Anti-Melanogenic Properties of Greek Plants. A Novel Depigmenting Agent from Morus alba Wood

Abstract

In therapeutic interventions associated with melanin hyperpigmentation, tyrosinase is regarded as a target enzyme as it catalyzes the rate-limiting steps in mammalian melanogenesis. Since many known agents have been proven to be toxic, there has been increasing impetus to identify alternative tyrosinase inhibitors, especially from natural sources. In this study, we investigated 900 extracts from Greek plants for potential tyrosinase inhibitive properties. Among the five most potent extracts, the methanol extract of Morus alba wood (MAM) demonstrated a significant reduction in intracellular tyrosinase and melanin content in B16F10 melanoma cells. Bioassay-guided isolation led to the acquisition of twelve compounds: oxyresveratrol (1), kuwanon C (2), mulberroside A (3), resorcinol (4), dihydrooxyresveratol (5), trans-dihydromorin (6), 2,4,3'-trihydroxydihydrostilbene (7), kuwanon H (8), 2,4-dihydroxybenzaldehyde (9), morusin (10), moracin M (11) and kuwanon G (12). Among these, 2,4,3'-trihydroxydihydrostilbene (7) is isolated for the first time from Morus alba and constitutes a novel potent tyrosinase inhibitor (IC50 0.8 ± 0.15). We report here for the first time dihydrooxyresveratrol (5) as a potent natural tyrosinase inhibitor (IC50 0.3 ± 0.05). Computational docking analysis indicated the binding modes of six tyrosinase inhibitors with the aminoacids of the active centre of tyrosinase. Finally, we found both MAM extract and compounds 1, 6 and 7 to significantly suppress in vivo melanogenesis during zebrafish embryogenesis.

Keywords: 2,4,3′-trihydroxydihydrostilbene; B16F10 melanoma cells; Greek flora; Morus alba; computational docking analysis; dihydroxyresveratrol; melanogenesis; tyrosinase inhibition; zebrafish.

Figure 1

Relative (%) cell survival (MTT assay) of B16F10 cells after treatment with increasing concentrations of five selected extracts for 48 h; Bars, ±SD (n = 3). *, p < 0.05; **, p < 0.01 vs. controls set to 100%.

Figure 2

Effect of the five selected extracts on (A) intracellular tyrosinase activity inhibition (Bars, ±SD (n = 3) *, p < 0.05; **, p < 0.01 vs. controls set to 100%) and (B) on suppressing melanin cellular accumulation.

Figure 3

Compounds isolated from Morus alba wood.

Figure 4

(A) Superposition of tropolone and low energy conformations of 1, 5 and 7 inside the binding cavity of mushroom tyrosinase. Low energy structures of mushroom tyrosinase in complex with (B) oxyresveratrol (1); (C) 2,4,3′-trihydroxydihydrostilbene (7) and (D) dihydrooxyresveratrol (5).

Figure 5

Melanogenesis inhibition on zebrafish embryos treated from 24 hpf to 48 hpf with Morus alba compounds. Bright field images of embryos treated with (A) PTU used as a positive control of melanogenesis inhibition; (B) DMSO used as negative control; (C) 70 μg/mL Morus alba methanol wood extract (MAM); (D) 70 μg/mL CPC F33 fraction that contains the compounds 2,4,3-trihydrodihydrostilbene (7), moracin M (11) and kuwanon G (12); (E) 10 μg/mL CPC F38-42 fraction that contains the compounds oxyresveratrol (1) and trans-dihydromorin (6); (F) 10 μg/mL CPC F43 fraction that contains the compounds oxyresveratrol (1) and dihydrooxyresveratrol (5); (G–L) purified compounds from CPC fractions: (G) 50 μg/mL oxyresveratrol (1); (H) 7 μg/mL kuwanon C (2); (I) 50 μg/mL dihydrooxyresveratrol (5); (J) 50 μg/mL trans-dihydromorin (6); (K) 50 μg/mL 2,4,3′-trihydrodihydrostilbene (7); (L) 10 μg/mL moracin M (11); Scale bar: 100μM.