Tyrosinase Inhibitors from the Stems of Streblus Ilicifolius

Two new stilbene derivatives, named strebluses C and D, were isolated from the EtOAc-soluble fraction of the stems of Streblus ilicifolius (Moraceae). Its absolute configuration was elucidated based on NMR spectroscopic data interpretation and optical rotation calculation. Streblus C possesses strong tyrosinase inhibitory activity with an IC50 value of 0.01 μM. Docking studies of 1 and 2 with oxy-tyrosinase were carried out to analyze their interactions. The analysis of the docked poses confirmed that 1 showed better binding affinity for oxy-tyrosinase than that of 2.


Introduction
Tyrosinase (EC 1.14.18.1), which is a binuclear coppercontaining monooxygenase, is a key enzyme in the oxidation of phenol to the corresponding o-quinone [1,2]. It plays a main factor causing freckles, brown age spots, and melasma. Some commercial compounds, such as hydroquinone, arbutin, kojic acid, azelaic acid, L-ascorbic acid, ellagic acid, and tranexamic acid, were reported as the well-known tyrosinase inhibitors. ese compounds have been used as skin whitening agents in cosmetic products, but they have certain drawbacks [3]. us, the finding of the new efficient and safe tyrosinase inhibitors is necessary for antihyperpigmentation product development.
Streblus ilicifolius (Vidal) Corner, which belongs to Moraceae family, was found and cultivated in Vietnam. Its barks have been traditionally used as an antipimple medicine. In a few published studies, some phenolic compounds have been reported in this plant [4][5][6][7]. In our continued studies on the screening of medicinal plants for tyrosinase inhibitory activity [8][9][10][11][12], it was found that a MeOH-soluble extract of the stems of Streblus ilicifolius showed a strong inhibitory effect, with an IC 50 value of 0.63 μg·mL −1 . us, our study on chemical constituents of the stems of S. ilicifolius was carried out, leading to the isolation of two undescribed stilbene derivatives, strebluses C (1) and D (2). Compound 1 showed a strong tyrosinase inhibitory activity with an IC 50 value of 0.01 μM, which makes it 4400 times more potent than that of kojic acid (IC 50 , 44.6 μM). In addition, molecular docking studies of 1 and 2 with the oxyform 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 32°C for 30 min, followed by addition of 1000 μL of L-DOPA 1.5 mM in pH 6.8 phosphate buffer, and incubated at 32°C for 7 min. e absorbance (A) at 475 nm was acquired on Shimadzu UV-1800 spectrophotometer. e inhibitory percentage (I%) was calculated according to the formula: 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.

HPLC Data of the EtOAc-Soluble Fraction from S. ilicifolius.
e concentrations of the EtOAc-soluble fraction and streblus C (1) were approximately 12,000 ppm and 200 ppm, respectively. e detection wavelength was set at 385 nm. An Agilent Zorbax SB-C18 column (150 × 4.6 × 5 mm) was used with a flow rate of 1 mL/min. e injection volume was 10 Μl, and the column temperature was maintained at 30°C. e mixtures of water and ACN were used as the mobile phase with gradient elution (20 ⟶ 40% ACN for 30 min).

Optical Rotation Calculation.
e conformational searches were performed on Spartan'18 (Wave function, Inc., Irvine, USA) by using Merck molecular force field (MMFF). All conformers with Boltzmann weight ˃10% were optimized using DFT method at the B3LYP/6-31G * level in the gas phase, to give the preferred conformers with the Boltzmann weight >90%. e optical rotation calculations at sodium D line frequency were carried out using the B3LYP functional and the 6-311++G(2d, 2p) basis set in IEFPCM solvation model for methanol. ese calculations were performed on Gaussian 09 (Gaussian, Inc., Wallingford, USA). e calculated optical rotation values were expressed as Boltzmann-weighted average of all output data.

Extraction and Isolation.
e dried powdered stems of S. ilicifolius were exhaustively extracted in a Soxhlet extractor with n-hexane, EtOAc, and MeOH to yield the corresponding fractions.
e EtOAc-soluble fraction was repeatedly chromatographed using silica gel CC and preparative TLC to obtain two undescribed stilbene derivatives, strebluses C (1) and D (2) (Figure 1).  Figure 2) from H-3 to C-1, C-2, and C-4, from H-5 to C-1 and C-4, from H-6 to C-2 and C-4, from H-α to C-2 and C-6, and from H-β to C-1, indicated that two hydroxy groups and Cα-Cβ double bond located at C-2, C-4, and C-1, respectively, of the 1,2,4-trisubstituted aromatic ring. e presence of the cyclohex-2-en-1-one 5,6-acetonide moiety in 1 was established based on the observed HMBC correlations. e HMBC correlations from H-α to C-1′ and from H-β to C-1′ and C-2′ were supportive of the Cβ-C1′ linkage. In addition, the prenyl group was determined to be located at C-4′ by the HMBC correlations from H-1″ to C�O, C-4′, and C-5′ and from H-5′ to C-1″. erefore, 1 was suggested to be a prenylated stilbene-like compound. e difference in chemical shifts of the methyl groups of the dimethylacetonide moiety in 1 is 0.14 ppm, which established the presence of the cis-acetonide [13]. Moreover, it was unambiguously confirmed based on the NOESY correlation between H-5′ and H 2 -1″ (Figure 2). e preferred conformations of the cis-(R, R)-acetonide 1 were generated by the MM2 calculation using MMFF94 force field [14]. ese conformers were reoptimized by DFT-B3LYP method using basis set 6-31G * , to obtain the most preferred conformer with 92.8% Boltzmann distribution (Table S1). e optical rotation value at sodium D line frequency was computed using B3LYP/6-311++G(2d, 2p) level with IEFPCM solvent model for methanol. e large basis set with diffuse functions such as 6-311++G(2d, 2p) was applied to give very consistent results [15,16]. us, a (R, R) absolute configuration was concluded for streblus C (1).

Structural Elucidation of Two
A careful HPLC analysis of the EtOAc-soluble fraction was accomplished, which revealed a peak at t R 20.766 min in the chromatogram in accord with that of 1 (t R 20.800 min) ( Figure S1). us, the presence of 1 in the EtOAc-soluble fraction from S. ilicifolius was confirmed, and the possibility of 1 being artifact could be ignored.
Compound 2, streblus D, showed a molecular formula to be C 19 H 20 O 5 based on the HRESIMS sodium adduct ion at m/z 351.1224 [M + Na] + (calcd for C 19 H 20 O 5 Na, 351.1208). e 1 H and 13 C NMR data of 2 (Table 1) resembled those of 1, except for the presence of the singlet olefinic proton at δ H 7.31 instead of two trans-coupling olefinic protons in 1 and disappearance of the acetonide group. Based on the 13 C NMR data and observed HMBC   Evidence-Based Complementary and Alternative Medicine correlations for 2 (Figure 2), the structure of 2 was assigned as a benzofuran-type stilbene. e NOESY correlations between H-5′ and H 2 -1″ indicated the presence of the cis-diol configuration. e 3 J H-5′/H-6′ coupling constants were 5.9 and 2.7 Hz, to suggest the equatorial configuration of H-5′ [17], which was supportive of the (R,R) or (S,S) absolute configurations for 2. e conformational search for (R,R)-2 was generated and optimized to obtain six conformers with total Boltzmann weight >90% (Table S1).
us, a (S, S) absolute configuration was concluded for streblus D (2).

Tyrosinase Inhibitory Activity of Isolated Compounds from S. ilicifolius.
Compounds 1 and 2 were tested for their tyrosinase inhibitory activities [18]. Kojic acid, a purported skin lightening agent, was used as a positive control. Streblus C (1) exhibited remarkable inhibitory effect with an IC 50 value of 0.01 μM, which was 4400 times more potent than that of kojic acid (IC 50 , 44.6 μM). Meanwhile, streblus D (2) was inactive with an IC 50 value > 100 μM. ese results were consistent with a previous report on the structure-activity relationships of stilbene derivatives. Compound 1 having 2,4-resorcinol subunit contributed the most to inhibitory activity [19]. In addition, the 2-arylbenzofuran derivatives showed lower tyrosinase inhibitory activities than the corresponding stilbene derivatives, suggesting that the formation of the five-membered ring led to the loss of inhibitory activity [20].

Docking Studies of Compounds 1 and 2.
Tyrosinase is an oxidase, which is represented as one of four possible forms (deoxy-, oxy-, met-, and deact-forms) [21]. Oxytyrosinase form oxidizes both phenols and catechols to o-quinones. Herein, mushroom tyrosinase (EC 1.14.18.1) plays the same role with respect to oxy-tyrosinase form. Two bound Cu 2+ ions bind to six histidine residues, and the peroxide group is in the binding site of oxy-tyrosinase, which has a role in the catalytic oxidation [22]. To explore the strong inhibitory activity of 1 against tyrosinase, the molecular docking studies of 1 and 2, respectively, with oxy-tyrosinase (PDB ID : 1WX2) were carried out [23]. e docking studies were performed with MOE. e topranked pose with the highest negative binding free energy value (S value) was selected for further interaction analysis with Discovery Studio Visualizer. Following our previous in silico study on tyrosinase inhibition, this docking procedure was already validated based on the docking results of the positive control (kojic acid) and the decoy (hypoxanthine) [12].
In the binding site, compound 1 showed the H-donor interaction between the C-4 hydroxy group and peroxide bridge PER404, presenting the distances of 1.85Å. e C-3′ carbonyl group formed the H-acceptor interaction with ASN188 residue (Figure 3). e aromatic ring exhibited the π-π stacking interaction with HIS194 residue localized in the active pocket. In addition, two methyls of the acetonide group showed the π-σ interactions with TRP184 residue. Compound 2 did not show any interaction with the catalytic site (i.e., Cu 2+ ions and peroxide bridge), whereas kojic acid showed the interactions with a Cu 2+ ion, HIS194, and THR203 residues in the binding site.
ree hydroxy groups of 2 interacted with ASP45, ALA202, and MET201 residues via the H-donor bonding. e furan ring formed the π-π and π-σ interactions with TRP184 and ILE42 residues, respectively. e S values and these interactions suggested that 1 showed high binding affinity for oxy-tyrosinase than that of 2 (Table 2). is result confirmed that the formation of furan ring in 2 led to the loss of inhibitory activity. Evidence-Based Complementary and Alternative Medicine

Conclusions
Two new stilbene derivatives were isolated from the stems of S. ilicifolius. eir structures were elucidated based on the NMR spectroscopic interpretation and optical rotation calculation. Compound 1 was found to possess strong tyrosinase inhibitory activity with an IC 50 value of 0.01 μM. Binding interaction analyses between the isolated compounds (1 and 2) and oxy-tyrosinase active site have been performed.

Data Availability
e NMR data used to support the findings of this study are included within the supplementary information file.

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