Two new derivatives of 8-prenyl-5,7-dihydroxycoumarin from the stems of Streblus ilicifolius (S.Vidal) Corn

Abstract From the EtOAc-soluble extract of the stems of Streblus ilicifolius (Moraceae), two new secondary metabolites named strebluses A (1) and B (2) were isolated. Their chemical structures have been concluded based on the chemical derivatisation and the spectroscopic interpretation. All compounds have been tested for their tyrosinase inhibitory activity. They showed weaker inhibitory activity than that of kojic acid (IC50, 44.6 µM).


Introduction
Streblus ilicifolius (S.Vidal) Corn. (Moraceae), known as a scandent spinous shrub, growing up to 2.5-3 m high, is widely distributed in India, China, and South Asia (Singh et al. 2012). In Vietnam, this plant which is locationally called the trivial name 'Duoi Oro', was planted as a fence and its bark is a folk medicine for treating pimples (Do 2006). Several previous phytochemical studies on S. ilicifolius stems revealed that it had contained some of coumarins, stilbenes, lignans, and polyphenols and its biological activities on anti-tyrosinase and antimicrobial activities had been reported (Dej-adisai et al. 2016;Zhang et al. 2019).
By a continuing effort on the inhibition of melanin synthesis of the indigenous medicinal plants of Vietnam (Nguyen et al. 2016;Nguyen et al. 2012), the MeOH-soluble extract of the stems of S. ilicifolius showed significant tyrosinase inhibitory activity with an IC 50 value of 6.3 mg mL À1 . Further biological study on the EtOAc-soluble fraction exhibited more potent inhibitory effect with an IC 50 value of 4.9 mg mL À1 . Thus, its phytochemical investigation was carried out, leading to the isolation of two new compounds, strebluses A (1) and B (2) (Figure 1). In this study, we have reported the isolation and the structure elucidation of both new compounds along with their tyrosinase inhibitory activity.

Results and discussion
Compound 1 was obtained as a white amorphous powder. The HRESIMS data gave a sodium adduct molecular ion peak at m/z 287.0900 [M þ Na] Table S1). On the other hand, the 13 C NMR spectrum of 1 showed resonance signals for 14 carbons including an ester carbonyl carbon (d C 160.7, C-2), three oxygenated aromatic carbons (d C 153.2, C-5; 159.4, C-7; 154.5, C-9), two tertiary aromatic carbons (d C 108.1, C-8 and 102.3, C-10), an methine aromatic carbon (d C 98.0, C-6), along with one oxymethylene (d H 67.4, C-4 0 ), one methine (d H 35.6, C-3 0 ), two methylenes (d H 19.7, C-1 0 and 33.2, C-2 0 ), and one methyl (d H 16.4, C-   Table S1). These 1 H and 13 C NMR data together with the observed HMBC correlations indicated 1 having the coumarin moiety. The location of two hydroxy groups was determined to be at C-5 and C-7 based on the HMBC correlations from H-6 to both C-5 and C-7. The presence of the 4-hydroxy-3-methylbutyl group was suggested on the basis of the HMBC correlations from the oxymethylene protons H 2 -4 0 to the methylene carbon C-2 0 , the methine carbon C-3 0 , and the methyl carbon C-5 0 . In addition, its position was determined at C-8 by the HMBC correlations from the oxymethylene protons H 2 -1 0 to the aromatic carbons C-7, C-8, and C-9, and from the oxymethylene protons H 2 -2 0 to the aromatic carbon C-8. The methylation derivatisation of 1 was carried out to obtain a dimethylated product 1 0 . The structure of 1 0 was concluded based on the 1 H and 13 C NMR data (Supplementary data Table  S1) and its HMBC correlations (Supplementary data Figure S1). The NOESY correlations of 1 0 were used to confirm the position of the 4-hydroxy-3-methylbutyl group in 1. Compound 1 0 showed the NOESY correlations between H-6/5-OMe, H-6/7-OMe, and H 2 -1 0 /7-OMe, which identified the location of the 4-hydroxy-3-methylbutyl group at C-8. Thus, the structure of streblus A (1) was concluded as 5,7-dihydroxy-8-(4 0 -hydroxy-3 0 -methylbutyl)coumarin.
Compound 2 was isolated as a white amorphous powder. The molecular formula of 2 was determined to be C 16 H 18 O 6 by the HRESIMS. The 1 H and 13 C NMR spectra of 2 resembled closely those of 1, except for the presence of the acetyl group (d H 1.98; d C 171.2, 20.8) (Supplementary data Table S1). The HMBC correlation between the oxymethylene protons H 2 -4 0 and the acetoxy carbonyl at d C 171.2 suggested that the location of acetylation is at C-4 0 (Supplementary data Figure S1). The methylation of 2 was carried out to afford a dimethylated product 2 0 . The NOESY spectra of 2 0 showed the correlations between H-6/5-OMe, H-6/7-OMe, and H 2 -2 0 /7-OMe. Thus, the 4-acetoxy-3-methylbutyl group was determined to be at C-8. The structure of streblus B (2) was concluded as 5,7-dihydroxy-8-(4 0 -acetoxy-3 0 -methylbutyl)coumarin. The HPLC data of 1 and the MeOH-soluble extract (Supplementary data Figure S24) showed the presence of 1 and 2 in this extract. Thus, possibility of 2 to be an artifact can be ignored.
Two isolated compounds were tested for their tyrosinase inhibitory activity according to the modified method of Arung et al. (2005). Kojic acid (IC 50 , 44.6 mM), which is a well-known tyrosinase inhibitor (Hae et al. 2019), was used as the positive control. The assay was carried out at various concentrations ranging from 10 to 100 mM. Compounds 1 and 2 at the concentration of 100 mM showed weak effect with the inhibitory percentage values of 26.9% and 21.1%, respectively.

General experimental procedures
Optical rotation was recorded on a Jasco P-2000 digital polarimeter (JASCO International Co., Ltd., Japan). HRESIMS was performed on a Bruker micrOTOF-QII mass spectrometer (Bruker Singapore Pte., Ltd., Singapore). IR spectra was measured with a JASCO FT/IR-6600 spectrometer (JASCO International Co., Ltd., Japan) with KBr as a carrier for the sample. NMR spectra was taken on a Bruker Avance III 500 spectrometer (Brucker BioSpin AG, Thailand) with acetone-d 6 as an internal standard, and chemical shifts are expressed in d values. HPLC data were carried out on Agilent 1260 Infinity II LC System with diode array detector (Agilent Technologies, Pte Ltd., Singapore). The absorbance (OD) was measured with a Shimadzu UV-1800 UV-Vis spectrophotometer (Shimadzu Pte., Ltd, Singapore). Column chromatography was carried out using Silica gel 60, (40-63 mm) (Scharlau, Spain). Analytical and preparative TLCs were carried out on precoated Kieselgel 60 F 254 plate (Merck KGaA, Germany). Mushroom tyrosinase (EC 1.14.18.1) (3933 U/mL) and L-3,4-dihydroxyphenylalanine (L-DOPA) were purchased from Sigma-Aldrich (Sigma-Aldrich Pte. Ltd., Singapore). Kojic acid and DMSO were purchased from Merck (Merck KGaA, Germany). Other chemicals were of the highest grade available.

Plant material
The stems of S. ilicifolius were collected at Hoai Nhon District, Binh Dinh Province, Vietnam in October 2017. It was identified by Dr. rer. nat. Anh Tuan Dang-Le, Faculty of Biology and Biotechnology, University of Science, Ho Chi Minh City, Vietnam. A voucher sample of the stems (MCE0052) has been deposited at the Department of Medicinal Chemistry, Faculty of Chemistry, University of Science, Ho Chi Minh City, Vietnam.

Hplc data of the MeOH-Soluble extract from S. ilicifolius
The concentrations of the MeOH-soluble extract and two new compounds (1 and 2) were approximately 10,000, 1000, and 1000 ppm, respectively. The detection wavelength was set at 330 nm. An Agilent Zorbax SB-C18 column (150 Â 4.6 Â 5 mm) was used with a flow rate of 1 mL/min. The injection volume was 20 mL and the column temperature was maintained at 30 C. The mixtures of water and ACN were used as the mobile phase with gradient elution (20 ! 30% ACN, for 15 min).

General procedure for O-methylation
Dissolved $5.0 mg of each compound in 3 mL acetone, then added 5.0 mg K 2 CO 3 . The mixture was subsequently treated with 30 mL CH 3 I and stirred for 2 h at room temperature in Ar atmosphere. The reaction mixture was added 10 mL water and 3 mL saturated NaCl solution, and then extracted three times with 3 mL EtOAc for each time. Finally, flash column chromatography was applied and eluted with EtOAc-n-hexane mixture (v/v, 20:80) to isolate the product.

Tyrosinase inhibitory activity assay
All the samples were first dissolved in DMSO and used for the actual experiment at concentrations of 1-100 mg/mL (or mM for pure compounds). The tyrosinase inhibitory activity assay was performed as previously described by Arung et al. (2005). The assay mixtures consisting of 1450 mL of test solution in 0.1 M phosphate buffer (pH ¼ 6.8) and 50 mL of enzyme solution (15 U/mL in 0.1 M phosphate buffer) was prepared immediately before use. After preincubation at room temperature for 30 min, the reaction was initiated by the addition of 500 mL of substrate solution (1.5 mM L-DOPA in 0.1 M phosphate buffer). The assay mixture was incubated at room temperature for 7 min, and then measured the absorbance (Abs) at 475 nm. Kojic acid, a known tyrosinase inhibitor, was used as a positive control. Tyrosinase inhibitory activity was expressed as the inhibitory percentage, calculated as [(1 À Abs sample /Abs control ) Â 100%]. Data were represented as means ± standard error (n ¼ 3).

Disclosure statement
No potential conflict of interest was reported by the authors.