Inhibitory Effect and Mechanism of Scutellarein on Melanogenesis

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 medicinal 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 explore (Figure their ef-1) fectsis used on asmelanin a positive production control to in examine B16 cells. the Du effectring ofthe scutellarein present study, on the 4-n-butylresorcinol tyrosinase activity, (Figurecytotoxicity, 1) is used and melaninas a positive content control of B16 to cells. examine The effectsthe effect of scutellareinof scutellarein on theon the expression tyrosi- naseof tyrosinase activity, alsocytotoxicity, are explored. and melanin Further, thecontent potential of B16 of cells. scutellarein The effects to cause of scutellarein leukoderma on is thepredicted expression by the of measurementtyrosinase also of are the levelsexplored. of free Further, hydroxyl the radicalspotential (˙OH) of scutellarein and, also, itsto causesafety leukoderma for use as a is skin-lightening predicted by the agent measuremen is evaluated.t of the levels of free hydroxyl radicals (˙OH) and, also, its safety for use as a skin-lightening agent is evaluated. 2. Materials and Methods 2.2.1. Materials Materials and Methods 2.1. MaterialsDulbecco’s modiﬁed Eagle’s medium (DMEM) + GlutaMAXTM and 0.05% trypsin- ethylenediaminetetraaceticDulbecco’s modified Eagle’s acid (EDTA) medium were (DMEM) purchased + GlutaMAX from GibcoTM (Thermo and 0.05% Fisher trypsin- Scien- ethylenediaminetetraacetictiﬁc, Waltham, MA, USA). Phosphate-bufferedacid (EDTA) were purchased saline (PBS) from was Gibco purchased (Thermo from Fisher TaKaRa Sci- entific,Bio (Shiga, Waltham, Japan). MA, Fetal USA). bovine Phosphate-buffered serum, α-melanocyte saline stimulating (PBS) was hormone purchased (α-MSH), from and mushroom tyrosinase were purchased from Sigma-Aldrich (St. Louis, MO, USA). The cell counting kit-8 was purchased from Dojindo Laboratories (Kumamoto, Japan)

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and 20% HCl, NaCl, NaOH, 2-amino-2-hydroxymethyl-1,3-propanediol (Tris), glycine, 3-(3,4-dihydroxyphenyl)-L-alanine (L-DOPA), sodium dodecyl sulfate (SDS), sodium deoxycholate, 2-mercaptoethanol, dimethyl sulfoxide (DMSO), and rhododendrol were purchased from FUJIFILM Wako Pure Chemical (Osaka, Japan). Scutellarein and 4-n- butylresorcinol were purchased from the Tokyo Chemical Industry (Tokyo, Japan), and Triton X-100 and Tween 20 were procured from Bio-Rad Laboratories (Alfred Nobel Drive Hercules, CA, USA).

2.2. Preparation of Test Compound Scutellarein and baicalein solutions and the 4-butylresorcinol solution were prepared with dimethyl sulfoxide (DMSO) as the experimental and positive control groups, respectively.

2.3. Cell Culture B16 mouse melanoma cells were cultured in Dulbecco’s modiﬁed Eagle’s medium ◦ (DMEM) containing 10% fetal bovine serum at 37 C in a 5% CO2 incubator. The cells were harvested using trypsin-EDTA.

2.4. Melanin Content Assay B16 cells were cultured in a 100-mm dish (AGC Techno Glass, Shizuoka, Japan) at a concentration of 2.5 × 105 cells/mL for 24 h, then treated with the speciﬁc test compound ◦ and 20 ng/mL α-melanocyte stimulating hormone (α-MSH) at 37 C in a 5% CO2 incubator for 72 h. Cells were exposed to trypsin and shaken gently to remove all cells from the dish. The obtained solution was clariﬁed by centrifugation at 10,000 rpm for 10 min, the supernatant was removed, and the pellet was dissolved in an NaOH solution per the number of cells. Absorbance of the cell suspension was measured at 475 nm using the multi-detection microplate reader.

2.5. Cellular Viability Assay B16 cells were cultured in a 24-well microplate (Corning Coster) at a concentration of 1.25 × 105 cells/mL for 24 h and treated with the speciﬁc test compound at 37 ◦C in a 5% CO2 incubator for 48 h. Cell counting kit-8 solution was added and the absorbance of the water-soluble formazan dye after 0 h and 2 h was measured at 450 nm using a multi-detection microplate reader. Cell viability was expressed as the difference between the two absorbance values.

2.6. Tyrosinase Activity Assay B16 cells were cultured in a 96-well microplate (Corning Coster, Corning, NY, USA) at a concentration of 1.0 × 105 cells/mL for 24 h and treated with the speciﬁc test com- ◦ pound at 37 C in a 5% CO2 incubator for 48 h. Triton X-100, 3-(3,4-dihydroxyphenyl)-L- alanine (DOPA), and phosphate-buffered saline (PBS) were added, then the absorbance of dopachrome after 0 h and 2 h was measured at 475 nm using a multi-detection microplate reader (PowerScan HT; DS Pharma Biomedical Co. Ltd., Osaka, Japan). Tyrosinase activity was expressed as the difference between the two absorbance values.

2.7. Mushroom Tyrosinase Activity Assay Phosphate-buffered saline (PBS) (79 µL), the test compound (1 µL), and 10 mM 3- (3,4-dihydroxyphenyl)-L-alanine (10 µL) with 100 U/mL mushroom tyrosinase (10 µL) were added to each 96-well plate. The absorbance of dopachrome after 0 min and 10 min was measured at 475 nm using a multi-detection microplate reader. Mushroom tyrosinase activity was expressed as the difference between the two absorbance values. Cosmetics 2021, 8, 15 4 of 12

2.8. Western Blotting Assay B16 cells cultured in a 100-mm dish at a concentration of 2.5 × 105 cells/mL for 24 h ◦ were treated with the specific test compound at 37 C in a 5% CO2 incubator for 72 h. Cells were washed with phosphate-buffered saline (PBS) three times and lysed with 2% sodium dodecyl sulfate (SDS) and 1% sodium deoxycholate (0.5 mL). Supernatants were collected as whole-cell lysates, and protein concentrations were determined using the BCA protein assay reagent (Thermo Fisher Scientific, Rockford, MA, USA). Each sample was adjusted to the same protein concentration and heated to 95 ◦C for 5 min. Proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) using a 4–20% gel (TEFCO, Hachioji, Japan), and then transferred to polyvinylidene fluoride (PVDF) membranes (Merck Milli- pore, Cork, Ireland). The membrane was blocked with 5% skim milk in tris-buffered saline (TBS) at room temperature for 30 min, then incubated with a mouse anti-β-actin antibody (diluted 1:1000; Proteintech Group, Rosemont, IL, USA) and a final anti-rabbit serum recognizing the anti-tyrosinase antibody (diluted 1:1000; Thermo Scientific) or anti-rabbit serum recognizing the microphthalmia-associated transcription factor (MITF) antibody (diluted 1:1000; Proteintech Group) with TBS at room temperature for 1 h. The membrane was washed three times with tris buffered saline with tween 20 (TBST) for 30 min and then incubated with a peroxidase-labeled anti-mouse antibody (GE Healthcare, Marlborough, MA, USA) for 30 min at room temperature. The membrane was washed three times with TBST for 3 h, and a western blotting detection reagent (GE Healthcare) was added; the membrane was then incubated for 5 min at room temperature. Finally, immunoreactive bands were visualized using a Lumino Graph (ATTO, Tokyo, Japan).

2.9. Reverse Transcription qPCR (PT-qPCR) Assay B16 cells cultured in 35-mm dishes at a concentration of 1.5 × 105 cells/mL for 24 h ◦ were treated with the speciﬁc test compound at 37 C in a 5% CO2 incubator. RNA was extracted from cells after 6, 12, and 24 h. Total RNA was prepared using the RNeasy Protect Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s protocol. Total RNA was reverse transcribed to cDNA using the One Step TB GreenTM PrimeScriptTM RT-PCR Kit II (TaKaRa Bio) and oligo (dT) primers (QIAGEN). Real-time PCR was performed using the QuantStudio® 5 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). A reverse transcription reaction was carried out under the following conditions: 42 ◦C for 5 min and 95 ◦C for 10 s; PCR ampliﬁcation: 95 ◦C for 5 s and 60 ◦C for 34 s; dissociation protocol: 95 ◦C for 15 s, 60 ◦C for 1 min, and 95 ◦C for 15 s. Expression levels of the tyrosinase gene were normalized to those of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH). Relative changes in mRNA expression levels were calculated with the relative calibration method, and the levels were normalized to that of GAPDH. The experiment was performed in triplicate.

2.10. Measurements of Hydroxyl Radical (˙OH) Phosphate-buffered saline (PBS) (78 µL), the specific test compound (2 µL), and hydroxyphenyl fluorescein (HPF; 5 µmol/L, Goryo Chemical, Sapporo, Japan), with or without 100 U/mL mushroom tyrosinase (10 µL), were added to each 96-well plate and allowed to react at 37 ◦C. The fluorescence intensity of the compound generated by free hydroxyl radical (˙OH) (excitation, 485 nm; fluorescence, 528 nm) was measured after 30 min using the multi-detection microplate reader.

2.11. Data Analysis Data are expressed as mean ± standard deviation (SD). An unpaired t-test (two-tailed) was performed to compare the data of each test compound to the control data using an Excel for Microsoft Ofﬁce Professional Plus 2016; p < 0.05 was considered statistically signiﬁcant. Cosmetics 2021, 8, x FOR PEER REVIEW 5 of 12

Cosmetics 2021, 8, 15 5 of 12 an Excel for Microsoft Office Professional Plus 2016; p < 0.05 was considered statistically significant.

3. Results 3.1. Effects of Scutellarein and Baicalei Baicaleinn on the Melanin Content of B16 Cells α When B16 cells were incubated with scutellarein and α-melanocyte-melanocyte stimulating stimulating hor- hor- α mone ( α-MSH), the cell pellet dissolved in the NaOH solution was visibly black. The color of the dissolved cell pellet was compared using NaOH at differentdifferent concentrations,concentrations, withwith the absorbance measured at 475 nm. Melanin content was signiﬁcantlysignificantly decreased in cellscells treated with 4-n-butylresorcinol and scutellarein compared with the untreated control; treated with 4-n-butylresorcinol and scutellarein compared with the untreated control; a a clear dose-dependent decrease in cellular melanogenesis was observed (Figure2a,b ). clear dose-dependent decrease in cellular melanogenesis was observed (Figure 2a,b). To To contrast, the color of the cell pellet dissolved in NaOH, corresponding to baicalein contrast, the color of the cell pellet dissolved in NaOH, corresponding to baicalein treat- treatment at different concentrations, did not show any signiﬁcant change, and there was ment at different concentrations, did not show any significant change, and there was no no difference in absorbance (Figure2c). difference in absorbance (Figure. 2c).

(a) (b) (c)

Figure 2. Effects of the tested compounds on the melanin contentcontent ofof B16B16 cells.cells. TheThe upperupper partpart ofof the theﬁgure figure shows shows the the appearance appearance of theof the cell cell suspension suspension after after the the addition addition of of the the NaOH NaOH solution. solution. The Thelower lower part part of the of ﬁgurethe figure represents represents the absorbancethe absorbance of the of cellthe pelletcell pellet dissolved dissolved in NaOH; in NaOH; absorbance ab- wassorbance measured was measured at 475 nm. at Data 475 nm. are expressedData are ex aspressed a percentage as a percentage of the control. of the (a control.): 4-n-butylresorcinol; (a): 4-n-butylresorcinol;(b): scutellarein; (b): ( cscutellarein;): baicalein. (c): baicalein.

3.2. Effects of Scutellarein on the Tyrosinase ActivityActivity inin thethe WellWell Tyrosinase is is an an important important enzyme enzyme for for melanin melanin production, production, and and its regulation its regulation is one is ofone the of factors the factors affecting affecting skin pigmentation skin pigmentation [25]. Tyrosinase [25]. Tyrosinase activity activity in the inwell the was well deter- was mineddetermined by adding by adding 3-(3,4-dihydroxyphenyl)-L- 3-(3,4-dihydroxyphenyl)-alanineL-alanine to the to cells the cellsand andmeasuring measuring the theab- sorbanceabsorbance of the of the produced produced dopachrome dopachrome at 475 at 475 nm. nm. Regarding Regarding the thepositive positive control, control, 4-n-bu- 4-n- tylresorcinolbutylresorcinol dose-dependently dose-dependently inhibited inhibited tyrosinase tyrosinase activity activity at 2.5, at 5, 2.5, and 5, and10 μg/mL 10 µg/mL (Fig- (Figureure 3a).3 Compareda). Compared to the to thecontrol control group, group, when when the theconcentration concentration of scutellarein of scutellarein in the in theme- diummedium was was 4 and 4 and 8 μg/mL, 8 µg/mL, scutellarein scutellarein dose-dependently dose-dependently inhibited inhibited tyrosinase tyrosinase activity activity (Fig- (Figureure 3b).3 b).

3.3. Effects of Scutellarein on the Viability of B16 Cells (a) Cell viability was determined by reacting the cells with the cell counting kit-8 solution and measuring the absorbance of the water-soluble formazan dye at 450 nm. After cells were cultured in media containing different concentrations of 4-n-butylresorcinol for 48 h, there was no tendency toward a decreased cell viability, indicating that 4-n-butylresorcinol and scutellarein exhibited no cytotoxicity within the experimental concentration range (Figure4a,b).

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an Excel for Microsoft Office Professional Plus 2016; p < 0.05 was considered statistically significant.

3. Results 3.1. Effects of Scutellarein and Baicalein on the Melanin Content of B16 Cells When B16 cells were incubated with scutellarein and α-melanocyte stimulating hormone (α-MSH), the cell pellet dissolved in the NaOH solution was visibly black. The color of the dissolved cell pellet was compared using NaOH at different concentrations, with the absorbance measured at 475 nm. Melanin content was significantly decreased in cells treated with 4-n-butylresorcinol and scutellarein compared with the untreated control; a clear dose-dependent decrease in cellular melanogenesis was observed (Figure 2a,b). To contrast, the color of the cell pellet dissolved in NaOH, corresponding to baicalein treatment at different concentrations, did not show any significant change, and there was no difference in absorbance (Figure. 2c).

(a) (b) (c)

Figure 2. Effects of the tested compounds on the melanin content of B16 cells. The upper part of the figure shows the appearance of the cell suspension after the addition of the NaOH solution. The lower part of the figure represents the absorbance of the cell pellet dissolved in NaOH; absorbance was measured at 475 nm. Data are expressed as a percentage of the control. (a): 4-n-butylresorcinol; (b): scutellarein; (c): baicalein.

3.2. Effects of Scutellarein on the Tyrosinase Activity in the Well Tyrosinase is an important enzyme for melanin production, and its regulation is one of the factors affecting skin pigmentation [25]. Tyrosinase activity in the well was determined by adding 3-(3,4-dihydroxyphenyl)-L-alanine to the cells and measuring the absorbance of the produced dopachrome at 475 nm. Regarding the positive control, 4-n-butylresorcinol dose-dependently inhibited tyrosinase activity at 2.5, 5, and 10 μg/mL (Fig- Cosmetics 2021, 8, 15 ure 3a). Compared to the control group, when the concentration of scutellarein in the me- 6 of 12 dium was 4 and 8 μg/mL, scutellarein dose-dependently inhibited tyrosinase activity (Fig- ure 3b).

(a) Cosmetics 2021, 8, x FOR PEER REVIEW 6 of 12

(b)

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(b)

Figure 3. The effects of the tested compounds on the tyrosinase activity of B16 cells. Tyrosinase activity was determined by calculating the absorbance change rate in 2 h at 475 nm. (a): 4-n-butylresorcinol; (b): scutellarein. Data are presented as the mean ± SD of experiments performed in triplicate. ∗ p < 0.05 compared to the control, ∗∗ p < 0.01 compared to the control.

3.3. Effects of Scutellarein on the Viability of B16 Cells Cell viability was determined by reacting the cells with the cell counting kit-8 solution and measuring the absorbance of the water-soluble formazan dye at 450 nm. After cellsFigure were 3. TheFigure cultured effects 3. Theofin themedia effects tested containing of compounds the tested differ compoundson theent tyrosinase concentrations on the activity tyrosinase of of 4-n-butylresorcinol B16 activity cells. Tyrosinase of B16 cells. for Tyrosinase activity was determined by calculating the absorbance change rate in 2 h at 475 nm. (a): 4-n-butyl- 48 h, thereactivity was no was tendency determined toward by calculating a decreased the cell absorbance viability, change indicating rate in that 2 h 4-n-butyl- at 475 nm. (a): 4-n- resorcinol; (b): scutellarein. Data are presented as the mean ± SD of experiments performed in trip- resorcinol∗ butylresorcinol; and scutellarein (b): exhibited scutellarein. ∗∗no Data cyto aretoxicity presented within as the the mean experimental± SD of experiments concentra- performed in licate. p < 0.05 compared to the control, p < 0.01 compared to the control. tion rangetriplicate. (Figure 4a,b).∗ p < 0.05 compared to the control, ∗∗ p < 0.01 compared to the control.

3.3. Effects of Scutellarein on the Viability of B16 Cells (a) Cell viability was determined by reacting the cells with the cell counting kit-8 solution and measuring the absorbance of the water-soluble formazan dye at 450 nm. After cells were cultured in media containing different concentrations of 4-n-butylresorcinol for 48 h, there was no tendency toward a decreased cell viability, indicating that 4-n-butylresorcinol and scutellarein exhibited no cytotoxicity within the experimental concentration range (Figure 4a,b).

(a)

(b)

Figure 4. TheFigure effects 4. ofThe the effects tested ofcompounds the tested on compounds the viability on of theB16 viabilitycells. Cell of viability B16 cells. was Cell deter- viability was mined by calculatingdetermined the by absorbance calculating change the absorbance rate in 2 changeh at 450 rate nm. in (a 2): h4-n-butylresorcinol; at 450 nm. (a): 4-n-butylresorcinol; (b): (b): scutellarein. Data are presented as the mean ± SD of experiments performed in triplicate. scutellarein. Data are presented as the mean ± SD of experiments performed in triplicate. 3.4. Effect of Scutellarein and Baicalein on Mushroom Tyrosinase Activity The inhibitory activity of scutellarein and baicalein on mushroom tyrosinase was investigated. 4-n-Butylresorcinol was used as a positive control. 4-n-Butylresorcinol showed

a dose-dependent inhibitory effect, but scutellarein and baicalein did not inhibit the activ- ityFigure of mushroom 4. The effects tyrosinase of the tested (Figure compounds 5). on the viability of B16 cells. Cell viability was determined by calculating the absorbance change rate in 2 h at 450 nm. (a): 4-n-butylresorcinol; (b): scutellarein. Data are presented as the mean ± SD of experiments performed in triplicate.

3.4. Effect of Scutellarein and Baicalein on Mushroom Tyrosinase Activity The inhibitory activity of scutellarein and baicalein on mushroom tyrosinase was investigated. 4-n-Butylresorcinol was used as a positive control. 4-n-Butylresorcinol showed a dose-dependent inhibitory effect, but scutellarein and baicalein did not inhibit the activity of mushroom tyrosinase (Figure 5).

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3.4. Effect of Scutellarein and Baicalein on Mushroom Tyrosinase Activity The inhibitory activity of scutellarein and baicalein on mushroom tyrosinase was Cosmetics 2021, 8, x FOR PEER REVIEW 7 of 12 investigated. 4-n-Butylresorcinol was used as a positive control. 4-n-Butylresorcinol showed a dose-dependent inhibitory effect, but scutellarein and baicalein did not inhibit the activity of mushroom tyrosinase (Figure5).

Figure 5. The effects of the tested compounds on mushroom tyrosinase activity. Mushroom ty- Figure 5. Therosinase effects of activity the tested was compounds determined on by mushroom calculating tyrosinase the absorbance activity. change Mushroom rate in tyrosi- 10 min at 475 nm. nase activity4-n-butylresorcinol: was determined by calculating 40 µmol/L the (6.64 absorbanceµg/mL), 20changeµmol/L rate (3.32 in 10µ ming/mL), at 475 10 nm.µmol/L 4-n- (1.66 µg/mL), butylresorcinol:5 µmol/L 40 μmol/L (0.83 (6.64µg/mL), μg/mL), 2.5 µ20mol/L μmol/L (0.42 (3.32µg/mL), μg/mL), 1.25 10 µμmol/Lmol/L (1.66 (0.21 μµg/mL),g/mL), 5 0.625 µmol/L (0.10 μmol/L (0.83µ μg/mL).g/mL), scutellarein:2.5 μmol/L (0.42 40 µ mol/Lμg/mL), (11.44 1.25 µμg/mL),mol/L (0.21 20 µ mol/Lμg/mL), (5.72 0.625µg/mL), μmol/L 10 (0.10µmol/L (2.86 µg/mL), μg/mL). scutellarein: 40 μmol/L (11.44 μg/mL), 20 μmol/L (5.72 μg/mL), 10 μmol/L (2.86 μg/mL), 5 5 µmol/L (1.43 µg/mL), 2.5 µmol/L (0.72 µg/mL), 1.25 µmol/L (0.36 µg/mL), 0.625 µmol/L (0.18 μmol/L (1.43 μg/mL), 2.5 μmol/L (0.72 μg/mL), 1.25 μmol/L (0.36 μg/mL), 0.625 μmol/L (0.18 µg/mL). baicalein: 40 µmol/L (10.8 µg/mL), 20 µmol/L (5.4 µg/mL), 10 µmol/L (2.7 µg/mL), μg/mL). baicalein: 40 μmol/L (10.8 μg/mL), 20 μmol/L (5. 4μg/mL), 10 μmol/L (2.7 μg/mL), 5 μmol/L (1.355 μµg/mL),mol/L 2.5 (1.35 μmol/Lµg/mL), (0.68 2.5 μg/mL),µmol/L 1.25 (0.68 μmol/Lµg/mL), (0.34 1.25 μg/mL),µmol/L 0.625 (0.34 μmol/Lµg/mL), (0.17 0.625 µmol/L (0.17 μg/mL). ∗p < µ0.05,g/mL). ∗∗p < * 0.01,p < 0.05, ∗∗∗p **< 0.001p < 0.01, compared *** p < 0.001to the compared control. Data to the are control. presented Data as are the presented mean ± as the mean SD of experiments± SD ofperformed experiments in triplicate. performed in triplicate. 3.5. Effects of Scutellarein on Tyrosinase and Microphthalmia-Associated Transcription Factor 3.5. Effects of Scutellarein on Tyrosinase and Microphthalmia-Associated Transcription Factor (MITF) Protein Expressions (MITF) Protein Expressions To further elucidate the mechanism of scutellarein in melanogenesis, we focused To furtheron tyrosinase elucidate andthe mechanism microphthalmia-associated of scutellarein in transcription melanogenesis, factor we (MITF)focused protein on expres- tyrosinase sions.and microphthalmia-associated Western blot analysis, using tran a specificscription antibody factor (MITF) against protein tyrosinase, expres- suggested that sions. Westerntyrosinase blot analysis, protein using expression a specific was antibody inhibited against in a dose-dependent tyrosinase, suggested manner that when scutel- tyrosinase proteinlarein concentration expression was in the inhibited medium in wasa dose-dependent 8 and 16 µg/mL manner (p < 0.05, whenp <0.05, scutel- respectively; larein concentrationFigure6a ).Additionally,in the medium the was expression 8 and 16 ofμg/mL MITF ( alsop < 0.05, showed p <0.05, in a dose-dependentrespectively; manner Figure 6a).Additionally,when scutellarein the expression concentration of MITF in the also medium showed was in a 4, dose-dependent 8, and 16 µg/mL manner (p < 0.05, p < 0.05, when scutellareinp < 0.01, concentration respectively; in Figure the medium6b). was 4, 8, and 16 μg/mL (p < 0.05, p <0.05, p < 0.01, respectively; Figure 6b). (a) 3.6. Effect of Scutellarein on Tyrosinase mRNA Expression After adding scutellarein for 6, 12, and 24 h, tyrosinase mRNA expression in B16 cells was detected. However, quantitative analysis showed that after 6 and 12 h, tyrosinase mRNA expression in each concentration condition did not change significantly. After 24 h, tyrosinase mRNA levels were significantly downregulated at a concentration of 8 and 16 µg/mL (p < 0.05) in B16 cells, compared to the untreated control (Figure7).

tyrosinase

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Figure 5. The effects of the tested compounds on mushroom tyrosinase activity. Mushroom tyrosinase activity was determined by calculating the absorbance change rate in 10 min at 475 nm. 4-n- butylresorcinol: 40 μmol/L (6.64 μg/mL), 20 μmol/L (3.32 μg/mL), 10 μmol/L (1.66 μg/mL), 5 μmol/L (0.83 μg/mL), 2.5 μmol/L (0.42 μg/mL), 1.25 μmol/L (0.21 μg/mL), 0.625 μmol/L (0.10 μg/mL). scutellarein: 40 μmol/L (11.44 μg/mL), 20 μmol/L (5.72 μg/mL), 10 μmol/L (2.86 μg/mL), 5 μmol/L (1.43 μg/mL), 2.5 μmol/L (0.72 μg/mL), 1.25 μmol/L (0.36 μg/mL), 0.625 μmol/L (0.18 μg/mL). baicalein: 40 μmol/L (10.8 μg/mL), 20 μmol/L (5. 4μg/mL), 10 μmol/L (2.7 μg/mL), 5 μmol/L (1.35 μg/mL), 2.5 μmol/L (0.68 μg/mL), 1.25 μmol/L (0.34 μg/mL), 0.625 μmol/L (0.17 To further elucidate the mechanism of scutellarein in melanogenesis, we focused on tyrosinase and microphthalmia-associated transcription factor (MITF) protein expressions. Western blot analysis, using a specific antibody against tyrosinase, suggested that tyrosinase protein expression was inhibited in a dose-dependent manner when scutellarein concentration in the medium was 8 and 16 Cosmetics 2021, 8, 15 μg/mL (p < 0.05, p <0.05, respectively; Figure 6a).Additionally, the expression of MITF also showed8 of 12 in a dose-dependent manner when scutellarein concentration in the medium was 4, 8, and 16 μg/mL (p < 0.05, p <0.05, p < 0.01, respectively; Figure 6b).

(a)

tyrosinase Cosmetics 2021, 8, x FOR PEER REVIEW 8 of 12

β-actin (b)

MITF

β-actin

FigureFigure 6. The 6. effectsThe effects of scutellarein of scutellarein on tyrosinase on tyrosinase protein protein (a) and (a microphthalmia-associated) and microphthalmia-associated tran- tran- scriptionscription factor factor (MITF) (MITF) protein protein (b) expressions. (b) expressions. B16 cells B16 were cells weretreated treated with withdifferent different concentrations concentrations of scutellareinof scutellarein for 48 for h. 48Western h. Western blot analysis blot analysis was then was thenconducted conducted using using an anti-tyrosinase an anti-tyrosinase anti- antibody bodyor or anti-MITFanti-MITF antibody;antibody;β β-actin-actin waswas usedused asas aa loadingloading control.control. FluorescenceFluorescence intensitiesintensities of of tyrosinase ty- rosinaseand andβ-actin β-actin were were quantiﬁed quantified using using ImageJ. ImageJ. Band Band intensities intensities were were normalized normalized to toβ -actin.β-actin. Data are ∗ Data presentedare presented as the as mean the mean± SD ± ofSD experiments of experiments performed performed in triplicate. in triplicate. * p < 0.05 p < compared 0.05 compared to the to control. the control. ∗∗ p < 0.01 compared to the control. ** p < 0.01 compared to the control. 3.6. Effect of Scutellarein on Tyrosinase mRNA Expression After adding scutellarein for 6, 12, and 24 h, tyrosinase mRNA expression in B16 cells was detected. However, quantitative analysis showed that after 6 and 12 h, tyrosinase mRNA expression in each concentration condition did not change significantly. After 24 h, tyrosinase mRNA levels were significantly downregulated at a concentration of 8 and 16 μg/mL (p < 0.05) in B16 cells, compared to the untreated control (Figure 7).

Cosmetics 2021, 8, x FOR PEER REVIEW 8 of 12

β-actin (b)

MITF

β-actin

Figure 6. The effects of scutellarein on tyrosinase protein (a) and microphthalmia-associated transcription factor (MITF) protein (b) expressions. B16 cells were treated with different concentrations of scutellarein for 48 h. Western blot analysis was then conducted using an anti-tyrosinase antibody or anti-MITF antibody; β-actin was used as a loading control. Fluorescence intensities of tyrosinase and β-actin were quantified using ImageJ. Band intensities were normalized to β-actin. Data are presented as the mean ± SD of experiments performed in triplicate. ∗ p < 0.05 compared to the control. ∗∗ p < 0.01 compared to the control.

3.6. Effect of Scutellarein on Tyrosinase mRNA Expression After adding scutellarein for 6, 12, and 24 h, tyrosinase mRNA expression in B16 cells was detected. However, quantitative analysis showed that after 6 and 12 h, tyrosinase mRNA expression in each concentration condition did not change significantly. After 24 Cosmetics 2021h,, 8 ,tyrosinase 15 mRNA levels were significantly downregulated at a concentration9 of of 12 8 and 16 μg/mL (p < 0.05) in B16 cells, compared to the untreated control (Figure 7).

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Figure 7. TheFigure effects 7. The of effects scutellarein of scutellarein on ontyrosinase tyrosinase mRNAmRNA expression. expression. B16 cellsB16 were cells treated were with treated with different concentrationsdifferent concentrations of scutellarein of scutellarein for 6, 6, 12, 12, and and 24 h. 24 RT-qPCR h. RT-qPCR analysis was analysis then performed was then using performed using the glyceraldehyde-3-phosphatethe glyceraldehyde-3-phosphate dehydrogenase dehydrogenasegene (GAPDH) gene (asGAPDH) a loading as control. a loading Data are control. presented Data are presented asas the the mean mean± SD ± ofSD experiments of experiments performed performed in triplicate. *inp

3.7. Hydroxyl Radical (˙OH) Generation from Scutellarein in the Presence of Tyrosinase A comparison of the fluorescence intensity of the tested compounds in the presence of tyrosinase after 30 min is shown in Figure 8. The amount of free hydroxyl radicals (˙OH) generated did not change in the absence of tyrosinase. Compared to the control group, rhododendrol was found to generate ˙OH at 10 μmol/L (1.66 μg/mL), 100 μmol/L (16.6 μg/mL), and 1000 μmol/L (166 μg/mL). To contrast, the generation of ˙OH at each concentration of scutellarein was significantly lower than the control level at 4 μg/mL (14 μmol/L), 8 μg/mL (28 μmol/L), and 16 μg/mL (56 μmol/L) and, as the concentration increased, the levels of ˙OH decreased.

Figure 8. A comparison of free hydroxyl radical (˙OH) generation by the tested compound in the presence of tyrosinase.

4. Discussion

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Figure 7. The effects of scutellarein on tyrosinase mRNA expression. B16 cells were treated with different concentrations of scutellarein for 6, 12, and 24 h. RT-qPCR analysis was then performed Cosmetics 2021, 8, 15using the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) as a loading control. Data are 10 of 12 presented as the mean ± SD of experiments performed in triplicate. ∗ p < 0.05 compared to the control.

3.7. Hydroxyl Radical3.7. Hydroxyl (˙OH) Generation Radical (˙OH) from Generation Scutellarein from in Scutellareinthe Presence inof theTyrosinase Presence of Tyrosinase A comparisonA of comparison the fluorescence of the intensity ﬂuorescence of the intensity tested compounds of the tested in compounds the presence in the presence of tyrosinase afterof tyrosinase 30 min is shown after 30 in minFigure is shown8. The amount in Figure of 8free. The hydroxyl amount radicals of free (˙ hydroxylOH) radicals generated did(˙OH) not change generated in the did absence not change of tyrosinase. in the absence Compared of tyrosinase. to the control Compared group, to the control rhododendrol group,was found rhododendrol to generate was ˙OH found at 10 to generateμmol/L (1.66 ˙OH atμg/mL), 10 µmol/L 100 μ (1.66mol/Lµg/mL), (16.6 100 µmol/L μg/mL), and 1000(16.6 μµmol/Lg/mL), (166 and μg/mL). 1000 µ Tomol/L contrast, (166 theµg/mL). generation To contrast, of ˙OH theat each generation concen- of ˙OH at each tration of scutellareinconcentration was significantly of scutellarein lower was signiﬁcantlythan the control lower level than at the 4 controlμg/mL level(14 at 4 µg/mL μmol/L), 8 μg/mL(14 µ mol/L),(28 μmol/L), 8 µg/mL and 16 (28 μµg/mLmol/L), (56 andμmol/L) 16 µg/mL and, as (56 theµmol/L) concentration and, as thein- concentration creased, the levelsincreased, of ˙OH the decreased. levels of ˙OH decreased.

Figure 8. A comparison of free hydroxyl radical (˙OH) generation by the tested compound in the presence of tyrosinase. Figure 8. A comparison of free hydroxyl radical (˙OH) generation by the tested compound in the presence of tyrosinase. 4. Discussion Since the molecular structure of melanin facilitates ultraviolet light absorption, it has the ability to resist ultraviolet light [26]. Melanocytes are generally distributed in the hair, eyes, and skin. Since the 21st century, researchers have discovered many whitening agents used in drugs for treating pigment diseases or in skin-whitening cosmetics. Most whitening agents may be classified based on their different mechanisms of action, including the interference with melanin synthesis and transport and the acceleration of melanin decomposition [27]. Additionally, using a novel 675 nm laser may be considered promising when treating benign pigmented lesions, due to a low risk of side effects and simple post-treatment management [28]. Scutellarein and baicalein are flavones widely found in perennial herbs [29]. Presently, many scientists have confirmed that scutellarein has high anticancer activity and can be used as a drug for clinical treatment [30]. This study used three experiments assessing melanin content to explore the effects of scutellarein and baicalein on melanin production. The melanin content of B16 cells treated with scutellarein was significantly reduced after three days of culture; however, there was no change in the melanin content following baicalein treatment. We observed that scutellarein had an inhibitory effect on tyrosinase activity and showed no cytotoxicity at concentrations of 2 µg/mL, 4 µg/mL, 8 µg/mL, and 16 µg/mL. No enzyme inhibitory effect on tyrosinase was observed in scutellarein and baicalein. Therefore, in subsequent experiments, baicalein was not used as the tested compound. The chemical structures of scutellarein and baicalein were compared, and it was found that scutellarein has a 4-hydroxyphenyl in the 2-position, which has a stronger reducibility than baicalein, which has a phenyl group at the 2-position. This may explain why baicalein did not show any melanin inhibitory effect. The 4-substituted phenolic (para-phenol) compounds were previously shown to be directly toxic to melanocytes in the presence of tyrosinase [31]. Measuring the amount of free hydroxyl radicals (˙OH) generated in the presence of tyrosinase is an in vitro method that predicts the risk of the development of chemical leukoderma due to a particular compound [10]. Compared to the positive control, that is rhododendrol, scutellarein showed no ˙OH generation activity. Moreover, as the concentration of scutellarein increased, the ˙OH generation activity Cosmetics 2021, 8, 15 11 of 12

showed a downward trend. Scutellarein has been reported to have good antioxidant and anti-inﬂammatory effects, which may be one of the reasons for the inhibition of ˙OH generation. 4-n-Butylresorcinol did not show ˙OH generation activity in the presence of tyrosinase as previously reported [10]. Additionally, the effect of scutellarein on tyrosinase and microphthalmia-associated transcription factor (MITF) expressions also were tested. When scutellarein concentrations were 8 µg/mL and 16 µg/mL, tyrosinase protein levels decreased, and MITF protein levels decreased at the concentrations of 4 µg/mL, 8 µg/mL, and 16 µg/mL. Concurrently, tyrosinase mRNA levels decreased in a dose-dependent manner in the culture medium at concentrations of 8 µg/mL and 16 µg/mL. These results show that scutellarein affects melanin synthesis by inhibiting the expression of MITF and its down regulation tyrosinase protein. Conversely, 4-n-butylresorcinol has been reported to suppress melanin production by a mechanism that directly inhibits tyrosinase [8]. These results show that scutellarein affects melanin synthesis by inhibiting tyrosinase expression. However, in terms of gene expression, other factors that affect melanin production require further research. Further- more, the effect of scutellarein on the synthesis and decomposition of melanosomes via different pathways is still being investigated by our group. To conclude, our results revealed that scutellarein has an inhibitory effect on melanin production at the concentration of any cytotoxicity; inhibition of ˙OH generation also was observed. While investigating the mechanism of scutellarein’s inhibition of melanin production, we found that tyrosinase expression was inhibited, which ultimately affected the synthesis of melanin in cells. To explore whether scutellarein could serve as a promising new skin-lightening agent, further experiments and research, such as the cutaneous absorption experiment, concentration setting experiment, and clinical trials, are needed.

Author Contributions: L.D., L.G. and K.M. performed the experiments. K.M. designed the study and performed the data analysis. L.D., L.G. and K.M. interpreted the data and drafted the manuscript. K.M. supervised the study and critically revised the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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