Kinase experiment:

Briefly, Mel-Ab cells are cultured in 60 mm dishes. After incubating with test substances (including 4-Butylresorcinol) in DMEM containing 2% FBS for 4d, the cells are washed with ice-cold PBS and lysed with phosphate buffer (pH 6.8) containing 1% Triton X-100. The cells are then disrupted by freeze-thawing, and lysates are clarified by centrifuging at 10000×g for 5 min. After quantifying protein levels and adjusting concentrations with lysis buffer, 90 μL of each lysate, is placed in a well of a 96-well plate, and 10 μL of 10 mM L-DOPA is then added. Control wells contain 90 μL of lysis buffer and 10 μL of 10mM L-DOPA[2].

Cell experiment:

Mel-Ab cell line is a mouse-derived spontaneously immortalized melanocyte cell line that produces large amounts of melanin. Mel-Ab cells are incubated in DMEM supplementing with 10% fetal bovine serum (FBS), 100 nM TPA, 1 nM CT, 50 μg/mL streptomycin, and 50 U/mL penicillin at 37 ℃ in 5% CO2. Cell viability is determined using a crystal violet assay. After incubating cells with test substances for 24 h, the medium is removed and stained with 0.1% crystal violet in 10% ethanol for 5 min at room temperature and then rinsed four times with distilled water[2].

4-Butylresorcinol is a phenol derivative which can inhibit tyrosinase with IC50 of 11.27 μM.

It shows that 4-Butylresorcinol (4-n-butylresorcinol) significantly inhibits melanin synthesis in a concentration-dependent manner. In addition, it is also found to inhibit the activity of tyrosinase, the rate-limiting melanogenic enzyme. 4-Butylresorcinol does not induce ERK, Akt activation, or MITF degradation, and has no effect on cAMP response element binding protein (CREB) phosphorylation, which stimulates MITF expression. 4-Butylresorcinol strongly reduces tyrosinase activity in a cell-free system. Moreover, 4-Butylresorcinol shows an additive effect in combination with hinokitiol, which reduces MITF expression[2].

[1]. Jiang Y, et al. Synthesis and biological evaluation of unsymmetrical curcumin analogues as tyrosinaseinhibitors. Molecules. 2013 Apr 3;18(4):3948-61. [2]. Kim DS, et al. Inhibitory effects of 4-n-butylresorcinol on tyrosinase activity and melanin synthesis. Biol Pharm Bull. 2005 Dec;28(12):2216-9.