Chemical Constituents from the Branches of Carpinus turczaninowii with Antioxidative Activities

EXPERIMENTAL

Reagents and Instruments

DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS [2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] were purchased from Aldrich. All solvents used were of analytical grade. A Biochrom Libra S22 UV-visible spectrophotometer was used for the screening of radical inhibition activities. 1H (400 MHz) and 13C (100.6 MHz) nuclear magnetic resonance (NMR) spectra were recorded on JNM-LA 400 instrument (JEOL) with chemical shift (δ) data were reported in ppm relative to the solvent used. Merck silica gel (0.063-0.2 mm) was used for normal phased column chromatography. Silica gel 60 F254 coated on aluminum plates by Merck were used for thin layer chromatography (TLC). Gel filtration chromatography (GFC) was performed using Sephadex LH-20 (25-100 μm) from Fluka.

Plant Material

The branches of Carpinus turczaninowii were collected in December 2009 from Halla Botanical Garden in Jeju Island, Korea. Voucher specimen (sample number 308) was deposited at the herbarium of Department of Chemistry, Jeju National University.

Extraction and Isolation

The shade dried C. turczaninowii (650.0 g) was cut into small pieces, and extracted with 70% ethanol (14 L) two times at room temperature for 24 h. The gummy extract (78.1 g) was obtained after concentration of the filtered solution. Part of the ethanol extract (26.5 g) was suspended in water (2 L), and fractionated into n-hexane (0.30 g), ethyl acetate (3.8 g), n-butanol (7.0 g) and water (16.1 g) portions. The ethyl acetate (EtOAc) layer was subjected to vacuum liquid chromatography (VLC) on silica gel, using step-gradients (n-hexane/EtOAc to EtOAc/MeOH, 300 mL each) to provide 30 fractions (V1-V30). The compounds 1 (13.3 mg), 3 (24.3 mg) and 4 (37.4 mg) were obtained from fractions V4, V5 and V8 respectively by recrystallization. Fraction V13 was purified by silica gel column chromatography (CC) using chloroform-EtOAc-methanol (2:3:1) to afford the compound 5 (26.9 mg). The fractions V17 and V18 were combined, and purified by silica gel CC with chloroform-EtOAc-methanol (3:3:1) to give compound 6 (36.0 mg). The fraction V19 was subjected to silica gel CC with chloroform-EtOAc-methanol (3:3:1) followed by Sephadex LH-20 CC chloroform-EtOAc-methanol (3:2:1) to isolate the compounds 7 (6.1 mg) and 8 (8.9 mg). The compound 2 (8.1 mg) was isolated from the fraction V21 as a methanol-insoluble substance.