A rare isoflavone-quinone and a new flavanone from the roots of Dalbergia stipulacea Roxb.

Abstract Two previously undescribed compounds, namely dalpulapans F and G (1 and 2), along with 11 known compounds were isolated from the MeOH crude extract of the roots of Dalbergia stipulacea. Dalpulapan F was found as a rare isoflavone-quinone derivative. Their structures and absolute configurations were supported by extensive spectroscopic data analysis, including 1 D and 2 D NMR, HRESIMS data, specific rotation data, and comparison of the experimental and calculated ECD data. Cytotoxicity evaluation of the isolated compounds against HepG2 and KKU-M156 cell lines revealed that isoflavonoid 9 and rotenoid 13 exhibited the most activity against the two cell lines. Graphical Abstract


Introduction
Dalbergia stipulacea Roxb. is an important member of Dalbergia genus in the family Fabaceae. Previous investigations of secondary metabolites from this genus have resulted in the isolations of various classes of compounds, such as flavonoids (Kaennakam et al. 2019), isoflavonoids (Songsiang et al. 2009;Umehara et al. 2009), neoflavonoids (Kumar et al. 2014;Lin et al. 2020), as well as rotenoids (Abou El-Kassem et al. 2020). Many of these compounds possess interesting pharmacological activities, including antioxidant (Liu et al. 2019), antimicrobial (Kumar et al. 2017), antifungal (Posri et al. 2021b), as well as anticancer (Cheenpracha et al. 2009;Umehara et al. 2009;Posri et al. 2021a). D. stipulacea, known locally in Thai as "Ma-Kham-Thao", is the genus of a large woody climber and a small tree. This medicinal plant has been used in oral healthcare (Nongmaithem et al. 2018), in addition, the roots and bark have been used to be poison fish (Bhatt and Dayal 1992). Previous phytochemical investigation of this plant resulted in a prenylated chalcone, luteolin 4 0 -rutinoside, from the fresh vegetative part (Bhatt and Dayal 1992;Borai and Dayal 1993). Recently, we discovered chalcone, isoflavonoid, pterocarphan, flavonoid, arylpropen together with courmarin derivatives from the stems of this plant, and reported their antifungal activity against Pythium insidiosum (Posri et al. 2021b). In 2021, we found that R,R-velucarpin A showed cytotoxicity against HeLa cells with IC 50 value of 10.9 ± 0.42 mM (Posri et al. 2021a). From further chemical study, thirteen metabolites from the methanol extract of the root of this plant are reported and cytotoxicity evaluation of the isolated compounds is made.

Biological activity
All compounds, except 7 and 12, were evaluated for cytotoxicity against HepG2 and KKU-M156 cell lines and the results are shown in Table S2. Most compounds showed moderate cytotoxicity (IC 50 > 50 mM) against these two cell lines. However, compounds 9 and 13 displayed cytotoxicity against HepG2 cell line with IC 50 values of 16.14 ± 4.78 and 18.01 ± 1.51 mM, respectively. Comparing between isoflavonoids 6, 8 and 9, hydroxyl group at C-5 position may play an important role for cytotoxicity toward two cancer cell lines. In the cases of 9 and 5, compound 5 showed less activity against HepG2 cells than 9. These results seem to indicate that the geranyl group at C-8 decreases the activity. Flavanone 11 exhibited more activity than flavanone 2 toward HepG2 and KKU-M156 cell lines; it may be concluded that the aromatic aldehyde at C-3 0 may decrease the activity. In the cases of isoflavonoids 1 and 9, the quinone form in the B-ring showed a dramatical decrease in activity against HepG2 cells.

General experimental procedures
Melting points were detected on a Sanyo Gallenkamp melting point apparatus. Optical rotations were determined on a JASCO P-1020 polarimeter. ECD and UV spectra were determined on a JASCO J-810 apparatus. A PerkinElmer Spectrum One FT-IR spectrophotometer was used to measure the IR spectra. The NMR spectra were obtained using a Varian Mercury plus 400 spectrometer and Bruker AVANCE NEO operating at 400 MHz ( 1 H) and at 100 MHz ( 13 C). HRESIMS data were performed on a Micromass Q-TOF-2 spectrometer. Column chromatography was applied via silica gel (40-63 lm) and Sephadex LH-20 (40-70 lm). Thin layer chromatography (TLC) was done using MERCK silica gel 60 F254 TLC aluminium sheets. Preparative layer chromatography (PLC) was carried out on Merck silica gel 60 PF254 glass plates (20 Â 20 cm). The organic solvents were distilled before use in the separation process.

Plant material
The roots of D. stipulacea (Voucher specimen KKU012018) were collected from Phuwieng District, Khon Kaen Province, Thailand, in February 2018. The plant was characterized by Assoc. Prof. Suppachai Tiyaworanant, Faculty of Pharmaceutical Sciences, Khon Kaen University, Thailand.

Ecd calculations
The conformers of compound 1 was obtained using the MM2 force field with ChemBio3D software and were optimized at the B3LYP/6-311g (d,p) basis set by density functional theory. The single point energy calculations were computed using timedependent density functional theory (TD-DFT) at the CAM-B3LYP/6-311þþg (d,p) level of theory. The bulk solvent effects were examined using the CPCM polarizable conductor calculation model. All calculations were performed with the Gaussian 09 program package.

Cytotoxicity assay
The effect of compounds on cell viability was determined using an MTS-based assay. The assay was performed using CellTiter 96 Aqueous One Solution reagents according to the manufacturer's protocol (Promega; Madison, WI, USA). Briefly, KKU-M156 and HepG2 cells were plated into 96-well plates overnight and incubated with the compound for 24 h. Absorbance of the formazan product was measured (k ¼ 490 nm) using a microplate reader. IC 50 values (mean ± 95% confidence intervals (CI)) were calculated by fitting the dose-response curve using SigmaPlot 12 Software. The positive control was a cisplatin standard and showed IC 50 values of 13.22 ± 2.64 and 30.88 ± 6.23 mM against HepG2 and KKU-M156 cell lines, respectively.

Conclusion
In this study, the isolation and purification of isolated compounds from the crude MeOH extract of the roots of D. stipulacea led to two new compounds, along with 11 known compounds. The cytotoxicity evaluation against HepG2 and KKU-M156 cell lines was studied. It was found that isoflavone 9 and rotenoid 13 showed the most activity against two cell lines. Isoflavonoid 9 exhibited IC 50 values of 16.14 ± 4.78 mM and 62.82 ± 10.54 mM toward HepG2 and KKU-M156 cell lines, respectively, while rotenoid 13 showed IC 50 values of 18.01 ± 1.51 mM and 24.97 ± 3.56 mM, respectively.