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4-4-3 (33 mg), Fr. 4-4-4 (35 mg), Fr. 4-4-5 (26 mg), Fr. 4-4-6 (70 mg), and treated with hesperidinase (75 mg) and the solution was stirred at 40 °C for
Fr. 4-4-7 (271 mg)]. Fraction 4-4-6 (70 mg) was further separated by HPLC 48 h. Work-up of the reaction mixture as described above gave 3a (31.0 mg,
[CH3CN–H2O (90 : 10, v/v)] to furnish cucurbitacin J 2-O-b-D-glucopyra-
noside (6, 30 mg, 0.0015%) and cucurbitacin K 2-O-b-D-glucopyranoside (7,
80%).
Colocynthol A (1a): A white powder, [a]D27 ꢃ12.7° (cꢁ0.05, MeOH).
15 mg, 0.0007%). Fraction 5 (4.1 g) was subjected to reversed-phase silica High-resolution positive-ion FAB-MS: Calcd for C32H44O9Na (MꢃNa)ꢃ
gel column chromatography [150 g, MeOH–H2O (15 : 85→30 : 70→50 : 50,
595.2883; Found 595.2891. CD [MeOH, nm (De)]: 227 (ꢂ0.80), 290
v/v)→MeOH] to afford five fractions {Fr. 5-1 (734 mg), Fr. 5-2 (765 mg), Fr. (ꢃ1.37), 328 (ꢂ1.32). UV [MeOH, nm (log e)]: 257 (3.58). IR (KBr, cmꢂ1):
5-3 (733 mg), Fr. 5-4 (150 mg), and Fr. 5-5 (942 mg). Fraction 5-3 (733 mg) 3432, 1736, 1701, 1664, 1648, 1086. 1H-NMR d: given in Table 2. 13C-
was purified by HPLC [MeOH–H2O (50 : 50, v/v)] to give isoorientin 3ꢀ- NMR dC: given in Table 3. Positive-ion FAB-MS m/z 595 (MꢃNa)ꢃ. Nega-
methyl ether (12, 69 mg, 0.0032%). Fraction 5-4 (485 mg) was separated by tive-ion FAB-MS m/z 571 (MꢂH)ꢂ.
HPLC [CH3CN–H2O (35 : 65, v/v)] to give three fractions [Fr. 5-4-1
(144 mg), Fr. 5-4-2 [ꢁ4 (25 mg, 0.0035%)], and Fr. 5-4-3 (317 mg)]. Frac-
tion 5-4-1 (144 mg) was further separated by HPLC [MeOH–H2O (55 : 45,
v/v)] to furnish khekadaengoside E (9, 10 mg, 0.0014%). Fraction 5-5
(942 mg) was purified by HPLC [MeOH–H2O (60 : 40, v/v)] to furnish 5
Enzymatic Hydrolysis of 2 with Cellulase A solution of 2 (10.0 mg) in
0.2 M acetate buffer (pH 5.0, 2.0 ml) was treated with cellulase (20 mg, from
Aspergillus niger, Sigma) and the solution was stirred at 37 °C for 15 h.
After EtOH was added to the reaction mixture, the solvent was removed
under reduced pressure and the residue was purified by normal-phase silica
(33 mg, 0.0015%). Fraction 6 (4.0 g) was further separated by reversed- gel column chromatography [1.5 g, CHCl3–MeOH (4 : 1, v/v)] to give colo-
phase silica gel column chromatography [150 g, MeOH–H2O (30 : 70→
40 : 60→60 : 40, v/v)→MeOH] to afford eight fractions [Fr. 6-1 (65 mg), Fr.
6-2 (256 mg), Fr. 6-3 (811 mg), Fr. 6-4 [ꢁisovitetin (11, 735 mg, 0.039%),
cynthol B 2-O-b-D-glucopyranoside (2a, 6.2 mg, 76%).
Colocynthol B 2-O-b-D-Glucopyranoside (2a): A white powder, [a]D27
ꢃ1.4° (cꢁ0.36, MeOH). High-resolution positive-ion FAB-MS: Calcd for
Fr. 6-5 (189 mg), Fr. 6-6 (1200 mg), Fr. 6-7 (416 mg), and Fr. 6-8 (328 mg)]. C36H54O11Na (MꢃNa)ꢃ 683.3407; Found 683.3398. CD [MeOH, nm (De)]:
Fraction 6-2 (256 mg) was purified by HPLC [CH3CN–H2O (10 : 90, v/v)] to
240 (ꢂ0.23), 274 (ꢃ0.31), 332 (ꢂ017). UV [MeOH, nm (log e)]: 257
give colocynthoside B (2, 78 mg, 0.0095%). Fraction 7 (10.3 g) was sub- (3.76). IR (KBr, cmꢂ1): 3569, 1686, 1637, 1078, 1032. 1H-NMR d: given in
jected to reversed-phase silica gel column chromatography [300 g, Table 2. 13C-NMR dC: given in Table 3. Positive-ion FAB-MS m/z 683
MeOH–H2O (15 : 85→30 : 70→50 : 50, v/v)→MeOH] to afford seven frac- (MꢃNa)ꢃ. Negative-ion FAB-MS m/z 659 (MꢂH)ꢂ.
tions [Fr. 7-1 (159 mg), Fr. 7-2 (1.80 g), Fr. 7-3 (730 mg), Fr. 7-4 (4.30,
218 mg), Fr. 7-5 (4.30 g), Fr. 7-6 (2.10 g), and Fr. 7-7 (13 mg)]. Fraction 7-2
(510 mg) was purified by HPLC [MeOH–H2O (15 : 85, v/v)] to give 4-(b-D-
glucopyranosyloxy)benzyl alcohol (17, 70 mg, 0.0015%). Fraction 7-5
Enzymatic Hydrolysis of 2a with Hesperidinase A solution of 2a
(6.0 mg) in 0.2 M acetate buffer (pH 3.8, 2.0 ml) was treated with hesperidi-
nase (20 mg) and the solution was stirred at 40 °C for 48 h. After EtOH was
added to the reaction mixture, the solvent was removed under reduced pres-
(870 mg) was purified by HPLC [MeOH–H2O (55 : 45, v/v)] to give 2 sure and the residue was purified by reversed-phase silica gel column chro-
(29 mg, 0.0085%). Fraction 8 (2.7 g) was subjected to reversed-phase silica matography [1.0 g, MeOH–H2O (50 : 50, v/v)] to give colocynthol B (2b,
gel column chromatography [300 g, MeOH–H2O (15 : 85→30 : 70→50 : 50,
v/v)→MeOH] to afford eight fractions [Fr. 8-1 (814 mg), Fr. 8-2 (453 mg),
4.0 mg, 89%).
Colocynthol B (2b): A white powder, [a]D27 ꢃ45.2° (cꢁ0.20, MeOH).
Fr. 8-3 (56 mg), Fr. 8-4 (200 mg), Fr. 8-5 (81 mg), Fr. 8-6 (604 mg), Fr. 8-7 High-resolution EI-MS: Calcd for C30H42O6 (Mꢃ) 498.2981; Found
(397 mg), and Fr. 8-8 (157 mg)]. Fraction 8-4 (200 mg) was purified by 498.2986. CD [MeOH, nm (De)]: 245 (ꢂ0.95), 285 (ꢃ2.28), 330 (ꢂ1.78).
HPLC [MeOH–H2O (55 : 45, v/v)] to give isosaponarin (13, 9 mg,
UV [MeOH, nm (log e)]: 268 (3.67). IR (KBr, cmꢂ1): 3440, 1686, 1655,
1
0.0005%).
1078, 1040. H-NMR d: given in Table 2. 13C-NMR dC: given in Table 3.
The known compounds were identified by comparison of their physical EI-MS m/z (%): 498 (Mꢃ, 9), 164 (100).
data ([a]D, IR, 1H-NMR, 13C-NMR, MS) with reported values7,18—28)
Colocynthoside A (1): A white powder, [a]D27 ꢂ13.5° (cꢁ0.58, MeOH).
Bioassay Method. Animals Male ddY mice weighing about 25—30 g
were purchased from Kiwa Laboratory Animal Co., Ltd., Wakayama, Japan.
High-resolution positive-ion FAB-MS: Calcd for C38H54O14Na (MꢃNa)ꢃ The animals were housed at a constant temperature of 23ꢄ2 °C and were fed
757.3411; Found 757.3418. CD [MeOH, nm (De)]: 229 (ꢂ1.12), 279 a standard laboratory chow (MF, Oriental Yeast Co., Ltd., Tokyo, Japan).
(ꢃ0.63), 330 (ꢂ0.64). UV [MeOH, nm (log e)]: 236 (4.04). IR (KBr, cmꢂ1): The animals were fasted for 24—26 h prior to the beginning of the experi-
3440, 1718, 1686, 1650, 1637, 1078. 1H-NMR d: given in Table 2. 13C- ment, but were allowed free access to tap water. All of the experiments were
NMR dC: given in Table 3. Positive-ion FAB-MS m/z 757 (MꢃNa)ꢃ. Nega- performed with conscious mice unless otherwise noted. The experimental
tive-ion FAB-MS m/z 733 (MꢂH)ꢂ.
protocol was approved by the Experimental Animal Research Committee at
Kyoto Pharmaceutical University.
Effects on Ear Passive Cutaneous Anaphylaxis (PCA) Reactions in
Colocynthoside B (2): A white powder, [a]D27 ꢂ26.7° (cꢁ0.58, MeOH).
High-resolution positive-ion FAB-MS: Calcd for C42H62O15Na (MꢃNa)ꢃ
829.3986; Found 829.3978. CD [MeOH, nm (De)]: 236 (ꢂ0.34), 271 Mice Experiments on the effects of the methanolic extract and its fractions
(ꢃ0.41), 338 (ꢂ0.36). UV [MeOH, nm (log e)]: 255 (3.82). IR (KBr, cmꢂ1): from the fruit of C. colocynthis on ear PCA reactions were performed ac-
3569, 1686, 1655, 1637, 1078, 1037. 1H-NMR d: given in Table 2. 13C- cording to the method reported previously36) with slight modification.
NMR dC: given in Table 3. Positive-ion FAB-MS m/z 829 (MꢃNa)ꢃ. Nega- Briefly, 10 ml of anti-DNP IgE diluted in PBS (20 mg/ml), or PBS alone
tive-ion FAB-MS m/z 805 (MꢂH)ꢂ.
Acid Hydrolysis of 1 and 2 A solution of colocynthosides (1 and 2,
2.0 mg each) in 1 M HCl (0.5 ml) was heated under reflux for 3 h. After cool-
(normal group) was injected intradermally into both ears of male ddY mice
(5—6 weeks old). Forty-seven hours later, test compounds suspended in 5%
acacia solution were administrated orally. After 1 h, 0.25 ml of PBS which
ing, the reaction mixture was poured into ice-water and neutralized with contained 2% Evans blue and 0.25 mg of DNP-BSA was injected into the
Amberlite IRA-400 (OHꢂ form), and the resin was removed by filtration. vein. Thirty minutes later, mice were killed by cervical dislocation and the
Then, the filtrate was extracted with EtOAc. The aqueous layer was sub- both ears were removed and incubated with 1 M KOH solution overnight at
jected to HPLC analysis under the following conditions: HPLC column, 37 °C to dissolve them. The solution was then mixed with 4.5 ml of a mix-
Kaseisorb LC NH2-60-5, 4.6 mm i.d.ꢆ250 mm (Tokyo Kasei Co., Ltd.,
ture of acetone–0.2 M H3PO4 (13 : 5, v/v). After centrifugation at 4000 rpm
Tokyo, Japan); detection, optical rotation [Shodex OR-2 (Showa Denko Co., for 10 min, absorbance was measured at 620 nm using a spectrophotometer
Ltd., Tokyo, Japan)]; mobile phase, CH3CN–H2O (85 : 15, v/v); flow rate
0.8 ml/min; column temperature, room temperature. Identification of L-
rhamnose (i) from 2 and D-glucose (ii) from 1 and 2 present in the aqueous
(Beckmann DU 530). An antiallergic agent, tranilast, was used as a refer-
ence compound.37,38)
Statistics Values were expressed as meansꢄS.E.M. One-way analysis of
layer were carried out by comparison of their retention time and optical rota- variance (ANOVA) followed by Dunnett’s test was used for statistical analy-
tion with those of an authentic samples. tR: (i) 7.8 min (negative optical rota- sis.
tion); (ii) 13.9 min (positive optical rotation).
Enzymatic Hydrolysis of 1 with Hesperidinase A solution of 1
(6.5 mg) in 0.2 M acetate buffer (pH 3.8, 2.0 ml) was treated with hesperidi-
nase (20 mg, from Aspergillus niger, Sigma) and the solution was stirred at
Acknowledgments This research was supported by the 21st COE Pro-
gram, Academic Frontier Project, and a Grant-in Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and Technology of
40 °C for 48 h. After EtOH was added to the reaction mixture, the solvent Japan.
was removed under reduced pressure and the residue was purified by re-
versed-phase silica gel column chromatography [1.0 g, MeOH–H2O (50 : 50,
v/v)] to give colocynthol A (1a) (4.2 mg, 83%). Through the similar proce-
dure, a solution of 3 (50.0 mg) in 0.2 M acetate buffer (pH 3.8, 10.0 ml) was
References and Notes
1) Part XXVI: Morikawa T., Matsuda H., Li N., Nakamura S., Li X.,
Yoshikawa M., Heterocycles, 68, 1139—1148 (2006).