Rutin of Moringa oleifera as a potential inhibitor to Agaricus bisporus tyrosinase as revealed from the molecular dynamics of inhibition

Abstract

Tyrosinase is a binuclear copper-containing enzyme that catalyzes the conversation of monophenols to diphenols via o-hydroxylation and then the oxidation of o-diphenols to o-quinones which is profoundly linked to eukaryotic melanin synthesis and fruits browning. The hyperpigmentation due to unusual tyrosinase activity has gained growing health concern. Plants and their metabolites are considered promising and effective sources for potent antityrosinase enzymes. Hence, searching for potent, specific tyrosinase inhibitor from different plant extracts is an alternative approach in regulating overproduction of tyrosinase. Among the tested extracts, the hydro-alcoholic extract of Moringa oleifera L. leaves displayed the potent anti-tyrosinase activity (IC₅₀ = 98.93 µg/ml) in a dose-dependent manner using _L-DOPA as substrate; however, the kojic acid showed IC₅₀ of 88.92 µg/ml. The tyrosinase-diphenolase (TYR-Di) kinetic analysis revealed mixed inhibition type for the Ocimum basilicum L. and Artemisia annua L. extracts, while the Coriandrum sativum L. extract displayed a non-competitive type of inhibition. Interestingly, the extract of Moringa oleifera L. leaves exhibited a competitive inhibition, low inhibition constant of free enzyme ( ${\text{K}}_{\text{ii}}^{\text{app}}$ ) value and no Pan-Assay Interfering Substances, hinting the presence of strong potent inhibitors. The major putative antityrosinase compound in the extract was resolved, and chemically identified as rutin based on various spectroscopic analyses using UV-Vis, FTIR, mass spectrometry, and ¹H NMR. The in silico computational molecular docking has been performed using rutin and A. bisporus tyrosinase (PDB code: 2Y9X). The binding energy of the predicted interaction between tropolone native ligand, kojic acid, and rutin against 2Y9X was respectively - 5.28, - 4.69, and - 7.75 kcal/mol. The docking simulation results revealed the reliable binding of rutin to the amino acid residues (ASN²⁶⁰, HIS²⁵⁹, SER²⁸²) in the tyrosinase catalytic site. Based on the developed results, rutin extracted from M. oleifera L. leaves has the capability to be powerful anti-pigment agent with a potential application in cosmeceutical area. In vivo studies are required to unravel the safety and efficiency of rutin as antityrosinase compound.

Keywords: Agaricus bisporus; Anti-pigment; Cosmetics; Flavonoid; Inhibition; Kinetics; Kojic acid; Rutin; Tyrosinase.

Figure 1

The structures of the examined tyrosinase inhibitory ligands: (A) tropolone (inherent ligand of target protein tyrosinase (PDB, 2Y9X), (B) rutin, and (C) kojic acid.

Figure 2

Tyrosinase inhibitory activity using the extract of different plants when _L-DOPA used as substrate. (A) Representation of the total flavonoids in the extract of various plants and the matching IC₅₀ of the inhibition for tyrosinase-diphenolase activity with respect to the concentration of inhibitor. (B) Representation of the correlation among the total flavonoids in the extract of the four tested plants and the activity of tyrosinase-diphenolase inhibition.

Figure 3

Tyrosinase inhibitory activity using the extract of Ocimum basilicum L. (A) and Artemisia annua L (B), Moringa oleifera L. leaves (C) and Coriandrum sativum L. (D) when l-DOPA used as substrate. Representation of the Lineweaver–Burk plots for tyrosinase-diphenolase activity inhibition in the absence of inhibitor () and in the presence of different concentration of inhibitor, namely 50 µg/ml (), 100 µg/ml (), and 150 µg/ml ().

Figure 4

Plot of the slope from the Lineweaver–Burk plots versus different inhibitor concentrations which used to detect inhibition constants (K_i) of tyrosinase by the extract of Ocimum basilicum L. (A), Artemisia annua L. (B), Moringa oleifera L. (C) and Coriandrum sativum L. (D). A linear regression fit was employed to determine the linear equation and regression coefficient.

Figure 5

Plot of the intercept from the Lineweaver–Burk plots versus different inhibitor concentrations which used to detect inhibition constants (K_ii) of tyrosinase by the extract of Ocimum basilicum L. (A), Artemisia annua L. (B), and Coriandrum sativum L. (C). A linear regression fit was employed to determine the linear equation and regression coefficient.

Figure 6

(A) The tyrosinase inhibitory activity represented in IC₅₀ (µg/ml) and the antioxidant activity displayed in IC₅₀ (µg/ml) of Moringa oleifera L. fractions. The IC₅₀ designates the concentration of fraction which inhibits 50% (B) The absorbance spectrum at 510 nm of flavonoids fractions derived from leaves of Moringa oleifera L. for ethylacetate fraction.

Figure 7

Representation of the HPLC chromatogram peaks of the crude Moringa oleifera L. leaves methanolic extract. The HPLC analysis was performed by injecting 20 µl of crude methanolic extract of leaves (10 mg/ml). The identity of the developed compounds was identified according to their retention time. The peaks # 1, 2, 3, 4 and 5 refers to the Gallic acid, Rutin, Kaempferol, Myricetin and Apigenin, respectively.

Figure 8

The chemical analyses of the putative rutin from Moringa oleifera. UV-Spectra (A), FTIR (B) and LC–MS (C), and 1H-NMR (D), of the purified rutin from Moringa oleifera.

Figure 9

The conformation depiction of the interaction between different inhibitors and the binding pocket of tyrosinase from Agaricus bisporus (2X9Y) using Molecular operating environment (MOE) version 10 program software. The 3D visualization of interaction of amino acid residues in the active site of tyrosinase (2Y9X) with the co-crystallized tropolone (represented in cyan sticks) as positive inhibitor (A), rutin (represented in cyan sticks) inhibitor (C) kojic acid (E). The depiction of 2D interaction of amino acid residues in the active site of tyrosinase (2Y9X) with the co-crystallized tropolone as positive inhibitor (B), rutin inhibitor (D), and kojic acid (F). The structure of target protein was obtained from the last step of molecular docking. The computational simulation of rutin was performed in the active site, where located two copper ions (cyan spheres). The depiction of the molecular analysis was carried out using Molecular Operating Environment (MOE, 2015.10).