A new flavonoid glycoside from Toxicodendron vernicifluum (Stokes) F.A. Barkley

Abstract Toxicodendron vernicifluum (Stokes) F.A. Barkley, also called ‘Qishu’, is a shrub belonging to the Anacardiaceae family and producing lacquer. In this work, a new flavonoid glycoside (1), was isolated from the heartwood of T. vernicifluum, together with four known compounds (2-5). The structure of the new compound was determined as 4′,7-dihydroxy-3′-methoxy-3-O-β-D-glucopyranosyl-flavonoid (1), on the basis of acidic hydrolysis, and spectroscopic analyses. Compound 1 showed significantly cytotoxic against A549 cell lines with the values of IC50 at 1.5 μM.


Introduction
Toxicodendron vernicifluum (Stokes) F.A. Barkley, commonly called Qishu, has been used as local medicine for treatment of cough (Antal et al. 2021, Lee et al. 2020). It has been reported that T. vernicifluum is rich in urushiols (Kim et al. 2019, Xie et al. 2016, sesquiterpenoids (He et al. 2013), flavonoids , Song et al. 2021, polysaccharides (Wiart 2014), and phenolic acids (Jin et al. 2015). In the present research, one new flavonoid glycoside (1) and four known compounds (2-5), was isolated from the n-butanol fraction of the heartwood of T. vernicifluum (Figure 1). Herein, we report the isolation, structure elucidation, as well as the cytotoxic activity of the compounds.

General experimental procedures
The NMR spectra were measured in the methanol-d 4 , on a Bruker AV600 instrument with TMS as an internal standard. ESI-MS spectra were recorded on Waters Quattro micro API-LC/MS/MS spectrometer (Waters, USA). HPLC was performed on JAI LC9103 Recycling preparative HPLC (Japan Analytical Industries, Japan) equipped with JAIGEL-ODS-AP-P column and JAIGEL-GS310 column using a JAI refractive index detector and a JAI UV-3702 detector with MultiChro 2000 workstation.

Plant material
The heartwood of T. vernicifluum were collected in October 2020 at Dandong, Liaoning, China, and authenticated by Prof. Guangtong Chen (School of Pharmacy, Nantong University). A voucher specimen has been deposited in our laboratory (voucher No. QSXY-2020-024).

Acid hydrolysis of compound 1
Compound 1 (2 mg) was treated with 1 M hydrochloric acid (4 mL) at 90 C for 2 h. Then the reaction mixture was extracted with chloroform (5 Â 3 mL). The aqueous layer was collected and evaporated under vacuum to remove the solvent. The residue was redissolved in anhydrous pyridine (2 mL) and mixed with a pyridine solution of L-cysteine methyl ester hydrochloride (2 mL). After the mixed solution was heated at 60 C for 1 h, trimethylchlorosilane (0.5 mL) was added and the resulting mixture was stirred at 60 C for another 30 min. Then the solution was concentrated to dryness and taken up in water (3 Â 1 mL), followed by extraction with n-hexane (3 Â 1 mL). The supernatant was analyzed by GC. Separations were carried out on HP-5 columns (320 mmÂ 30 cm, 0.25 mm). Highly pure N 2 was employed as a carrier gas (1.0 mL/min), and the FID detector operated at 280 C (column temperature 160-200 C). The retention times of the monosaccharide derivatives were as follows: and D-glucopyranosyl moiety (14.43 min).

Cytotoxicity
Human lung cancer cell (A549) was provided by the American Type Culture Collection (ATCC). The cells were cultured in medium (RPMI1640 for A549 and DMEM for HeLa) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and antibiotics antimycotics (PSF; 100 units/mL penicillin G sodium, 100 mg/mL streptomycin, and 250 ng/ mL amphotericin B). The cells were incubated at 37 C and 5% CO 2 in a humidified atmosphere. Etoposide (Sigma, purity > 98%) was used as a positive control. Cell viability was determined by the sulforhodamine B (SRB) protein staining method. Cells were seeded in 96-well plates and incubated for 24 h, and were fixed (for zero day controls) or treated with test compounds for 72 h. All compounds were solved in DMSO (final concentration of 0.1% [v/v]), stored at À20 C and diluted to desired concentration (0.01, 0.1, 1, 10, 100 lM) in normal saline immediately prior to each experiment. Each concentration was tested thrice. At least three experiments were performed. After incubation, cells were fixed with 10% trichloroacetic acid (TCA), dried and stained in 0.4% sulforhodamine B (SRB) in 1% acetic acid solution. Unbound dye was washed and stained cells were dried and dissolved in 10 mM Tris (pH 10.0). Absorbance was measured at 515 nm and cell proliferation was determined as follows: cell proliferation (%) ¼ (average absorbance compoundaverage absorbance zero day )/ (average absorbance controlaverage absorbance zero day ) Â 100%. GI 50 values were calculated by non-linear regression analysis using the Table Curve 2

Conclusion
A new flavonoid glycoside (1), 4 0 ,7-dihydroxy-3 0 -methoxy-3-O-b-D-glucopyranosyl-flavonoid, along with four known compounds were isolated from 95% ethanol from the the heartwood of T. vernicifluum. Compound 1 showed significantly cytotoxic against A549 cell lines with the values of IC 50 at 1.5 lM. In this contribution, our findings would both enrich chemical context of genus Toxicodendron and expand the variety of flavonoid glucosides.

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