Antioxidant and Antimelanogenic Activities of Kimchi-Derived Limosilactobacillus fermentum JNU532 in B16F10 Melanoma Cells

Abstract

Melanin is a natural skin pigment produced by specialized cells called melanocytes via a multistage biochemical pathway known as melanogenesis, involving the oxidation and polymerization of tyrosine. Melanogenesis is initiated upon exposure to ultraviolet (UV) radiation, causing the skin to darken, which protects skin cells from UVB radiation damage. However, the abnormal accumulation of melanin may lead to the development of certain skin diseases, including skin cancer. In this study, the antioxidant and antimelanogenic activities of the cell-free supernatant (CFS) of twenty strains were evaluated. Based on the results of 60% 2,2-diphenyl-1-picrylhydrazyl scavenging activity, 21% 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) scavenging capacity, and a 50% ascorbic acid equivalent ferric reducing antioxidant power value, Limosilactobacillus fermentum JNU532 was selected as the strain with the highest antioxidant potential. No cytotoxicity was observed in cells treated with the CFS of L. fermentum JNU532. Tyrosinase activity was reduced by 16.7% in CFStreated B16F10 cells (but not in the cell-free system), with >23.2% reduction in melanin content upon treatment with the L. fermentum JNU532-derived CFS. The inhibitory effect of the L. fermentum JNU532-derived CFS on B16F10 cell melanogenesis pathways was investigated using quantitative reverse transcription polymerase chain reaction and western blotting. The inhibitory effects of the L. fermentum JNU532-derived CFS were mediated by inhibiting the transcription of TYR, TRP-1, TRP-2, and MITF and the protein expression of TYR, TRP-1, TRP-2, and MITF. Therefore, L. fermentum JNU532 may be considered a potentially useful, natural depigmentation agent.

Keywords: Limosilactobacillus; Melanin; antioxidant; microphthalmia-associated transcription factor; tyrosinase.

Fig. 1

(A) DPPH radical scavenging activity. (B) ABTS radical scavenging activity. (C) Ascorbic acid equivalent FRAP value of cell-free supernatants of tested strains incubated at 37°C for 18 h in MRS medium. Ascorbic acid (0.05 mg/ml) was used as a positive control. The mean values of samples indicated with different letters are significantly different. Data are presented as the mean ± standard deviation (n = 3). DPPH, 2,2-diphenyl-1-picrylhydrazyl; ABTS, (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)); FRAP, ferric reducing antioxidant power.

Fig. 2

(A) Inhibition of tyrosinase activity by tested strains derived CFS. (B) Tyrosinase activity in B16F10 cells treated with CFS. Arbutin (500 μM) was used as a positive control. The mean values of samples indicated with different letters are significantly different. Significant differences are indicated by different letters according to melanin content from low to high. Experimental groups were normalized to control groups, and the data were analyzed using the t-test. *** ρ < 0.001 for 500 μM Arbutin versus sample, respectively. Data are presented as mean ± standard deviation (n = 3). CFS, cell-free supernatant.

Fig. 3

(A) Viability and (B) melanin content of B16F10 cells treated with CFS. DMEM was used as a control. Arbutin (500 μM) was used as a positive control. Significant differences are indicated using different letters according to melanin content from low to high. Experimental groups were normalized to control groups, and the data were analyzed using a t-test. *ρ < 0.05, **ρ < 0.01, ***ρ < 0.001 for control versus sample, respectively. Data are presented as the mean ± standard deviation (n = 3). CFS, cell-free supernatant; DMEM, Dulbecco’s modified Eagle’s medium.

Fig. 4

(A) Relative mRNA expression of TYR, TRP1, TRP2, and MITF in B16F10 cells treated with the CFS of Limosilactobacillus fermentum JNU532. DMEM was used as a control. Arbutin (500 μM) was used as a positive control. The mean values of the samples indicated with different letters are significantly different. (B) Inhibitory effects of L. fermentum JNU532-derived CFS on protein expression of TYR, TRP1, TRP2, and MITF in B16F10 cells incubated at 37°C for 18 h in MRS medium. DMEM was used as a control. The protein levels of the tyrosinase family of enzymes were analyzed via western blotting, and protein loading amounts were confirmed via β-actin expression. Experimental groups were normalized to control groups, and the data were analyzed using the t-test. **ρ < 0.01, ***ρ < 0.001 for control versus sample, respectively. Data are presented as the mean ± standard deviation (n = 3).CFS, cell-free supernatant; DMEM, Dulbecco’s modified Eagle’s medium.

Fig. 5

Acid and bile acid tolerances of Limosilactobacillus fermentum JNU532.