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Vol. 57, No. 2
Table 4. Inhibitory Effects of 4, 5, and 8 on Plasma Triglyceride Elevation in Olive Oil-Treated Mice
Plasma triglyceride (mg/dl)a)
Dose
(mg/kg, p.o.)
Treatment
n
2.0 h
4.0 h
6.0 h
Normal
Control
4
—
—
50
100
200
—
—
50
100
200
200
7
9
7
7
7
7
9
7
7
7
7
7
9
7
7
7
119.3ꢃ11.8**
565.8ꢃ74.0
623.8ꢃ71.2
401.1ꢃ81.1
288.2ꢃ73.4*
142.8ꢃ20.7**
513.0ꢃ83.0
377.5ꢃ75.9
354.3ꢃ87.7
177.7ꢃ23.6**
595.0ꢃ82.0
91.9ꢃ9.4**
440.3ꢃ60.2
371.3ꢃ41.5
203.8ꢃ52.1**
198.6ꢃ24.1**
132.5ꢃ10.6**
635.3ꢃ84.4
650.5ꢃ50.4
563.0ꢃ93.2
344.9ꢃ36.3*
121.8ꢃ12.7**
397.2ꢃ63.4
367.5ꢃ43.0
321.1ꢃ46.7
178.4ꢃ21.5**
480.6ꢃ61.0
97.3ꢃ7.4**
393.2ꢃ60.1
297.0ꢃ67.4
160.4ꢃ47.7**
131.0ꢃ16.8**
101.1ꢃ5.2**
458.3ꢃ91.8
325.8ꢃ37.7
557.6ꢃ88.4
384.5ꢃ52.8
123.7ꢃ73.1**
259.3ꢃ48.8
287.2ꢃ30.1
450.5ꢃ44.2
169.9ꢃ28.9
350.0ꢃ24.1
90.6ꢃ9.4**
263.3ꢃ45.0
171.9ꢃ24.9
129.1ꢃ16.6**
114.5ꢃ7.6**
Normal
Control
5
Mukurozioside IIb (8)
Normal
Control
—
5
10
20
Orlistat
a) Values represent the meansꢃS.E.M. Significantly different from the control group, ∗ pꢄ0.05, ∗∗ pꢄ0.01.
give rarasaponin IV (1, 8.6 mg, 0.02%) and hederagenin 3-O-(3,4-di-O- Kyoto Pharmaceutical University.
Inhibitory Effect on Plasma TG Elevation in Olive Oil-Treated Mice
acetyl-a-L-arabinopyranosyl)-(1→3)-a-L-rhamnopyranosyl-(1→2)-a-L-ara-
binopyranoside (5, 22.3 mg, 0.06%). Fraction 4-3 (113.6 mg), which was de- The experiments were performed as described in our previous method with a
scribed previously,1) was further separated by HPLC [Wakopak Navi C30-5, slight modification.26—28) Each test sample was administered orally to fasted
CH3CN–H2O (50 : 50, v/v)] to furnish rarasaponin VI (3, 6.2 mg, 0.12%).
Fraction 5 (670.0 mg) was further purified by HPLC [Cosmosil HILIC, sample (ca. 0.25 ml) was collected from the infraorbital venosus plexus
CH3CN–H2O (92 : 8, v/v)] to afford rarasaponin V (2, 42.2 mg, 0.01%). under ether anesthesia at 2, 4, and 6 h after oral administration of olive oil.
Fraction 6 (250.0 mg) was separated by HPLC [Wakopak Navi C30-5, The collected blood was immediately mixed with heparin sodium
mice and olive oil (5 ml/kg) was administered p.o. 30 min thereafter. Blood
CH3CN–H2O (50 : 50, v/v)] to give 2 (18.0 mg, 0.19%).
(5 units/tube). After centrifugation of blood sample, plasma TG was deter-
Rarasaponin IV (1): An amorphous powder, [a]D27 ꢀ25.6° (cꢂ0.43, mined by enzymatic method using Triglyceride E test Wako (Wako Pure
MeOH). High-resolution positive-ion FAB-MS: Calcd for C52H80O19Na Chemical Ind., Ltd., Osaka, Japan).
(MꢀNa)ꢀ: 1031.5191. Found: 1031.5183. IR (KBr): 3488, 1736, 1655,
Statistics Values were expressed as meansꢃS.E.M. For statistical analy-
1
1254, 1086, 1053 cmꢁ1. H-NMR (600 MHz, pyridine-d5) d: given in Table sis, one-way analysis of variance followed by Dunnett’s test was used.
2. 13C-NMR data (150 MHz, pyridine-d5) dC: given in Table 3. Positive-ion
FAB-MS m/z: 1031 (MꢀNa)ꢀ. Negative-ion FAB-MS m/z: 1007 (MꢁH)ꢁ,
Acknowledgements M. Yoshikawa, and H. Matsuda were supported by
965 (MꢁC2H3O)ꢁ, 791 (MꢁC9H13O6)ꢁ, 749 (MꢁC11H15O7)ꢁ, 603 (Mꢁ the 21st COE Program, Academic Frontier Project, and a Grant-in Aid for
C17H25O11)ꢁ, 471 (MꢁC22H33O15)ꢁ.
Scientific Research from The Ministry of Education, Culture, Sports, Sci-
ence and Technology of Japan (MEXT). O. Muraoka and T. Morikawa were
Rarasaponin V (2): An amorphous powder, [a]D29 ꢁ8.3° (cꢂ1.00, MeOH).
High-resolution positive-ion FAB-MS: Calcd for C48H76O17Na (MꢀNa)ꢀ: supported by High-tech Research Center Project (2007—2011) and a Grant-
947.4981. Found: 947.4987. IR (KBr): 3569, 1734, 1717, 1656, 1250, 1053 in Aid for Scientific Research from MEXT.
1
cmꢁ1. H-NMR (600 MHz, pyridine-d5) d: given in Table 1. 13C-NMR data
(150 MHz, pyridine-d5) dC: given in Table 2. Positive-ion FAB-MS m/z: 947 References and Notes
(MꢀNa)ꢀ. Negative-ion FAB-MS m/z: 923 (MꢁH)ꢁ, 881 (MꢁC2H3O)ꢁ,
749 (MꢁC7H11O5)ꢁ, 603 (MꢁC13H21O9)ꢁ, 471 (MꢁC18H29O13)ꢁ.
Rarasaponin VI (3): An amorphous powder, [a]D26 ꢀ1.9° (cꢂ0.41,
MeOH). High-resolution positive-ion FAB-MS: Calcd for C50H78O18Na
(MꢀNa)ꢀ: 989.5086. Found: 989.5082. IR (KBr): 3568, 1736, 1719, 1655,
1240, 1055 cmꢁ1. 1H-NMR (600 MHz, pyridine-d5) d: given in Table 1. 13C-
NMR data (150 MHz, pyridine-d5) dC: given in Table 2. Positive-ion FAB-
MS m/z: 989 (MꢀNa)ꢀ. Negative-ion FAB-MS m/z: 965 (MꢁH)ꢁ, 749
(MꢁC9H13O6)ꢁ, 603 (MꢁC15H23O10)ꢁ, 471 (MꢁC20H31O14)ꢁ.
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Deacylation of Rarasaponins IV (1), V (2), and VI (3) A solution of
rarasaponins IV (1, 3.1 mg), V (2, 8.0 mg), and VI (6, 3.0 mg) in 0.5%
sodium methoxide (NaOMe)–MeOH (1.0 ml) was stirred at room tempera-
ture for 3 h, respectively. The reaction mixture was neutralized with Dowex
HCR-W2 (Hꢀ form) and the resin was removed by filtration. Evaporation of
the solvent from the filtrate under reduced pressure gave a residue, which
was purified by HPLC [Cosmosil HILIC, CH3CN–H2O (85 : 15, v/v)] to fur-
nish 4 (from 1, 1.9 mg, 71.0%), 6 (from 2, 5.3 mg, 73.3%), and sapindoside
B (7, 2.1 mg from 3, 75.8%).
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Bioassay Method. Animals Male ddY mice weighing about 25—30 g
were purchased from Kiwa Laboratory Animal Co., Ltd., Wakayama, Japan. 10) Morikawa T., Ando S., Matsuda H., Kataoka S., Muraoka O.,
The animals were housed at a constant temperature of 23ꢃ2 °C and were fed Yoshikawa M., Chem. Pharm. Bull., 53, 625—630 (2005).
a standard laboratory chow (MF, Oriental Yeast Co., Ltd., Tokyo, Japan). 11) Morikawa T., Matsuda H., Yamaguchi I., Pongpiriyadacha Y.,
The animals were fasted for 20—24 h prior to the beginning of the experi- Yoshikawa M., Planta Med., 70, 152—159 (2004).
ment, but were allowed free access to tap water. All of the experiments were 12) Matsuda H., Ninomiya K., Morikawa T., Yasuda D., Yamaguchi I.,
performed with conscious mice unless otherwise noted. The experimental Yoshikawa M., Bioorg. Med. Chem. Lett., 18, 2038—2042 (2008).
protocol was approved by the Experimental Animal Research Committee at 13) Matsuda H., Tewtrakul S., Morikawa T., Nakamura A., Yoshikawa M.,