Original article Compounds isolated from the aerial part of Crataegus azarolus inhibit growth of B16F10 melanoma cells and exert a potent inhibition of the melanin synthesis Nadia Mustapha a,b , Ime `n Mokdad Bze ´ ouich a,b , Kamel Ghedira b , Thierry Hennebelle c , Leila Chekir-Ghedira a,b, * a Laboratoire de biologie cellulaire et mole ´culaire, faculte ´ de me ´decine dentaire, universite ´ de Monastir, rue Avicenne, 5000 Monastir, Tunisia b Unite ´ de substances naturelles bioactives et biotechnologie UR12ES12, faculte ´ de pharmacie de Monastir, universite ´ de Monastir, rue Avicenne, 5000 Monastir, Tunisia c E.A. 4481, laboratoire de pharmacognosie, faculte ´ de pharmacie, universite ´ de Lille 2, B.P. 83, 59006 Lille cedex, France 1. Introduction Melanin is the major pigment for color of human skin. It is secreted by melanocyte cells in basal layer of the epidermis [1]. Melanin may be overproduced with chronic sun exposure, melasma, or other hyper pigmentation diseases [2]. Therefore, a number of depigmenting agents have been developed for cases of undesirable skin discoloration. Tyrosinase is a key enzyme that catalyzes melanin synthesis in melanocytes [3]. Melanin biosynth- esis can be inhibited by avoiding ultraviolet (UV) exposure, by inhibition of melanocyte metabolism and proliferation [4], by inhibition of tyrosinase, or by removal of melanin by corneal ablation. Tyrosinase inhibitors therefore can be clinically useful for the treatment of some dermatological disorders associated with melanin hyper pigmentation. They also find uses in cosmetics for whitening and depigmentation after sunburn. Traditional herbal medicines provide an interesting, largely unexplored source for development of potential new drugs. The potential use of traditional herbal medicines for development of new skin-care cosmetics has been emphasized recently [5]. In the continuing search for new effective melanogenesis inhibitors as skin-whitening agents from natural sources, the focus was on the Crataegus azarolus leaves extracts and compounds by checking their influence on the protein expression levels of melanin biosynthesis enzymes. 2. Material and methods 2.1. Reagents and solvents Trypsin, penicillin, streptomycin, vitamins, sodium pyruvate, RPMI-1640 medium, non-essential amino acids and fetal bovine serum were purchased from Sigma Cell Culture (Courtaboeuf, France). 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Biomedicine & Pharmacotherapy 69 (2015) 139–144 A R T I C L E I N F O Article history: Received 8 October 2014 Accepted 5 November 2014 Keywords: B16F10 melanoma cells Crataegus azarolus Tyrosinase Melanin A B S T R A C T Poor therapeutic results have been reported for treatment of malignant melanoma; therefore in this study, we have investigated inhibitory capacity of vitexin-2 00 -O-rhamnoside as well as the extract from which it was isolated, i.e. the ethyl acetate extract obtained from the leaves of Crataegus azarolus, on mouse melanoma (B16F10) proliferation. Cell viability was determined using the 3-(4, 5-dimethylthia- zol-2-yl)-2,5-diphenyltetrazolium bromide assay. In addition, amounts of melanin and tyrosinase were measured spectrophotometrically at 475 nm. Ethyl acetate extract and vitexin-2 00 -O-rhamnoside exhibited significant anti-proliferative activity against B16F10 melanoma cells after incubation for 48 hours with IC 50s of 50 mg/mL and 20 mM, respectively. Furthermore, these two compounds have the ability to reduce the melanin content by inhibiting the tyrosinase activity of B16F10 cells. Thus, further investigations are merited to ascertain their potential application in treating hyperpigmentation disorders. ß 2014 Published by Elsevier Masson SAS. * Corresponding author. Laboratory of Cellular and Molecular Biology, Faculty of Dental Medicine, University of Monastir, rue Avicenne, Monastir 5000, Tunisia. Tel./fax: +216 73 461 150. E-mail address: Leila.chekir@laposte.net (L. Chekir-Ghedira). Available online at ScienceDirect www.sciencedirect.com http://dx.doi.org/10.1016/j.biopha.2014.11.010 0753-3322/ß 2014 Published by Elsevier Masson SAS.