cosmetics Article Inhibitory Effect and Mechanism of Scutellarein on Melanogenesis Liyun Dai 1, Lihao Gu 1 and Kazuhisa Maeda 1,2,* 1 Bionics Program, Tokyo University of Technology Graduate School, 1404-1 Katakuramachi, Hachioji City, Tokyo 192-0982, Japan; [email protected] (L.D.); [email protected] (L.G.) 2 School of Bioscience and Biotechnology, Tokyo University of Technology, 1404-1 Katakuramachi, Hachioji City, Tokyo 192-0982, Japan * Correspondence: [email protected]; Tel.: +81-42-637-2442 Abstract: Fairer skin is preferred in many Asian countries and there is a high demand for skin whitening and lightening products. However, in recent years, problems related to the safety of using whitening agents have emerged. This study demonstrates that plant-derived scutellarein effectively inhibits melanogenesis in B16 melanoma cells. However, baicalein, which is similar to scutellarein in its chemical structure, does not show any inhibitory effect on melanogenesis. Cellular tyrosinase activity is decreased by scutellarein in a dose-dependent manner. No cytotoxicity is observed at the effective concentration range. Additionally, both the protein and mRNA levels of tyrosinase are significantly decreased by scutellarein. Further, the risk of leukoderma development also is determined by evaluating the production of free hydroxyl radicals (˙OH); scutellarein treatment does not induce ˙OH production. Scutellarein shows no risk of causing leukoderma. Our results suggest that scutellarein or plant extracts containing high concentrations of scutellarein have the potential to inhibit melanin production and serve as cosmetic skin-lightening agents. Keywords: scutellarein; melanogenesis; tyrosinase; hydroxyl radical Citation: Dai, L.; Gu, L.; Maeda, K. Inhibitory Effect and Mechanism of 1. Introduction Scutellarein on Melanogenesis. Accompanying an increase in people’s awareness of their own beauty and esthetic Cosmetics 2021 8 , , 15. https:// appeal, an increasing number of people are pursuing cleaner and fairer skin [1]. Recently, doi.org/10.3390/cosmetics8010015 the demand for whitening products, mainly skin-lightening cosmetics, has increased [2]. According to the results of a cosmetics market survey conducted by Japan’s Fuji Keizai Received: 9 January 2021 in 2018 [3], consumers have high expectations for medicinal cosmetics with anti-freckle Accepted: 9 February 2021 Published: 15 February 2021 and anti-pigmentation properties, and the market demand for skin-lightening cosmetics is expected to gradually increase in the future. However, the leukoderma incident of Kanebo Publisher’s Note: MDPI stays neutral in 2013 [4] brought more attention to the safety of cosmetic whitening ingredients. Approx- β with regard to jurisdictional claims in imately 20 active ingredients, including hydroquinone- -D-glucoside (arbutin, ARB), 4-n- published maps and institutional affil- butylresorcinol (4BR), 3-O-ethyl ascorbic acid, tranexamic acid, and 4-methoxysalicylic acid iations. potassium salt (4MSK), have been approved for use in quasi-drug cosmetics in Japan [5–9]. However, rhododendrol [(±) 4-(3-hydroxybutyl) phenol; 4HP], and magnolignan (2,2- dihydroxy-5,5-dipropyl-biphenyl; ML) react with tyrosinase substrates to generate high amounts of free hydroxyl radicals (˙OH), thereby exerting toxic effects on melanocytes [10]. Thus, over time, melanocyte destruction could occur, and leukoderma may become per- Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. manent [11]. Therefore, there is currently an increased interest in the development of new, This article is an open access article safe, and effective whitening agents from traditional herbs [12]. Recently, researchers have distributed under the terms and focused on the whitening properties of plant-derived compounds and have found that conditions of the Creative Commons compounds such as the ethanol extract of Angelica gigas [13] and plant-derived luteolin [14] Attribution (CC BY) license (https:// have an inhibitory effect on melanogenesis. creativecommons.org/licenses/by/ Scutellarein and baicalein (Figure1) are bioactive flavones purified from the medicinal 4.0/). plant Scutellaria baicalensis Georgi (SBG) [15], which has been used for the treatment of Cosmetics 2021, 8, 15. https://doi.org/10.3390/cosmetics8010015 https://www.mdpi.com/journal/cosmetics Cosmetics 2021, 8, x FOR PEER REVIEW 2 of 12 Cosmetics 2021, 8, 15 2 of 12 Scutellarein and baicalein (Figure 1) are bioactive flavones purified from the medici- nal plant Scutellaria baicalensis Georgi (SBG) [15], which has been used for the treatment of variousvarious inflammatory inflammatory diseases,diseases, hepatitis,hepatitis, tumors, tumors, and and diarrhea, diarrhea, in Eastin East Asian Asian countries countries [16]. [16].Low Low levels levels of scutellarein of scutellarein are observed are observed in the in aerial the aerial part part of SBG; of SBG; scutellarein scutellarein exhibits exhibits high highantioxidant antioxidant activity activity [17] and,[17] consequently,and, consequent is usedly, is asused a medicine as a medicine to treat to inflammation treat inflamma- and tionneurological and neurological diseases diseases [18]. Baicalein [18]. Baicalei mainlyn accumulatesmainly accumulates in the roots in the of roots SBG andof SBG exhibits and exhibitsfree radical-scavenging free radical-scavenging activity activity [19]. Additionally, [19]. Additionally, studies studies have shownhave shown that SBG that plantSBG plantextracts extracts inhibit inhibit melanogenesis melanogenesis [20]. However, [20]. Howe therever, is nothere related is no research related onresearch the whitening on the whiteningeffects of scutellareineffects of scutellarein and baicalein. and baicalein. FigureFigure 1. 1. The Thechemical chemical structure structure of 4-n- ofbutylresorcinol, 4-n-butylresorcinol, scutellarein scutellarein [5,6,7-trihydroxy-2-(4-hydrox- [5,6,7-trihydroxy-2-(4- yphenyl)-chromen-4-one]hydroxyphenyl)-chromen-4-one] and baicalein and baicalein (5,6,7-tri (5,6,7-trihydroxy-2-phenyl-chromen-4-one)hydroxy-2-phenyl-chromen-4-one) derived derivedfrom Scutellariafrom Scutellaria baicalensis baicalensis GeorgiGeorgi (SBG). (SBG). HyperpigmentationHyperpigmentation of of solar solar lentigo lentigo arises arises primarily primarily from from increased melanogenesis ofof existing existing melanocytes melanocytes in in the the basal basal layer layer of the of theepidermis, epidermis, as well as wellas from as fromincreased increased mel- anosomemelanosome complexes complexes in keratinocytes in keratinocytes [21]. [Alth21].ough Although melanin melanin is the ismain the mainculprit culprit in skin in darkening,skin darkening, the precise the precise reason reasonis the accumula is the accumulationtion of melanin of melaninin the keratinocytes in the keratinocytes and mel- anocytesand melanocytes in the perinuclear in the perinuclear area—like area—like "caps" on “caps”the nucleus—which on the nucleus—which helps in protecting helps in theprotecting DNA from the DNAultraviolet from rays ultraviolet [22]. The rays biosynthesis [22]. The biosynthesis process is controlled process is by controlled a cascade by of a tyrosinase,cascade of tyrosinase,tyrosinase related tyrosinase protein-1, related and protein-1, tyrosinase and related tyrosinase protein-2 related (TRP-1 protein-2 and (TRP-1 TRP- 2)and and TRP-2) is very and complicated. is very complicated. Tyrosine Tyrosineis hydroxylated is hydroxylated to 3-(3,4-dihydroxyphenyl)-alanine to 3-(3,4-dihydroxyphenyl)- alanine (DOPA) by tyrosinase, which is the rate-limiting step in melanogenesis [23]. DOPA (DOPA) by tyrosinase, which is the rate-limiting step in melanogenesis [23]. DOPA is then is then oxidized to dopaquinone, which undergoes autoxidation to form dopachrome, oxidized to dopaquinone, which undergoes autoxidation to form dopachrome, then cata- then catalyzed by TRP-2, resulting in exhibition of dopachrome tautomerase activity, to lyzed by TRP-2, resulting in exhibition of dopachrome tautomerase activity, to dihydrox- dihydroxyindole carboxylic acid (DHICA). DHICA is oxidized by TRP-1 to indolequinone, yindole carboxylic acid (DHICA). DHICA is oxidized by TRP-1 to indolequinone, which which eventually forms eumelanin. Concurrently, pheomelanin synthesis is accomplished eventually forms eumelanin. Concurrently, pheomelanin synthesis is accomplished through cysteine. Additionally, researchers found that unlike tyrosinase and TRP-1, which through cysteine. Additionally, researchers found that unlike tyrosinase and TRP-1, are mostly distributed in mature melanosomes, TRP-2 is concentrated in the perinuclear which are mostly distributed in mature melanosomes, TRP-2 is concentrated in the peri- area [24]. Microphthalmia-associated transcription factor (MITF) is a transcription fac- nuclear area [24]. Microphthalmia-associated transcription factor (MITF) is a transcription tor known to be essential for melanocyte development. Additionally, it regulates the factor known to be essential for melanocyte development. Additionally, it regulates the transcription of three major pigmentation enzymes: tyrosinase, TRP-1, and TRP-2 [25]. transcription of three major pigmentation enzymes: tyrosinase, TRP-1, and TRP-2 [25]. Here, scutellarein and baicalein are used as experimental objects to explore their effects on melaninHere, scutellarein production and in B16 baicalein cells. During are used the as present experimental study, 4-n-butylresorcinol objects to