K. Bae et al. / Fitoterapia 78 (2007) 409–413
411
same method, compounds 3 (25 mg) and 4 (299 mg) were isolated from 60% MeOH eluted fraction (35 g). Compound
(350 mg) was purified from 80% MeOH eluted fraction (46 g). These compounds showed a satisfactory purity,
N95%, as evidenced by NMR and HPLC analysis.
5
2
5
Compound 1. Yellow amorphous powder; mp 183–184 °C; [α] −22.5° (c 0.4, MeOH); UVmax (MeOH): 263,
D
−
3
28 nm; IR bands (KBr): 3400, 2930, 1720, 1650, 1610, 1510, 1455, 1360, 1260, 1180, 1080, 835 cm 1; HR-FAB-
MS m/z: 601.1536 [M+Na]+. Calc. for C27H30O14Na: 601.1533; 1H-NMR (600 MHz, CDCl3): δ: 7.85 (2 H, d, J
.7 Hz, H-2', 6'), 6.91 (2 H, d, J 8.7 Hz, H-3', 5'), 6.59 (1 H, d, J 2.1 Hz, H-8), 6.52 (1 H, d, J 2.1 Hz, H-6), 6.50
1 H, s, H-3), 5.28 (1 H, d, J 6.6 Hz, H-1q), 5.21 (1 H, d, J 0.6 Hz, H-1q'), 3.65–3.68 (2 H, m, H-6q), 3.66 (2 H, m, H-
8
(
3
3
q, 3q'), 3.64 (1 H, m, H-5q'), 3.46 (1 H, m, H-5q), 3.41 (1 H, m, H-2q), 3.37 (1 H, m, H-2q'), 3.33 (1 H, m, H-4q),
.10 (1 H, m, H-4q'), 1.02 (3 H, d, J 6.0 Hz, H-6q'); 13C-NMR (150 MHz, CDCl3): 176.3 (C-4), 162.9 (C-7), 161.3
(
(
(
C-2), 160.9 (C-4'), 159.5 (C-9), 158.4 (C-5), 128.7 (C-2', 6'), 122.3±C-1'), 116.8 (C-3', 5'), 108.3 (C-10), 106.8
C-3), 100.5 (C-1q'), 99.7 (C-6), 98.5 (C-1q), 97.1 (C-8), 77.8 (C-5q), 77.6 (C-2q), 77.5 (C-3q), 73.1 (C-4q'), 71.4
C-3q'), 71.2 (C-2q'), 70.6 (C-4q), 68.3 (C-5q'), 61.5 (C-6q), 18.7 (C-6q').
1
13
Compound 2. H and C-NMR spectral data were in accordance with published data [10,11].
Compound 3. H and C-NMR spectral data were in accordance published data [12,13].
Compound 4. H- and C-NMR spectral data were in accordance with published data [14,15].
Compound 5. H- and C-NMR spectral data were in accordance with published data [16].
.4. Enzymatic hydrolysis of 1
1
13
1
13
1
13
2
Naringinase (200 mg, from Penicillium decumbens) was added to a suspension of 1 (10.0 mg) in 50 mM acetate
buffer (pH 5.5) and the mixture was stirred at 37 °C for 5 h. The reaction mixture was extracted with EtOAc
3×1000 ml), and the organic layer was evaporated to dryness. The residue was Si–gel CC using a gradient of CHCl3-
(
MeOH (20:1 to 10:1) to give apigenin (4.8 mg) as a yellow amorphous powder. The water layer was passed through a
Sep–Pak C18 cartridge (Waters, Milford, MA), which was then analyzed by HPLC under the following condition:
solvent MeCN–H O (3:1); flow rate 0.5 ml/min; detection RI and OR. The identification of D-glucose and L-rhamnose
2
present in the water layer were carried out by the comparison of their retention times and polarities with those of
authentic samples: t 17.5 min (d-glucose, positive polarity) and t 11.7 min (l-rhamnose, negative polarity).
R
R
2
.5. Bioassys
Free radical (DPPH) scavenging activity was examined as previously reported [17]. In brief, 20 μl of each sample in
DMSO was seeded to 980 μl of 150 μM methanolic DPPH and incubated at r.t. After 30 min, the absorbance of the
mixture was measure at 517 nm on a Shimazu UV-1240 spectrometer.
Superoxide radical scavenging activity was assayed by NBT reduction method [18]. The 495 μl assay mixture
consisted of 50 mM sodium carbonate buffer (pH 10.2), 0.1 mM xanthine and 25 μM nitro blue tetrazolium (NBT). The
reaction was initiated by addition of 5 μl 20 nM xanthine oxidase in the presence or absence of each compound. The
increase in absorbance at 560 nm was read after 5 min on a spectrophotometer Shimadzu UV-1240. Superoxide radical
scavenging activity was expressed by the degree of NBT reduction decrease of test group in comparison with that of the
control group [19].
3
. Results and discussion
Repeated column chromatography (including normal silica gel, Diaion HP-20, and Sephadex LH-20) of the water
soluble fraction from EtOH extract of leaf of C. koreana led to the isolation of a new flavone glycoside (1) together
with four known flavonol glycosides (2–5). Compounds 2–5 were identified as quercetin 3,7-di-O-glucoside (2)
[
10,11], quercetin 3-O-α-L-rhamnopyranoside (3) [12,13], quercetin-3-O-β-D-glucopyranoside (4) [14,15], and
quercetin 3-O-(6q-O-acetyl)-β-d-glucoside (5) [16] (Fig. 1).
2
5
Compound 1 was obtained as light yellowish amorphous powder with [α] −22.5° (c 0.4, MeOH). It showed
D
greenish brown color in FeCl test and red color in HCl–Mg reaction. Bands were observed at 263 and 328 nm in UV
3
−
1
−1
spectrum. The IR spectrum of 1 showed the bands for hydroxyl group at 3400 cm , carbonyl group at 1720 cm
,