Human skin, hair, and eye coloration are intricately governed by a biological process called melanogenesis. This process involves synthesizing and distributing melanin, a polymerized pigment originating from the amino acid tyrosine. Beyond its role in coloration, melanin is pivotal in safeguarding the skin against harmful ultraviolet (UV) radiation and contributing to overall homeostasis [1]. In addition to being a fundamental interest, understanding the intricate mechanisms of melanogenesis has important implications in various fields, including dermatology, genetics, and cosmetic research.

Melanogenesis begins with the activation of specialized pigment-producing cells known as melanocytes, located in the basal layer of the epidermis, hair follicles, and uveal tract of the eye [2]. Melanocytes derive from melanoblasts originating from embryonic neural crest cells [3] and, in the skin, one melanocyte is surrounded by approximately 36 keratinocytes [4]. Melanogenesis is regulated by a complex interplay of genetic, cellular, and environmental factors. Upon stimulation, melanocytes synthesize and deposit melanin within specialized organelles called melanosomes. These melanosomes are subsequently transported and transferred to adjacent keratinocytes, ultimately enhancing the pigmentation of tissues [4]. The production of eumelanin, which gives brown to black color, and pheomelanin, which imparts yellow to red hues, occurs through additional enzymatic processes. This outcome depends on factors such as the enzyme ratios, substrate availability, and the pH conditions inside melanosomes [4].

Several regulatory mechanisms control melanogenesis, ensuring its fine-tuned regulation in response to physiological and environmental cues. The most notable regulators include $\alpha$-melanocyte-stimulating hormone ($\alpha$-MSH), derived from proopiomelanocortin (POMC), and its receptor, melanocortin 1 receptor (MC1R) [5]. The binding of $\alpha$-MSH to MC1R leads to increased tyrosinase activity and melanin synthesis [6]. Importantly, genetic factors play a substantial role in determining the regulation and variation of melanogenesis in humans. Moreover, mutations in genes involved in melanin synthesis can lead to various disorders, including albinism, vitiligo, and melanoma, highlighting the clinical relevance of understanding melanogenesis [7].

Understanding the molecular intricacies of melanogenesis has been the subject of intense scientific investigation. Researchers have identified numerous factors involved in melanocyte development, differentiation, and pigment production. Elucidating the signaling pathways, transcription factors, and regulatory elements that govern melanogenesis has provided valuable insights into the complex interplay between genetic determinants and environmental influences [8]. Melanogenesis goes beyond determining pigmentation; it has clinical significance linked to skin disorders. Conditions like melasma and post-inflammatory hyperpigmentation, for example, stem from excessive melanin production and uneven distribution, leading to hyperpigmentation issues [9].

As per the reports, laboratory scale-70% ethanol extracts of Sargassum spp. found around the Korean Sea indicated tyrosinase inhibitory effects [10]. Another interesting study conducted by S.H. CHA and the team found that Sargassum silquastrum reduced cellular melanin synthesis and tyrosinase activity along with the inhibitory effects on the pigmentation of zebrafish in vivo model [11]. Moreover, M.S. Azam et al. [12] reported that ethanolic extract from Sargassum serratifolium indicated hypo pigmenting properties on B16F10 murine melanocytes. In addition, Sargassum cristaefolium and Sargassum thunbergii have exhibited the inhibitory potential of melanin production in B16F10 murine melanocytes [13, 14]. By considering the potential of Sargassum spp. on skin whitening, Sargassum horneri 70% ethanol extract (SHE70), S. horneri 50% ethanol extract (SHE50), and S. horneri ultrasonic-assisted ethanol extract (SHUE) were screened for their melanin inhibitory potential in $\alpha$-MSH-stimulated B16F10 murine melanocytes in this study. Moreover, by taking screening findings into account, an extensive study was designed to reveal whether SHUE indicates potent anti-melanogenic activity on $\alpha$-MSH-stimulated B16F10 murine melanocytes, hypothesizing that SHUE regulates cellular tyrosinase activity as well as melanogenesis-related signaling.

Skin whitening products have gained popularity among consumers worldwide, with an estimated 15% of the global population engaging in their use. As reported in 2022, approximately $13 billion was expended on skincare and cosmetic products alone in the Asia-Pacific region [22]. Unfortunately, the global circulation of skin-lightening cosmetics containing illicit and hazardous ingredients is readily available in various countries and sold through both conventional and online ways [23]. With health concerns, the quest for naturally available multidisciplinary drugs for skin whitening is of paramount significance in dermatological research [24]. Such compounds hold the potential to address hyperpigmentation and other skin-related issues while minimizing adverse effects [24]. Natural ingredients offer a promising avenue to develop safe and effective cosmeceutical agents, as they often encompass a range of bioactive compounds with complementary mechanisms of action [25]. By revealing the power of natural sources, we can tap into a wealth of biodiversity and traditional knowledge, accelerating the discovery of novel compounds for achieving even skin tone. This pursuit aligns with the growing demand for safer alternatives in the cosmetics and dermatology industries, emphasizing the need for a holistic approach to skin whitening that prioritizes both efficacy and safety.

In the present study, we prepared the three different extracts (SHE50, SHE70, and SHUE) based on two extraction approaches, room temperature S. horneri ethanol extraction and ultrasonic assisted ethanol extraction. The ultrasonic-assisted ethanol extraction method and room temperature ethanol extraction method which were used in this study are both implemented in extracting compounds from botanical materials, yet they diverge in efficiency and the techniques used. The ultrasonic-assisted ethanol extraction method utilizes high-frequency sound waves to enhance the extraction process. This results in increased penetration of the solvent into the plant matrix [26]. It facilitates the extraction of target compounds more rapidly and efficiently compared to room temperature extraction, which relies solely on the natural diffusion of the solvent. Moreover, ultrasonic-assisted extraction typically requires shorter extraction times and lower temperatures, thus preserving thermally sensitive compounds better [27]. On the other hand, room-temperature ethanol extraction is simpler and more accessible, requiring no specialized equipment. Results obtained from the compositional analysis as well as HLPC analysis for the target compound (Fucosterol) indicated that SHUE consists of a higher yield and a better intracellular melanin reduction potential in contrast to the other two extracts in the preliminary investigation.

When discussing melanogenesis, the key enzyme namely tyrosinase which is a copper-containing glycoprotein catalyzes the hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA). Subsequently, L-DOPA is converted to dopaquinone [28]. Recent studies have shed light on the role of microenvironmental factors in melanogenesis. For instance, UV radiation is a potent inducer of melanin production, acting through the activation of various signaling cascades, including the mitogen-activated protein kinase (MAPK) pathway [4, 29]. UV exposure triggers DNA damage and oxidative stress, leading to the activation of melanocyte-stimulating factors and the upregulation of melanogenic enzymes. These adaptive responses serve as a protective mechanism against UV-induced DNA damage by increasing the production and distribution of melanin [30]. In addition to UV radiation, other environmental factors, such as hormonal changes, inflammation, and chemical exposure, can influence melanogenesis. Hormones, particularly estrogens, have been shown to modulate melanocyte function and melanin synthesis [31]. Inflammatory mediators, such as prostaglandins and cytokines, can disrupt melanogenesis by altering the expression and activity of melanogenic enzymes [9]. A neuropeptide hormone, $\alpha$-MSH, plays a pivotal role in the regulation of melanogenesis, particularly in B16F10 murine melanocytes. It has the ability to exert its effects through binding to the MC1R on the surface of melanocytes [29]. As reported, activation of MC1R triggers a signaling cascade that culminates in increased production of melanin [5]. So far, research on the regulation of melanin production has implemented $\alpha$-MSH as a positive inducer [32]. Furthermore, various cytokines, growth factors, and signaling pathways modulate melanogenesis. For instance, endothelin-1 (ET-1), secreted by keratinocytes and fibroblasts, enhances the production and release of $\alpha$-MSH and other melanogenic factors [33]. As per the analysis results obtained from the present study, $\alpha$-MSH has no cytotoxic effect on the B16F10 murine melanocytes. Interestingly, the treatment of B16F10 murine melanocytes with SHUE did not show any signs of cytotoxicity up to the concentration of 125 µg/mL. Based on the statistical analysis, 15.6, 31.3, and 62.5 µg/mL have no significant cytotoxicity, therefore, further investigations were conducted with these concentrations on the $\alpha$-MSH-stimulated B16F10 murine melanocytes. With satisfactory results, the study was extended to demonstrate the inhibitory mechanism of SHUE in $\alpha$-MSH-stimulated B16F10 murine melanocytes.

Intracellular melanin content correlates directly with the activity of tyrosinase [17]. To begin assessing the impact of SHUE on $\alpha$-MSH-induced melanogenesis, our initial step involved examining whether SHUE could inhibit melanin synthesis in $\alpha$-MSH-stimulated B16F10 murine melanocytes. Results indicated that SHUE significantly suppressed the $\alpha$-MSH-induced melanin production and simultaneously indicated the notable reduction of intracellular tyrosinase activity in B16F10 murine melanocytes in contrast to the control group. Tyrosinase serves as a crucial rate-limiting enzyme that regulates the pace of melanin synthesis. Consequently, a reduction in the activity of tyrosinase leads to a decrease in melanin production [34]. Numerous prior research studies have documented compounds extracted from natural sources as well as brown seaweed extracts effectively curbed tyrosinase activity, resulting in the inhibition of melanin production [35].

Many similar studies about natural extracts or compounds have shown that they directly inhibit the catalytic activity of tyrosinase by regulating the molecular structure [36, 37, 38]. However, the cell lysate contained tyrosinase as well as TRP-1, TRP-2, and various other signaling molecules. Following the reveal of the potential of SHUE to suppress tyrosinase activity in this study, we resolved to investigate how SHUE regulates the expression of proteins involved in melanin synthesis. Moreover, the mechanism behind its suppression of melanin production was investigated. TRP-2, one of the enzymes involved in melanin synthesis, catalyzes the reaction from DOPA chrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) [39]. Therefore, like tyrosinase, a decrease in the protein expression level of TRP-2 is considered a cause of melanin production suppression. MITF is the transcription factor of tyrosinase, TRP-1, and TRP-2; and it is known as the most important transcription factor in melanin production. Protein expression analyzed by western blotting revealed that the expression levels of tyrosinase, TRP-1, and TRP-2 were significantly decreased by the SHUE treatment in stimulated B16F10 murine melanocytes. Furthermore, the expression of MITF was also decreased by the SHUE treatment in stimulated B16F10 murine melanocytes. Findings of the study suggest that SHUE suppresses tyrosinase activity, based on the downregulation of tyrosinase and MITF expression levels, resulting in the suppression of melanin synthesis. Recent studies indicate that the ERK kinase cascade and the AKT pathway play roles in regulating melanin synthesis. As reported, they trigger MITF phosphorylation, which breaks it down and ultimately reduces melanin production when ERK and AKT are activated [40]. Moreover, the melanocyte differentiation program activates the MITF expression. Therefore, this cascade is consistent with the idea that MITF is a master regulator of melanogenesis. In addition, MITF is a nuclear mediator of Wnt signals during melanocyte differentiation by inhibiting Glycogen synthase kinase 3$\beta$ (GSK3$\beta$), and stimulation of $\beta$-catenin accumulation. Furthermore, GSk3$\beta$ is implicated in regulating melanogenesis. Inhibition of GSk3$\beta$ activity could increase melanin synthesis through Wnt/$\beta$-catenin pathway activation. Therefore, activation of Wnt/$\beta$-catenin signaling pathway may increase melanin production [41]. As an extension to the study, we focused on the ERK and AKT pathways, which promote the degradation of MITF or inhibit the transcriptional activity; and examined their relationship with the SHUE inhibitory effect on melanin production. In this experiment, we identified that the most important factors in the SHUE downregulating effects were AKT and ERK phosphorylation in $\alpha$-MSH-stimulated B16F10 murine melanocytes. Further, we verified the involvement of ERK with the ERK inhibitor in $\alpha$-MSH-stimulated B16F10 murine melanocytes. The results suggest that although a higher dose of SHUE has some cytotoxicity, it still has the potential as an effective whitening agent for in vivo studies.

In summary, our study indicates that SHUE effectively inhibits melanin synthesis and tyrosinase activity by targeting the ERK pathway, leading to the suppression of MITF, tyrosinase, TRP-1, and TRP-2 in $\alpha$-MSH-stimulated B16F10 murine melanocytes. This suggests the potential utility of SHUE as a therapeutic agent for treating hyperpigmentation and as an effective ingredient in skin whitening and lightening cosmetics. Nonetheless, it is essential to emphasize that safety should remain paramount in future practical applications, particularly in in vivo studies.

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

All authors contributed to the conception and design of the research study. KGISK, AMKJ, CIK, and YSA performed the research. SJH, JYO, EAK, SHC and GA provided help and advice on the experiments. KGISK, AMKJ, SJH, and EAK performed data analysis, curation, and visualization. SHC confirmed the reproduction of the results. CIK, YSA, JYO, and GA provided funding and resources. CIK, YSA, and GA were responsible for overall project supervision. KGISK wrote the draft manuscript, and AMKJ, CIK, YSA, SJH, JYO and EAK contributed to its review and editing. SHC and GA conceived the manuscript and contributed to its review and editing. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects.

S. horneri was collected along the West coast of Jeju island. And sample was identified by the Biodiversity Research Institute in Jeju, South Korea (voucher specimen number is SH2017J005).

Not applicable.

This work was supported by the Technology Development Program (S3266086) funded by the Ministry of SMEs and Startups (MSS, Korea).

Given Dr. Seon-Heui Cha’s role as an editorial board member, Dr. Seon-Heui Cha was not involved in the peer review of this article and has no access to information regarding its peer review. Full responsibility for the editorial process for this article was delegated to Marcello Iriti. Mr. Chang-Ik Ko and Yong-Seok Ahn work researchers at Choung Ryong Fisheries Co., Ltd. as researchers and were involved ultrasonic extract preparation and provided it for the study. All responsibility for this article was delegated to Dr. Seon-Heui Cha and Dr. Ginnae Ahn. Other authors declare no conflict of interest.