Avaliação de derivados de curcumina visando aplicações biotecnológicas: inibição de enzimas digestivas de carboidratos, capacidade antioxidante, estudos teóricos e biofísicos

Abstract

The transient inhibition of carbohydrate digestive enzymes has been widely used as an adjuvant therapeutic strategy for the treatment of diabetes mellitus (type II), with acarbose (AC), miglitol and voglibol being the drugs sold worldwide. However, the low anti-glycating potential and the presence of several side effects, such as gastrointestinal complications associated with these medications, have motivated the search for new carbohydrate digestive enzyme inhibitors. The inhibitory potential of curcumin derivatives (natural product) on the action of enzymes such as α-amylase and α-glucosidase has been reported in the specialized literature, therefore, the evaluation of similar compounds becomes a promising strategy for this purpose. Therefore, eleven compounds derived from curcumin were synthesized and characterized in relation to their action as inhibitors of α-amylase and α-glucosidase. Thus, a screening of curcumin derivatives was carried out through inhibitory activity against enzymes, classical kinetic and inhibition studies (in vitro) and evaluation of antioxidant capacity (DPPH• and ABTS•+), being the most promising compound (6) selected for biophysical studies exploring theoretical (molecular docking) and experimental tests. Initially, a screening assay using 120 µM for all compounds was carried out in order to verify their potential to inhibit α-amylase (1 U mL-1 ). In this assay, only four derivatives showed inhibition greater than 50% and of these, only 6 had an inhibitory potential similar to AC, which presented IC50 = 18.50 ± 0.31 µM. In a similar assay for α-glucosidase (0.02 U mL-1 ) applying a single dose of 250 µM of the compounds, it was possible to verify that only six compounds expressed inhibitory activity above 50%, with derivative 7 being more active with IC50 = 17 .67 ± 0.58 µM, while AC presented IC50 of 19± 03 µM (α-amylase) and 205 ± 0.9 µM (α-glucosidase). Derivatives 6 and 7 were classified as mixed inhibitors based on classical kinetics assays for both enzymes evaluated. In the antioxidant assays, curcumin derivative 6 was the most active, presenting the lowest IC50 values for both radicals evaluated (DPPH• and ABTS•+), compared to the other derivatives. Theoretical studies using molecular docking were in agreement with experimental enzymatic tests, with 6 and 7 being the compounds with the best interaction with the enzymes α-amylase and α-glucosidase, respectively. In this sense, 6 was selected for biophysical studies using α-amylase as model enzymes. Therefore, with the increase in temperature (22 to 38ºC), inversely proportional values of the Stern-Volmer constant (KSV) were observed, characterizing a process profile by static quenching, which was confirmed based on the values of the extinction rate constant. biomolecular, since Kq > 2.0×1010 M-1 s-1 . The main intermolecular interactions present between α-amylase and 6 were through hydrogen bonding and Van der Waals forces. Furthermore, it was possible to verify that the interaction process between the enzyme and the ligand was thermodynamically spontaneous (ΔG < 0). Finally, compound 6 demonstrated to be a molecule with potential inhibitory action on carbohydrate digestive enzymes.