2- S-Lipoylcaffeic Acid, a Natural Product-Based Entry to Tyrosinase Inhibition via Catechol Manipulation

Abstract

Conjugation of naturally occurring catecholic compounds with thiols is a versatile and facile entry to a broad range of bioinspired multifunctional compounds for diverse applications in biomedicine and materials science. We report herein the inhibition properties of the caffeic acid- dihydrolipoic acid S-conjugate, 2-S-lipoylcaffeic acid (LC), on mushroom tyrosinase. Half maximum inhibitory concentration (IC₅₀) values of 3.22 ± 0.02 and 2.0 ± 0.1 µM were determined for the catecholase and cresolase activity of the enzyme, respectively, indicating a greater efficiency of LC compared to the parent caffeic acid and the standard inhibitor kojic acid. Analysis of the Lineweaver⁻Burk plot suggested a mixed-type inhibition mechanism. LC proved to be non-toxic on human keratinocytes (HaCaT) at concentrations up to 30 µM. These results would point to LC as a novel prototype of melanogenesis regulators for the treatment of pigmentary disorders.

Keywords: ">l-DOPA; caffeic acid; depigmenting agents; dihydrolipoic acid; dopachrome; keratinocytes; lipoic acid; melanin; tyrosinase.

Figure 1

Structure of 2-S-lipoylcaffeic acid (LC).

Scheme 1

Dopachrome formation by tyrosinase-catalyzed oxidation of 3,4-dihydroxy-l-phenylalanine (l-DOPA). λ_max: Wavelength of maximum absorbance.

Figure 2

Time course of the absorbance change at 475 nm in the oxidation mixture of l-DOPA (1 mM) with mushroom tyrosinase in the absence (control (ctrl)) or presence of different concentrations of LC. Reported are the mean values of at least three experiments (standard deviation (SD) < 5%). AU: Arbitrary units.

Figure 3

Percent of inhibition of mushroom tyrosinase activity vs. LC concentration using l-DOPA (1 mM) as substrate. Reported are the mean ± SD values of at least three experiments.

Figure 4

Tyrosinase-catalyzed oxidation mixtures of l-DOPA (1 mM) in the absence (B) or in the presence of 10 µM inhibitor (LC: 2-S-lipoylcaffeic acid; CAF: Caffeic acid; KOJ: Kojic acid).

Figure 5

Percent of inhibition of mushroom tyrosinase activity vs. LC concentration using l-tyrosine (1 mM) as the substrate. Reported are the mean ± SD values of at least three experiments.

Figure 6

Lineweaver–Burk plot for the inhibition of mushroom tyrosinase-catalyzed l-DOPA oxidation by LC at 0 (control (ctrl)), 3 or 5 µM. Data were obtained as mean ± SD values of 1/V, inverse of the increase of absorbance at 475 nm per min (ΔA₄₇₅/min), of three independent experiments with different concentrations of l-DOPA.

Figure 7

The effect of LC on the enzymatic kinetics for the mushroom tyrosinase-induced oxidation of l-DOPA. Data were obtained as mean ± SD values of the increase of absorbance at 475 nm per min (ΔA₄₇₅/min) (V) of three independent experiments with different concentrations of l-DOPA.

Figure 8

Effect of LC on HaCaT cell viability determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Cells were cultured in normal growth medium and then subjected to treatment with LC (black: Control; dark grey: 0.3 µM; grey: 3 µM; white: 30 µM) for 24, 48, and 72 h. Cell viability was evaluated by measuring the A_570nm. Results are expressed as the percentage (means ± SD from at least three experiments) compared to the control.