Three new sulphur glycosides from the seeds of Descurainia sophia

Abstract Three new sulphur glycosides, raphanuside B–D (1–3), together with a known sulphur glycoside, raphanuside (4) were isolated from the decoction of the seeds of Descurainia sophia (L.) Webb ex Prantl, and the compound 4 was reported for the first time from this plant. Their structures were identified by means of UV, IR, 1D, 2D NMR (HSQC, HMBC and NOESY) and HR–ESI–MS spectroscopic data.


Introduction
Descurainia sophia (L.) Weeb ex Prantl, which belongs to the Cruciferae family, distributed in north-eastern China and has been widely used as a folk medicine. The decoction of the aerial part is used for throat diseases and as an antipyretic for measles and smallpox (Bekker et al. 2005). Besides, its seeds have been used to relieve cough, prevent asthma, reduce oedema, promote urination and also have an effect of cardiotonic (Sun et al. 2005). In addition, the plant is also reported to display potent cytotoxicity against diverse human cancer cells, which is a potential anticancer drug (Kim et al. 2013). For medicinal materials, it is very important to study its chemical compositions. At present, according to the results of previous chemical studies, some secondary metabolites have been identified already, such as cardiac glycosides (Chen et al. 1981), glucosinolate degradation products and alkanes (Afsharypuor & Lockwood 1985), nor-lignan (Sun et al. 2006), phenylpropanoids (Mohamed & Mahrous 2009), lactones (Sun et al. 2004), flavonol glycosides and phenols ). But only one sulphur glycoside (Sun et al. 2005) was reported from this plant. As our current interest was in finding more sulphur glycosides of the seeds of D. sophia, we carried out a phytochemical investigation on the seeds. Based on the use of Traditional Chinese Medicine, we took water decocting method in the extraction and obtained four sulphur glycosides, of which were three new sulphur glycosides (1-3) and a known sulphur glycoside (4). Herein, we reported the isolation and structures elucidation of these new compounds.

Results and discussion
The water decoction from the seeds of D. sophia was subjected to column chromatography over Diaion HP20, silica gel, Sephadex LH-20, Toyopearl HW-40 and purified by preparative HPLC to afford four sulphur glycosides (1-4).
Raphanuside (4) (Figure 1) was characterised by comparison of its MS and NMR spectra with reported previously as raphanuside (Feng et al. 2008). These sulphur glycosides were also evaluated for their in vitro cytotoxicities against Eca109, NCI-H460 and MCF-7 cell lines using MTT assay. All of them showed an IC 50 values higher than 100 μM and were considered inactive against these cell lines. However, the findings can enrich the number and structures of sulphur glycosides in this plant and lay the foundation for investigating other pharmacological activities of these compounds.

General experimental procedures
NMR spectra (500 MHz for 1 H-NMR and 125 MHz for 13 C-NMR) were recorded on a Bruker AVANCE III 500 spectrometer using tetramethylsilane as internal standard. HR-ESI-MS data were measured using a Bruker maxis HD mass spectrometer. Analytical HPLC was carried out on a Waters Alliance 2695 system with a Platisil ODS C 18 column (5 μm, 4.6 × 250 mm). IR spectrum was determined on a Nicolet is 10 Microscope Spectrometer. UV spectra were recorded in methanol on a Shimadzu UV-2401PC apparatus. Optical rotation was measured with AP-IV (Rudolph Research Analytical, USA). Semi-preparative HPLC was conducted using a Saipuruisi LC-50 instrument with a UV200 detector and YMC-Pack ODS-A column (250 × 10 mm, 5 μm), using a flow rate of 3 mL/min at a column temperature of 25 °C. Column chromatography was performed on Diaion HP20 (Mitsubishi Chemical Corp., Tokyo, Japan), silica gel (160-200 mesh, Marine Chemical Industry, Qingdao, China). Toyopearl HW-40 (TOSOH Corp., Tokyo, Japan), Sephadex LH-20 (40-70 μm, Amersham Pharmacia Biotech AB, Uppsala, Sweden), TLC was carried out on self-made silica gel G and GF 254 plates (Qingdao Marine Chemical Industry, Qingdao, China).

Plant material
The dried seeds of D. sophia were purchased from herbal market in Zhengzhou, Henan province and identified by Prof Cheng-ming Dong (Henan University of Traditional Chinese Medicine). A voucher specimen (No. 20130715A) was deposited in the Research Department of Natural Medicinal Chemistry, School of Pharmacy, Henan University of Traditional Chinese Medicine.

Acid hydrolysis and HPLC analysis of 1-3
Compounds 1-3 (2.0 mg) were dissolved in 2 mol/L HCl (5.0 mL) and refluxed in water bath (90 °C) for 2.5 h. After cooling, the solution was evaporated under reduced pressure, CHCl 3 (1.0 mL) was added, and the CHCl 3 solution was extracted with H 2 O (1.0 mL). The aqueous fraction was evaporated to dryness, the residue (including d-glucose, l-glucose) was dissolved in pyridine (1.0 mL) and heated with L-cysteine methyl ester (2.5 mg) at 60 °C for 1.5 h, and then O-tolyl isothiocyanate (15.0 μL) was added to the mixture and heated at 60 °C for an additional 1.5 h. The reaction mixture was analysed by HPLC: column Platisil C 18 , 250 mm × 4.6 mm, 5 μm; 25% acetonitrile-0.2% TFA water, 0.8 mL/min; UV detection at 250 nm; temperature at 25 °C. The sugar of 1-3 was identified as d-glucose (t R , 19.7 min) [authentic samples, d-glucose (t R , 19.6 min) and l-glucose (t R , 16.8 min)] (Xiao et al. 2014).

Cytotoxic activity
The cytotoxic activity of compound 1-4 was tested by MTT method (Xiong et al. 2010). The experiment was conducted in RPMI 1640 medium (Hyclone, Logan, UT) containing 10% foetal bovine serum (Hyclone, Logan, UT) and antibiotic (100 U/mL penicillin and 100 μg/mL streptomycin) with the cells being grown 24 h prior to treatment. MTT assay was carried out after 48-h cultivation. The absorbencies were measured at 490 nm. Three human cancer cell lines including human lung adenocarcinoma NCI-H460, oesophageal carcinoma Eca109, and human breast adenocarcinoma cell line MCF-7 were used.