폴리페놀의 물리, 화학, 생물학적 특성을 이용한 효과적인 미백성분의 개발

Abstract

Research and development of functional cosmetics using natural resources are highly demanding in academics and industrial sectors due to increase in interest of skin health and personal beauty. Among various natural resources showing antioxidant activity, phenolic compounds (known as polyphenols) have been frequently utilized as cosmetic ingredients. Particularly, quercetin as a model polyphenol compound has been well known to be a strong antioxidant as well as anti-whitening agent in many literatures. Some researchers addressed that quercetin enhanced melanin biosynthesis and showed toxicity in skin cells and others did not. Therefore, an efficacy of quercetin for a cosmetic ingredient is still debating and the role of it needs to be further investigated by suitable materials and methods. In order to show whether quercetin is suitable as anti-melanogenic agent or not, firstly, the toxicity test of quercetin and its derivatives has been performed using various cell lines and zebrafish models before anti-melanogenesis study. The synthesis of quercetin derivatives was done to reduce toxicity of quercetin and to increase anti-melanogenesis activity. Several quercetin derivatives have been synthesized by SN2 reaction with quercetin and appropriate reagents, then their cytotoxicities and anti-melanogenesis activities were investigated. In addition to quercetin and its derivatives, a cyanidin-3-o-galactoside (C3G) in extracts of aronia berry (known as chokeberry) has been examined for its activity because of strong antioxidant activity and little literature of C3G. In this study, some assays about cytotoxicity and anti-melanogenesis for quercetin, quercetin derivatives, aronia extracts, and C3G were done in B16F10 melanoma cells with and without induction of melanin formation after α–melanocyte stimulating hormone (MSH) treatment. Cytotoxic concentration levels of quercetin, quercetin derivatives, aronia extracts, and C3G were > 10 µM, > 50 µg/mL, > 50 µg/mL, and > 50 µM, respectively. Importantly, mushroom inhibitory tyrosinase assay in in-vitro cell free system showed anti-melanogenic effect while in in-vitro B16F10 melanoma cell system it did not. Mortality rate (%) of quercetin in in-vivo zebrafish displayed about 8-12% in all conditions and tyrosinase activity increased with quercetin concentration. Moreover, melanin formation was promoted in 10-200 µmol/L range. Therefore, it is believed that quercetin is not a good whitening agent. Next, quercetin derivatives were successfully prepared using seven substrates. Among them, oleyl bromide-substituted quercetin exhibited low cytotoxic concentration level at > 50 µM, which is five-fold less toxic compared to original quercetin. Expressions of tyrosinase, tyrosinase-related protein-1 (TRP-1) and TRP-2 were considered to be inhibited in in vitro B16F10 melanoma cells. Total polyphenol contents of aronia extracts were analyzed to be 115.23 mg/g as gallic acid equivalent. The total anthocyanin contents were measured to be 4.19 mg/g as C3G equivalent. Antioxidant assay for DPPH and ABTS radical scavenging activity showed IC50 value of 9.63 µg/mL and 42.20 µg/mL, respectively. While, IC50 value in ORAC assay was 140.25 µM TE/mL. The IC50 values of SOD and catalase activity assay were 28.25 µg/mL and 25.52 µg/mL, respectively. Cytotoxic concentration level of aronia extracts in B16F10 melanoma cells was > 100 µg/mL and protein expression variations in tyrosinase and TRP-1 were negligible but TRP-2 expression was moderately inhibited by the extracts. For C3G compound, cytotoxic concentration level of C3G was > 50 µM. Expression levels in tyrosinase, TRP-1 and TRP-2 were decreased which implies potential skin whitening agents. Mortality rate (%) of C3G in in vivo zebrafish was from 100 µmol/L. At 30-100 µmol/L concentration range, the antioxidant activity levels were dose-dependently increased but it was gradually decreased again up to 200 µmol/L. For anti-melanogenesis assay using in vivo zebrafish model with C3G of 10-100 µmol/L range, it showed skin whitening effect but at > 150 µmol/L, it did not show the effect. Based on the results from in vitro and in vivo assay using quercetin derivatives and C3G summary, these compounds could be used for potential skin-whitening agents in functional cosmetics. Purification and structure elucidation of the synthesized quercetin derivatives are under way and future clinical trials should be done to launch it in the market.