Article pubs.acs.org/JAFC
Demethyleugenol β‑Glucopyranoside Isolated from Agastache rugosa Decreases Melanin Synthesis via Down-regulation of MITF and SOX9 Taek Hwan Lee,† SeonJu Park,† Guijae Yoo,† Cheongyun Jang,‡ Mi-hyun Kim,‡,# Seung Hyun Kim,† and Sun Yeou Kim*,‡,Δ,# †
College of Pharmacy, Yonsei University, 85 Songdogwahak-ro, Yeonsu-gu, Incheon 21983, Republic of Korea Laboratory of Pharmacognosy, College of Pharmacy, Gachon University, 191 Hambakmoero, Yeonsu-gu, Incheon 21936, Republic of Korea Δ Gachon Medical Research Institute, Gil Medical Center, Incheon 21565, Republic of Korea # Gachon Institute of Pharmaceutical Science, Gachon University, 191 Hambakmoero, Yeonsu-gu, Incheon 21936, Republic of Korea ‡
S Supporting Information *
ABSTRACT: Agastache rugosa (Fisch. & C. A. Mey.) Kuntze has been well-known for its antioxidative properties. This study investigated the anti-melanogenesis effect of demethyleugenol β-D-glucopyranoside (1) from A. rugosa by studying molecular regulation of melanogenesis in melan-a mouse melanocytes and normal human epidermal melanocytes (NHEMs) and in in vivo models. The SRY (sex-determining region on the Y chromosome)-related high-mobility group (HMG) box 9 (SOX9), one of the critical factors that affect skin pigmentation, is up-regulated. Interestingly, 1 down-regulated the expression of SOX9 and microphthalmia-associated transcription factor (MITF). Reduction of these two transcription factors resulted in a decrease in melanogenic enzymes such as tyrosinase, tyrosinase-related protein 1, and dopachrome tautomerase. As a result, 1 significantly inhibited melanin synthesis in melan-a mouse melanocytes and NHEMs. In addition, the anti-melanogenic effect of 1 was confirmed in zebrafish and reconstructed skin tissue models. In conclusion, 1, as a potent SOX9 regulator, ameliorates skin pigmentation. KEYWORDS: demethyleugenol β-glucopyranoside, SOX9, melanogenesis, NHEM, reconstructed skin
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INTRODUCTION Melanin accumulation is caused by increased numbers or activities of melanocytes. Excess production of melanin sometimes induces hyperpigmentary disorders such as postinflammatory hyperpigmentation, melasma, solar lentigo, and geriatric pigment spots.1,2 Therefore, it is important to find depigmenting agents for the prevention and treatment of irregular skin hyperpigmentation. Until now, regulations of melanogenic enzymes and melanosome transfer have been the most common approaches to achieving skin pigmentation improvement.3 There are many reports on depigmentation by the suppression of melanogenic enzymes such as tyrosinase, tyrosinaserelated protein 1 (TRP-1), dopachrome tautomerase (DCT), or microphthalmia-associated transcription factor (MITF).4−7 In addition, it has been reported that melanogenic proteins are regulated by the SRY (sex-determining region on the Y chromosome)-type high-mobility group (HMG) box 9 (SOX9) protein.8 SOX proteins, transcription factors that share a similar HMG-box domain, which mediates sequence-specific DNA binding, are involved in various developmental stages, particularly in melanocyte development.9 Furthermore, one of the SOX factors, SOX9, is expressed by melanocytes in human skin, and UVR overexposure up-regulates it.8 There is no doubt that there is exact parallelism between the regulation of SOX9 and expression of tyrosinase, DCT, and MITF.8 Nevertheless, there are no reports on candidates that protect against melanogenesis, possibly through regulation of SOX9. © 2016 American Chemical Society
Agastache rugosa (Fisch. & C. A. Mey.) Kuntze, commonly called Korean mint in Korea and pinyin in China, has been reported to possess antibacterial,10 antifungal,11 and antiatherogenic properties.12 The plant has been well-known for its antioxidative properties. In this study, we evaluate its protective role against skin hyperpigmentation using various cell lines and reconstructed skin. In our prescreening study, n-hexane-, chloroform-, ethyl acetate (EA)-, and water-soluble fractions from a total extract were evaluated for their effect on melanin synthesis in mouse melan-a cells. Interestingly, the EA-soluble fraction decreased melanin synthesis in melan-a cells. Using an activity-guided isolation technique, compounds 1−7 were isolated from the EA fraction, and then we further evaluated the effects of the compounds on melanogenesis in melan-a cells. Our findings suggested that a potential SOX9 regulator, demethyleugenol β-D-glucopyranoside (compound 1), could be useful as an antimelanogenic agent for the prevention and treatment of diseases associated with skin pigmentation.
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MATERIALS AND METHODS
Materials. Phenylthiourea (PTU) was purchased from SigmaAldrich (St. Louis, MO, USA). The isolated compounds were each dissolved in dimethyl sulfoxide (DMSO) to make a stock solution. Received: Revised: Accepted: Published: 7733
July 21, 2016 September 27, 2016 September 27, 2016 September 27, 2016 DOI: 10.1021/acs.jafc.6b03256 J. Agric. Food Chem. 2016, 64, 7733−7742
Article
Journal of Agricultural and Food Chemistry
Figure 1. Structures of compound 1−7 isolated from Agastache rugosa extract. Antibodies against tyrosinase and SOX9 were purchased from Abcam (Cambridge, MA, USA). Antibodies against TRP-1, DCT, and MITF were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against cyclic AMP response element-binding protein (CREB) and phosphorylated CREB (p-CREB) were purchased from Cell Signaling Technology (Danvers, MA, USA). Chemical shifts are reported in parts per million from TMS. All NMR spectra were recorded on an Agilent 400-MR-NMR spectrometer operated at 400 and 100 MHz for hydrogen and carbon, respectively. Data processing was carried out with the MestReNova ver. 6.0.2 program. HR-ESI-MS spectra were obtained using an Agilent 6550 iFunnel Q-TOF LC/MS system. Preparative HPLC was carried out using an Agilent 1200 HPLC system. Column chromatography was performed on silica gel (Kieselgel 60, 70−230 and 230−400 mesh, Merck) or YMC RP-18 resins (30−50 μm, Fujisilisa Chemical Ltd.). For thin layer chromatography (TLC), precoated silica gel 60 F254 (0.25 mm, Merck) and RP-18 F254S plates (0.25 mm, Merck) were used. Compounds 1−7 were isolated from A. rugosa, and by comparison of their spectroscopic data with those reported in the literature, these compounds were identified as demethyleugenol β-D-glucopyranoside and benzyl β-glucopyranoside (1 and 5, respectively),13 5β,6αdihydroxy-3β-(β-glucopyranosyloxy)-7-megastigmen-9-one (2), 14 (E)-4-[3′-(β-glucopyranosyloxy)butylidene]-3,5,5-trimethyl-2-cyclohexen-1-one (3),15 (6R,9R)-3-oxo-α-ionol-9-O-β-glucopyranoside (4),16 salidroside (6),17 and citrusin C (7)18 (Figure 1). Extraction and Isolation of Bioactive Compounds. The dried leaves of A. rugosa (4.0 kg) were extracted with methanol under sonication for 4 h to yield 400.0 g of extract. This extract was suspended in H2O and successively partitioned with CHCl3 and EtOAc to obtain CHCl3 (AR1, 119.8 g), EtOAc (AR2, 156.3 g), and aqueous (AR3, 130.0 g) fractions. The AR2 fraction (156.3 g) was chromatographed on a silica gel column and eluted with a gradient of CHCl3/MeOH (20:1 → 1:1, v/v) to obtain five subfractions, AR1A (11.4 g), AR1B (8.7 g), AR1C (3.5 g), AR1D (3.9 g), and AR1E (55.8 g). The AR1C fraction was chromatographed on a silica gel column eluting with CHCl3/MeOH (20:1, v/v) to give three smaller fractions, AR1C1 (1.7 g), AR1C2 (1.0 g), and AR1C3 (0.8 g). The AR1C2 fraction was chromatographed on HPLC using a J’sphere ODS H-80 (250 mm × 20 mm, 4 μm, 8 nm) column eluting with 25% aqueous acetonitrile at a flow rate of 3 mL/min to yield 5 (16.2 mg) and 6 (7.5 mg). The AR1C3 fraction was also chromatographed on HPLC using a J’sphere ODS H-80 (250 mm × 20 mm, 4 μm, 8 nm) column eluting with 22% aqueous acetonitrile at a flow rate of 3 mL/min to yield 1 (17.0 mg), 3 (6.1 mg), and 4 (2.9 mg). The AR1E fraction was chromatographed on a silica gel column eluting with CHCl3/MeOH (6:1, v/v) to yield 2 (2.6 mg) and 7 (4.8 mg). Cell Culture. Normal human epidermal melanocytes (NHEMs) were purchased from CEFObio Corp. (Seoul, Korea) and maintained in melanocyte growth medium (CEFObio). Melan-a murine melanocytes
were provided by Dr. Byeong Gon Lee (Skin Research Institute, AmorePacific Co., Yongin, Korea) and maintained in RPMI 1640 medium containing 10% fetal bovine serum, a 1% penicillin−streptomycin solution, and 200 nM tissue plasminogen activator at 37 °C in an atmosphere of 5% CO2. MTT Assay. The colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was carried out according to the method described by Ferrari et al.19 The medium was removed, and the cells were washed with phosphate-buffered saline (PBS). Then 400 μL of 0.1% MTT reagent was added, and the plate was incubated for 1 h. After removal of the solution, DMSO was added to each well. The absorbance was measured at 570 nm using a microplate reader. Melanin Content Assay and in Situ Tyrosinase Activity. Measurement of the melanin content was carried out according to the method described by Kumar et al.20 Briefly, after removal of the medium, the cells were washed twice with PBS. A sodium hydroxide solution (1 N, 1 mL) was added to each well to dissolve melanin. The melanin absorbance was measured at 405 nm using a microplate reader. In situ tyrosinase activity was assessed according to the method described by Newton at al.21 NHEMs were fixed with 4% paraformaldehyde in PBS for 15 min at 37 °C. After the fixation, the cells were permeabilized by 0.1% Triton X-100 treatment and then washed with PBS. The cells were incubated with 0.1% L-3,4-dihydroxyphenylalanine (L-DOPA) for 3 h at 37 °C, then rinsed with PBS, and observed under a microscope (Olympus Corp., Tokyo, Japan). PCR and Western Blot Analysis. Expression of tyrosinase, TRP-1, DCT, MITF, and SOX9 mRNA was measured by RT-PCR in melan-a cells. Extraction of total RNA and preparation of reverse-transcribed cDNA were performed as previously described.22 The sequences of the PCR primers used were based on published sequences for the tyrosinase, TRP-1, DCT, MITF,22 and SOX9 genes.23 PCR products were separated by gel electrophoresis in a 1.0% agarose gel containing ethidium bromide. To assess the protein expression of tyrosinase, TRP-1, DCT, MITF, and SOX9, Western blotting was conducted using a cell lysate. After the treatment, the cells were harvested and washed with PBS. Then, a lysis buffer [50 mM Tris-HCl, pH 8.0, 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl, 1% NP-40, 0.02% sodium azide, 0.5% sodium deoxycholate, 100 pg/mL phenylmethanesulfonyl fluoride, and 1 pg/mL aprotinin] was added. The protein concentration was measured using the Bradford reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of total protein were separated on 10% SDS− polyacrylamide gels. Separated proteins were transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, UK) and blocked with a solution containing 5% nonfat milk in Tris-buffered saline containing Tween 20 (TBST) for 1 h at room temperature. The membranes were incubated with primary antibodies against tyrosinase, TRP-1, DCT, MITF, and SOX9 at 4 °C overnight, then washed with TBST, and incubated with secondary antibodies (Santa Cruz Biotechnology) for 1 h at room temperature. Finally, immune complexes were detected using a 7734
DOI: 10.1021/acs.jafc.6b03256 J. Agric. Food Chem. 2016, 64, 7733−7742
Article
Journal of Agricultural and Food Chemistry
Figure 2. Effects of solvent fractions of an Agastache rugosa extract on (A) melanin content and (B) cell viability of melan-a cells. Four fractions, including n-hexane-, chloroform-, ethyl acetate-, and water-soluble fractions, were tested at concentrations of 1 and 10 μg/mL. Experiments were carried out in triplicate. The data are expressed as the mean ± SD. EPI-100-NMM medium). The medium was changed with a fresh one every 2 days. Compound 1 was dissolved in a mixture of PBS and propylene glycol (5:5, v/v) and then topically applied to the skin tissue once every 2 days for 14 days. At the end of the treatment, the tissue was washed with PBS and then fixed with 10% formalin. The fixed tissue was embedded in paraffin and sectioned at 4 μm. The sections were stained with hematoxylin−eosin (H&E) and a Fontana−Masson silver kit (IHC World, GA, USA) according to the manufacturer’s protocol. The stained slides were examined under a light microscope. Statistical Analysis. The data are expressed as the means ± standard deviation (SD). Statistical comparisons among different treatment groups were performed using one-way analysis of variance (ANOVA) with Tukey’s multiple-comparisons test. The level of statistical significance was set at P < 0.05.
ChemiDoc XRS+ imaging system (Bio-Rad). Densitometry analysis of the bands was performed using ImageQuant TL software, version 8.1 (GE Healthcare, Madison, WI, USA). Docking Simulations. The 3D X-ray protein structure of a complex of the HMG domain of the transcription factor SOX9 bound to DNA (SOX9/DNA) of Homo sapiens was obtained at 2.77 Å resolution from the Protein Data Bank (PDB code 4EUW) and prepared using the protein preparation wizard of the Schrödinger Maestro program. 3D conformations of compound 1 were generated using the Schrödinger LigPrep program with the optimized potentials for liquid simulations (OPLS) 2005 force field. For our docking study, the Schrödinger Glide program was used. A grid of 20 × 20 × 20 Å along the XYZ directions could cover the whole PDB structure, and the grid (20 Å cubic region) was generated under default conditions (scaling factor for van der Waals interactions, 0.7; partial charge cutoff of nonpolar atom, 0.25). The standard precision mode was chosen for docking scoring; flexible ligand sampling was considered (with nitrogen inversion permitted and with sampling of ring conformation), and penalties on the Epik state (ionization and tautomeric state) and nonpolar conformation of the amide group were assigned. After generation of docking poses, postdocking minimization of the top 100 poses according to a docking score was performed. As a result, the acquired 100 docking poses presented an extremely dominant binding region near the major groove consisting of DA 7, 8, 9, and 10. After 1 of the 100 poses had been chosen on the basis of frequency, the chosen pose was used as a centroid for a new grid (10 Å cubic region) and, subsequently, extra precision docking was conducted under the conditions of the first docking (except scoring function). To compare the docking mode of compound 1 with that of compound 7, docking of compound 7 was conducted under the same conditions. Additional docking work of compounds 2−6 was conducted using the grid files of compounds 1 and 7. Immunofluorescence. NHEMs were cultured on coverslips and treated with 10 μM compound 1. After 48 h, the cells were washed with PBS and then fixed in 4% paraformaldehyde for 15 min. The cells were permeabilized and blocked by incubation with 0.1% Triton X-100 in 1% horse serum-containing PBS for 30 min at room temperature. Then, the slides were incubated with a primary antibody at 4 °C overnight. After a washing with PBS, the slides were incubated with the appropriate fluorescence-conjugated secondary antibody. Finally, the slides were mounted and analyzed by using a Nikon inverted confocal microscope Eclipse Ti-E (Nikon, Tokyo, Japan). Zebrafish and Reconstructed Skin Tissue Studies. Embryos of zebrafish were obtained from the Zebrafish Resource Bank (Daegu, South Korea). Embryos were treated with compound 1 for 9−72 h postfertilization. After the treatment, the depigmentation effect of compound 1 on zebrafish was observed under a stereomicroscope. The reconstructed skin, MelanoDerm (MatTek, Ashland, MA, USA), a highly differentiated model of the human epidermis, consists of normal human epidermal keratinocytes and melanocytes. Reconstructed skin tissue was maintained in a 6-well plate format (5% CO2, 37 °C, 5 mL of
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RESULTS AND DISCUSSION Effects of Compounds from A. rugosa on Melanin Production in Melan-a Cells. A methanolic extract of A. rugosa was first fractionated into n-hexane-, chloroform-, EA-, and water-soluble fractions. These four fractions were tested for their inhibitory effects on melanin synthesis in melan-a cells. Melan-a cells, an immortalized pigmented cell line from nontumorigenic mouse melanocytes, have been widely used for pigmentation studies.24,25 In our prescreening study, the EA-soluble fraction showed the highest inhibitory effect on melanin synthesis, without any cytotoxicity (Figure 2). In an attempt to isolate an active compound from the EA-soluble fraction, we isolated seven compounds using various chromatographic techniques and identified those by extensive spectroscopic methods, including one-dimensional (1D) and 2D nuclear magnetic resonance spectroscopy and mass spectrometry. Subsequently, compounds 1−7 were tested for their inhibitory effects on melanin synthesis in melan-a cells. As a result, compound 1 at 10 μM concentration Table 1. Effects of Compounds 1−7 on Melanin Content and Cell Viability in Melan-a Cells control 1 2 3 4 5 6 7 PTU 7735
melanin content (%)
cell viability (%)
100.0 ± 3.4 61.6 ± 0.1 114.1 ± 4.5 105.8 ± 4.7 107.4 ± 0.2 109.9 ± 2.7 98.0 ± 6.8 120.8 ± 2.7 68.8 ± 5.2
100.0 ± 0.8 103.6 ± 3.8 92.4 ± 0.4 75.9 ± 5.3 79.7 ± 2.9 96.0 ± 3.2 87.4 ± 3.0 97.8 ± 3.1 97.9 ± 0.6 DOI: 10.1021/acs.jafc.6b03256 J. Agric. Food Chem. 2016, 64, 7733−7742
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Journal of Agricultural and Food Chemistry
Figure 3. Effects of compound 1 on tyrosinase activity in NHEMs. (A) Cells were incubated with L-DOPA for evaluating in situ tyrosinase activity after treatment with compound 1 (1, 5, and 10 μM) for 48 h. PTU was used as a positive control. Images were captured under bright-field microscopy. (B) Densitometric analysis of stained melanocytes was performed with the NIS-Elements imaging software, version 4.0 (Nikon, Tokyo, Japan). (C) Cell viability was measured by the MTT assay.
Figure 4. Effects of compound 1 on mRNA expression of melanogenesis genes in melan-a cells. Cells were treated with 5 and 10 μM compound 1 for 6 h. (A) Tyrosinase, TRP-1, DCT, MITF, and SOX9 mRNA expressions were examined by RT-PCR. The levels of gene expression of (B) tyrosinase, (C) TRP-1, (D) DCT, (E) MITF, and (F) SOX9 were normalized to that of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Statistical comparisons among the different treatment groups were performed using ANOVA with Tukey’s multiple-comparisons test. The level of statistical significance was set at (∗) P < 0.05, (∗∗) P < 0.01, and (∗∗∗) P < 0.001.
significantly inhibited melanin production by 61.6 ± 0.1% compared to that in an untreated control. At the same time, compound 1 had no effect on cell viability (Table 1). To compare potential effects of compound 1 on melanin synthesis, PTU was used positive control,26 which also inhibited melanin production at 68.8 ± 5.2%. Inhibitory Effects of Compound 1 on in Situ Tyrosinase Activity in Normal Human Epidermal Melanocytes. To elucidate whether compound 1 has a hypopigmentation effect on a normal cell line, we used a human-originated cell line of NHEMs. NHEMs were treated with compound 1 for 48 h and then incubated in the presence of L-DOPA, which has been
used for observation of tyrosinase activity in situ,21 because melanin production is related to tyrosinase activity in human melanocytes. Measuring tyrosinase activity is a sensitive method for melanogenesis.27,28 The results showed that compound 1 suppressed tyrosinase activity at concentrations of 5 and 10 μM (Figure 3A,B) without cytotoxicity (Figure 3C). These results supported the suggestion that the decrease of melanin synthesis by compound 1 was caused by regulation of the tyrosinase enzyme. PTU as positive control also suppressed tyrosinase activity at a concentration of 10 μM. Down-regulation of Melanogenic Genes by Treatment of Melan-a Cells with Compound 1. Tyrosinase, TRP-1, and 7736
DOI: 10.1021/acs.jafc.6b03256 J. Agric. Food Chem. 2016, 64, 7733−7742
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Journal of Agricultural and Food Chemistry
Figure 5. Docking simulations of the binding complex of SOX9−DNA with compounds 1 and 7. (A) SOX9−DNA complex with compound 1. (B) SOX9−DNA complex with compound 7. Hydrogen bonds are indicated by yellow-dot lines, and pi−pi interactions are indicated by cyan-dot lines.
regulation of these target genes, including MITF, which is a transcriptional factor regulating the three melanogenic enzymes.34 Interestingly, researchers have found that the SOX family is related to the melanocyte development and melanogenesis through MITF regulation.9 Thus, SOX5 participates in MITF balancing in human melanoma cells,35 whereas SOX10 is a well-known target gene associated with melanogenesis, which directly activates
DCT have already been proven to be the key enzymes for melanin biosynthesis.29,30 These three enzymes control melanin synthesis in organelles called melanosomes.31 Tyrosinase converts L-tyrosine into L-DOPA in an early step, and subsequently L-DOPA is converted into dopaquinone. DCT and TRP-1 participate in the next step of melanogenesis, from dopachrome to a melanin polymer.32,33 Many studies have been focused on the 7737
DOI: 10.1021/acs.jafc.6b03256 J. Agric. Food Chem. 2016, 64, 7733−7742
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Journal of Agricultural and Food Chemistry
Figure 6. Compound 1 down-regulated the expression of SOX9 and MITF, resulting in a decrease of expression of melanogenesis-related proteins. (A) After 48 h, SOX9 (green) and MITF (red) were detected by immunocytochemistry. The proteins were strongly expressed in the nucleus of the control cells. However, a decrease of SOX9 and MITF expression (indicated by arrows) was observed in the cells treated with compound 1. (B−D) Tyrosinase, TRP-1, and DCT were detected by Alexa 488 (green) fluorescence after incubation with respective antibodies.
MITF transcription.36 In various animal models, a mutation in SOX10 caused pigmentary defects.37,38 In a recent paper, SOX9 silencing by small interfering RNA knockdown resulted in decreased MITF expression and led to a conclusion that SOX9 regulates the MITF expression,8 even though SOX9 did not regulate SOX10. Although no mutual correlation among members of the SOX family has been clearly proven yet, many studies have demonstrated that the SOX9 gene is a key factor regulating melanogenesis. In this study, to investigate the mechanism of the depigmentation action of compound 1, we performed reverse transcription polymerase chain reaction (RT-PCR) experiments to determine mRNA levels of tyrosinase, TRP-1, DCT, MITF, and SOX9. Our results showed that treatment with compound 1 at a concentration of 10 μM significantly down-regulated the key melanogenic genes, including SOX9, in melan-a cells (Figure 4). Docking Simulations between Complex of SOX9−DNA and Compound 1. Through docking simulations, we were able
to propose a binding complex of SOX9−DNA with compound 1. Compound 1 formed multiple hydrogen bonds with DNA and a hydrogen bond network with DNA and Lys106 in the HMG-box domain of SOX9 (Figure 5A). In particular, (1) the tetrahydropyran oxygen in compound 1 formed a hydrogen bond with the NH2 group in DA 9; (2) the primary alcohol formed a hydrogen bond with guanosine N-7 (sp2) in DA 10; (3) the ortho-phenolic OH group in compound 1 formed two hydrogen bonds with the adenosine N-7 (sp2) and adenosine 6-NH2 groups in DA 7; and (4) the tetrahydropyran 4-OH group in compound 1 formed a hydrogen bond network with DA 9 and Lys106. In the binding mode, the phenolic OH group existed in a very narrow proximity to DA 7, with two noncovalent interactions. Even though the X-ray crystal structure used in our study could show one conformation of the SOX9−DNA complex and the current docking study did not show any direct noncovalent interaction between SOX9 and compound 1, the 7738
DOI: 10.1021/acs.jafc.6b03256 J. Agric. Food Chem. 2016, 64, 7733−7742
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Journal of Agricultural and Food Chemistry
Figure 7. Depigmentation effects of compound 1 in zebrafish and reconstructed skin. (A) Synchronized zebrafish embryos were treated with 5, 10, and 30 μM compound 1 and PTU, respectively, and observed under a stereomicroscope after 63 h. (B) 0.1% compound 1 (w/v) and 1% PTU (w/v) were applied, respectively, to reconstructed skin tissue for 14 days. Following the incubation period, the tissue was photographed using a digital camera. (C) Reconstructed tissue was fixed and stained with H&E (top) and Fontana−Masson silver stain (bottom).
Figure 8. Schematic representation of the mechanism of inhibitory action of compound 1 on melanogenesis. Compound 1 down-regulates MITF, which is known as a transcriptional factor regulating melanogenesis-related enzymes such as tyrosinase, TRP-1, and DCT. Compound 1 does not affect the phosphorylation of CREB. Reduction of the MITF expression induces down-regulation of tyrosinase, TRP-1, and DCT, resulting in a decrease of melanin synthesis.
DNA-binding inhibitors of transcription factors among three classes of inhibition modes of a transcription factor.39 In addition, our docking simulation supports the data of our experiments on
hydrogen bond network and position of compound 1 strongly support the possibility that compound 1 is a small-molecule DNA binder regulating transcription factors based on reported 7739
DOI: 10.1021/acs.jafc.6b03256 J. Agric. Food Chem. 2016, 64, 7733−7742
Article
Journal of Agricultural and Food Chemistry
To measure its depigmentation effect in 3D epidermal skin tissue, compound 1 was directly applied to skin tissue for 14 days. In the absence of compound 1, the color of the skin tissue changed to black due to the production of melanin in melanocytes. However, upon treatment with compound 1, the skin tissue was light-colored compared to the black-colored control skin tissue. Moreover, compound 1 and PTU showed equivalent efficacies in this experiment (Figure 7B). In a histological analysis, compound 1 did not show any toxic effect on tissue. The melanin content in the basal layer, where melanocytes are located in the epidermis (Figure 7C), was decreased by the compound. In summary, the present study demonstrated that demethyleugenol β-D-glucopyranoside (compound 1) isolated from A. rugosa inhibited melanin synthesis both in vitro and in vivo. Interestingly, compound 1 down-regulated SOX9 expression and MITF levels, which resulted in the suppression of the melanogenic enzymes tyrosinase, TRP-1, and DCT (Figure 8). These findings suggest that compound 1 may be a potential candidate for the treatment of hyperpigmentary disorders, such as postinflammatory hyperpigmentation, melisma, solar lentigo, and geriatric pigment spots.
SOX9 inhibition. Our docking study could elucidate why compound 1 can inhibit SOX9, whereas compound 7 cannot, despite a very small difference in substituents between the two compounds. The distance between the 6-NH2 group in DA 7 and the OH group in compound 1 was shown to be
