In vitro and in silico studies of the inhibitory effects of some novel kojic acid derivatives on tyrosinase enzyme

Document Type: Original Article


1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Department of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

3 Department of Biochemistry, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran


Objective(s): Tyrosinase is a key enzyme in pigment synthesis. Overproduction of melanin in parts of the skin results in hyperpigmentation diseases. This enzyme is also responsible for the enzymatic browning in fruits and vegetables. Thus, its inhibitors are of great importance in the medical, cosmetic and agricultural fields.
Materials and Methods: A series of twelve kojic acid derivatives were designed to be evaluated as tyrosinase activity inhibitors. The potential inhibitory activity of these compounds was investigated in silico using molecular docking simulation method. Four compounds with a range of predicted tyrosinase inhibitory activities were prepared and their inhibitory effect on tyrosinase activity was evaluated. The antioxidant properties of these compounds were also investigated by in vitro DPPH (2,2-diphenyl-1-picrylhydrazyl) and hydrogen peroxide scavenging assays.
Results: Compound IIId exhibited the highest tyrosinase inhibitory activity with an IC50 value of 0.216 ± 0.009 mM which was in accordance with the in silico ΔGbind results (-13.24 Kcal/mol).
Conclusion: Based on the docking studies, from the twelve compounds studied, one (IIId) appeared to have the highest inhibition on tyrosinase activity. This was confirmed by enzyme activity measurements. Compound IIId has an NO2 group which binds to both of Cu2+ ions located inside the active site of the enzyme. This compound appeared to be even stronger than kojic acid in inhibiting tyrosinase activity. The DPPH free radical scavenging ability of all the studied compounds was more than that of BHT. However, they were not as strong as BHT or gallic acid in scavenging hydrogen peroxide.


1. Chang TS. An updated review of tyrosinase inhibitors. Int J Mol Sci 2009; 10:2440-2475.

2. Muñoz-Muñoz JL, García-Molina Mdel M, Garcia-Molina F, Berna J, Garcia-Ruiz PA, García-Moreno M, et al. Catalysis and inactivation of tyrosinase in its action on o-diphenols, o-aminophenols and o-phenylendiamines: Potential use in industrial applications. J Mol Catal B Enzym 2013; 91:17-24.

3. Therdphapiyanak N, Jaturanpinyo M, Waranuch N, Kongkaneramit L, Sarisuta N. Development and assessment of tyrosinase inhibitory activity of liposomes of Asparagus racemosus extracts. Asian J Pharm Sci2013; 8:134-142.

4. Li ZC, Chen LH, Yu XJ, Hu YH, Song KK, Zhou XW, et al. Inhibition kinetics of chlorobenzaldehyde thiosemicarbazones on mushroom tyrosinase. J Agric Food Chem 2010; 58:12537-12540.

5. Guerrero A, Rosell G. Biorational approaches for insect control by enzymatic inhibition. Curr Med Chem 2005; 12:461-469.

6. Loizzo MR, Tundis R, Menichini F. Natural and synthetic tyrosinase inhibitors as antibrowning agents: An update. Compr Rev Food Sci Food Saf 2012; 11:378-398.

7. Decker H, Schweikardt T, Nillius D, Salzbrunn U, Jaenicke E, Tuczek F. Similar enzyme activation and catalysis in hemocyanins and tyrosinases. Gene 2007; 398:183–191.

8. De Faria RO, Moure VR, Lopes MA, Krieger N, Mitchell DA. The biotechnological potential of mushroom tyrosinases. Food Technol Biotech 2007; 45:287-294.

9. Aytemir MD, Karakaya G, Ekinci D. Kojic acid derivatives. In: Ekinci Deniz., editor. Medicinal Chemistry and Drug Design. 2012. pp. 1–26.

10. Noh JM, Kwak SY, Seo HS, Seo JH, Kim BG, Lee YS. Kojic acid–amino acid conjugates as tyrosinase inhibitors. Bioorg Med Chem Lett 2009; 19:5586-5589.

11. Burdock GA, Soni MG, Carabin IG. Evaluation of health aspects of kojic acid in food. Regul Toxicol Pharmacol 2001; 33:80-101.

12. Rho HS, Lee CS, Ahn SM, Hong YD, Shin SS, Park YH, et al.  Studies on tyrosinase inhibitory and antioxidant activities of benzoic acid derivatives containing kojic acid moiety. Bull Korean Chem Soc 2011; 32:4411-4414.

13. Rho HS, Ahn SM, Yoo DS, Kim MK, Cho DH, Cho JY. Kojyl thioether derivatives having both tyrosinase inhibitory and anti-inflammatory properties. Bioorg Med Chem Lett 2010; 20:6569–6571.

14. Noh JM, Kwak SY, Kim DH, Lee YS. Kojic acid–tripeptide amide as a new tyrosinase inhibitor. Biopolymers 2007; 88:300-307.

15. Lee YS, Park JH, Kim MH, Seo SH, Kim HJ. Synthesis of tyrosinase inhibitory kojic acid derivatives. Arch Pharm Chem Life Sci 2006; 339:111-114.

16. Wempe M, Fitzpatrick M. 5-Hydroxy-2-methyl-4H-pyran-4-one-esters as novel tyrosinase inhibitors, WO 2009/108271 A1.

17. Mohammadpour M, Behjati M, Sadeghi A, Fassihi A. Wound healing by topical application of antioxidant iron chelators: kojic acid and deferiprone. Int Wound J 2012; 10:260-264.

18. Mohammadpour M, Sadeghi A, Fassihi A, Saghaei L, Movahedian A, Rostami M. Synthesis and antioxidant evaluation of some novel orthohydroxypyridine-4-one iron chelators. Res Pharm Sci 2012; 3:171-179

19. Ahn SM, Rho HS, Baek HS, Joo YH, Hong YD, Shin SS,et al.  Inhibitory activity of novel kojic acid derivative containing trolox moiety on melanogenesis. Bioorg Med Chem Lett 2011; 24:7466-7469.

20. Lajis AF,  Hamid M,  Ariff  AB. Depigmenting effect of kojic acid esters in hyperpigmented B16F1 melanoma cells. J Biomed Biotechnol 2012; 1-9.

21. Huang SY, Zou X. Advances and challenges in protein-ligand docking. Int J Mol Sci 2010; 11:3016–3034.

22. Mukesh B, Rakesh K. Molecular docking: A review. Int J Res Ayurveda Pharm 2011; 2:746– 1751.

23. Saghaie L, Pourfarzam M, Fassihi A, Sartippour B. Synthesis and tyrosinase inhibitory properties of some novel derivatives of kojic acid. Res Pharm Sci 2013; 8:233-242.

24. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, et al. AutoDock4 and AutoDockTools4: Auto-mated docking with selective receptor flexibility. J Comput Chem 2009; 30:2785-2791.

25. Morris GM, Goodsell DS, Halliday RS, Huey R, Hart WE, Belew RK, et al. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. J Comput Chem 1998; 19:1639-1662.

26. Gasteiger J, Marsili M. Iterative partial equalization of orbital electronegativity-a rapid access to atomic charges. Tetrahedron 1980; 36:3219-322.

27. Ismaya WT, Rozeboom HJ, Weijn A, Mes JJ, Fusetti F, Wichers HJ, et al.  Crystal structure of Agaricus bisporus mushroom tyrosinase: Identity of the tetramer subunits and interaction with tropolone. Biochemistry 2011; 50:5477-5486.

28. Discovery Studio Visualizer. Release 3.5, Accelrys Software Inc., San Diego CA USA, 2012.

29. Weiner SJ, Kollman PA, Case DA, Singh UC, Ghio C, Alagona G, et al. A new force field for molecular mechanical simulation of nucleic acids and proteins. J Am Chem Soc 1984; 106:765-784.

30. Wallace AC, Laskowski RA, Thornton JM . LIGPLOT: A program to generate schematic diagrams of protein-ligand interactions. Protein Eng 1995; 8:127-134.

31. Iida K, Hase K, Shimomura K, Sudo S, Kadota S, Namba T. Potent inhibitors of tyrosinase activity and melanin biosynthesis from Rheum officinale. Planta Med 1995; 161:425-428.

32. Blois MS. Antioxidant determinations by the use of a stable free radical. Nature 1958; 181:1199-1200.

33. Ruch RJ, Cheng SJ, Klaunig JE. Prevention of cytotoxicity and inhibition of intracellular communication by antioxidant catechins isolated from Chinese green tea. Carcinogenesis 1989; 10:1003-1008.

34. Wen KC, Chang CS, Chien YC, Wang HW, Wu WC, Wu CS, et al.  Tyrosol and its analogues inhibit alpha-melanocyte-stimulating hormone induced melanogenesis. Int J Mol Sci 2013; 14:23420-23440.

35. Vontzalidou A, Zoidis G, Chaita E, Makropoulou M, Aligiannis N, Lambrinidis G, et al.  Design, synthesis and molecular simulation studies of dihydrostilbene derivatives as potent tyrosinase inhibitors. Bioorg Med Chem Lett 2012; 22:5523–5526.

36. Chung KW, Jeong HO, Jang EJ, Choi YJ, Kim DH, Kim SR, et al. Characterization of a small molecule inhibitor of melanogenesis that inhibits tyrosinase activity and scavenges nitric oxide (NO). Biochim Biophys Acta 2013; 1830:4752–4761.

37. Pei CJ, Lee J, Si YX, Oh S, Xu WA, Yin SJ, et al. Inhibition of tyrosinase by gastrodin: An integrated kinetic-computational simulation analysis. Proc Biochem 2013; 48:162–168.

38. Wang Z, Lee J, Si YX, Oh S, Yang JM, Shen D, et al. Toward the inhibitory effect of acetylsalicylic acid on tyrosinase: Integrating kinetics studies and computational simulations. Proc Biochem 2013; 48:260–266.

39.Ferreira ICFR, Baptista P, Vilas-Boas M, Barros L. Free-radical scavenging capacity and reducing power of wild edible mushrooms from northeast Portugal: Individual cap and stipe activity. Food Chem 2007; 100:1511–1516.

40. Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine. 4th ed. Oxford, UK: Clarendon Press; 2006.

41. Pise NM, Jena KB, Maharana D, Sabale AB, Jagtap TG. Free radical scavenging, reducing power, phenolic and biochemical composition of Porphyra species. J Algal Biomass Utiln 2010; 1:60-73.