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Caffeoylglucosides and Hypouricemic Activity of Aster glehni
KOREAN CHEMICAL SOCIETY
purified bypreparative TLC on RP-18 developed with MeOH/
H2O (40:60, v/v) to yield the roseoside 1a (1.0 mg) from the
CH2Cl2 residue, andaglycone1b(0.5 mg)and(E)-caffeicacid
from the EtOAc residue. Compound 3 (3.0 mg) was hydro-
lyzed in the same way to yield the aglycone 3a (1.6 mg) from
CH2Cl2 residue and (E)-caffeic acid from EtOAc residue. The
aqueous layer was passed through Sephadex LH-20 (MeOH)
to give D-glucose as a syrup, which was identified by compar-
ison of the 1H NMR spectral data and the optical rotation value
4), 2.273 (3H, s, H-10), 1.940 (1H, br t, J = 13.2 Hz, H-2β),
1.753 (3H, s, H-13), 1.495 (1H, ddd, J = 13.2, 3.6, 1.2 Hz,
H-2α), 1.136 (3H, s, H-11), 1.101 (3H, s, H-12).
Compound 3c: 1H NMR (CD3OD, 600 MHz) δ 7.252 (1H,
d, J = 16.8 Hz, H-7), 6.152 (1H, d, J = 16.2 Hz, H-8), 5.176
(1H, dt, J = 12.6, 4.2 Hz, H-3), 4.103 (1H, d, J = 4.2 Hz, H-
4), 2.314 (3H, s, H-10), 2.075 (1H, br t, J = 12.6 Hz, H-2β),
1.857 (3H, s, H-13), 1.702 (1H, ddd, J = 12.6, 3.6, 1.2 Hz,
H-2α), 1.136 (3H, s, H-11), 1.121 (3H, s, H-12).
25
{½αꢀD + 51:3ꢁ (c 0.3, H2O)} with the authentic sample.
Superoxide Anion Radical Scavenging Assay. The super-
oxide anion radical scavenging activity was determined by
the method previously described.23 The reaction mixture con-
tained 0.1 M potassium phosphate buffer (pH 7.5), 1 mM xan-
thine, 1 mM EDTA, 250 μM NBT, and various concentrations
of extracts or compounds in the 96-well plates. Reaction was
started by adding 0.05 U/mL XOD (EC 1.2.3.2), and the reac-
tion mixture was incubated at 37 ꢁC for 20 min. The absorb-
ance of each sample was measured spectrophotometrically
at 540 nm, and IC50 values denote the concentration of sam-
ples required to scavenge 50% superoxide anion radicals.
XOD Inhibitory Assay. The inhibitory activity on XOD was
measured spectrophotometrically at 295 nm under aerobic
condition according to the method described with slight mod-
ifications.24 The assay mixture consisting of 10 μL of sample
solution, 50 μL of 0.1 M phosphate buffer (pH 7.5), and 10 μL
of enzyme solution was prepared immediately before use.
After preincubation at 25 ꢁC for 10 min, the reaction was
initiated by adding 30 μL of 0.1 mM xanthine solution. This
assay mixture was incubated at 25 ꢁC for 30 min. The reaction
was stopped by adding 10 μL of 1 N HCl, and the absorbance
of the mixture at 295 nm was measured spectrophotometri-
cally. The inhibitory activity on XOD was assessed as the per-
centage inhibition, and the IC50 values denote the
concentration of samples required to inhibit 50% XOD
activity.
1
Compound 1a: Colorless amorphous powder, H NMR
(pyridine-d5, 400 MHz) δ 6.29 (1H, dd, J = 6.8, 16.0 Hz, H-
8), 6.10 (1H, d, J = 16.0 Hz, H-7), 6.04 (1H, br s, H-4),
4.98 (1H, overlapped, H-10), 4.70 (1H, quintet, J = 6.8 Hz,
H-9), 4.52 (1H, dd, J = 1.6, 12.0 Hz, H-60a), 4.27 (1H, dd, J
= 6.0, 12.0 Hz, H-60b), 4.22 (1H, t, J = 9.0 Hz, H-30), 4.13
(1H, t, J = 8.8 Hz, H-40), 4.01 (1H, t, J = 8.4 Hz, H-20), 3.88
(1H, t, m, H-50), 2.64 (1H, d, J = 16.4 Hz, H-2b), 2.36 (1H,
d, J = 16.4Hz, H-2a), 1.97 (3H, d, J = 1.2 Hz, H-13), 1.34
(3H, d, J = 6.4 Hz, H-10), 1.22, 1.12 (each 3H, s, H-11, 12).
Compound 1b: Colorless, 1H NMR (CD3OD, 600 MHz) δ
5.82 (1H, br s, H-4), 5.75 (1H, dd, J = 4.8, 15.6 Hz, H-8), 5.71
(1H, d, J = 15.6 Hz, H-7), 4.26 (1H, dd-like, J = 4.4, 6.8 Hz, H-
9), 2.42 (1H, d, J = 17.2 Hz, H-2), 2.10 (1H, d, J = 17.2 Hz, H-
2), 2.85 (3H, d, J = 1.2 Hz, H-13), 1.18 (3H, d, J = 6.4 Hz, H-
10), 0.98, 0.96 (each 3H, s, H-11, 12).
Compound 3a: Colorless amorphous powder, [α]25 –42.5ꢁ
(c 0.08, MeOH); CD (MeOH) Δε (nm): +3.65 (223.8), +5.29
(275.0), −3.30 (314.4); 1H NMR (CD3OD, 400 MHz) δ: 7.21
(1H, d, J = 16.8 Hz, H-7), 6.08 (1H, d, J = 16.4 Hz, H-8), 3.81
(1H, d, J = 4.0 Hz, H-4), 3.72 (1H, dt, J = 12.8, 4.0 Hz, H-3),
2.25(3H, s, H-10), 1.76(1H, brt, J=12.4 Hz, H-2β), 1.81(3H,
s, H-13), 1.41 (1H, dd, J = 12.4, 2.0 Hz, H-2α), 1.06 (3H, s, H-
11), 1.02 (3H, s, H-12); HR-ESI-MS m/z 247.1311 [M + Na]+
(calcd for C13H20O3Na, 247.1310).
Hypouricemic Effects of AGEF onSerum Uric Acid Levels
in Potassium Oxonate-induced Hyperuricemic Rats. Male
Sprague–Dawley rats of age 12–16 weeks (200 g 20% in
weight), purchased from Oriental-Bio Co. (Gyeonggi, Korea),
were kept in the animal polycarbonate house with three ani-
mals per cage. The animals were maintained on a 12-h light/
dark cycle, at a temperature of 23 3 ꢁC and a relative humid-
ity of 55 15%, and were allowed free access to standard food
pellets (DreamBio Co., Seoul, Korea) and tap water for 1 week
prior to the experiment. All animals were housed and all
experiments performed according to the policies and guide-
lines of the Institutional Animal Care and Use Committee of
the Korea Animal Medicinal Science Institute (KAMSI-
IACUC), Gyeonggi, Korea (publication no: 14-KE-161).
Following adaption for a week, the animals were treated
orally once a day with vehicle (sterile water for injection),
AGEF, or allopurinol for 6 days. On the seventh day, potas-
sium oxonate (250 mg/kg, i.p.) was injected to induce hyper-
uricemia according to the method described with slight
modifications7,8 1 hbefore finaladministrationofthesamples.
Rats were randomly divided into six groups of 10 animals
Preparation of (R)-MTPA Ester (3b) and (S)-MTPA
Ester (3c) of 3a. A stirred solution of 3a (0.8 mg) in
pyridine (500 μL) was treated with (S)-(+)-α-methoxy-
α-trifluoromethylphenyl acetic acid chloride (MTPA-Cl, 5 μ
L) in the presence of 1-ethyl-3-(3-dimethoxylaminopropyl)-
carbodiimide hydrochloride (EDCꢂHCl, 1.5 mg) and 4-
methylaminopyridine (4-DMAP, 0.7 mg), and the mixture
was stirred at room temperature for 14 h. After cooling, the
reaction mixture was poured into water and extracted with
CH2Cl2. The CH2Cl2 extract was successively washed with
5% aqueous HCl, saturated aqueous NaHCO3, and brine, then
dried over anhydrousNa2SO4 and filtered. Removal ofthe sol-
vent from the filtrate under reduced pressure furnished a res-
idue, which was separated by preparative TLC on silica gel
developed with CH2Cl2–MeOH (30:1, v/v) to give the (R)-
MTPA ester 3b (0.4 mg). The (S)-MTPA ester 3c (0.4 mg)
was obtained through a similar procedure from 3a using
(R)-(−)-MTPA-Cl, EDC ꢂ HCl and 4-DMAP.
Compound 3b: 1H NMR (CD3OD, 600 MHz) δ 7.203 (1H,
d, J = 16.2 Hz, H-7), 6.111 (1H, d, J = 16.2 Hz, H-8), 5.121
(1H, dt, J = 12.6, 4.2 Hz, H-3), 4.165 (1H, d, J = 4.2 Hz, H-
Bull. Korean Chem. Soc. 2015, Vol. 36, 503–512
© 2015 Korean Chemical Society, Seoul & Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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