Hyperpigmentary disorders like melasma, actinic and senile lentigines and postinflammatory hyperpigmentation are a major cosmetic problem for which patients seek medical advice. These disorders affect populations with darker skin complexion, like Asians and Hispanics, with greater frequency and severity.1 Numerous topical products are available, containing diverse active ingredients to reduce melanin production and distribution. In principle, skin depigmentation can be achieved by regulating the transcription and activity of tyrosinase and other melanosomal proteins, inhibition of melanocyte activation, interference with the uptake and distribution of melanosomes in keratinocytes and an accelerated turnover of pigmented keratinocytes.2 Tyrosinase is the key regulator of melanin production and, consequently, the most prominent target for inhibitors of hyperpigmentary disorders. To date, many substances have been described in the literature as inhibitors of tyrosinase; however, most of them lack the efficacy and specificity necessary for practical applications and, thus, only a few compounds are being used in topical products.3–5 Among these, kojic acid and hydroquinone arbutin are most widely used.6 Skin‐whitening actives have to be applied as a long‐term treatment and may cause various side‐effects, like skin irritation and dyschromia; therefore, it is important to find an effective and well‐tolerable alternative to reduce pigment irregularities and protect against the reappearance. We were interested in evaluating the inhibition of skin pigmentation by well‐known compounds with skin‐whitening activity like hydroquinone, arbutin kojic acid and 4‐n‐butylresorcinol, and therefore compared the efficacy in various in vitro and in vivo assay systems.
Inhibition of human tyrosinase
All four compounds were tested over a wide range of concentrations, up to four orders of magnitude, for tyrosinase inhibition (Fig. 1). 4‐butylresorcinol proved to be a highly effective inhibitor of human tyrosinase with an IC50 of 21 μmol/L and complete enzyme inhibition at concentrations above 100 μmol/L. The resorcinol derivatives 4‐hexylresorcinol and 4‐phenylethylresorcinol showed an IC50 of 94 and 131 μmol/L respectively (data not shown). Kojic acid was more than 20 times less potent with an IC50 at 500 μmol/L and maximum inhibition (89%) at 5.6 mmol/L concentration. Arbutin and hydroquinone are only poor inhibitors of human tyrosinase with IC50 values in the millimolar range, i.e. approximately 6500 μmol/L for arbutin and 4400 μmol/L for hydroquinone. Neither arbutin nor hydroquinone completely inhibited human tyrosinase.
Inhibition of human tyrosinase by 4‐butylresorcinol, kojic acid, arbutin and hydroquinone. The L‐DOPA oxidase activity of tyrosinase was determined at various concentrations of the inhibitors to allow for the calculation of IC50 values. Data represent the mean of three independent experiments.
Reduction of melanin production
Arbutin showed only marginal efficacy on melanin production in MelanoDerm skin models with an IC50 for inhibition of > 5000 μmol/L (Fig. 2). Kojic acid inhibited melanin synthesis with an IC50 > 400 μmol/L and showed a surprisingly steep dose–response curve. Concentrations below 200 μmol/L only marginally inhibited melanin production, i.e. 5% inhibition at 150 μmol/L. Interestingly, hydroquinone inhibited melanin production in MelanoDerms with an IC50 below 40 μmol/L, pointing towards a mechanism different from tyrosinase inhibition. 4‐butylresorcinol was the most potent inhibitor with an IC50 of 13.5 μmol/L. A comparison of the dose–response curves of hydroquinone and 4‐butylresorcinol reveals that at concentrations above 20 μmol/L, 4‐butylresorcinol is slightly more effective than hydroquinone, at concentrations below 20 μmol/L hydroquinone is slightly more effective than 4‐butylresorcinol.
Inhibition of melanin production in MelanoDerm™ skin models by 4‐butylresorcinol, kojic acid, arbutin and hydroquinone. Melanin content of skin models was determined after 13 days of cultivation in the presence of various inhibitor concentrations. Data represent the mean of five independent experiments.
Elderly subjects treated age spots twice daily either with a formula containing 4‐butylresorcinol, 4‐hexylresorcinol or 4‐phenylethylresorcinol (Fig. 3). Within 8 weeks, 4‐butylresorcinol significantly reduced the appearance of age spots while 4‐hexylresorcinol or 4‐phenylethylresorcinol showed significant effects after 12 weeks (all in comparison to vehicle).
Age spot lightening by 4‐butylresorcinol, 4‐hexylresorcinol and 4‐phenylethylresorcinol. The spots were treated twice daily for 12 weeks with a formula containing the respective inhibitor. Efficacy was evaluated after 4, 8 and 12 weeks. Data represent the mean of 14 subjects. *P < 0.05: statistically significant vs. the untreated control age spots.
Epi‐Flash photographs revealed visible improvement in the appearance of age spots after 12 weeks of treatment with 4‐butylresorcinol. Control age spots remained unchanged (not shown).
In a second study, subjects applied 1% 4‐butylresorcinol to age spots of the volar forearm using a spot applicator. Control age spots were treated with a spot applicator containing the vehicle. Already after 4 weeks of treatment, the treated spots were lighter than the control spots. Improvement continued over the entire treatment period, and after 16 weeks some of the spots were undistinguishable from the surrounding skin (Fig. 4). After treatment, the age spots were monitored for several weeks. Even after 4 weeks without treatment, the age spots previously treated with 4‐butylresorcinol were still significantly lighter than the vehicle‐treated spots.
Clinical Study – monitoring of a treated age spot during treatment with a spot applicator. Photographs of age spots were taken at baseline and after 8, 12 and 16 weeks of treatment. The arrows mark the treated age spot. For comparison, untreated spots were included in the photographed area.
References source: JEADV (https://doi.org/10.1111/jdv.12051)