Melanogenesis Inhibitors from the Rhizoma of Ligusticum Sinense in B16-F10 Melanoma Cells In Vitro and Zebrafish In Vivo

The rhizoma of Ligusticum sinense, a Chinese medicinal plant, has long been used as a cosmetic for the whitening and hydrating of the skin in ancient China. In order to investigate the antimelanogenic components of the rhizoma of L. sinense, we performed an antimelanogenesis assay-guided purification using semi-preparative HPLC accompanied with spectroscopic analysis to determine the active components. Based on the bioassay-guided method, 24 compounds were isolated and identified from the ethyl acetate layer of methanolic extracts of L. sinense, and among these, 5-[3-(4-hydroxy-3-methoxyphenyl)allyl]ferulic acid (1) and cis-4-pentylcyclohex-3-ene-1,2-diol (2) were new compounds. All the pure isolates were subjected to antimelanogenesis assay using murine melanoma B16-F10 cells. Compound 1 and (3S,3aR)-neocnidilide (8) exhibited antimelanogenesis activities with IC50 values of 78.9 and 31.1 μM, respectively, without obvious cytotoxicity. Further investigation showed that compound 8 demonstrated significant anti-pigmentation activity on zebrafish embryos (10‒20 μM) compared to arbutin (20 μM), and without any cytotoxicity against normal human epidermal keratinocytes. These findings suggest that (3S,3aR)-neocnidilide (8) is a potent antimelanogenic and non-cytotoxic natural compound and may be developed potentially as a skin-whitening agent for cosmetic uses.


Introduction
Melanin is a brown black pigment that is principally responsible for the color of skin, hair, and eyes [1]. Melanin is a biopolymer and includes two major classes of pigments in human skin, brownish black eumelanin and reddish yellow pheomelanin [1]. Melanin biosynthesis is a complex multistep process called melanogenesis, which is a physiological response of human skin to prevent deleterious effects of ultraviolet (UV) radiation and environmental pollutants. However,

Effects of Compounds 1 and 8 on Melanin Content in α-MSH-Stimulated B16-F10 Cells
To ascertain the depigmentation constituents in rhizoma of L. sinense, all the pure isolates were subjected to antimelanogenesis assay in B16-F10 melanoma cells. Murine melanoma B16-F10 cells are a well-established model for antimelanogenic principles discovery, and have been widely adopted in previous studies [48]. α-Melanocyte-stimulating hormone (α-MSH) is a peptide hormone and responsible for the production of melanin by melanocytes through activating melanocortin 1 receptor [49]. In this research, B16-F10 cells were stimulated with α-MSH (100 nM) and simultaneously treated with each compound at concentrations of 25, 50, or 100 µM. The melanin content of B16-F10 melanoma cells without compound treatment was assigned as 100%. Arbutin, a common skin whitening agent in cosmetic products, was used as positive control. Of these compounds, 1 and 8 inhibit α-MSH-induced melanin production in a dose-dependent manner. The IC 50 values of compounds 1 and 8 were 78.9 and 31.1 µM, respectively ( Figure 3B). Compound 1 and 8 at the effective concentrations did not show obvious effects on MTT assay ( Figure 3A). The MTT results may arise from the combined effects of cell proliferation reduction and cell viability inhibition according to the experimental conditions. These results suggested that the anti-melanogenic effects of compound 1 and 8 did not attribute to the cell death or growth inhibition.
Using the HPLC-DAD method, the main effective constituents (1 and 8) were characterized from the crude extract ( Figure 4) by comparing with the retention time of pure 1 (from laboratory synthesis, data not published yet) and 8 (from Sigma) as standards. Stock solutions of 1, 10, 20, 40, 60, 80 and 100 µg/mL were utilized. Each concentration was injected in triplicate. The content ratios of 1 and 8 in the extract of dried material were quantified to be 0.009% and 0.15% (w/w) by linear regression of the respective peak areas.

In Vivo Zebrafish Pigmentation Assay
In addition to evaluate the effect of 8 on in vitro antimelanogenesis, its in vivo anti-pigmentation ability through zebrafish pigmentation assay was further investigated. Zebrafish has been considered as an advantageous vertebrate model organism due to its small size, high fecundity, and similar gene sequences and organ systems to human beings. Additionally, the melanin pigmentation process on its surface allowing easy observation makes zebrafish a particular useful model for investigating in vivo melanogenic inhibitors or stimulators [50]. In this study, arbutin at 20 mM was used as positive control, and arbutin of 20 µM was included to compare with compound 8 at same concentration level. After the incubation of zebrafish embryos from 7 hpf to 72 hpf, compound 8 exhibited higher pigmentation inhibitory activity at concentrations of 10 and 20 µM compared to arbutin (20 µM) (

In Vivo Zebrafish Pigmentation Assay
In addition to evaluate the effect of 8 on in vitro antimelanogenesis, its in vivo anti-pigmentation ability through zebrafish pigmentation assay was further investigated. Zebrafish has been considered as an advantageous vertebrate model organism due to its small size, high fecundity, and similar gene sequences and organ systems to human beings. Additionally, the melanin pigmentation process on its surface allowing easy observation makes zebrafish a particular useful model for investigating in vivo melanogenic inhibitors or stimulators [50]. In this study, arbutin at 20 mM was used as positive control, and arbutin of 20 µM was included to compare with compound 8 at same concentration level. After the incubation of zebrafish embryos from 7 hpf to 72 hpf, compound 8 exhibited higher pigmentation inhibitory activity at concentrations of 10 and 20 µM compared to arbutin (20 µM) ( Figure 5). Upon the treatment of 20 µM compound 8, the pigmentation level of zebrafish markedly decreases about 31%; while compound 8 at 10 µM decreases 26.2% pigmentation level.

Viability Assay of Human Epidermal Skin Equivalents
The safety of cosmetic products is a serious concern. For instance, hydroquinone, an effective skin lightening agent, has been banned from the market because of numerous adverse reactions and controversy over the potential carcinogenic risk [7]. Kojic acid, a potent tyrosinase inhibitor, may induce contact dermatitis and potential genotoxicity [51,52]. In order to assess the safety of

Viability Assay of Human Epidermal Skin Equivalents
The safety of cosmetic products is a serious concern. For instance, hydroquinone, an effective skin lightening agent, has been banned from the market because of numerous adverse reactions and controversy over the potential carcinogenic risk [7]. Kojic acid, a potent tyrosinase inhibitor, may induce contact dermatitis and potential genotoxicity [51,52]. In order to assess the safety of compounds 1 and 8 on human skin, we performed a cell viability assay on Human skin equivalents (HSEs) model. HSEs are three-dimensional culture systems that are generated by seeding human keratinocytes onto an appropriate dermal substrate pre-seeded with human fibroblasts. HSEs are physiologically comparable to the natural skin and provide suitable alternatives for animal testing. Under controlled culture conditions, the HSEs demonstrate high similarity with the native tissue from which it was derived [53]. In this study, viability assays on the normal human epidermal keratinocytes (NHEKs) in Leiden epidermal models (LEMs) were performed. Compounds 1 and 8 did not affect the cell viability at the concentrations of 10-100 µM. The cell viabilities of compound 1 are 98% and 106 % at the concentrations of 10 and 100 µM, respectively. The cell viabilities of compound 8 are 97% and 92% at the concentrations of 10 and 100 µM, respectively. Accordingly, compounds 1 and 8 did not exert cytotoxicity against NHEKs in the Leiden epidermal models at concentrations below 100 µM ( Figure 6). Since the effective concentration of 8 is below 100 µM, which suggests that 8 might be safe for skin whitening below the tested concentrations. However, in practice, cosmetic products may be applied on human skin for a long time, thus an extended experimental period of tested compounds on HSEs has to be further conducted. in practice, cosmetic products may be applied on human skin for a long time, thus an extended experimental period of tested compounds on HSEs has to be further conducted.

Molecular Docking Study of B16-Mus Musculus Tyrosinase
Tyrosinase catalysis is the rate-limiting step of melanin biosynthesis. Thus, inhibition of tyrosinase is the most common approach to achieve skin whiteness [54,55]. In order to determine whether L. sinense suppressed melanogenesis in B16-F10 cells through tyrosinase inhibition, 3D stick models ( Figure 7A) and a 2D diagram ( Figure 7B) of molecular docking using DS software were performed, which revealed the possible inhibitory mechanism of compound 8 on mouse (Mus musculus) tyrosinase. It was shown that the active site of mouse tyrosinase was located at a domain surrounded by amino acids His377, Asn378, His381, Gly389, Thr391, Ser394 together with two copper ions. The CDOCKER interaction energy between the enzyme and inhibitor was −46.0067 kcal/mol. The simulation results showed that hydrophobic amino acids including Gly389, Asn 378, Thr391 Ser394, His 404, and His192 around the catalytic site formed van der Waals forces with compound 8. Additionally, the oxygen atom of γ-lactone moiety of 8 forms hydrogen bonds with His377 and His215, while its carbonyl group forms coordination bonds with two copper ions. It was Figure 6. The effect of compounds 1 and 8 on cell viability in normal human epidermal keratinocytes (NHEKs). NHEKs were exposed to 1, 8, and vehicle at the indicated concentrations for 24 h. Cell viability was determined by MTT assay.

Molecular Docking Study of B16-Mus Musculus Tyrosinase
Tyrosinase catalysis is the rate-limiting step of melanin biosynthesis. Thus, inhibition of tyrosinase is the most common approach to achieve skin whiteness [54,55]. In order to determine whether L. sinense suppressed melanogenesis in B16-F10 cells through tyrosinase inhibition, 3D stick models ( Figure 7A) and a 2D diagram ( Figure 7B) of molecular docking using DS software were performed, which revealed the possible inhibitory mechanism of compound 8 on mouse (Mus musculus) tyrosinase. It was shown that the active site of mouse tyrosinase was located at a domain surrounded by amino acids His377, Asn378, His381, Gly389, Thr391, Ser394 together with two copper ions. The CDOCKER interaction energy between the enzyme and inhibitor was −46.0067 kcal/mol. The simulation results showed that hydrophobic amino acids including Gly389, Asn 378, Thr391 Ser394, His 404, and His192 around the catalytic site formed van der Waals forces with compound 8. Additionally, the oxygen atom of γ-lactone moiety of 8 forms hydrogen bonds with His377 and His215, while its carbonyl group forms coordination bonds with two copper ions. It was also observed that the cyclohexene ring and butane exerted weak π-alkyl interaction with His381 and His215, respectively. Previous studies have demonstrated that L. sinense extract displayed mushroom tyrosinase inhibition and down-regulation of tyrosinase mRNA expression in B16-F10 cells [21,56]. Since the molecular docking illustrated a strong interaction between the active domain of mouse tyrosinase and compound 8, it was thus proposed that (3S,3aR)-neocnidilide (8) exhibited antimelanogenesis activity due to tyrosinase activity attenuation and further decreasing melanin production. However, in addition to tyrosinase inhibition, the other underlying mechanisms of antimelanogenesis activity of (3S,3aR)-neocnidilide (8) remains to be further investigated.

HPLC-DAD Analysis
Chromatographic analyses were performed on a Hitachi HPLC system consisting of L-7100 pump, L-7200 autosampler, L-7455 detector and D-7000 system manager data acquisition software, on an XBridge TM C18 column (250 mm length, 4.6 mm internal diameter, 5 um particle size; Waters).

Cell Culture
The B16-F10 murine melanoma cells (CRL6475) were purchased from the Food Industry Research and Development Institute (FIRDI, Hsinchu, Taiwan). The cells were cultured in 90% Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, St. Louis, MO, USA) and 1% penicillin-streptomycin solution in culture flasks in a CO 2 incubator with a humidified atmosphere containing 5% CO 2 in the air at 37 • C. The culture medium was changed every two days. The cells were harvested by trypsinization when they were about 85% confluent, counted with a haemocytometer (Neubauer Improved., Marienfeld, Germany) and seeded at the appropriate numbers into wells of cell culture plates for further experiments.

Cell Viability Assay
To determine the safety of the various extracts the viability of cells following treatment with extracts was determined by the MTT assay. This method is based on the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) to formazan by mitochondrial enzymes in viable cells [57]. The quantity of formazan formed is proportional to the number of viable cells present and can be measured spectrophotometrically. Briefly, 100 nM α-melanocyte stimulating hormone (α-MSH)-pretreated cells seeded at a density of 1 × 10 4 cells/well in a 12-well plate were left to adhere overnight. Pure isolates or arbutin were then added to each well and incubated for another 72 h. Then, the treated cells were labelled with MTT dye reagent (Applichem, Denmark) in PBS (2 mg/mL) for 3 h. The formazan precipitates were dissolved by DMSO and the concentrations were measured at 570 nm in a microplate reader. Cell viability was calculated using the following formula: cell viability (%) = (A sample/A control) × 100, where A sample and A control are the absorbances from the mixture with, or without the addition of test sample, respectively.

Melanin Content Assay
Melanin content was measured as described previously with slight modifications [58]. The B16-F10 melanoma cells were seeded with 1 × 10 4 cells/well in 3 mL of medium in 6-well culture plates and incubated overnight to allow cells to adhere. The cells were exposed to various concentrations (25,50 and 100 µM) of the pure isolates or arbutin for 72 h in the presence of 100 nM α-MSH. At the end of the treatment, the cells were washed with PBS and lyzed with 150 µL of 1 N NaOH (Merck, Germany) containing 10% DMSO for 1 h at 80 • C. The absorbance at 405 nm was measured using a microplate reader. The melanin content of B16-F10 melanoma cells without compound treatment was assigned as 100%, and the melanin content of compound-treated cells was calculated relative to the control group.

In Vivo Zebrafish Pigmentation Assay
The animal use protocol has been reviewed and approved by the Institutional Animal Care and Use Committee or Panel (IACUC/IACUP) of Taipei Medical University (No. LAC-2017-0311). The methods were carried out in compliance with the relevant laws and the approved guidelines. Wild-type zebrafish embryos were collected from Zebrafish Core Facility of Taipei Medical University. Phenotype-based evaluation of zebrafish embryo was performed according to the previous study with slight modification [50]. The embryos were incubated at 28 • C with 1% ethanol as control and a different concentration of compounds from 7 hpf (post-fertilization) to 72 hpf. To evaluate the anti-melanogenesis effects of melanogenic modulators on zebrafish developmental process, the pigmentation of zebrafish was analyzed at 72 hpf. The embryos were mounted in 1% Low Melting Agarose (Bioshop Canada, Burlington, ON, Canada) and captured images with a ZEISS Stemi 508 stereomicroscope (ZEISS, Oberkochen, Germany). Images pixel measurement analysis was carried out by Fiji package of ImageJ (http://rsb.info.nih.gov/ij/index.html, NIH, USA). The quantification of pigmentation data was analyzed (the area below the eyes of the zebrafish) and compared to control group.

Viability Assay on Normal Human Epidermal Keratinocytes
All primary human skin cells from healthy donors used by the Department of Dermatology of the Leiden University Medical Center are isolated from surplus tissue collected according to article 467 of the Dutch Law on Medical Treatment Agreement and the Code for proper Use of Human Tissue of the Dutch Federation of Biomedical Scientific Societies. According to article 467 surplus tissues can be used if no objection is made by the patient. This means that the patient who will undergo plastic surgery is well informed on the research. None of the authors were involved in the tissue sampling. The Declaration of Helsinki principles were followed when working with human tissues.
The fresh mamma reduction surplus skin of a single female individual was used for isolation of normal human epidermal keratinocytes (NHEKs) as described previously [59]. The NHEKs in Leiden epidermal models (LEMs) were incubated overnight under submerged conditions in keratinocyte medium. Within four days, fetal bovine serum was gradually omitted and the NHEKs in LEMs were cultured serum-free at the air-liquid interface for seven days, while culture medium was refreshed twice a week. Viability assays were performed by adding 0.5 mL of 1 mg/mL MTT to each of the NHEKs in LEMs for 3 h, after 24 h exposure to the test compounds 1, 8, and DMSO (negative control). The precipitated blue formazan product was extracted from the cells within 2 h with 0.5 mL isopropanol per well. The concentration of formazan was measured by determining the OD at 570 nm using a Tecan Infinite F50 microplate reader.

Statistical Analysis
All the data in our study were obtained as averages of experiments that were performed at least in triplicate and expressed as means ± SD (Standard deviation). Statistical analysis was performed by Student's t-test. The statistical significance of results was set at p < 0.05 (*) and p < 0.01 (**).

Homology Modeling
As no three-dimensional structures for Mus musculus tyrosinase are available now, Homology modeling is the most assured method for prediction of three-dimensional structures of unknown protein based on the assumption that the structure of the unknown protein is similar to the known structures of some homologous reference proteins. We acquired the Mus musculus tyrosinase amino acid sequence from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm. nih.gov/) protein sequence database. A homolog protein template of the query protein Mus musculus tyrosinase was identified by Phyre2 (Protein Homology/analog Y Recognition Engine V 2.0) [60]. The 446 residues (81% of Mus musculus sequence) have been modelled with 100.0% confidence by the single highest scoring template as PDB code: c5m8pA was chosen as a receptor for the docking calculation studies.

Analysis of Ligand-Protein Interaction
The binding site of the Mus musculus tyrosinase was determined based on reference human tyrosinase (PDB 5M8M) six amino acid residues and the binding pocket have two copper. Therefore, the binding site sphere was defined. Subsequently, the 3D structure of the compound 8 was prepared and optimized by energy minimization docked into the binding pocket of the Mus musculus tyrosinase using the CDOCKER program and the number of docking runs was set to 50 for inhibitor. All other parameters were set as default to the analysis of Ligand-Protein Interaction through using Biovia Discovery Studio v.4.0 (Accelrys Software Inc., San Diego, CA, USA). Finally, from the 50 docking conformations, the top one with the highest CDOCKER energy score was chosen to explore the binding mode of docked compound in the Mus musculus tyrosinase active site.

Conclusions
In the present study, we adopted a bioassay-guided method using B16-F10 cells to isolate the antimelanogenesis constituents from L. sinense rhizoma extracts. The active constituents were determined to be 5-[3-(4-hydroxy-3-methoxyphenyl)allyl]ferulic acid (1) and (3S,3aR)-neocnidilide (8). According to the HPLC analysis, the content of compound 8 accounts for 0.15% of the crude extract. The antimelanogenesis activity of 8 was verified by both in vitro B16-F10 cells and in vivo zebrafish assays. All these findings suggested that 8 may, at least, provide a rationale for the potential antimelanogenesis effect of L. sinense for its high potency and quantity. The cell viability data on B16-F10 cells and NHEKs imply that 8 could be developed potentially as an antimelanogenesis agent. The mode of action of 8 on antimelanogenesis was speculated to be the inhibition of the tyrosinase activity based on the results of molecular docking; however, that remains to be further confirmed. Our finding revealed that compound 8 exhibited anti-melanogenesis effects and safety through in vitro, in vivo, and ex vivo studies, however, the depigmenting efficacy and biosafety on human skin need to be evaluated and validated by clinical trials in the future.

Patents
An earlier version of the manuscript has been published as a patent (patent number: EP 2832719, TW I507390).