Animals
ether-EtOAc (9:1) yielded oleanolic acid 3 (2300 ± 3300 mL,
The rats (Sprague-Dawley, 250 20 g) were bred at the Animal 100 mg) and ursolic acid 4 (3600 ± 5200 mL, 200 mg).
Station of Natural Products Research Institute, Seoul National
University. They were fed with a commercial solid food (Sa- Alkaline hydrolysis and methylation of compounds 1 and 2 were
myang Yuji Co. Ltd., Seoul) and tap water and were housed at 23 performed by standard procedures. IR, MS and NMR allowed
0.58C and 60% humidity in a 12 hours light-dark cycle in identification of aglycones 1a and 2a, and dimethyl esters 1b
accordance with the Guide for the Care and Use of Laboratory and 2b.
Animals by Seoul National University.
Sugar moiety of 2 and the absolute configuration were determined
General experimental procedures
to be D-glucose according to the Hara's method [16].
Melting point was determined on a Büchi B-540 apparatus and
was uncorrected. UV spectra were recorded on a Hitachi U-3210 Trachelosperoside A-1 (1) [17]: white powder, [a]D13: + 7.5 (c 0.1,
UV-VIS spectrometer. IR spectra were obtained on a Jasco FT/IR- MeOH).
5300 spectrometer. FAB-MS were measured on a Jeol JMS-SX
102A mass spectrometer and EI-MS on a Hewlett Packard model Trachelosperogenin A (1a) [17]: white powder, [a]D22: + 25.0 (c 0.1,
5989B GC/MS spectrometer. NMR spectra were recorded on a CHCl3-MeOH, 4:1).
Varian Gemini-2000 spectrometer or on a Bruker Advance 400
spectrometer operating at 300 or 400 MHz for 1H and 75 or 100 Trachelosperogenin A dimethyl ester (1b) [17], [18]: white pow-
MHz for 13C-NMR. Optical rotations were determined on a Jasco der, [a]D19: + 17.1 (c 0.1, CHCl3).
P-1020 polarimeter. Centrifugations were taken at Sorvall RT
6000 centrifuge and Sorvall OTD 65B ultracentrifuge, rotor T 28-O-b-D-Glucopyranosyl-2a,3b-dihydroxyolean-12-ene-24,28-dioic
865. GC analysis was carried out on Hewlett Packard 5890 Series acid (2): white powder, m.p. > 3008C, Rf = 0.46 on silica gel (CHCl3-
II, HP-5 (30 m0.32 mm0.25 mm). Silica gel (63 ± 200 mm, MeOH-H2O, 70:30:4) and 0.07 on RP-18 F254 s (60% MeOH), [a]1D3:
Merck KGaA), Sephadex LH-20 (25 ± 100 mm, Sigma Fluka) and + 15.5 (c 0.1, MeOH), UV (MeOH): lmax = end absorption only, IR
LiChroprep RP-18 (40 ± 63 mm, Merck KGaA) were used for open (KBr): nmax = 3434, 1720, 1701, 1637, 1458, 1275, 1069 cm±1, 1H-
chromatography. TLC was performed on silica gel 60 F254 (Merck) and 13C-NMR: see Table 1, FAB-MS: m/z = 687.48 [M + Na]+.
and RP-18 F254 s (Merck).
2a,3b-Dihydroxyolean-12-ene-24,28-dioic acid (2a): white
Determination of tissue factor activity by single-stage clotting powder, m.p. > 3008C, [a]D22: + 30.5 (c 0.1, CHCl3-MeOH 4:1);
1
assay
IR (KBr): nmax = 3436, 1700, 1630, 1064 cm±1, H-NMR (CDCl3,
A microsomal fraction of rat lung tissue was used as a tissue fac- 300 MHz): d = 4.00 (1H, ddd, J = 10.4, 9.9, 4.5 Hz, H-2), 2.83
tor source [13]. A TF stock solution from 5 g of rat lung taken from (1H, d, J = 9.9 Hz, H-3), 5.21 (1H, brs, H-12), 2.76 (2H, dd,
4 rats (250 10 g) contained 0.539 mg protein/0.1 mL, when pro- J = 14.7, 3.9 Hz, H-18), 1.06 (3H, s, H-23), 0.84 (3H, s, H-25),
tein concentration was determined according to the Lowry's 0.72 (3H, s, H-26), 1.39 (3H, s, H-27), 0.84 (3H, s, H-29), 0.86
method [14] using bovine serum albumin as a standard protein. (3H, s, H-30); 13C-NMR (CDCl3, 75 MHz): d = 46.0 (C-1), 67.7
Dilution of the stock solution by 100 ± 200 times was used for as- (C-2), 82.7 (C-3), 49.1 (C-4), 56.0 (C-5), 19.6 (C-6), 32.5 (C-7),
say, which gave 5 to 10 units of TF per 100 mL. Prothrombin time 38.8 (C-8), 46.2 (C-9), 38.1 (C-10), 22.6 (C-11), 121.8 (C-12),
was measured to determine the TF activity by the single-stage 143.5 (C-13), 41.5 (C-14), 27.2 (C-15), 23.0 (C-16), 46.6 (C-17),
328
clotting assay, using citrated plasma from rats [13], [15].
41.0 (C-18), 45.5 (C-19), 30.2 (C-20), 33.5 (C-21), 32.2 (C-22),
25.2 (C-23), 180.4 (C-24), 14.2 (C-25), 16.3 (C-26), 23.7 (C-27),
179.6 (C-28), 32.5 (C-29), 23.2 (C-30), EI-MS: m/z = 502 [M]
+
Extraction and isolation
Dried pieces of the fruits of C. sinensis (20 kg) were three times (18.9%), 456 (18.9), 248 (100), 203 (40.2).
extracted with MeOH (54 L). The methanolic extract (4.6 kg)
was successively partitioned between n-hexane (8.4 L), EtOAc 2a,3b-Dihydroxyolean-12-ene-24,28-dioic acid dimethyl ester
(8.4 L), n-BuOH (8.4 L) and H2O (7 L) to afford 140, 350, 973 and (2b): white powder, [a]D19: + 35.4 (c 0.1, CHCl3); H-NMR (CDCl3,
1
2960 g of residues, respectively. The EtOAc (350 g) fraction was 300 MHz): d = 4.02 (1H, ddd, J = 11.1, 9.9, 5.1 Hz, H-2), 2.87
divided into two parts according to its solubility in 50% MeOH (1H, m, H-3), 5.28 (1H, t like, H-12), 2.87 (2H, m, H-18), 1.11 (3H,
(1200 mL); 50% MeOH soluble (79 g) and insoluble (223 g) parts. s, H-23), 0.79 (3H, s, H-25), 0.72 (3H, s, H-26), 1.43 (3H, s, H-27),
Repetitive chromatography of the former part (79 g) over Sepha- 0.89 (3H, s, H-29), 0.91 (3H, s, H-30), 3.60 (3H, s, OCH3±24), 3.66
dex LH-20 (500 g) eluting with MeOH-H2O (1:1) (14,000 mL) af- (3H, s, OCH3±28); 13C-NMR (CDCl3, 75 MHz): d = 46.1 (C-1), 68.2
forded an active fraction A (19 g) (3800 ± 8300 mL) and an inac- (C-2), 83.5 (C-3), 49.1 (C-4), 56.5 (C-5), 20.1 (C-6), 32.7 (C-7),
tive fraction B (38 g, 8500 ± 12,000 mL). The active fraction was 39.2 (C-8), 46.7 (C-9), 38.3 (C-10), 23.0 (C-11), 122.2 (C-12),
subjected to column chromatography on silica gel (600 g) eluting 143.6 (C-13), 41.7 (C-14), 27.6 (C-15), 23.6 (C-16), 46.9 (C-17),
with CHCl3-MeOH-H2O (5:1:0.1, 3:1:0.1, each 6000 mL) to give 41.3 (C-18), 45.7 (C-19), 30.7 (C-20), 33.8 (C-21), 32.3 (C-22),
an active sub-fraction A1 (6600 ± 9000 mL, 1.9 g), which by fur- 25.7 (C-23), 178.5 (C-24), 14.5 (C-25), 16.7 (C-26), 23.8 (C-27),
ther chromatography on reverse phase C-18 (350 mL volume; 178.2 (C-28), 33.1 (C-29), 23.6 (C-30), 51.5 (OCH3±24), 51.5
MeOH-H2O, 1:1) yielded compounds 1 (705 ± 900 mL, 160 mg) (OCH3±28), EI-MS: m/z = 530 [M]+ (1.7%), 470 (1.7), 262 (34.7),
and 2 (1140 ± 1190 mL, 180 mg). Column chromatography of an 203 (100).
aliquot (20 g) of the 50% MeOH insoluble part (223 g) of the
EtOAc fraction on silica gel (600 g) eluting with petroleum
Lee MH, Han YN. A New in vitro¼ Planta Med 2003; 69: 327±331