Evaluation of antioxidant and anti-melanogenic activities of different extracts from aerial parts of Nepeta binaludensis Jamzad in murine melanoma B16F10 cells

Document Type : Original Article


1 Department of Pharmacodynamics and Toxicology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran

2 Department of Pharmacognosy, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran


Objective(s): Nepeta binaludensis Jamzad (Lamiaceae) has been used in folk medicine of Iran to cure various diseases. The plant is an endemic species to the country that has recently been identified in Razavi Khorasan province. To evaluate the antioxidant and anti-melanogenesis of N. binaludensis, in this study the inhibitory activity of different extracts of N. binaludensis in murine melanoma B16F10 cells is investigated. 
Materials and Methods: The effects of petroleum ether, dichloromethane, ethyl acetate, and methanol extracts isolated from the plant on melanogenesis in B16 melanoma cells were investigated.To assess the inhibitory effects of this plant on melanogenesis, various assays were used including cytotoxicity, inhibition of mushroom tyrosinase and cellular tyrosinase, determination of melanin content, the effect of extracts on reactive oxygen species and western blot analysis of proteins involved in melanogenesis process.
Results: The content of melanin and the activity of tyrosinase were significantly reduced with different extracts of N. binaludensis in cells. Reactive oxygen species was also significantly decreased following the treatment of cell with the mentioned extracts, while a resazurin assay showed no cytotoxicity. Furthermore, we have found that the plant decreased the amount of tyrosinase and microphthalmia-associated transcription factor proteins, which verify the role of suppression of microphthalmia-associated transcription factor protein in melanogenesis inhibition.
Conclusion: Taken together the data indicate that N. binaludensis has inhibitory activity on melanin synthesis with no cytotoxic effects in B16 melanoma cells. Therefore, it merits future investigations to apply as whitening agent in hyperpigmentation.


1.   Fang D, Kute T, Setaluri V. Regulation of Tyrosinase-related Protein-2 (TYRP2) in Human Melanocytes: Relationship to Growth and Morphology. Pigment Cell Res 2001; 14:132-139.
2.   Fang D, Setaluri V. Role of microphthalmia transcription factor in regulation of melanocyte differentiation marker TRP-1. Biochem Biophys Res Commun 1999; 256: 657-663.
3.   Kameyama K, Sakai C, Kuge S, Nishiyama S, Tomita Y, Ito S, et al. The expression of tyrosinase, tyrosinase-related proteins 1 and 2 (TRP1 and TRP2), the silver protein, and a melanogenic inhibitor in human melanoma cells of differing melanogenic activities. Pigment Cell Res 1995; 8:97-104.
4.   Michaela Brenner M, Hearing VJ. The Protective Role of Melanin Against UV Damage in Human Skin.Photochem Photobiol 2008; 84:539–549.
5.   Busca R, Ballotti R. Cyclic AMP a key messenger in the regulation of skin pigmentation. Pigment Cell Res 2000; 13:60-69.
6.   Costin GE, Hearing VJ. Human skin pigmentation: melanocytes modulate skin color in response to stress. FASEB J 2007; 21:976–994.
7.   Ortonne JP, Nordlund JJ. Mechanisms That Cause Abnormal Skin Color, The Pigmentary System. Blackwell Publishing Ltd; 2007.p. 521-537.
8.   Im S, Kim J, On WY, Kang WH. Increased expression of alpha-melanocyte-stimulating hormone in the lesional skin of melasma. Br J Dermatol 2002; 146:165-167.
9.   Kang WH, Yoon KH, Lee ES, Kim J, Lee KB, Yim H, et al. Melasma: histopathological characteristics in 56 Korean patients. Br J Dermatol 2002; 146:228-237.
10. Maeda K, Fukuda M. Arbutin: mechanism of its depigmenting action in human melanocyte culture. J Pharmacol Exp Ther 1996; 276:765-769.
11. Ando H, Watabe H, Valencia JC, Yasumoto K, Furumura M, Funasaka Y, et al. Fatty acids regulate pigmentation via proteasomal degradation of tyrosinase: a new aspect of ubiquitin-proteasome function. J Biol Chem 2004; 279:15427-15433.
12. Mabberley DJ. The Plant-Book. Cambridge: Cambridge University Press; 2008.
13. Rechinger KH. Nepeta. In: Rechinger KH, editors. Flora Iranica, Labiatae. 30. Graz: Akademische Druc-u Verlagsanstalt; 1982.p. 108-216.
14. Taskina A, Javan M, Sonboli A, Semnanian S. Evaluation of the anti-nociceptive and anti-inflammatory effects of essential oil of Nepeta pogonosperma Jamzad et Assadi in rats. Daru 2012; 20:48.
15. Emami SA, Aghazari F. Les Phanerogames endemiques de la flore d’Iran. Teheran: Publications de I’Université de’Iran des Sciences Médicales; 2011.
16. Ghahreman A, Attar F. Biodiversity of plant species in Iran. Tehran: Tehran University Publication; 1999.
17. Jamzad Z. Nepeta menthoides Boiss. & Buhse and species allied to it in Iran. Iran J Bot 1991; 5:17-27.
18. Nadjafi F, Koocheki A, Honermeier B, Asili J. Autecology, ethnomedicinal and phytochemical studies of Nepeta binaludensis Jamzad a highly endangered medicinal plant of Iran. J Essent Oil Bear Pl 2009; 12:97-110.
19. Phillips R, Rix M. The Botanical Garden. London: Mac Milan; 2002.
20. García-Gavín J, González-Vilas D, Fernández-Redondo V, Toribio J. Pigmented contact dermatitis due to kojic acid. A paradoxical side effect of a skin lightener. Contact Dermat 2010; 62: 63–64.
21. Ali L, Ali S, Rizvi TS, Khan AL, Hassan Z, Al-Harrasi A, et al. Antioxidant flavonoids from Nepeta floccosa benth. Rec Nat Prod 2015; 9: 567-571.
22. Kraujalis P, Venskutonis PR, and Ragazinskiene O. Antioxidant activities and phenolic composition of extracts from Nepeta plant species. in 6th Baltic Conference on Food Science and Technology: Innovations for Food Science and Production, FOODBALT-2011. 2011. Jelgava.
23. Mothana RA. Chemical composition, anti-microbial and antioxidant activities of the essential oil of Nepeta deflersiana growing in Yemen. Rec Nat Prod 2012;  6: 189-193.
24. Pandey AK, Mohan M, Singh P, and Tripathi NN. Chemical Composition, Antioxidant and Anti-microbial Activities of the Essential Oil of Nepeta hindostana (Roth) Haines from India. Rec Nat Prod 2015; 9: 224-233.
25. Kurek-Górecka A, Rzepecka-Stojko A, Górecki M, Stojko J, Sosada M, and Swierczek-Zieba G. Structure and antioxidant activity of polyphenols derived from propolis. Molecules 2014;  19: 78-101.
26. Lambert JD and Elias RJ. The antioxidant and pro-oxidant activities of green tea polyphenols: A role in cancer prevention. Arch Biochem Biophys 2010; 501: 65-72.
27. Raederstorff D. Antioxidant activity of olive polyphenols in humans: A review. Int J Vit Nutr Res 2009; 79: 152-165.
28. O'Brien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 2000; 267:5421-5426.
29. Hosoi J, Abe E, Suda T, Kuroki T. Regulation of melanin synthesis of B16 mouse melanoma cells by 1 alpha, 25-dihydroxyvitamin D3 and retinoic acid. Cancer Res 1985; 45:1474-1478.
30. Kim YJ, No JK, Lee JS, Kim MS, Chung HY. Anti-melanogenic activity of 3,4-dihydroxyacetophenone: inhibition of tyrosinase and MITF. Biosci Biotechnol Biochem 2006; 70:532-534.
31. No JK, Kim YJ, Lee JS, Chung HY. Inhibition of melanogenic activity by 4,4'-dihydroxybiphenyl in melanoma cells. Biol Pharm Bull 2006; 29:14-16.
32. Ohguchi K, Banno Y, Nakagawa Y, Akao Y, Nozawa Y. Negative regulation of melanogenesis by phospholipase D1 through mTOR/p70 S6 kinase 1 signaling in mouse B16 melanoma cells. J Cell Physiol 2005; 205:444-451.
33. Kageyama A, Oka M, Okada T, Nakamura S, Ueyama T, Saito N, et al. Down-regulation of melano-genesis by phospholipase D2 through ubiquitin proteasome-mediated degradation of tyrosinase. J Biol Chem 2004; 279:27774-27780.
34. Kim DS, Kim SY, Park SH, Choi YG, Kwon SB, Kim MK, et al. Inhibitory effects of 4-n-butylresorcinol on tyrosinase activity and melanin synthesis. Biol Pharm Bull 2005; 28:2216-2219.
35. Mun YJ, Lee SW, Jeong HW, Lee KG, Kim JH, Woo WH. Inhibitory effect of miconazole on melanogenesis. Biol Pharm Bull 2004; 27:806-809.
36. Usuki A, Ohashi A, Sato H, Ochiai Y, Ichihashi M, Funasaka Y. The inhibitory effect of glycolic acid and lactic acid on melanin synthesis in melanoma cells. Exp Dermatol 2003; 2:43-50.
37. Slominski A, Tobin DJ, Shibahara S, Wortsman J. Melanin Pigmentation in Mammalian Skin and Its Hormonal Regulation. Physiol Rev 2004; 84:1155-1228.
38. Otreba M, Rok J, Buszman E, Wrzesniok D. Regulation of melanogenesis: the role of cAMP and MITF. Postepy Hig Med Dosw 2012; 66:33-40.
39. Li J, Song JS, Bell RJA, Tran TNT, Haq R, Liu H, et al. YY1 regulates melanocyte development and function by cooperating with MITF. PLoS Genetics 2012; 8: e1002688.
40. Levy C, Khaled M, and Fisher DE. MITF: master regulator of melanocyte development and melanoma oncogene. Trends Mol Med 2006; 12: 406-414.
41. Paillerets BBD, Lesueur F, and Bertolotto C. A germline oncogenic MITF mutation and tumor susceptibility. Eur J Cell Biol 2014; 93: 71-75.
42. Bertolotto C, Lesueur F, Giuliano S, Strub T, De Lichy M, Bille K, et al. A SUMOylation-defective MITF germline mutation predisposes to melanoma and renal carcinoma. Nature 2011; 480: 94-98.            
43. Hartman ML and Czyz M. Pro-survival role of MITF in melanoma. J Invest Dermatol 2015; 135: 352-358.
44. Rudloff U and Samuels Y. TYRO3-mediated regulation of MITF: A novel target in melanoma? Pigment Cell Melanoma Res 2010; 23: 9-11.
45. Kawano M, Matsuyama K, Miyamae Y, Shinmoto H, Kchouk ME, Morio T, et al. Anti-melanogenesis effect of Tunisian herb Thymelaea hirsuta extract on B16 murine melanoma cells. Exp. Dermatol 2007; 16:977-984.
46. Wu M, Hemesath TJ, Takemoto CM, Horstmann MA, Wells AG, Price ER, et al. c-Kit triggers dual phosphorylations, which couple activation and degradation of the essential melanocyte factor Mi. Genes Dev 2000; 14:301-312.
47. Yamahara M, Sugimura K, Kumagai A, Fuchino H, Kuroi A, Kagawa M, et al. Callicarpa longissima extract, carnosol-rich, potently inhibits melanogenesis in B16F10 melanoma cells. J Nat Med 2016; 70: 28-35.
48. Huang HC, Ho YC, Lim JM, Chang TY, Ho CL, and Chang TM. Investigation of the Anti-Melanogenic and Antioxidant Characteristics of Eucalyptus camaldule-nsis Flower Essential Oil and Determination of Its Chemical Composition. Int J Mol Sci 2015; 16: 10470-10490.