Epidermal hydrating and anti-melanogenic effects of rice-derived glucosylceramides and elasticamide on cell basis evaluation
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Presented by: Hiroshi Shimoda
Introduction: Skin ceramides (Cer) are lipids mainly existing in stratum corneum and play pivotal rolls on epidermal hydrating and barrier function with other lipids and moisturizing proteins such as filaggrin. Especially 12 major Cer form intracellular lipids in stratum corneum. In terms of plant-derived major Cer, glucosylceramides (GlcCer) dominantly exist in wide variety of botanical resources. Among them, GlcCer are well studied about clinical effects on skin barrier function. As other biological effects of GlcCer on skin condition, maize cerebroside mixture has been reported to suppress melanogenesis in B16 melanoma cells. Rice GlcCer consist of multiple molecules which are composed of different types of sphingoid bases and multiple length of free fatty acids. Beside GlcCer fraction, there are free Cer as minor constituents including elasticamide (ceramide[AP]). However, no study results have been reported regarding epidermal hydrating and anti-melanogenic effects of these single molecules of GlcCer and elasticamide.
Purpose: In this study we have isolated 13 GlcCer and elasticamide (Cer[t:18:0/24:0]) from gummy by-products of rice bran oil and evaluated epidermal moisturizing activity and anti-melanogenic effects.
Methods: GlcCer and elasticamide were purified from GlcCer-rich fraction of the by-product of rice bran oil by a normal phase middle pressure column chromatography and reversed-phase HPLC. Epidermal hydrating effects on the reconstructed human epidermal keratinization (RHEK) model (J-TEC) were evaluated by measuring transepidermal water loss (TEWL) chronologically after adding samples (10 μM) to the membrane side of RHEK. For evaluation of elasticamide in RHEK, we determined Cer contents by TLC and measured mRNA and protein expressions of Cer synthesizing enzymes by real time PCR and western blotting analysis. On the other hand, anti-melanogenic effect in B16 melanoma cells was performed inducing melanogenesis by theophylline. For elasticamide and GlcCer[d18:2 (4E,8Z)/20:0], we also evaluated anti-melanogenic effects in a human 3D cultured epidermal melanocytes (MEL-300-A, Kurabo Ind. Ltd.), and mRNA expressions of melanin synthesizing enzymes and ATP concentration in normal melanocytes.
Results: As a result of evaluation of TEWL from the surface of RHEK, 10 μM of 4E,8Z type GlcCer including GlcCer[d18:2(4E,8Z)/18:0], GlcCer[d18:2(4E,8Z)/20:0], GlcCer[d18:2(4E,8Z)/22:0], GlcCer[d18:2(4E,8Z)/24:0], GlcCer[d18:2(4E,8Z)/26:0] and elasticamide decreased TEWL by 7-day treatment. Among GlcCer, TEWL was improved depending on the length of fatty acids and GlcCer[d18:2(4E,8Z)/26:0] exhibited most potent hydrating effect. The hydrating effect of elasticamide was stronger than GlcCer[d18:2(4E,8Z)/26:0]. Moreover, elasticamide (10 μg/mL, 14.3 μM) significantly increased total Cer and Cer[NS/NDS] with enhancing mRNA and protein expressions of GlcCer synthase. On melanogenesis in B16 melanoma cells, GlcCer[d18:2(4E,8Z)/18:0], GlcCer[d18:2(4E,8Z)/20:0], and elasticamide significantly suppressed melanin production with IC50 values: 6.6, 5.2, and 3.9 μM. Elasticamide but not GlcCer[d18:2 (4E,8Z)/20:0] suppressed melanogenesis in a human 3D cultured epidermal melanocytes and tyrosinase related protein 1 (TYRP-1) expression in normal human melanocytes.
Discussion: Rice-derived GlcCer were found to exhibit fatty acid length-dependent epidermal hydrating effects. Thus, longer fatty acids are appeared to enhance hydrating effects of GlcCer. Elasticamide, a Cer[AP] showed strongest hydrating effect with enriching Cer[NS/NDS] by enhancing GlcCer synthase expression. However, it did not increase Cer[AP]. Further studies are required to find the detailed metabolism and Cer increase mechanism of elasticamide. On the other hand, GlcCer[d18:2(4E,8Z)/18:0] and GlcCer[d18:2(4E,8Z)/20:0] suppressed melanogenesis in B16 melanoma cells. However, the structure-activity relationship of GlcCer was not clarified except the fact that longer fatty acid length potentiated cytotoxicity. Elasticamide suppressed melanogenesis without cytotoxicity in both melanoma cells and normal melanocytes. Suppression of TYRP-1 and ATP were involved in the suppressive mechanism of elasticamide.
Conclusion: We demonstrated that some rice GlcCer and Cer suppressed epidermal dehydration in RHEK. ω-Hydroxy type Cer such as Cer[EOS] have been reported to possess stronger hydrating effect. However, detailed reports regarding GlcCer were not available. Thus this study is the first report which examined hydrating effects of GlcCer in single molecule level. Anti-melanogenic effects of rice-derived GlcCer and Cer had not been clarified too. Our result showed that elasticamide is one of the promising skin whitening compound in rice Cer with strong barrier function.
Purpose: In this study we have isolated 13 GlcCer and elasticamide (Cer[t:18:0/24:0]) from gummy by-products of rice bran oil and evaluated epidermal moisturizing activity and anti-melanogenic effects.
Methods: GlcCer and elasticamide were purified from GlcCer-rich fraction of the by-product of rice bran oil by a normal phase middle pressure column chromatography and reversed-phase HPLC. Epidermal hydrating effects on the reconstructed human epidermal keratinization (RHEK) model (J-TEC) were evaluated by measuring transepidermal water loss (TEWL) chronologically after adding samples (10 μM) to the membrane side of RHEK. For evaluation of elasticamide in RHEK, we determined Cer contents by TLC and measured mRNA and protein expressions of Cer synthesizing enzymes by real time PCR and western blotting analysis. On the other hand, anti-melanogenic effect in B16 melanoma cells was performed inducing melanogenesis by theophylline. For elasticamide and GlcCer[d18:2 (4E,8Z)/20:0], we also evaluated anti-melanogenic effects in a human 3D cultured epidermal melanocytes (MEL-300-A, Kurabo Ind. Ltd.), and mRNA expressions of melanin synthesizing enzymes and ATP concentration in normal melanocytes.
Results: As a result of evaluation of TEWL from the surface of RHEK, 10 μM of 4E,8Z type GlcCer including GlcCer[d18:2(4E,8Z)/18:0], GlcCer[d18:2(4E,8Z)/20:0], GlcCer[d18:2(4E,8Z)/22:0], GlcCer[d18:2(4E,8Z)/24:0], GlcCer[d18:2(4E,8Z)/26:0] and elasticamide decreased TEWL by 7-day treatment. Among GlcCer, TEWL was improved depending on the length of fatty acids and GlcCer[d18:2(4E,8Z)/26:0] exhibited most potent hydrating effect. The hydrating effect of elasticamide was stronger than GlcCer[d18:2(4E,8Z)/26:0]. Moreover, elasticamide (10 μg/mL, 14.3 μM) significantly increased total Cer and Cer[NS/NDS] with enhancing mRNA and protein expressions of GlcCer synthase. On melanogenesis in B16 melanoma cells, GlcCer[d18:2(4E,8Z)/18:0], GlcCer[d18:2(4E,8Z)/20:0], and elasticamide significantly suppressed melanin production with IC50 values: 6.6, 5.2, and 3.9 μM. Elasticamide but not GlcCer[d18:2 (4E,8Z)/20:0] suppressed melanogenesis in a human 3D cultured epidermal melanocytes and tyrosinase related protein 1 (TYRP-1) expression in normal human melanocytes.
Discussion: Rice-derived GlcCer were found to exhibit fatty acid length-dependent epidermal hydrating effects. Thus, longer fatty acids are appeared to enhance hydrating effects of GlcCer. Elasticamide, a Cer[AP] showed strongest hydrating effect with enriching Cer[NS/NDS] by enhancing GlcCer synthase expression. However, it did not increase Cer[AP]. Further studies are required to find the detailed metabolism and Cer increase mechanism of elasticamide. On the other hand, GlcCer[d18:2(4E,8Z)/18:0] and GlcCer[d18:2(4E,8Z)/20:0] suppressed melanogenesis in B16 melanoma cells. However, the structure-activity relationship of GlcCer was not clarified except the fact that longer fatty acid length potentiated cytotoxicity. Elasticamide suppressed melanogenesis without cytotoxicity in both melanoma cells and normal melanocytes. Suppression of TYRP-1 and ATP were involved in the suppressive mechanism of elasticamide.
Conclusion: We demonstrated that some rice GlcCer and Cer suppressed epidermal dehydration in RHEK. ω-Hydroxy type Cer such as Cer[EOS] have been reported to possess stronger hydrating effect. However, detailed reports regarding GlcCer were not available. Thus this study is the first report which examined hydrating effects of GlcCer in single molecule level. Anti-melanogenic effects of rice-derived GlcCer and Cer had not been clarified too. Our result showed that elasticamide is one of the promising skin whitening compound in rice Cer with strong barrier function.