Chemical constituents from the fruits of Cullen corylifolium (L.) Medik. by the targeted separation mode

Abstract Two new compounds, corylifol H (1) and epi-bavacoumestan C (2), together with a new natural product named 8-geranyl daidzein (3), were isolated from the fruits of Cullen corylifolium (L.) Medik. (syn. of Psoralea corylifolia L.) by the targeted separation mode. The structures of these compounds were elucidated on the basis of spectroscopic methods and by comparison with literature properties. The anti-inflammatory effects of the two new compounds were also evaluated by activity assay in vitro. The results showed that compounds 1 and 2 inhibited nitric oxide production in LPS-activated RAW 264.7 macrophages in a dose dependent manner. Graphical Abstract


General experimental procedures
The optical rotation data were measured on an Autopol IV digital polarimeter (Rudolph, Wilmington, MA, USA). The UV spectra were determined on a UV-2102PCS UV-Vis spectrophotometer (UNICO, Dayton, NJ, USA). The IR spectra were carried out on a Spectrum 65 spectrometer (PerkinElmer, Waltham, MA, USA). The NMR spectra were recorded on Bruker AVIII 400 and 600 MHz spectrometers (Bruker, Zurich, Switzerland). The HR-ESI-MS data were recorded on LCMS-IT-TOF (Shimadzu, Kyoto, Japan). Silica gel (200300 mesh,Qingdao Marine Chemical Co. Ltd.,Qingdao,China) was used for open column chromatograph. Preparative HPLC system was performed using an Agilent Technologies 1260 system (Agilent, Santa Clara, CA, USA), composed of a quaternary pump, a DAD detector and an Eclipse XDB-C18 PrepHT column (21.2 × 250 mm, 7 μm). UPLC analysis was carried out using an ACQUITY UPLC instrument equipped with a quaternary pump, a DAD detector, an autosampler, a column heater and an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm; Waters, Milford, MA, USA). The optical density was determined on a microplate reader (Tecan spark 10M, Männedorf, Switzerland).

Plant material
The dried fruits of Cullen corylifolium (L.) Medik. were purchased from Anguo Chinese medicine market (Hebei province, China), which was identified by Prof.
Tian-Xiang Li, Tianjin University of Traditional Chinese Medicine. A voucher specimen (B20523289) was deposited in Research and Development Center of TCM (Tianjin University of Traditional Chinese Medicine, Tianjin, China).

Extraction and enrichment
The dried fruits of C. corylifolium (4 kg) were refluxed twice with 32 L water in 1.5 h per time. The water extract was discarded, and then the dried herb residue was powdered and macerated twice with 20 L petroleum ether in 24 h per time. The treated powder was dried and collected, then refluxed twice with 15 L 95% EtOH in 2 h per time. Moreover, the extracting solvent was removed to obtain a crude extract (590 g), which was subjected to silica gel column chromatography with an isocratic elution of petroleum ether/EtOAc (9:1). Bakuchiol, psoralen and isopsoralen in the eluate were monitored by TLC. Until these three compounds have almost not been detected in the eluate, EtOH was further employed to elute. The EtOH eluent was collected and concentrated to yield the target extract (419 g).

Isolation of compounds
3.2.1. UPLC conditions. UPLC analysis was performed on an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 µm) with the column heater fixed at 60C. The mobile phase consisted of methanol (A) and 0.1% formic acid aqueous solution (B): 1050% A at 05 min, 5095% A at 525 min for detecting the samples in the preparation of the target extract, and 595% A at 020 min for detecting the fractions produced by the preparative HPLC. The flow rate was 0.3 mL/min. The detection wavelength was 246 nm and the injection volume was 2 L.

LCMS-IT-TOF conditions.
Detection was performed using a LCMS-IT-TOF mass spectrometer. The source parameters were set as follows: CDL temperature at 200 C; spray voltage in positive ion mode at 4.5 kV; heat block temperature at 200 C; nitrogen flow rate at 1.5 L/min; drying gas pressure at 113.0 kPa; input pressure of high purity argon as CID collision and cooling gas at 2.4 bar; detector voltage at 1.58 kV; collision energy at 40%; collision gas ratio at 60%; ion accumulation time for 30 msec; the mass range within m/z 1001000.

Compounds preparation.
Further isolation of the target extract was carried out on an Agilent 1260 Infinity preparative HPLC system. Chromatographic separation was performed on an Eclipse XDB-C18 PrepHT column (21.2 × 250 mm, 7 μm) at ambient temperature and the flow rate was 10 mL/min. The optimal mobile phase was

Cytotoxicity assay
The cell viability test was determined by CCK-8 (Dojindo Molecular Technologies, Inc., Tokyo, Japan) assay. RAW 264.7 macrophages in good condition were seeded in 96 well plates (6×10 3 cells per well) after cells were mechanically scraped. Cells cultured for 12 h in condition of adherence growth were pretreated with 100 μL solution at the different concentrations of compound 1 (37.0, 74.0, 148 μM) and compound 2 (28.3, 56.6, 113 μM) for 12 h. Then, LPS was added to reach 1 µg/mL in culturing medium and cells were continually cultured for 24 h. After that, the CCK-8 (10 μL) was added into per well and cells were incubated for 2 h. The optical density at 450 nm was measured by a microplate reader to calculate the cell viability according to the formula, CV = (OD experiment -OD blank ) / (OD control -OD blank ) × 100%, for the evaluation of compound cytotoxicity. The independent experiments were performed in triplicate.

Anti-inflammatory assay
In order to investigate the effect of compounds 1 and 2 on LPS-induced nitric oxide (NO) production, RAW 264.7 macrophages mechanically scraped in good condition were seeded in 24 well plates (1×10 5 cells per well) and cultured for 12 h. The control, model, and experimental groups were assigned by employing dexamethasone (DEX) as positive control. Cells were treated with 100 μL solution at the different concentrations of compound 1 (37.0, 74.0, 148 μM) and compound 2 (28.3, 56.6, 113 μM), and DEX (97.1 μM) for 12 h. After stimulation with LPS at 1 µg/mL for 24 h, Griess I and Griess II (50 μL) were added to 24 well plates in turn, respectively. The optical density was measured at 540 nm on a microplate reader. The NO concentration was calculated by using standard curve of sodium nitrite. All tests were performed in triplicate.