Dalvelutinoside, a new isoflavone glycoside from the methanol extract of Dalbergia velutina roots

Abstract A new isoflavone glycoside, dalvelutinoside (1), together with one known isoflavone (2) and five known isoflavone glycosides (3–7) were isolated from the methanol extract of the roots of Dalbergia velutina. Their structures were determined by spectroscopic analysis. All isolated compounds were evaluated for their cytotoxicity against KB and HeLa cell lines.


Results and discussion
The MeOH extract from the roots of D. velutina was separated by silica gel (Merck Art 7730) and Sephadex LH-20 column chromatography to obtain the compounds 1-7. Their structures were elucidated on the basis of detailed spectroscopic analysis including 1D and 2D NMR spectroscopy and mass spectrometry techniques.

General experimental procedures
1D and 2D NMR spectra were recorded on a Bruker 400 AVANCe spectrometer. Melting points were obtained using Fisher-Johns Melting Point apparatus. HReSIMS spectra were obtained using a Bruker MICROTOF model mass spectrometer. IR data were obtained using a Nicolet 6700 FT-IR spectrometer using KBr discs. UV-visible absorption spectra were taken on a UV-2550 UV-vis spectrometer (Shimadzu, Kyoto, Japan). Optical rotation was recorded by Jasco P-1010 Polarimeter. HPLC analysis were obtained using a Alltech System equipped with model 626 binary gradient pumps, select TM degasser, 580 autosampler, 2000 eS evaporative light scattering detector (Alltech) and peak simple chromatography data system software.

Plant material
The roots of D. velutina were collected from Sahatsakhan district, Kalasin province, Thailand, in October 2014. The plant material was identified by Ms Suttira Khumkratok, and a voucher specimen was deposited as a reference  at the Walai Rukhavej Botanical Research Institute, Mahasarakham University.

Acidic hydrolysis and HPLC analysis
A solution of dalvelutinoside (1) (2 mg) in 1 M HCl (1.0 mL) was heated at reflux for 1 h and then the reaction mixture was neutralised with an equal volume of 1 M NaOH and extracted with CH 2 Cl 2 (5 mL). The sugar moiety was identified as glucose by co-TLC analysis (etOAC: MeOH: H 2 O, 1:8:1) of the aqueous solution in comparison with an authentic glucose. In addition, the glucose was identified as D-glucose by HPLC analysis (column: lichrocart-NH 2 (250 × 4.0 mm), carrier: 82% ACN in H 2 O (1.5 mL/min), retention time: 8.133 min) in comparison with an authentic D-glucose.

Cytotoxicity assay
All isolated compounds (1-7) were subjected to cytotoxic evaluation against KB (human epidermoid carcinoma) and HeLa (human cervical carcinoma) cell lines employing the colorimetric method (Skehan et al. 1990;Kongkathip et al. 2003;Kaennakam et al. 2015). Adriamycin was used as the reference substance which exhibited activity against KB and HeLa cell lines.

Conclusion
The MeOH crude extract from the roots of D. velutina comprises one new dalvelutinoside (1), one known isoflavone (2) and five known isoflavone glycosides (3-7). Compound 3 was isolated for the first time from this genus. Compounds 2 and 4 showed weak cytotoxicity against KB and HeLa cells with IC 50 values of 48.06, 63.77 μM and 59.82, 86.36 μM, respectively. Therefore, we believe that this plant is an important source for the diverse structure of isoflavone glycosides and should be further investigated for other biological activities.

Supplementary material
Supplementary material relating to this paper is available online, along with Figures S1-S6.