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A New Tigliane-Type Diterpenoid from Daphne genkwa
A New Tigliane-Type Diterpenoid from Daphne genkwa
Bulletin of the Korean Chemical Society. 2014. Feb, 35(2): 669-671
Copyright © 2014, Korea Chemical Society
  • Received : October 14, 2013
  • Accepted : December 04, 2013
  • Published : February 20, 2014
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About the Authors
Da Yu Li
Chul Lee
Qinghao Jin
Jin Woo Lee
Mi Kyeong Lee
Bang Yeon Hwang

Abstract
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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 CD 3 OD 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 F 254 (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 H 2 O (1.5 L) and then successively partitioned with n -hexane (3 × 1.5 L), CH 2 Cl 2 (3 × 1.5 L), EtOAc (3 × 1.5 L) to give n -hexane (24.6 g), CH 2 Cl 2 (21.5 g), EtOAc (27.5 g), and water-soluble extract (46.4 g), respectively. The CH 2 Cl 2 -soluble extract (21.5 g) was then subjected to column chromatography on silica gel eluted with CH 2 Cl 2 -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 CH 2 Cl 2 -EtOAc gradient system (100:0 to 0:100), and further purified by semipreparative HPLC (MeCN-H 2 O 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-H 2 O gradient (from 20:80 to 100:0) to obtain four fractions (C321- C324). Fraction C323 (0.3 g) was further purified by HPLC (MeCN-H 2 O 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 CH 2 Cl 2 -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 CH 2 Cl 2 -MeOH gradient system (100:0 to 0:100). Fraction C35 (2.3 g) was further chromatographed over silica gel column chromatography with CH 2 Cl 2 - EtOAc gradient system (100:0 to 0:100) to give compound 7 (38 mg).
Daphwanin (1) : White amorphous powder;
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+102 ( c 0.01, MeOH), UV max (MeOH) λ max (log ε) 213 (3.38) nm; IR (KBr) v max 3350, 1725, 1680, 1620, 1465 cm −1 ; 1 H-NMR (500 MHz, CD 3 OD) and 13 C-NMR (125 MHz, CD 3 OD), see Table 1 ; HRESIMS m/z 573.3054 [M-H] - (calcd for C 32 H 45 O 9 : 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 × 10 5 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.
Acknowledgements
This work was supported by the research grant of the Chungbuk National University in 2012.Supporting Information.1H-,13C-NMR, DEPT, HMQC, HMBC, NOESY, and HRESIMS spectra of1are available as Supporting Information.
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