Condensed tannins from Ficus virens as tyrosinase inhibitors: structure, inhibitory activity and molecular mechanism

Abstract

Condensed tannins from Ficus virens leaves, fruit, and stem bark were isolated and their structures characterized by 13C nuclear magnetic resonance spectrometry, high performance liquid chromatography electrospray ionization mass spectrometry, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results showed that the leaves, fruit, and stem bark condensed tannins were complex mixtures of homo- and heteropolymers of B-type procyanidins and prodelphinidins with degrees of polymerization up to hexamer, dodecamer, and pentadecamer, respectively. Antityrosinase activities of the condensed tannins were studied. The results indicated that the condensed tannins were potent tyrosinase inhibitors. The concentrations for the leaves, fruit, and stem bark condensed tannins leading to 50% enzyme activity were determined to be 131.67, 99.89, and 106.22 μg/ml on monophenolase activity, and 128.42, 43.07, and 74.27 μg/ml on diphenolase activity. The inhibition mechanism, type, and constants of the condensed tannins on the diphenolase activity were further investigated. The results indicated that the condensed tannins were reversible and mixed type inhibitors. Fluorescence quenching, copper interacting, and molecular docking techniques were utilized to unravel the molecular mechanisms of the inhibition. The results showed that the hydroxyl group on the B ring of the condensed tannins could chelate the dicopper irons of the enzyme. Moreover, the condensed tannins could reduce the enzyme product o-quinones into colourless compounds. These results would contribute to the development and design of antityrosinase agents.

Figure 1. Catalytic cycles of the hydroxylation of monophenol and oxidation of o-diphenol to o-quinone by tyrosinase².

Figure 2. Structure of the condensed tannins, flavan-3-ol monomer units, and substrates of tyrosinase.

(1) Chemical structure of the condensed tannins. The 4 to 6 linkage (dotted line) is an alternative interflavan bond. The terminal unit is at the bottom the multi-unit structure. (2) Structure similarities between the flavan-3-ol monomer units and the substrates of tyrosinase.

Figure 3. ¹³C NMR (150 MHz) spectra of the condensed tannins in DMSO-d ₆.

PC: procyanidin; PP: propelargonidin; a, b, and c represented the condensed tannins from leaves fruit, and stem bark of F. virens, respectively.

Figure 4. HPLC chromatograms and MALDI-TOF MS of the condensed tannins.

(1) Reversed-phase HPLC chromatograms of the condensed tannins. Terminal units: epicatechin (EC). Extender units: afzelechin (AF-thio), epiafzelechin (EAF-thio), catechin (C-thio), epicatechin (EC-thio), BM, benzyl mercaptan. (2) MALDI-TOF positive reflectron mode mass spectra of the condensed tannins. DP, degree of polymerization. (3) Enlarged picture of the DP 4 of the condensed tannins. a, b, and c represented the condensed tannins from leaves fruit, and stem bark of F. virens, respectively.

Figure 5. Determination of the inhibitory activity, inhibition mechanism, type, and constants.

(1) Inhibitory activity of the condensed tannins on monophenolase activity of mushroom tyrosinase. (I) Progress curves for the oxidation of L-Tyr by the enzyme. (II) Effects on the oxidation rate of L-Tyr by the enzyme. (III) Effects on the lag time of monophenolase. The concentrations of the condensed tannins for curves 0–5 were 0, 30, 60, 90, 120, and 180 μg/ml, respectively. (2) Inhibitory activity of the condensed tannins on diphenolase activity of mushroom tyrosinase. (3) Inhibition mechanism of the condensed tannins on mushroom tyrosinase. The concentrations of the condensed tannins for curves 0–4 were 0, 20, 40, 60, and 80 μg/ml, respectively. (4) Determination of the inhibition type and constants of the condensed tannins on mushroom tyrosinase. (I) Lineweaver-Burk plots for diphenolase activity. (II) The plot of slope versus the concentration of the condensed tannins for determining the inhibition constants K_I. (III) The plot of intercept versus the concentration of the condensed tannins for determining the inhibition constants K_IS. Assay conditions: 3 ml reaction system containing 50 mM phosphate sodium buffer (pH 6.8) and 3.3% DMSO. a, b, and c represented the condensed tannins from leaves, fruit, and stem bark of F. virens, respectively.

Figure 6. Changes in tyrosinase intrinsic fluorescence.

The concentration of catechin and condensed tannins from the leaves, fruit, and stem bark was 85 μg/ml. (I) Intrinsic fluorescence changes. (II) Maximum fluorescence intensity changes. Values were expressed as mean±standard deviation (n = 3). (III) Maximum peak wavelength changes. Values were expressed as mean±standard deviation (n = 3). Different letters in the column denoted significantly different at P<0.05.

Figure 7. Absorption spectra for test samples and the Cu-test samples complex at pH 7.4.

a, b, and c, represented the condensed tannins from the leaves, fruit, and stem bark of F. virens; d, e, and f represented catechin, L-DOPA, and L-Try.

Figure 8. Binding mode of the lowest energy docked conformation found for the ligand with tyrosinase residues.

The receptor exposure differences were shown by the size and intensity of the turquoise discs surrounding the residues. The red arrows indicated the interaction of the ligand with the copper iron. a, b, c, d, and e represented L-Tyr, L-DOPA, catechin, afzelechin, and PCs dimmers, respectively.

Figure 9. UV-Vis spectra for the oxidation of L-DOPA.

Black, 100 μg/ml L-DOPA; Pink, 100 μg/ml L-DOPA+50 μg/ml CT; Blue, 100 μg/ml L-DOPA +100 μg/ml CT; Purple, 100 μg/ml L-DOPA+100 μg/ml NaIO₄; Green, 100 μg/ml L-DOPA+100 μg/ml NaIO₄+50 μg/ml CT; Red, 100 μg/ml L-DOPA+ 100 μg/ml NaIO₄+100 μg/ml CT. The green and red spectra of the insert is corresponding to the green and red spectra of the main figure after subtraction of the corresponding condensed tannins absorption from the spectra, while the purple spectra of the insert is the same as the purple in the main figure. a, leaves condensed tannins; b, fruit condensed tannins; c, stem bark condensed tannins; CT, condensed tannins. The assay was performed in 3 ml of 50 mM sodium phosphate buffer (pH 6.8) at 30°C.