Introduction
!Melanin is a biopigment synthesized in melanosomes. Melanosomes enclosing melanin polymers are transferred from melanocytes to surrounding keratinocytes in the human epidermis. The color of human skin is determined primarily by the quantity, type, and distribution of melanin in keratinocytes [1,2]. Melanin has a critical role in the protection of the skin from solar ultraviolet (UV) radiation [3], but excess synthesis of melanin leads to hyperpigmentation disorders such as melasma, lentigo, and age spots [4]. Hence, the inhibitors of melanin biosynthesis have received considerable attention in clinical and cosmetic research, and melanin inhibitors such as hydroquinone [5], kojic acid [6,7], arbutin [8,9], and linoleic acid [10,11] have been discovered. "Sappan Lignum" is the heartwood of sappanwood (Caesalpinia sappan Linn.; Fabaceae) and is used both as a dyestuff and as an herbal medicine [12]. It has been used to treat wounds, skin diseases, leprosy, dysentery, menorrhagia, menoxenia, leukorrhea, and diabetic complications [13]. Several pharmacological studies have been undertaken focusing on the extracts and constituents of this plant as well as on the various activities found (e.g., immunomodulatory, . However, the effect on melanin synthesis of the extracts and constituents of this plant has not been studied in detail. In the present study, we investigated the inhibitory effects of the methanol (MeOH) extract of sappanwood on melanin synthesis in human melanoma HMV-II cells stimulated with forskolin. The MeOH extract showed inhibitory activity, and six compounds (one of which is novel) were isolated as active principles from the extract. Furthermore, the mechanism of action for the most potent inhibitory compound, brazilin 5, was investigated.
Abstract! Sappanwood (Caesalpinia sappan Linn.) is used as an herbal medicine. It is sometimes used to treat skin damage or as a facial cleanser. In the present study, the methanol (MeOH) extract of sappanwood was found to inhibit melanin synthesis in cultured human melanoma HMV-II cells stimulated with forskolin, and six active compounds (1-5 and 7) were isolated from the extract along with a non-active compound (6). Compounds 2-7 were identified as sappanchalcone (2), 3′-deoxy-4-Omethylsappanol (3), brazilein, (4), brazilin (5), sappanol (6), and 4-O-methylsappanol (7). Compound 1 was a new compound, and its structure was determined to be (6aS,11bR)-7,11b-dihydro-6H-indeno[2,1-c]chromene-3,6a,10,11-tetrol by spectroscopic analyses. Among the six active compounds, brazilin (5) (EC 50 : 3.0 ± 0.5 µM) and 4-O-methylsappanol (7) (EC 50 : 4.6 ± 0.7 µM) strongly suppressed melanin synthesis in HMV-II cells. Bioactive compounds showed moderate cytotoxicities against HMV-II cells with IC 50 values of 83.1 ± 4.0 µM (for 2), 72.0 µM ± 2.4 (for 3), 33.8 ± 1.1 µM (for 4), 18.4 ± 0.8 µM (for 5), and 20.2 ± 0.8 (for 7), respectively. Brazilin (5) selectively suppressed the expression of mRNAs for tyrosinase-related protein (TYRP) 2 and tyrosinase but did not influe...
