Strebluses E–H, four new stilbene-like derivatives from the stems of Streblus ilicifolius

Following bioactivity-guided isolation, four new stilbene-like derivatives, named Strebluses E–H, were isolated from the EtOAc-soluble fraction of the stems of Streblus ilicifolius (Moraceae). Their chemical structures were elucidated based on NMR spectroscopic data interpretation and optical rotation calculation. Streblus E possesses potent tyrosinase inhibitory activity with an IC50 value of 0.1 μM. Oxy-tyrosinase has two bound Cu2+ ions and a peroxide group in the binding site, which has a role in the catalytic oxidation. Thus, a docking study of Streblus E with oxy-tyrosinase was performed to analyze the ligand–protein interactions. With in silico modelling, the S value and the ligand–protein interactions suggested that Streblus E showed lower binding affinity for oxy-tyrosinase than that of Streblus C.


Introduction
Streblus ilicifolius (S. Vidal) Corn. (Moraceae) is widely distributed in India, China, and South Asia. 1 Several phytochemical studies of S. ilicifolius have been carried out, leading to the identication of amide glycosides, coumarins, stilbenes, lignans, and polyphenols. In addition, the anti-tyrosinase, antimicrobial, and anti-inammatory activities of these chemical constituents has been reported. [2][3][4] Our continued studies on the bioactivity-guided phytochemical investigation of medicinal plants for tyrosinase inhibitory activity [5][6][7][8][9] has led to the identication of two coumarins (Strebluses A and B) and two stilbene-like derivatives (Strebluses C and D) from the stems of Streblus ilicifolius, among which Streblus C exhibited a remarkable inhibitory effect with an IC 50 value of 0.01 mM. 10,11 Thus, this phytochemical study was continuously performed, leading to the isolation of four new stilbene-like derivatives, Strebluses E-H (1)(2)(3)(4). Their structures were determined by NMR spectroscopic interpretation. In addition, their absolute congurations were identied based on the optical rotation calculation. Herein, the tyrosinase inhibitory activity assays and the molecular docking studies with the oxy-tyrosinase were performed.

Results and discussion
Structural elucidation of four new isolated compounds from S. ilicifolius The EtOAc-soluble fraction of S. ilicifolius stems was chromatographed to obtain four undescribed stilbene-like derivatives, Strebluses E-H (1-4) (Fig. 1 The 1 H and 13 C NMR spectra showed signals of the prenylated stilbene-like feature having the 5,6-dihydroxycyclohex-2-en-1one moiety (Tables 1 and 2), which resembled those of Streblus C except for the absence of the acetonide group. 10 The observed HMBC correlations ( Fig. 2) indicated the presence of the 2,4-dihydroxyphenyl group in 1. The 5,6-dihydroxycyclohex-2-en-1-one substructure was established based on the HMBC Fig. 1 The structure of compounds 1-4.

Tyrosinase inhibitory activity and docking studies
All isolated compounds were tested for their tyrosinase inhibitory activities. 17 Kojic acid was used as a positive control. Streblus E (1) showed potent inhibitory effect with an IC 50 value of 0.1 mM as compared to that of kojic acid (IC 50 , 44.6 mM). All remaining compounds were inactive (IC 50 > 100 mM). The docking study of 1 was performed with MOE following our previous procedure. 9 In the binding pocket of the top-rank pose, 1 showed the H-donor interaction from the C-4 ′ hydroxy group to peroxide bridge PER404, and from the C-2 hydroxy group to ASP45 residue (Fig. 4). The aromatic ring had the p-cation interaction with ARG55 residue. In addition, the prenyl group showed the s-s and p-s interactions with VAL195 and TRP184, respectively.
The genus Streblus is a small deciduous shrub, which includes 20 species and mainly distributed in South China and South Asia. The phytochemical studies of Streblus ilicifolius were carried out to obtain various class of compounds. In our previous studies, we reported the structure and the antityrosinase evaluation of four new compounds, in which Streblus C showed noteworthy inhibitory activity. 10,11 With these   interesting results, we continued to carry out the bioactivityguided isolation, leading to the identication of four new stilbene-like derivatives were isolated as well as their in vitro and in silico tyrosinase inhibitory activity were reported. In this study, Streblus E (1) showed potent inhibitory effect with an IC 50 value of 0.1 mM. The 2,4-resorcinol subunit highly contributed to inhibitory activity. 18 In addition, the formation of the vemembered ring gave rise to the loss of inhibitory effect. 19 With in silico modelling, the S value and the ligand-protein interactions suggested that 1 showed lower binding affinity for oxy-tyrosinase than that of Streblus C. 10 This result was used to clarify the remarkable difference in IC 50 values of Strebluses C (0.01 mM) and E (0.1 mM).

Conclusions
Four new stilbene-like derivatives, named Strebluses E-H (1-4), were isolated from the EtOAc-soluble fraction of the stems of Streblus ilicifolius (Moraceae). Their structures were elucidated based on NMR spectroscopic data interpretation with the aid of optical rotation calculation. Streblus E (1) showed potent tyrosinase inhibitory effect with an IC 50 value of 0.1 mM.

Plant material
The stems of Streblus ilicifolius were collected at Hoai Nhon District, Binh Dinh Province, Vietnam, in October 2017. Its scientic name was identied by Dr Rer. Nat. Anh Tuan Dang-Le, Faculty of Biology and Biotechnology, University of Science, Ho Chi Minh City, Vietnam. A sample (MCE0052) has been deposited at the Department of Medicinal Chemistry, Faculty of Chemistry, University of Science, Ho Chi Minh City, Vietnam.

Tyrosinase inhibitory assay
All pure compounds were dissolved in DMSO and tested at concentrations of 0.01-100 mM. Assay mixtures in 0.1 M phosphate buffer pH 6.8 were prepared immediately before use, consisting of 100 mL of tyrosinase solution (15 U mL −1 ) and 1900 mL of test solution. These mixtures were preincubated at 32°C for 30 min, followed by 1000 mL of L-DOPA 1.5 mM in pH 6.8 phosphate buffer, and incubated at 32°C for 7 min. The absorbance (A) at 475 nm was acquired on Shimadzu UV-1800 spectrophotometer. The inhibitory percentage (I%) was calculated according to the formula: I% = [(A control − A sample )/A control ] × 100%. All experiments were performed in triplicate and data were represented as the mean of three samples with standard deviation.

Optical rotation calculation
The conformational searches were performed on Spartan'18 (Wave function, Inc., Irvine, USA) using Merck molecular force eld (MMFF). All conformers with Boltzmann weight > 1% were optimized using DFT method at the B3LYP/6-31G* level in the gas phase. The optical rotation calculations at 589.3 nm were carried out using the DFT-B3LYP with 6-311++G(2d,2p) basis set in IEFPCM solvation model for methanol. These calculations were performed on Gaussian 16 Rev. C.01 (Gaussian, Inc., Wallingford, USA). The calculated optical rotation values were expressed as the Boltzmann-weighted average of all output data.

Molecular docking
Docking studies were performed with Molecular Operating Environment 2019 (MOE 2019.0102) suite (Chemical Computing Group ULC, Montreal, Canada). All structures were minimized up to 0.0001 gradients using the Amber12:EHT force eld. The oxy-tyrosinase structure (1WX2) was prepared following our previous procedure. The molecular docking procedure was carried out with Triangle Matcher placement, Induced Fit renement, and two scoring methods (London dG and GBVI/WSA dG). Five top results with the negative binding free energy value (S value) were selected to show up. The ligand interactions were carried out using BIOVIA Discovery Studio Visualizer 2016 (Dassault Systèmes Americas Corp., Waltham, USA).

Author contributions
MTTN and NTN designed the work. THL, HXN, and TNVD directed the experiments. THL and TNVD performed tyrosinase inhibitory assay. PHD performed molecular docking and optical rotation calculations. The manuscript was written by THL, PHD, MTTN, and NTN.

Conflicts of interest
There are no conicts to declare.