arbutin and Hypopigmentation

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is synthesized in response to multiple cellular and environmental factors. Melanin protects skin cells from ultraviolet damage, but also has biophysical and biochemical functions. Excessive production or accumulation of melanin in melanocytes can cause dermatological problems, such as freckles, dark spots, melasma, and moles. Therefore, the control of melanogenesis with hypopigmentation agents is important in individuals with clinical or cosmetic needs. Melanin is primarily synthesized in the melanosomes of melanocytes in a complex biochemical process called melanogenesis, which is influenced by extrinsic and intrinsic factors, such as hormones, inflammation, age, and ultraviolet light exposure. We describe three methods to determine the hypopigmentation activity of chemicals or natural substances in melanocytes: measurement of the 1) cellular tyrosinase activity and 2) melanin content, and 3) staining and quantifying cellular melanin with image analysis. In melanogenesis, tyrosinase catalyzes the rate-limiting step that converts L-tyrosine into 3,4-dihydroxyphenylalanine (L-DOPA) and then into dopaquinone. Therefore, the inhibition of tyrosinase is a primary hypopigmentation mechanism. In cultured melanocytes, tyrosinase activity can be quantified by adding L-DOPA as a substrate and measuring dopaquinone production by spectrophotometry. Melanogenesis can also be measured by quantifying the melanin content. The melanin-containing cellular fraction is extracted with NaOH and melanin is quantified spectrophotometrically. Finally, the melanin content can be quantified by image analysis following Fontana-Masson staining of melanin. Although the results of these in vitro assays may not always be reproduced in human skin, these methods are widely used in melanogenesis research, especially as the initial step to identify potential hypopigmentation activity. These methods can also be used to assess melanocyte activity, growth, and differentiation. Consistent results with the three different methods ensure the validity of the effects.

Topics: Animals; Arbutin; Humans; Hypopigmentation; Melanins; Melanocytes; Tyrosine

Arbutin (Arb) and deoxyArbutin (dA) are both effective hypopigmentation agents. However, they are glucoside derivatives of hydroquinone (HQ), which may be decayed into HQ under higher energy environments. Therefore, safety and toxicity are very important issues when considering the usage of these compounds. However, no study has verified the properties of Ultra-Violet B (UVB)-irradiated Arb and dA. In this work, we investigated the cytotoxicity and hypopigmentation effects of UVB-irradiated Arb and dA in Detroit 551 human fibroblast cells and B16-F10 mouse melanoma cells. The results showed that UVB-irradiated Arb and dA have strong cytotoxicity for the fibroblast cells, especially for dA, the caspase-3 is also activated by the treatment of UVB-irradiated dA in Detroit 551 cells. The results correlated with the produced HQ. In addition, UVB-irradiated Arb and dA suppressed the production of melanin in melanoma cells; this is due to the release of HQ that compensates for the UVB triggered Arb and dA decomposition.

Topics: Animals; Arbutin; Caspase 3; Cell Survival; Cells, Cultured; Fibroblasts; Glucosides; Humans; Hydroquinones; Hypopigmentation; Melanins; Melanocytes; Melanoma, Experimental; Mice; Ultraviolet Rays