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Biochemistry

Quantification of Hypopigmentation Activity In Vitro

Published: March 6th, 2019

DOI:

10.3791/58185

1Department of Biotechnology, Graduate School of Life Sciences & Biotechnology, College of Life Sciences and Biotechnology, Korea University, 2JinWoo Bio Co. Ltd

We describe three experimental methods for evaluating the hypopigmentation activity of chemicals in vitro: quantification of 1) cellular tyrosinase activity and 2) melanin content, and 3) measurement of melanin by cellular melanin staining and image analysis.

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.

Melanin plays a critical role in the physiology, pathology, and toxicology of several organs including the skin, eyes, and brain1. Major functions of melanin are photo-screening and biochemical effects. Melanin absorbs near-infrared and visible light as well as ultraviolet (UV) radiation, with increased absorption rates at shorter wavelengths of light; thus, melanin protects tissues from damage caused by visible light or UV radiation2. Melanin is an antioxidant and has an affinity for metals and other toxic chemicals; therefore, it can protect tissues from oxidative and chemical stress3. However, ....

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1. Preparation of Medium, Compounds, and Reagents

  1. Prepare B16F10 growth complete medium. Supplement Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PEST).
    1. To make 500 mL of complete medium, mix 445 mL of Dulbecco’s modified Eagle’s medium (DMEM) with 50 mL of FBS and 5 mL of PEST in a 1 L sterilized glass bottle. After mixing gently, filter the medium through a 0.2-µm bottle-top filter to a new sterilized glas.......

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Representative results for the hypopigmentation activity of arbutin, an anti-melanogenic compound, in B16F10 melanocytes are shown below. Figure 1A shows that arbutin significantly suppressed the cellular tyrosinase activity compared with the vehicle-treated control. Similarly, the melanin content of cells stimulated with arbutin was significantly reduced compared with the controls (Figure 1B). Microscopic images of cells stained.......

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We presented protocols for evaluating the hypopigmentation activity of test compounds using cultured melanocytes. The representative results showed the hypopigmentation effect of arbutin, a tyrosinase inhibitor that inhibited tyrosinase activity and cellular melanin content. These methods are widely used in anti-melanogenic activity research. Using these assays, we have also successfully identified several bioactive compounds that have melanogenesis inhibitory effects on B16F10 cells in the past decade9.......

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This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through the Agri-Bioindustry Technology Development Program, funded by the Ministry of Agriculture, Food, and Rural Affairs (MAFRA) (116159-02-2-WT011) and School of Life Sciences and Biotechnology for BK21 PLUS, Korea University.

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Name Company Catalog Number Comments
3,4-Dihydroxy-l-phenylalanine Sigma D9628
Ammonium hydroxide solution Sigma #320145
Arbutin Fluka #10960
DMEM HyClone SH30243.01
FBS HyClone SH30084.03
Formalin Yakuri Pure Chemicals #16223 37%
Gold chloride American MasterTech AHG0226 0.10%
HCl Samchun chemical H0255
KCl Bio basic Canada Inc. PB0440
KH2PO4 Sigma #60218
Na2HPO4 J.T.Baker #3817-01
NaCl Duksan pure chemicals #81
NaOH Sigma 655104
Nuclear fast red Merck 100121 Nuclear fast red 0.1% in 5% aluminum sulfate.
Penicillin and streptomycin solution HyClone SV30010
Silver nitrate Duksan Pure Chemicals #900
Sodium phosphate dibasic (Na2HPO4) J.T. Baker #3817-01
Sodium phosphate monobasic (Na2HPO4) Sigma S5011
Sodium thiosulfate pentahydrate Duksan Pure Chemicals #2163
Tris(hydroxymethyl)aminomethane Bio Basic Canada TB0196
Triton X-100 Union Carbide T8787
Tyrosinase from mushroom Sigma T3824 25 KU

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