at least two inhibitor molecules is allowed. For the pancreatic enzyme the complexes EI, IEI and ESI were formed, with dissociation constants of 78.51±29.02, 36.41±15.57 and 780.29±589.87 μM, respectively. With the human salivary α-amylase only complexes EI and IEI were significant, with dissociation constants of 15.07±2.68 and 122.30±53.29 μM, respectively. Comparison with literature data on the classical α-amylase inhibitor acarbose reveals that PGG is less effective than the former as the latter presents dissociation constants ranging from 2.7 to 10.2 µM. Computer simulations were done in order to obtain details about the behaviour of PGG at its binding site and to get insight into the molecular events determining the differences between PGG and acarbose. PGG inserts less deeply than acarbose into the binding site at the human salivary αamylase. With this, parts of the molecule are exposed to the solvent, what results in higher mobility. The calculated binding free energies were −56.92 and −40.18 kcal/mol for acarbose and PGG, respectively. PGG interacts strongly with His 305, whereas acarbose binds more strongly to the nearby Asp 300. Contrary to acarbose, PGG shows a relatively weak interaction with Trp 59. Finally, PGG diminished the hyperglycemic excursion normally caused by starch administration when it was given to rats simultaneously to the polysaccharide. This suggests that PGG or foods and infusions rich in PGG could be helpful as adjuvants for maintaining normal glycemia. In article 2, both salivary and pancreatic amylases were inhibited by the extract. However, the salivary amylase was more intensely inhibited if one considers the IC50 values (concentrations of extract causing 50% inhibition) which were 250±15 µg/mL and 750±15 µg/mL for the salivary and pancreatic amylase. The inhibitory effects of the PPSB extract on the activities of the intestinal disaccharidase enzymes was also investigated. The extract effectively inhibited the invertase and βgalactosidase. It was a more efficient inhibitor of β-galactosidase than of invertase, with IC50 values of 25±5 µg/mL and 75±8 µg/mL, respectively. No inhibition was found in the case of the α-glucosidase (not shown) even at the high concentration of 250 µg/mL (p≥ 0.05).The results suggest that the stem bark of P. pluviosa presents elevated antioxidant activity as well as significant inhibition on both salivary and pancreatic amylases and on the intestinal disaccharidases β-galactosidase and invertase. The P. pluviosa extract may be useful for the development of new natural antidiabetic agents. Keywords: α-amylase inhibitors; diabetes; tannins; PGG, sibipiruna.

Artigos Publicados Vinculados a Tese:

https://www.sciencedirect.com/science/article/pii/S1756464618301117?via%3Dihub