Sage extract and ascorbic acid derivative inhibit melanogenesis via downregulating keratinocyte-derived GM-CSF

Abstract

Salvia officinalis (sage) extract has demonstrated potential as a functional ingredient for skin care application. However, its effect and mechanism in regulating skin pigmentation remain largely unclear. This study investigated the effects of sage ethanol extract (SGE) on melanogenesis and its underlying molecular mechanisms. Treatment with SGE in a human skin equivalent model (3D-skin) suppressed melanin production. To clarify the mechanism of action, the study focused on senescence-associated secretory phenotype (SASP) factors, which are implicated in age-related pigmentation changes. q-PCR and ELISA analyses showed that SGE inhibits melanogenesis by suppressing the expression of granulocyte-macrophage colony-stimulating factor (GM-CSF), a known SASP factor in keratinocytes. Interestingly, a similar effect was observed with L-ascorbic acid 2-glucoside (AG), previously identified as a tyrosinase inhibitor. Importantly, p38 and JNK MAP-kinase were identified as upstream regulators of GM-CSF that are suppressed by SGE. These findings provide new insights into how SGE and AG regulate pigmentation via keratinocyte-derived GM-CSF, highlighting their potential in modulating skin tone and pigmentation through cellular signaling pathways.

Fig 1. SGE and AG additively reduce melanin production.

Human full-thickness skin equivalents containing melanocytes were treated with 0.1 μg/mL SGE and/or 2.5 mM AG for 11 days. UVB irradiation was applied every 2 days during the treatment period. Afterward, melanin content in the skin equivalents was measured and normalized to protein content. Data are presented as mean ± SEM values from three independent experiments. Statistical significance was calculated using one-way ANOVA followed by Dunnett’s test.

Fig 2. SGE inhibits melanogenesis indirectly through its effects on keratinocytes, rather than acting directly on melanocytes.

(a) NHEMs were treated with SGE or 2.5 mM AG for 3 days, after which melanin content was measured and normalized to protein content. Data are presented as mean ± SEM values from four independent experiments. (b) HPEKs were treated with SGE and AG, incubated for 30 min, and then exposed to UVB irradiation at a dose of 5 mJ/cm². Co-culturing with NHEMs was subsequently initiated in CnT-PR medium. Melanin contained in NHEMs was measured and normalized to protein content. Data are presented as mean ± SEM values from five independent experiments. Statistical significance was calculated using one-way ANOVA followed by Dunnett’s test.

Fig 3. SGE suppresses melanogenesis by inhibiting GM-CSF production in keratinocytes.

HPEKs were treated with SGE, AG, GM-CSF, and anti-GM-CSF antibody, incubated for 30 min, and then exposed to UVB irradiation at a dose of 5 mJ/cm². Subsequently, co-culturing with NHEMs was initiated in CnT-PR medium. Forty-eight hours after starting the co-culture, the following experiments were performed. (a) Quantitative PCR (q-PCR) analysis of CSF2 expression in HPEKs. Data are presented as mean ± SEM values from five independent experiments. (b) ELISA analysis of GM-CSF released from HPEKs. Data are presented as mean±SEM values from four independent experiments. (c) Measurement of melanin content in HPEKs, normalized to protein content. Data are represented as mean ± SEM values from four independent experiments. Statistical significance was calculated using one-way ANOVA followed by Dunnett’s test for (a) and (b), and Tukey’s test for (c).

Fig 4. CSF2 expression is regulated by p38 and JNK MAPK in response to UVB exposure.

(a) HPEKs were exposed to UVB radiation (5 mJ/cm²) and incubated for the indicated times. The extracted proteins were then immunoblotted with the indicated antibodies. Graphs show the relative band intensities as determined by ImageJ and plotted as the mean ± SEM values of five independent experiments. (b) q-PCR analysis of CSF2 expression in HPEKs. HPEKs were exposed to UVB radiation (5 mJ/cm²) and incubated for 72 h. SP600125 (10 μM) and SB203580 (10 μM) were added 1 h before UVB exposure. Data are presented as mean ± SEM values from six independent experiments. Statistical significance was calculated using two-way ANOVA followed by Šídák's test (a) or one-way ANOVA followed by Dunnett's test (b).