May 2007
937
afford compounds 2 (550 mg) and 3 (400 mg). Fraction 1 was 48.1 (C-9), 48.1 (C-1), 48.4 (C-17), 53.2 (C-18), 61.3 (C-6ꢂ),
further separated by chromatography on ODS [Rp-8 66.5 (C-23), 68.9 (C-2), 69.3 (C-6ꢁ), 70.3 (C-5ꢅ), 71.0 (C-
(H2O : MeOHꢀ5 : 2)] and Sephadex LH-20 (H2O : MeOHꢀ 4ꢁ), 72.5 (C-2ꢅ), 72.7 (C-3ꢅ), 73.7 (C-2ꢁ), 73.9 (C-4ꢅ), 75.2
1 : 1) to purify 2 (900 mg), 3 (800 mg), 4 (45 mg), and 5 (C-2ꢂ), 76.4 (C-3ꢂ), 77.1 (C-5ꢂ), 77.8 (C-5ꢁ), 78.2 (C-3), 78.2
(25 mg).
(C-4ꢂ), 78.7 (C-3ꢁ), 95.6 (C-1ꢁ), 102.6 (C-1ꢅ), 104.8 (C-1ꢂ),
The structures of these compounds were elucidated on the 126.0 (C-12), 138.5 (C-13), 176.3 (C-28).
basis of following data and comparison with authentic sam-
ples.
Compound 7 (Kaempferol) Yellow amorphous powder;
IR nmax (KBr) cmꢃ1: 3385 (OH), 1660 (CꢀO), 1602, 1503,
Compound 1 (Chlorogenic Acid) Colorless needles 1450 (aromatic ring); UV lmax (MeOH) nm (log e): 256
from water and methanol, [a]D26 ꢃ28° (cꢀ0.8, H2O); IR nmax (4.14 ), 371 (4.15); H-NMR (d, DMSO-d6): 6.18 (1H, d,
1
(KBr) cmꢃ1: 3350 (OH), 1685 (CꢀO), 1652, 978 (trans Jꢀ2.1 Hz, H-6), 6.38 (1H, d, Jꢀ2.1 Hz, H-8), 8.06 (2H, d,
CꢀC), 1600, 1516, 1445 (aromatic ring); UV lmax (MeOH) Jꢀ9.1 Hz, H-2ꢁ, H-6ꢁ), 6.90 (2H, d, Jꢀ9.1 Hz, H-3ꢁ, H-5ꢁ),
1
nm (log e): 332 (4.60), 328 (4.55), 301 (4.51); H-NMR 13C-NMR (d, DMSO-d6): 148.03 (C-2), 137.06 (C-3), 177.30
(400 MHz, CD3OD): Table 1; 13C-NMR (100 MHz, CD3OD): (C-4), 162.42 (C-5), 99.27 (C-6), 165.49 (C-7), 94.49 (C-8),
Table 2.
158.20 (C-9), 104.52 (C-10), 123.71 (C-1ꢁ), 130.65 (C-2ꢁ, C-
Acid Hydrolysis of Compound 1 Compound 1 (15 mg) 6ꢁ), 116.29 (C-3ꢁ, C-5ꢁ).
was hydrolyzed with 3% HCl (5 ml) at 90 °C for 3 h under
Compound 8 (Quercetin) Yellow amorphous powder;
stirring. The reaction mixture, after cooling, was neutralized IR nmax (KBr) cmꢃ1: 3380 (OH), 1652 (CꢀO), 1613, 1512,
with Ag2CO3, and the insoluble material was filtered off. The 1450 (aromatic ring); UV lmax (MeOH) nm (log e): 255
1
filtrate was evaporated to dryness and the residue was sub- (4.36), 373 (4.38); H-NMR (d, DMSO-d6): 6.20 (1H, d,
jected to chromatography on Sephadex LH-20 (MeOH) to Jꢀ2.1 Hz, H-6), 6.42 (1H, d, Jꢀ2.1 Hz, H-8), 7.69 (1H, d,
give L-quinic acid, [a]D26 ꢃ38° (cꢀ0.3, MeOH).
Jꢀ2.2 Hz, H-2ꢁ), 6.90 (1H, d, Jꢀ8.5Hz, H-5ꢁ), 7.55 (1H, dd,
Compound 2 (3,5-Di-O-caffeoylquinic Acid) Colorless Jꢀ8.5, 2.2 Hz, H-6ꢁ); 13C-NMR (d, DMSO-d6): 146.79 (C-
amorphous powder, [a]D26 ꢃ175.8° (cꢀ0.84, MeOH); nega- 2), 135.69 (C-3), 175.69 (C-4), 160.71 (C-5), 98.16 (C-6),
tive-FAB-MS m/z: 515 [MꢃH]ꢃ; IR nmax (KBr) cmꢃ1: 3400 163.85 (C-7), 93.33 (C-8), 156.13 (C-9), 103.00 (C-10),
(OH), 1683 (CꢀO), 1652, 978 (trans CꢀC), 1602, 1516, 121.95 (C-1ꢁ), 115,05 (C-2ꢁ), 145.01 (C-3ꢁ), 147.65 (C-4ꢁ),
1447 (aromatic ring); UV lmax (MeOH) nm (log e): 332 115,58 (C-5ꢁ), 119.97 (C-6ꢁ).
(4.60), 297 (4.40), 245 (4.28), 219 (4.44); 1H-NMR
Compound 9 (Kaempferol 3-O-b-D-Glucuronide) Ye l -
(400 MHz, CD3OD): Table1; 13C-NMR (100 MHz, CD3OD): low amorphous powder, IR nmax (KBr) cmꢃ1: 3380 (OH),
Table 2.
1652 (CꢀO), 1613, 1512, 1450 (aromatic ring); 1H-NMR (d,
Compound 3 (1,5-Di-O-caffeoylquinic Acid) Colorless DMSO-d6): 6.10 (1H, d, Jꢀ2.0 Hz, H-6), 6.31 (1H, d,
amorphous powder, [a]D26 ꢄ204° (cꢀ0.68, MeOH); positive- Jꢀ2.0 Hz, H-8), 8.02 (2H, d, Jꢀ8.9 Hz, H-2ꢁ, H-6ꢁ), 6.85
FAB-MS m/z: 517 [MꢄH]ꢄ; IR nmax (KBr) cmꢃ1: 3400 (2H, d, Jꢀ8.9 Hz, H-3ꢁ, H-5ꢁ), 5.52 (1H, d, Jꢀ7.5 Hz, H-1ꢂ),
(OH), 1692 (CꢀO), 1650, 979 (trans CꢀC), 1602, 1522, 3.26—3.29 (2H, m, H-2ꢂ, H-3ꢂ), 3.22 (1H, m, H-4ꢂ), 3.40
1447 (aromatic ring); UV lmax (MeOH) nm (log e): 329 (1H, d, Jꢀ9.4 Hz, H-5ꢂ); 13C-NMR (d, DMSO-d6): 156.24
(4.60), 302 (4.42), 245 (4.28), 219 (4.44); 1H-NMR (C-2), 133.48 (C-3), 177.38 (C-4), 160.14 (C-5), 98.81 (C-6),
(400 MHz, CD3OD): Table 1; 13C-NMR (100 MHz, CD3OD): 164.96 (C-7), 93.86 (C-8), 156.37 (C-9), 103.60 (C-10),
Table 2.
120.85 (C-1ꢁ), 130.93 (C-2ꢁ, C-6ꢁ), 115.16 (C-3ꢁ, C-5ꢁ),
Compound 4 (3,4-Di-O-caffeoylquinic Acid) Colorless 160.98 (C-4ꢁ), 101.29 (C-1ꢂ), 74.48 (C-2ꢂ), 76.40 (C-3ꢂ),
amorphous powder, [a]D26 ꢃ134.7° (cꢀ0.63, MeOH); posi- 71.94 (C-4ꢂ), 74.06 (C-5ꢂ), 172.74 (C-6ꢂ).
tive-FAB-MS m/z: 517 [MꢄH]ꢄ; IR nmax (KBr) cmꢃ1: 3370
b
Compound 10 (Quercetin 3-O- -D-Glucuronide) Ye l -
(OH), 1693 (CꢀO), 1640, 978 (trans CꢀC), 1605, 1522, low amorphous powder, IR nmax (KBr) cmꢃ1: 3380 (OH),
1447 (aromatic ring); UV lmax (MeOH) nm (log e): 328 1652 (CꢀO), 1613, 1512, 1450 (aromatic ring); 1H-NMR (d,
(4.50), 245 (4.28), 218 (4.42); 1H-NMR (400 MHz, CD3OD): DMSO-d6): 6.17 (1H, d, Jꢀ2.0 Hz, H-6), 6.36 (1H, d,
Table 1; 13C-NMR (100 MHz, CD3OD): Table 2.
Jꢀ2.0 Hz, H-8), 8.47 (1H, d, Jꢀ2.2 Hz, H-2ꢁ), 6.82 (1H, d,
Compound 5 (4,5-Di-O-caffeoylquinic Acid) Colorless Jꢀ8.4 Hz, H-5ꢁ), 7.29 (1H, dd, Jꢀ8.4, 2.2 Hz, H-6ꢁ), 5.23
amorphous powder, [a]D26 ꢃ232.4° (cꢀ0.43, MeOH); posi- (1H, d, Jꢀ7.3 Hz, H-1ꢂ), 3.24—3.27 (2H, m, H-2ꢂ, H-3ꢂ),
tive-FAB-MS m/z: 517 [MꢄH]ꢄ, IR nmax (KBr) cmꢃ1: 3265 3.20 (1H, dd, Jꢀ9.7, 9.7 Hz, H-4ꢂ), 3.39 (1H, d, Jꢀ9.7 Hz,
(OH), 1695 (CꢀO), 1640, 978 (trans CꢀC), 1601, 1516, H-5ꢂ); 13C-NMR (d, DMSO-d6): 157.55 (C-2), 133.97 (C-3),
1447 (aromatic ring); UV lmax (MeOH) nm (log e): 329 177.50 (C-4), 160.95 (C-5), 99.00 (C-6), 165.14 (C-7), 93.85
(4.51), 245 (4.27), 218 (4.40); 1H-NMR (400 MHz, CD3OD): (C-8), 156.56 (C-9), 103.51 (C-10), 120.61 (C-1ꢁ), 115.44
Table 1; 13C-NMR (100 MHz, CD3OD): Table 2.
(C-2ꢁ), 144.80 (C-3ꢁ), 148.38 (C-4ꢁ), 115.44 (C-5ꢁ), 119.97
Compound 6 (Asiaticoside) Colorless needles from (C-6ꢁ), 102.94 (C-1ꢂ), 74.31 (C-2ꢂ), 76.73 (C-3ꢂ), 71.85 (C-
MeOH, mp 230—232 °C (decomp.), [a]D26 ꢃ16° (cꢀ0.52, 4ꢂ), 74.00 (C-5ꢂ), 172.20 (C-6ꢂ).
MeOH); negative-FAB-MS m/z: 957 [MꢃH]ꢃ; IR nmax (KBr)
Acid Hydrolysis of 9 and 10 for Analysis of Sugar Moi-
cmꢃ1: 3246 (OH), 2878 (CH3, CH2), 1734 (CꢀO), 1636 ety Refluxing of each compound 9 and 10 (10 mg) in 5%
(CꢀC); 13C-NMR (d, C5D5N): 14.4 (C-24), 17.3 (C-29), HCl for 4 h gave quercetin and kaempferol as aglycon moi-
17.6 (C-25), 17.8 (C-26), 18.4 (C-6ꢅ), 18.5 (C-6), 21.3 (C- ety, respectively and D-glucuronic acid as sugar moiety (iden-
30), 23.7 (C-27), 23.8 (C-11), 24.5 (C-16), 28.7 (C-15), 30.8 tified by comparison with authentic sample).
(C-21), 33.1 (C-7), 36.8 (C-22), 38.3 (C-10), 39.1 (C-19),
Oral-Administration of Samples To evaluate the anti-
39.3 (C-20), 40.2 (C-8), 42.5 (C-14), 43.6 (C-4), 47.9 (C-5), thrombotic effect on rat blood, the MeOH extract, EtOAc sol-