A New Tigliane-Type Diterpenoid from Daphne genkwa

Experimental

General Procedures. Optical rotations were determined with a JASCO DIP-1000 polarimeter. UV spectra were obtained on JASCO UV-550 spectrometer. NMR spectra were recorded on a Bruker DRX 500 MHz NMR spectrometer using CD3OD as a solvent. High resolution electrospray ionization mass (HRESIMS) spectra were measured on a Waters QTOF micromass spectrometer. Semipreparative HPLC was performed using a Waters HPLC system equipped with two Waters 515 pumps and a 2996 photodiode array detector using a YMC J’ sphere ODS-H80 column (4 µm, 150 × 20 mm, i.d.). Open column chromatography was performed using a silica gel (70-230 mesh, Merck) and Lichroprep RP-18 (40-63 µm, Merck). Thin-layer chromatography (TLC) was performed using precoated silica gel 60 F254 (0.24 mm, Merck) plates.

Plant Materials. The flower buds of D. genkwa were purchased from Kyung-dong market, Seoul, Korea, in March 2012. The origin of the herbal material was identified by on of the authors (B. Y. Hwang) and a voucher specimen was deposited at the Herbarium of College of Pharmacy, Chungbuk National University, Korea.

Extraction and Isolation. The dried and powdered flower buds of D. genkwa (2 kg) were extracted with MeOH three times at room temperature. The combined MeOH extracts were concentrated in vaccuo at 40 °C to yield 120 g of residue. The residue was suspended in H2O (1.5 L) and then successively partitioned with n-hexane (3 × 1.5 L), CH2Cl2 (3 × 1.5 L), EtOAc (3 × 1.5 L) to give n-hexane (24.6 g), CH2Cl2 (21.5 g), EtOAc (27.5 g), and water-soluble extract (46.4 g), respectively. The CH2Cl2-soluble extract (21.5 g) was then subjected to column chromatography on silica gel eluted with CH2Cl2-methanol gradient system (100:0 to 0:100) to yield five subfractions (C1-C5). Fraction C3 (6.1 g) was further subjected to column chromatography over silica gel eluted with n-hexane-EtOAc (100:0 to 0:100) to yield five subfractions (C31-C35). Fraction C33 (0.8 g) was chromatographed over silica gel chromatography by using CH2Cl2-EtOAc gradient system (100:0 to 0:100), and further purified by semipreparative HPLC (MeCN-H2O gradient from 30:70 to 100:0, flow rate 6.0 mL/min) to afford compound 1 (3.5 mg). Fraction C32 (0.7 g) was chromatographed on a RP-18 column chromatography eluted with MeOH-H2O gradient (from 20:80 to 100:0) to obtain four fractions (C321- C324). Fraction C323 (0.3 g) was further purified by HPLC (MeCN-H2O gradient from 40:60 to 100:0, flow rate 6.0 mL/min) to yield compounds 2 (4 mg), 3 (34 mg), 4 (5 mg). Fraction C31 (0.8 g) was chromatographed over silica gel column chromatography with CH2Cl2-MeOH gradient system (100:0 to 0:100) to afford compound 5 (500 mg). Compound 6 (17 mg) was obtained from fraction C34 by silica gel column chromatography with CH2Cl2-MeOH gradient system (100:0 to 0:100). Fraction C35 (2.3 g) was further chromatographed over silica gel column chromatography with CH2Cl2- EtOAc gradient system (100:0 to 0:100) to give compound 7 (38 mg).

Daphwanin (1) : White amorphous powder; +102 (c 0.01, MeOH), UVmax (MeOH) λmax (log ε) 213 (3.38) nm; IR (KBr) vmax 3350, 1725, 1680, 1620, 1465 cm −1 ; 1H-NMR (500 MHz, CD3OD) and 13C-NMR (125 MHz, CD3OD), see Table 1; HRESIMS m/z 573.3054 [M-H]- (calcd for C32H45O9: 573.3064).

Measurement of NO Production and Cell Viability Assay . Nitric oxide production was determined by measuring the amount of nitrite from cell culture supernatant as previously described.22 Briefly, RAW 264.7 cells (1 × 105 cells/well) were cultured in flat bottom 96 well microtitre plate in quadruplicate for 12 h. Thereafter, 100 µL of media were replaced with fresh medium containing either compound, 1 µg/mL of LPS (Sigma Chemical Co., St.Louis, MO, USA) and further cultured for 24 h. The culture supernatant was collected at the end of culture for nitrite assay, which was used as a measurement of NO production. The culture supernatant (100 µL) was mixed with equal volume of Griess reagent at room temperature for 10 min. The absorbance was measured at 540 nm by a microplate reader. The remaining cells after Griess assay were used for cell viability with the MTT-based colorimetric assay.