Effects Of Hydroquinone And Its Structural Analogues On Melanogenesis And Antioxidation

Background and objectivesHydroquinone, a monophenic compound, is the earliest skin depigmenting agent prescribed to treat disorders of hyperpigmentation in the clinic. The mechanism underlying skin whitening contributes to the competitive inhibition against tyrosinase because hydroquinone shares structural homologies with the substrate tyrosine. However, the physicochemical property of the topical preparations made by hydroquinone and other phenolic chemicals are unstable, they are liable to discoloration while exposure to the ambient atmospheric air. It had been reported that permanent leucoderma (artificial vitiligo) was found in the patients who accepted long-term hydroquinone therapy.In 1998, arbutin was successfully isolated from the leaves of Arctoataphylos uva-ursi by a Japanese research group and found as a potent tyroisnase inhibitor for wide uses of skin whitening products in cosmetics. Since then, the non-toxic, non-irritant, and efficacious skin whitening agent screened from the natural botanical source is highly desirable and becomes a hot-point for both dermatologist and cosmetic chemist. Deoxy-arbutin is a new glucoside derivative of hydroquinone by the removal of pendant hydroxyls from the glucose side-chain of arbutin. Besides the increase of hydrophobic property to penetrate the cellular membrane, it retains the inhibition against tyrosinase, the same as its precursor, arbutin.In this study, we determined the effects of hydroquinone, arbutin, and deoxy-arbutin on melanogenesis and anti-oxidation in cultured melanocytes. Meanwhile, to explore the mechanism of melanosomal acidification in tyrosinase activity regulation, acridine orange, a nonselective fluorochrome for acidic organelles, was introduced to label cultured human melanocytes after treated with hydroquinone and its glucoside derivants, followed by monitoring the tyrosinase activity and melanosome pH.Methods 1) To emerge an oxidative stress, mouse melan-a melanocytes were exposed to nontoxic UVA (3J/cm2), then the effects of hydroquinone, arbutin and deoxyarbutin on tyrosinase activity and cell viability were monitored.2) cell viability was determined by MTT assay.3) Tyrosinase activity was measured by L-Dopa oxidation and 14C-tyrosine incorporation.4) Intracellular reactive oxygen species (ROS) level was monitored by H2DCFDA fluorescence labeling.5) The pH in acidic organelles, including melanosome, was monitored by acridine orange labeling.6) Changes in tyrosinase acitivity inhibition of whitening agents were observed after melonosomes were neutralized by ammonium chloride, a weak base.ResultsThe cytotoxicity of hydroquinone and arbutin except for D-arb was increased while the cells exposed to a nontoxic dose (3 J/cm2) of UVA irradiation. Suppressed ROS generation was noted by the treatment of D-arb to compare with arbutin and hydroquinone. All three compounds had a similar inhibition on tyrosinase activity in dose-dependent manners with two-to three-fold decreases over the untreated control. There was no change in expression of tyrosinase protein in cells treated with arbutin or hydroquinone, but a decreased protein expression of tyrosinase was seen in deoxyarbutin-treated cells. Melanosomes were acidic with pH 5.5-6.8 in normal human melanocyte and the optimal pH for tyrosinase was 6.8. After treated with ammonium chloride for 5 days, the fluorescence of acridine orange turned to green from orange since intracellular acidic organelles including melanosomes were neutralized. The results showed that melanosomes neutralization did not invert low tyrosinase activity after treated with resorcinum, comparing to treated with 3μM hydroquinone or 30μM arbutin.ConclusionsDeoxyarbutin exerts potent tyrosinase inhibition, lessened cytotoxicity, and certain antioxidation potential, may serve as an effective and safe alternative to hydroquinone for use in skin whitening. Once acidic melanosomes are neutralized, the pre-existing tyrosinase therein are activated rapidly to promote melanogenesis. On the contrary, acidification of melanosomes will inhibit tyrosinase activity reversiblely.