Anti-Pigmentary Effect of (-)-4-Hydroxysattabacin from the Marine-Derived Bacterium Bacillus sp

Abstract

Bioactivity-guided isolation of a crude extract from a culture broth of Bacillus sp. has led to the isolation of (-)-4-hydroxysattabacin (1). The inhibitory effect of (-)-4-hydroxysattabacin (1) was investigated on melanogenesis in the murine melanoma cell line, B16F10, and human melanoma cell line, MNT-1, as well as a pigmented 3D-human skin model. (-)-4-Hydroxysattabacin treatment decreased melanin contents in a dose-dependent manner in α-melanocyte stimulating hormone (α-MSH)-stimulated B16F10 cells. Quantitative real time PCR (qRT-PCR) demonstrated that treatment with (-)-4-hydroxysattabacin down-regulated several melanogenic genes, including tyrosinase, tyrosinase-related protein 1 (TRP-1), and tyrosinase-related protein 2 (TRP-2) while their enzymatic activities were unaffected. The anti-melanogenic effects of (-)-4-hydroxysattabacin were further demonstrated in a pigmented 3D human epidermal skin model, MelanodermTM, and manifested as whitening and regression of melanocyte activation in the tissue.

Keywords: (-)-4-hydroxysattabacin; Bacillus sp.; anti-pigmentary effect; marine-derived; melanogenesis.

Figure 1

Effects of (-)-4-hydroxysattabacin (4OH-ST, 1) and (-)-sattabacin (2) on melanin contents and cell viability on B16F10 cells. (a) Chemical structures of (-)-4OH-ST (1) and (-)-sattabacin (2); (b) Melanin contents were measured by melanin assay; (c) Cell viability was determined by WST-1 assay. The cells were treated with arbutin at 50 µg/mL, (-)-4OH-ST (1) or (-)-sattabacin (2) at the indicated concentrations for 72 h. Data are presented as the mean ± standard deviation (SD) (n = 3–5).

Figure 2

Effects of (-)-4-hydroxysattabacin (4OH-ST), 1) treatment on B16F10 cells with various concentrations of conventional agents. (a) Melanin contents were measured by melanin assay; (b) Cell viability was determined by MTT assay. The cells were treated with (-)-4OH-ST (1), arbutin, or kojic acid at the indicated concentrations for 48 h; (c) Morphological changes of B16F10 cells were observed under optical microscopy (400×). Data are presented as the mean ± SD (n = 3).

Figure 3

Effects of (-)-4-hydroxysattabacin (4OH-ST), 1) on mushroom tyrosinase activity. Tyrosinase enzymatic activity was investigated using a mushroom tyrosinase assay with (a) l-tyrosine and (b) l-dopa (3,4-dihydroxyphenylalanine) as substrates. Data are presented as the mean ± SD (n = 3).

Figure 4

Effect of (-)-4-hydroxysattabacin (4OH-ST, 1) on mRNA and protein level of B16F10 cells. mRNA expression levels of (a) tyrosinase; (b) TRP-2; and (c) TRP-1 in B16F10 cells were determined by real-time PCR; (d) Protein levels were determined by Western blotting. The cells were treated with bisabolol at 50 µM and (-)-4OH-ST (1) at the indicated concentrations for 24 h. Data are presented as the mean ± SD (n = 4, * p < 0.05 and ** p < 0.01).

Figure 5

Effects of (-)-4-hydroxysattabacin (4OH-ST), 1) treatment on MNT-1 cells. (a) Cellular tyrosinase activity data using l-dopa as substrate; (b) Macroscopic view of MNT-1 cell pellets; (c) Cell viability was determined by MTT assay. MNT-1 cells were stimulated by indicated materials with various concentrations for 72 h. Data are presented as the mean ± SD (n = 3).

Figure 6

Effect of (-)-4-hydroxysattabacin (4OH-ST, 1) on the Melanoderm^TM 3D skin model. (a) Color of 3D human skin tissue model (Melanoderm^TM; MatTek) and hematoxylin and eosin (H & E) stained tissues; (b) The degree of hypopigmentation in Melanoderm^TM as measured by the ∆L value in skin tissue between baseline and 14 days after (-)-4OH-ST (1) or vehicle treatment; (c) Cell viability was further confirmed in a 3D reconstituted human epidermis model, Keraskin™, using WST-1 assay. Data are presented as the mean ± SD (n = 3).