Kojic Acid Showed Consistent Inhibitory Activity on Tyrosinase from Mushroom and in Cultured B16F10 Cells Compared with Arbutins

Abstract

Kojic acid, β-arbutin, α-arbutin, and deoxyarbutin have been reported as tyrosinase inhibitors in many articles, but some contradictions exist in their differing results. In order to provide some explanations for these contradictions and to find the most suitable compound as a positive control for screening potential tyrosinase inhibitors, the activity and inhibition type of the aforementioned compounds on monophenolase and diphenolase of mushroom tyrosinase (MTYR) were studied. Their effects on B16F10 cells melanin content, tyrosinase (BTYR) activity, and cell viability were also exposed. Results indicated that α-arbutin competitively inhibited monophenolase activity, whereas they uncompetitively activated diphenolase activity of MTYR. β-arbutin noncompetitively and competitively inhibited monophenolase activity at high molarity (4000 µM) and moderate molarity (250-1000 µM) respectively, whereas it activated the diphenolase activity of MTYR. Deoxyarbutin competitively inhibited diphenolase activity, but could not inhibit monophenolase activity and only extended the lag time. Kojic acid competitively inhibited monophenolase activity and competitive-noncompetitive mixed-type inhibited diphenolase activity of MTYR. In a cellular experiment, deoxyarbutin effectively inhibited BTYR activity and reduced melanin content, but it also potently decreased cell viability. α-arbutin and β-arbutin dose-dependently inhibited BTYR activity, reduced melanin content, and increased cell viability. Kojic acid did not affect cell viability at 43.8-700 µM, but inhibited BTYR activity and reduced melanin content in a dose-dependent manner. Therefore, kojic acid was considered as the most suitable positive control among these four compounds, because it could inhibit both monophenolase and diphenolase activity of MTYR and reduce intercellular melanin content by inhibiting BTYR activity without cytotoxicity. Some explanations for the contradictions in the reported articles were provided.

Keywords: cell viability; deoxyarbutin; diphenolase activity; inhibition type; kojic acid; melanin content; monophenolase activity; tyrosinases; α-arbutin; β-arbutin.

Figure 1

Chemical structure of α-arbutin, β-arbutin, deoxyarbutin, and kojic acid.

Figure 2

Progress curves of kojic acid, α-arbutin, β-arbutin, and deoxyarbutin on monophenolase and diphenolase of mushroom tyrosinase (MTYR). (A) Progress curves of kojic acid (7.5–120 µM), α-arbutin (480–8000 µM), β-arbutin (240–4000 µM), and deoxyarbutin (3.75–60 µM) on monophenolase of MTYR. (B) Progress curves of kojic acid (7.5, 30, and 120 µM), α-arbutin (500, 2000, and 8000 µM), β-arbutin (500, 2000, and 8000 µM), and deoxyarbutin (7.5, 30, and 120 µM) on diphenolase of MTYR.

Figure 3

IC₅₀ value of kojic acid, α-arbutin, β-arbutin, and deoxyarbutin on monophenolase and diphenolase of MTYR. (A) Inhibition rate of monophenolase by compounds; (B) Inhibition rate of diphenolase by compounds; (C) IC₅₀ value of compounds on monophenolase and diphenolase. If the inhibition rate was negative, it meant the tyrosinase was activated. The activation rate was the absolute value of inhibition rate.

Figure 4

Determination of the inhibition type of kojic acid, α-arbutin, β-arbutin, and deoxyarbutin on monophenolase and diphenolase from MTYR. (A) The Lineweaver–Burk plot for kojic acid, α-arbutin, β-arbutin, and deoxyarbutin on monophenolase; (B) The Lineweaver–Burk plot for kojic acid, α-arbutin, β-arbutin, and deoxyarbutin on diphenolase; (C) Kinetic parameters and inhibition type of kojic acid, α-arbutin, β-arbutin, and deoxyarbutin on monophenolase and diphenolase. Changes of V_max and K_m with the increasing compound molarity were shown.

Figure 5

Effect of four compounds on tyrosinase activity, melanin content and cell viability of B16F10 cells. (A) Effect of four compounds on BTYR activity; (B) Effect of four compounds on melanin content; (C) Effect of four compounds on cell viability. Control 1 was blank group, while control 2 was control group.