Quantification of hypopigmentation activity in vitro

Yeon Ji Kim, Min Jung Kim, Dong Keon Kweon, Seung Taik Lim, Sung Joon Lee

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)


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.

Original languageEnglish
Article numbere58185
JournalJournal of Visualized Experiments
Issue number145
Publication statusPublished - 2019 Mar

Bibliographical note

Funding Information:
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.


  • Biochemistry
  • Fontana-Masson stain
  • Hypopigmentation
  • Issue 145
  • Melanin contents
  • Melanocytes
  • Melanogenesis
  • Tyrosinase activity

ASJC Scopus subject areas

  • General Neuroscience
  • General Chemical Engineering
  • General Biochemistry,Genetics and Molecular Biology
  • General Immunology and Microbiology


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