Ishophloroglucin A Isolated From Ishige Okamurae Suppresses Melanogenesis Induced By α-MSH: In Vitro And In Vivo Part 1
Apr 03, 2023
Abstract: Diphlorethohydroxycarmalol (DPHC) isolated from Ishige Okamura (IO) showed potential whitening effects against UV-B radiation. However, the components of IO and their molecular mechanism against α-melanocyte-stimulating hormone (α-MSH) have not yet been investigated. Thus, this study aimed to investigate the inhibitory effects of Ishophloroglucin A (IPA), a phlorotannin isolated from brown algae IO, and its crude extract (IOE), in melanogenesis in vivo in an α-MSH-induced zebrafish model and in B16F10 melanoma cells in vitro. Molecular docking studies of the phlorotannins were carried out to determine their inhibitory effects and elucidate their interaction mode with tyrosinase, a glycoprotein related to melanogenesis. In addition, morphological changes and melanin content decreased in the α-MSH-induced zebrafish model after IPA and IOE treatment. Furthermore, Western blotting revealed that IPA upregulated the extracellular-related protein expression in α-MSH-stimulated B16F10 cells. Hence, these results suggest that IPA isolated from IOE has the potential for use in the pharmaceutical and cosmetic industries.
According to relevant studies,cistanche is a common herb known as "the miracle herb that prolongs life". Its main component is cistanoside, which has various effects such as antioxidant, anti-inflammatory, and immune function promotion. The mechanism between cistanche and skin whitening lies in the antioxidant effect of cistanche glycosides. Melanin in human skin is produced by the oxidation of tyrosine catalyzed by tyrosinase. The oxidation reaction requires the participation of oxygen, so the oxygen-free radicals in the body become an important factor affecting melanin production. Cistanche contains cistanoside, which is an antioxidant and can reduce the generation of free radicals in the body, thus inhibiting melanin production.
1. Introduction
Marine algae have gained immense attention from the functional food, cosmetics, and pharmaceutical industries as they utilize bioactive compounds such as phlorotannins, fucoidans, polysaccharides, and proteins [1–7]. According to a previous study, Ishige Okamurae (IO) was screened for its inhibitory effects on tyrosinase activity [8]. Although its phlorotannin, diphlorethohydroxycarmalol (DPHC), was assessed for anti-melanogenesis activity induced by UV-B radiation in an in vitro model [8], no further studies were performed to explain the interaction between DPHC and melanogenesis-related proteins. In this study, a novel polyphenol compound, Ishophloroglucin A (IPA) [9], was evaluated for its interaction with tyrosinase in a molecular docking study, compared with that of DPHC.
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Molecular docking is an efficient computational method to predict the potential binding mode of small molecules or ligands within the active site of a protein or receptor of a known three-dimensional structure for studying their interaction patterns or for drug design [10–12]. Previous studies have reported tyrosinase enzyme's interaction site and energy value as investigated by molecular docking studies [13,14]. Functionally, tyrosinase is a rate-limiting enzyme that catalyzes a relatively slow reaction rate in the signaling pathway and plays a pivotal role in melanin biosynthesis in specialized organelles called melanosomes, specifically synthesized by melanocytic cells [15]. Thus, the enzymatic activity of tyrosinase as modeled through in silico molecular docking simulations has been the target for investigating inhibitors to prevent cutaneous hyperpigmented disorders. Based on this, in experimental and computational fields, many phenolic compounds or inhibitors possessing metal chelating ability have been widely studied as tyrosinase inhibitors [16].
Melanin primarily modulates skin color and acts as a protective pigment when the skin is exposed to UV radiation [17]. Exposure to UV radiation causes the release of α-melanocyte-stimulating hormone (α-MSH) from cutaneous keratinocytes and melanocytes to induce melanin synthesis and subsequently protect the skin from the damaging effects of solar radiation [18]. However, long-term exposure to UV radiation induces signifificant melanin synthesis by α-MSH, resulting in skin damage such as freckles, sunspots, and black spots on the epidermis, leading to DNA photodamage and skin cancer [19]. Following this, the administration of α-MSH into melanocytes has been widely studied to induce considerable changes in the tyrosinase activity for melanogenesis in melanoma [20].
Zebrafish display variations in skin color like humans. They are an animal model used for understanding the genetic origin of human skin color, connecting basic model organism biology with human genetics [21]. In addition, the pigments on the ventral and lateral regions of the zebrafish gastrula and their transparency during larval stages allow simple observation of the pigmentation process, providing an opportunity to develop viable models for understanding skin cell disorders resulting from defects in melanocyte development [22–24].
Melanin synthesis occurs via the activity of several melanogenesis-related proteins, such as tyrosinase, microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (Trp-2), and tyrosinase-related protein-1 (Trp-1) [15]. In this study, we aimed to investigate the inhibitory effect of IPA and IO crude extract (IOE) in tyrosinase activity and melanogenesis on α-MSH-induced zebrafish in vivo and B16F10 melanoma cells in vitro to evaluate their potential use in treating skin pigmentation and melanoma.
2. Results
2.1. Molecular Docking Study
A docking method was carried out to simulate the binding of the commercial tyrosinase enzyme with IPA, DPHC, and arbutin [25]. The binding modes of IPA, DPHC, or arbutin with mushroom tyrosinase are presented in Figure 1A–C. As shown in the 3D diagram of the tyrosinase–IPA complex (Figure 1A), tyrosinase–DPHC (Figure 1B), and tyrosinase–arbutin (Figure 1C), although IPA, DPHC, and arbutin docked to the active site, the tyrosinase–IPA complex showed the largest binding surface area. In addition, as shown in a 2D diagram, the tyrosinase–IPA complex showed more interactions with the various amino acids of tyrosinase, compared to either tyrosinase–DPHC or tyrosinase–arbutin. Seven benzene rings (1st, 2nd, 3rd, 8th, 9th, 12th, and 16th) and their oxygen atoms have π–π interactions with Histidine60, Methionine61, Lysine157, Glutamate158, Proline160, Proline201, Arginine206, and Valine218. In particular, the 16th benzene ring was combined with Histidine60 and Histidine204, the main amino acids of the active site. In addition, many oxygen atoms in IPA interacted via hydrogen bonds with Glutamate158, Proline160, Aspartate167, Methionine184, Phenylalanine197, Asparagine199, Glutamine202, Asparagine205, and Arginine209. Furthermore, based on the docking analysis results, it was calculated that the total binding energy and CDOCKER interaction energy using the CDOCKER interaction energy program of DS 3.0 (Figure 1D) were as follows: interaction energy with tyrosinase: Ishophloroglucin A (IPA): −152.154 kcal/mol, diphlorethohydroxycarmalol (DPHC): −65.5221 kcal/mol and arbutin: −33.6835 kcal/mol and the binding energy with tyrosinase: Ishophloroglucin A (IPA): −546.504 kcal/mol, diphlorethohydroxycarmalol (DPHC): −407.706 kcal/mol and arbutin: −79.0913 kcal/mol.
2.2. Effects of Melanogenic Inhibitors on Melanin Synthesis in Zebrafish Larvae
The purpose of this study was to determine whether IPA and IOE can inhibit melanogenesis in zebrafish larvae in vivo. In our previous study, IPA and IOE showed no toxicity in zebrafish larvae [26]. We treated zebrafish larvae with zebrafish (0.05, 0.15, and 0.5 nM) and IOE (3, 10, and 30 µg/mL) to determine their melanin content. To estimate the inhibitory activities, we measured the total melanin content of the zebrafish extracts. The phenotypic effect on zebrafish melanin was observed by analyzing their body pigmentation under a microscope at room temperature. Surface melanin was significantly reduced by various concentrations of the target inhibitors (Figure 2). Treatment with 3 nM α-MSH significantly increased the melanin content of zebrafish compared with the untreated group. The highest concentrations of IPA (0.5 nM) and IOE (30 µg/mL) decreased the total melanin content by almost 28.56% and 30.36%, respectively, which presented similar effects as the arbutin (200 µM)-treated group (Figure 2A, B).
2.3. Effects of IPA and IOE on Melanin Synthesis in B16F10 Cells
The cytotoxic effects of IPA and IOE on B16F10 cells were measured via the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. We initially examined the cytotoxic effects of IPA and IOE on B16F10 cells treated with different concentrations without α-MSH stimulation. IPA (0.15, 0.5, 1.5, and 5 nM) did not reveal any signifificant cytotoxicity on B16F10 cells, whereas IOE (1, 3, 10, and 30 µg/mL) showed no cytotoxicity (Figure 3A, B). The concentrations of IPA and IOE with no toxicity were used for further study.
We examined whether the effect of melanin inhibition from either IPA or IOE treatment in vivo was reversible without α-MSH stimulation in B16F10 cells. Melanin content was significantly decreased upon treatment with IPA (5 nM) or IOE (30 µg/mL) compared to the control group (Figure 3C, D). These results suggested that IPA and IOE inhibited melanin synthesis without α-MSH stimulation in B16F10 cells, thus contributing to its anti-melanogenesis effect.
2.4. Effects of IPA and IOE on Tyrosinase Activity and Melanin Synthesis in α-MSH-Stimulated B16F10 Cells
Hence, doses of IPA (0.5, 1.5, and 5 nM) and IOE (3, 10, and 30 µg/mL) were incubated with B16F10 melanoma cells to determine their bioactivity in cellular tyrosinase activity and α-MSH-mediated melanogenesis.
The concentrations of α-MSH and arbutin were determined based on the results of their cytotoxicity and melanin production in B16F10 cells (Figure S2A–D). By analyzing the data on survival rate, 1 nM of α-MSH and 100 µM of arbutin were used for further experiments.
As for the cellular tyrosinase activity, although the inhibitory effects of 1.5 nM of IPA were relatively lower than those depicted in the positive control, the concentration differed nearly 10 times, with the higher concentration of arbutin at 100 µM (Figure 4A). IOE reduced tyrosinase activity with a slight fluctuation between the effects of different concentrations compared to the α-MSH-treated group (Figure 4B). As for α-MSH-mediated melanogenesis, signifificant reductions in melanin content were observed in the 1.5 and 5 nM IPA-treated groups (Figure 4C). In addition, 30 µg/mL IOE inhibited pigmentation to the point that resembled the blank group (Figure 4D). These results suggest that IPA and IOE downregulated tyrosinase activity and that this inhibitory effect may lead to decreased cellular melanin synthesis in B16F10 cells.
2.5. Effects of IPA and IOE on the Molecular Mechanism in α-MSH-Stimulated B16F10 Cells
To elucidate the mechanism responsible for their melanogenesis inhibitory effect, we determined the influence of IPA and IOE on the expression levels of signaling molecules involved in melanin synthesis (Figure 5). ERK (extracellular signal-regulated kinase), JNK (Jun N-terminal kinase), and p38 belong to the mitogen-activated protein kinase (MAPK) intracellular signal transduction cascade [27,28]. As presented in Figure 5A, B, ERK phosphorylation was enhanced with IPA treatment, whereas the phosphorylation of JNK was slightly decreased after treatment with IPA (1.5 and 5 nM) or IOE (30 µg/mL). In addition, as presented in Figure 5C, p-p38 levels were highly increased in the α-MSH-stimulated groups, at around 80% compared to the control. Furthermore, after treatment with IPA, IOE, or arbutin, the phosphorylation of p38 was slightly suppressed. These findings suggest that the melanogenic inhibitory effects of IPA and IOE may be related to the JNK and p38 MAPK signaling pathways.
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