Diarylalkanoids as Potent Tyrosinase Inhibitors from the Stems of Semecarpus caudata

From a CHCl3-soluble extract of the stems of Semecarpus caudata (Anacardiaceae), two new diarylalkanoids, semedienone (1) and semetrienone (2), were isolated. Their structures were elucidated based on NMR spectroscopic data interpretation. These compounds possess strong tyrosinase inhibitory activity with the IC50 values of 0.033 and 0.11 μM, respectively. Docking studies of 1 and 2 with oxy-tyrosinase were carried out to analyze their interactions. Accordingly, semedienone (1) showed good interactions with the peroxide group and amino acid residues. The biosynthesis of the isolated diarylalkanoids was proposed.


Introduction
Melanin is a pigment that is essential for protecting human skin against UV radiation. However, the abnormal accumulation of melanin induced skin pigmentation disorders. Melanogenesis is a complex process to produce melanin under control of tyrosinase. Tyrosinase (EC 1.14.18.1) is a binuclear copper-containing monooxygenase, which catalyzes the oxidation of phenol to the corresponding o-quinone [1,2]. Tyrosinase is the main factor causing some dermatological diseases including freckles, age spots, and melasma. Hydroquinone, arbutin, kojic acid, azelaic acid, L-ascorbic acid, ellagic acid, and tranexamic acid are commercial tyrosinase inhibitors, which have been used as skin-whitening agents, but these compounds have certain drawbacks [3]. us, the finding of new efficient and safe antityrosinase agents is necessary for anti-hyperpigmentation drug development.
A previous study on the chemical constituents of Semecarpus caudata (Anacardiaceae), collected at Dong Nai Province in Vietnam, led to the isolation of six flavonoid derivatives and the evaluation of their tyrosinase inhibitory activity [4]. Our continued phytochemical study on the stems of S. caudata was carried out, leading to the isolation of seven compounds (1-7) including two new diarylalkanoids named semedienone (1) and semetrienone (2). ese compounds were found to possess tyrosinase inhibitory activity. Semedienone (1) showed a strong effect with an IC 50 value of 0.033 μM, which makes it 1300 times more potent than that of kojic acid (IC 50 , 44.6 μM). In addition, molecular docking studies of 1 and 2 with the oxy-form of the copper-bound Streptomyces castaneoglobisporus tyrosinase were performed.

Tyrosinase Inhibitory Assay.
All pure compounds were dissolved in DMSO and tested at concentrations ranging from 0.01 to 100 μM. Assay mixtures in 0.1 M phosphate buffer pH 6.8 were prepared immediately before use, consisting of 100 μL of tyrosinase solution (15 U/mL) and 1900 μL of test solution. ese mixtures were preincubated at room temperature for 30 min, followed by addition of 1000 μL of L-DOPA 1.5 mM in pH 6.8 phosphate buffer and incubated at room temperature for 7 min. e absorbances (A) at 475 nm were acquired on Shimadzu UV-1800 spectrophotometer. e inhibitory percentage (I%) was calculated according to the formula: I % � ((A control − A sample )/A control ) × 100%. Data were represented as means ± standard error (n � 3). e IC 50 values were determined by using GraphPad Prism software with multivariate nonlinear regression and R 2 > 0.9. Kojic acid was used as positive control. e structures of these compounds were constructed by using the Builder module. Subsequently, all compounds were minimized up to 0.0001 gradients using the Amber12 : EHT force field. e crystal structure of the oxy-tyrosinase was taken from the Protein Data Bank (PDB code 1WX2). e caddie protein (ORF378) and water molecules were removed. e enzyme structure was prepared using the QuickPrep module. e binding site was determined based on the PLB (Propensity for Ligand Binding) score in the Site Finder module. e molecular docking was performed by Dock module, using Triangle Matcher placement, Induced Fit refinement, London dG, and GBVI/WSA dG scoring methods. Five top poses showed up based on the negative binding free energy value (S value). e best pose was selected to analyze the receptor-ligand interactions by using BIOVIA Discovery Studio Visualizer 2016.

Extraction and Isolation.
e dried powdered stems of S. caudata were exhaustively extracted in a Soxhlet extractor with MeOH to yield MeOH-soluble extract (700 g).

Tyrosinase Inhibitory Activity of Isolated Compounds from S. caudata.
Compounds (1-7) were tested for their tyrosinase inhibitory activity [13]. Kojic acid, a purported skin-lightening agent, was used as a positive control. 2,4,2′,4′-Tetrahydroxychalcone (8), which was synthesized following our previous procedure [14], showed potent activity with an IC 50 value of 0.016 μM ( Table 2). Semedienone (1) and semetrienone (2) exhibited remarkable inhibitory effect with the IC 50 values of 0.033 and 0.11 μM, respectively, more potent than that of kojic acid (IC 50 , 44.6 μM). Additionally, compounds 4 and 6 were found to possess tyrosinase inhibitory activity with the IC 50 values of 2.35 and 27.0 μM, respectively. e presence of α,β-unsaturated hydroxycarbonyl groups in cinnamic acid derivatives were found to enhance activity (2 ≫ 3). Additionally, the occurrence of a C-3 methoxy group decreased the inhibitory activity (2 ≫ 5) [15,16]. Diarylalkanoids with 2,4-disubstituted resorcinol subunit on ring B contributed the most to inhibitory activity [17]. Moreover, the length of the conjugated carbon chain in diarylalkanoids led to a change of activity (8 > 1 > 2). is result reaffirmed the (Z)-β-phenyl-α,β-unsaturated carbonyl scaffold plays an important role for tyrosinase inhibition [18,19]. In previous reports, diarylpentanoids such as diarylpentadiene-3-one were not significantly inhibiting tyrosinase activity [20], but some analogues showed moderate antimelanogenesis activity [21]. Some cyclic diarylheptanoids were found to have melanogenesis-inhibitory activity [22]. In this regard, semedienone (1) and semetrienone (2) could be the potent structural templates for developing new skin-lightening agents. Active Compounds 1, 2, and 8. Tyrosinase has four possible oxidation states (deoxy-, oxy-, met-, and deact-form) [23]. Met-tyrosinase, having a hydroxy and the two Cu 2+ ions in the binding site, is responsible for the oxidation of catechols. In this oxidizing process, met-tyrosinase is reduced to deoxy-tyrosinase which rapidly binds dioxygen to give oxy-tyrosinase form. Oxytyrosinase, which is the primary form of the enzyme, oxidizes both phenols and catechols to o-quinones by the monooxygenase and oxidase mechanisms, respectively. In the active site of oxy-tyrosinase, two bound Cu 2+ ions and the peroxide group play a catalytic oxidation role. Mushroom tyrosinase (EC 1.14.18.1), which was used in the inhibitory assay, plays the same role with respect to oxytyrosinase form. us, in this study, the molecular docking studies of 1, 2, and 8, respectively, with oxy-tyrosinase (PDB ID : 1WX2) [24] were carried out to explore their interactions and inhibition mechanisms.

Docking Study of the
In molecular docking study, the imperfect scoring results (false-positive hits), which may be considered as decoys, can be occurred by predicting incorrect ligand geometries or by applying nonbinding molecules. e active and decoy ligands are similar according to some physicochemical properties (molecular weight, number of rotational bonds, total hydrogen bond donors, total hydrogen bond acceptors, topological polar surface area, and the octanol-water partition coefficient), but decoy was presumed to be inactive against a target. According to Choi et al. [25], kojic acid and hypoxanthine showed the tyrosinase inhibitory constant (K i ) values of 13 μM and >1000 μM, respectively [25]. us, in this docking study, kojic acid and hypoxanthine was selected as the active inhibitor and the decoy molecule, respectively, to validate our docking protocol. e docking studies were performed with Molecular Operating Environment 2016 (MOE 2016.0802) suite [26]. e top-ranked pose with the highest negative binding free energy value (S value) was selected for further interaction analysis with BIOVIA Discovery Studio Visualizer 2016 [27].
Compounds 1, 2, and 8 showed an H-donor interaction between a hydroxy group and a peroxide bridge PER404, presenting the distances of 1.85, 1.88, and 1.78Å, respectively, whereas kojic acid showed the interactions with a Cu 2+ ion, HIS194, and THR203 residues (Figure 3)  binding pocket, compounds 1 and 8 showed more interactions with targeting residues than those of 2 (Table 3). ese analysis results were consistent with their experimental inhibitory activities (8 > 1 > 2). e C-2 hydroxy group of 1 exhibited H-bonding interactions with ASN191 and GLY183 residues. Moreover, the aromatic ring A of 1 formed π-π T-shaped and π-σ interactions with TRP184 and ILE42 residues, respectively. Compound 2 showed an H-acceptor interaction between C�O group and ASN188 residue. In addition, the aromatic ring B of 2 interacted with HIS194 residue via a π-π stacking interaction. us, the S values and these interactions suggested that 1 and 2 showed high binding affinity for oxy-tyrosinase than those of kojic acid. Hypoxanthine showed the lower negative S value and the longer-distance interactions than that of kojic acid. Apparently, these results could be used to validate the abovementioned docking procedure in this study.     acetate pathways [28]. α-Ketoglutarate-dependent hydroxylase is responsible for the C-2 hydroxylation of p-coumaroyl-CoA to give 2,4-dihydroxycinnamoyl-CoA [29]. It is condensed with four or five malonyl-CoA moieties to afford the corresponding polyketides, which undergo the intramolecular ring closure via Claisen reaction. After that, reduction, dehydration, and enolization must occur to give rise to 1 and 2.

Conclusions
From the CHCl 3 -soluble extract of the stems of S. caudata, two new diarylalkanoids were isolated together with five known compounds. Compounds 1 and 2 were found to possess potent tyrosinase inhibitory activity with the IC 50 values of 0.033 and 0.11 μM, respectively. Binding interaction analyses between the oxy-tyrosinase active site and the active compounds (1 and 2) have been performed. Plausible biogenetic pathways for formation of two new diarylalkanoids (1 and 2) were also proposed.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.