Inhibition of human monoamine oxidase A and B by flavonoids isolated from two Algerian medicinal plants

Abstract

Background: Monoamine oxidases (MAOs) are outer mitochondrial membrane flavoenzymes. They catalyze the oxidative deamination of a variety of neurotransmitters. MAO-A and MAO-B may be considered as targets for inhibitors to treat neurodegenerative diseases and depression and for managing symptoms associated with Parkinson's and Alzheimer's diseases.

Purpose: The objective was to evaluate the inhibitory effect of Hypericum afrum and Cytisus villosus against MAO-A and B and to isolate the compounds responsible for the MAO-inhibitory activity.

Methods: The inhibitory effect of extracts and purified constituents of H. afrum and C. villosus were investigated in vitro using recombinant human MAO-A and B, and through bioassay-guided fractionation of ethyl acetate fractions of areal parts of the two plants collected in northeastern Algeria. In addition, computational protein-ligand docking and molecular dynamics simulations were carried out to explain the MAO binding at the molecular level.

Results: The ethyl acetate (EtOAc) fractions of H. afrum and C. villosus showed the highest MAO inhibition activity against MAO A and B with IC₅₀ values of 3.37 µg/ml and 13.50 µg/ml as well as 5.62 and 1.87 µg/ml, respectively. Bioassay-guided fractionation of the EtOAc fractions resulted in the purification and identification of the known flavonoids quercetin, myricetin, genistein and chrysin as the principal MAO-inhibitory constituents. Their structures were established by extensive 1 and 2D NMR studies and mass spectrometry. Quercetin, myricetin and chrysin showed potent inhibitory activity towards MAO-A with IC₅₀ values of 1.52, 9.93 and 0.25 µM, respectively, while genistein more efficiently inhibited MAO-B (IC₅₀ value: 0.65 µM). The kinetics of the inhibition and the study of dialysis dissociation of the complex of quercetin and myricetin and the isoenzyme MAO-A showed competitive and mixed inhibition, respectively. Both compounds showed reversible binding. Molecular docking experiments and molecular dynamics simulations allowed to estimate the binding poses and to identify the most important residues involved in the selective recognition of molecules in the MAOs enzymatic clefts.

Conclusion: Quercetin and myricetin isolated from H. afrum together with genistein and chrysin isolated from C. villosus have been identified as potent MAO-A and -B inhibitors. H. afrum and C. villosus have properties indicative of potential neuroprotective ability and may be new candidates for selective MAO-A and B inhibitors.

Keywords: Antidepressant; Bioassay-guided fractionation; HR-ESI-MS; Molecular docking; Monoamine oxidase; NMR.

Fig. 1

Structures of the identified compounds isolated from H. afrum and C. villosus

Fig. 2

Inhibition dose response curves (IC₅₀ values) of recombinant human monoamine amine oxidase-A by quercetin, myricetin and phenelzine (% activity vs. concentration).

Fig. 3

Kinetic characteristics of inhibition of recombinant human MAO-A with [A] phenelzine [B] quercetin; [C] myricetin; (V = nmoles/min/mg protein and S = substrate kynuramine concentration (μM).

Fig. 4

The inhibition of recombinant human monoamine amine oxidase-A with Myricetin

Fig. 5

The inhibition of recombinant human monoamine amine oxidase-A with Quercetin

Fig. 6

Analysis of nature of binding of quercetin and myricetin with recombinant human MAO-A by recovery of catalytic activity of the enzyme after dialysis dissociation. Each bar shows mean ± S.D. of triplicate values.

Fig. 7

The binding modes of quercetin (Panel A), myricetin (Panel B), genistein (Panel C) and chrysin (Panel D) are shown in MAO-A, and the binding modes of genistein (Panel E) and chrysin (Panel F) are displayed in MAO-B. Ligands are shown as pink balls and sticks. The interacting amino acids are shown as grey sticks. Protein is shown as cartoon with yellow helices, pink strands and green loops. All possible hydrogen bonds in the range of 3.5 Å are displayed as yellow dots.

Fig. 8

Protein-ligand contacts of quercetin (Panel A). Four types of protein-ligand interactions were monitored throughout the simulation: hydrogen bond, hydrophobic, ionic and water bridges. 2D interaction diagram (Panel B) of the detailed ligand atom interactions of quercetin with the surrounding amino acid residues of MAO-A.

Fig. 9

Protein-ligand contacts of myricetin (Panel A). Hydrogen bonds, hydrophobic, ionic and water bridges were monitored throughout the simulation. 2D interaction diagram of myricetin (Panel B) with the amino acid residues of binding site of MAO-A.