Two New Acylated Flavonol Glycosides from the Seeds of Lepidium sativum

Two new acylated flavonol glycosides named kaempferol-3-O-(2-O-sinapoyl)-β-d-galactopyranosyl-(1→2)-β-d-glucopyranoside-7-O-α-l-rhamnopyranoside (1) and quercetin-3-O-(6-O-benzoyl)-β-d-glucopyranosyl-(1→3)-β-d-galactopyranoside-7-O-α-l-rhamnopyranoside (2), were isolated together with six known compounds from the seeds of L. sativum. Their structures were elucidated on the basis of spectroscopic analysis and chemical methods. In vitro 1 and 2 inhibited nitric oxide production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, with IC50 values of 25.36 and 25.08 µM, respectively.


General Procedures
Optical rotations were obtained on a Perkin-Elmer 341 digital polarimeter (Waltham, MA, USA).UV spectra were recorded on Shimadzu UV2550 (Tokyo, Japan).NMR spectra were obtained with a Bruker AV β 600 NMR spectrometer (chemical shift values are presented as δ values with TMS as the internal standard; Munich, Germany).HR-ESI-MS spectra were performed on a LTQ-Orbitrap XL spectrometer.GC analysis was carried out on a GC-7890: column, DB-5 (30 m × 0.32 mm × 0.25 mm); detector, FID-6850 (Agilent, Santa Clara, CA, USA).ODS gel (50 µm, YMC, Kyoto, Japan), Sephadex LH-20 (Pharmacia, Uppsala, Sweden), and MDS gel (Beijing Medicine Technology Center, Beijing, China) were used for column chromatography.HPLC separations were performed using a Waters 2535 series pump equipped with a PDA detector and a YMC (250 × 10 mm, 5 μm) semi-preparative column.TLC was carried out on silica gel GF 254 (Yantai Chemical Inst., Yantai, China) plates, and spots were visualized under UV light (254 or 365 nm) or by spraying with 5% H 2 SO 4 in 95% EtOH followed by heating.

Plant Material
The seeds of L. sativum were purchased from the Xinjiang Uygur Autonomous Region in August 2010.The plant material was authenticated by one of the authors (B.-L.Guo).A voucher specimen is deposited at the Natural Medicine Research Center of Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College.

Extraction and Isolation
The dried seeds of L. sativum (45 kg) were chopped and extracted with 95% EtOH (270 L) three times (each time 8 h) under percolation and then concentrated under vacuum.The residue was extracted three times under reflux by 50% EtOH for 1.5 h.The dried 95% EtOH extract was further suspended in water and partitioned successively with petroleum ether, CHCl 3 , EtOAc and n-BuOH (50 L, 80 L, 100 L, 100 L).After concentration the n-BuOH layer (400 g) was subjected to column chromatography on MDS gel column (15 cm × 47 cm, 75-150 μm) eluted with a gradient of MeOH-H The 50% EtOH extract which was dissolved in water and chromatographed on a D101 macroporous adsorptive resin column eluting with a gradient of EtOH-H 2 O (0:100, 10:90, 20:80, 30:70, 50:50, 95:5, v/v), the eluates were concentrated under reduced pressure to dryness and six fractions were obtained.
The 30% ethanol eluate was subjected to MDS-gel chromatography (

NO Inhibition Assay
Inhibition of NO production and cell viability of LPS-stimulated RAW 264.7 macrophage cells were determined.The NO production assay was carried out according to the method described before [22].The murine monocytic RAW 264.7 macrophages were dispensed into 96-well plates (2 × 10 5 cells/well) containing RPMI 1640 medium (Hyclone, Logan, UT, USA) with 10% FBS under humidified atmosphere of 5% CO 2 at 37 °C.After 24 h of preincubation, cells were treated with serial dilutions of compounds 1 and 2 with the maximum concentration of 50 µM in the presence of 1 µg/mL LPS for 18 h.Each compound (purity > 95%) was dissolved in DMSO and further diluted in the medium to produce different concentrations.NO production in each well was assessed by adding 100 µL of Griess reagents A and B to 100 µL of each supernatant from LPS or the compound-treated cells in