Glucose oxidase as a model enzyme for antidiabetic activity evaluation of medicinal plants: <i>In vitro</i> and <i>in silico</i> evidence

Abstract

Diabetes mellitus is a major public health problem in the world. In Africa, more than 80% of patients use plants for their treatment. However, the methods of validation of endogenous knowledge usually used are costly. The alternative method developed in this study aims at creating hyperglycemia <i>in vitro</i> and exploiting the metabolic pathway involving glucose oxidase for UV-visible spectrophotometric screening of medicinal plants’ antidiabetic activity. The evolution of glucose oxidation as a function of drug concentration is followed by UV-visible spectrophotometry. The formation of the stable complex between the enzyme and the inhibitor is studied using molecular docking. Drugs used (Gliben) and plant extracts exhibited an <i>in vitro</i> hypoglycemic effect by reducing exponentially, <i>in vitro</i>, the level of free glucose. The results also showed that <i>L. multiflora</i> is more active than <i>V. amygdalina</i> (IC<sub>50</sub>: 1.36 ± 0.09 mg/mL Vs IC<sub>50</sub>: 3.00 ± 0.54 mg/mL). Gliben (0.5 mg/mL) and <i>L. multiflora</i> (2 mg/mL) reduced both the rate of oxidation of glucose by glucose oxidase (catalytic power V<sub>max</sub>: 0.84 ± 0.11 mg*mL<sup>-1</sup>*min<sup>-1</sup> for Gliben and 1.72 ± 0.13 mg*mL<sup>-1</sup>*min<sup>-1</sup> for <sup>L. multiflora</sup>); and the affinity of this enzyme for its substrate-glucose (K<sub>M</sub>: 15.11 ± 2.72 mg*mL<sup>-1</sup> for Gliben and 9.17 ± 1.56 mg*mL<sup>-1</sup> for <i>L. multiflora</i>) when these results are compared to enzyme catalysis in the absence of inhibitor (V<sub>max</sub>: 2.86 ± 0.44 mg*mL<sup>-1</sup>*min-1; K<sub>M</sub>: 8.07 ± 1.96 mg*mL<sup>-1</sup>). The binding of GOX (1GAL) to selected phytocompounds derived from <i>L. multiflora</i> was confirmed by molecular docking. The most stable complexes were obtained for four compounds; <b>8</b> (-10.1±0.0 Kcal/mol), <b>6</b> (-9.5±0.1 Kcal/mol), <b>3</b> (-8.3±0.0 Kcal/mol) and <b>9</b> (-8.2±0.1 Kcal/mol). Among these, compounds <b>8</b> and <b>6</b> formed complexes with the enzyme stabilized by hydrogen bonds, the compound <b>8</b> forms 5 hydrogen bonds (<b>ASN514</b>, <b>ASP424</b>, <b>ARG95</b>, <b>TYP68</b>, <b>LEU65</b>) while compound <b>6</b> forms 2 hydrogen bonds (<b>ASN514</b> and <b>SER422</b>). However, no H-bonding interaction occurs in the complex that involves ligands <b>9</b> and <b>3</b> despite their high binding energy (-8.2±0.1 Kcal/mol and -8.3±0.0 Kcal/mol respectively). Glucose oxidase can serve as a marker enzyme for<i> in vitro</i> antidiabetic activity evaluation of medicinal plants.