November 2004
1389
Fig. 2
11.9 Hz), Glc-6-H2], 4.55 (1H, dd, Jꢃ8.8, 9.5 Hz, 2ꢂ-H), 4.86 (1H, d,
Jꢃ7.3 Hz, Glc-1-H), 6.43 (1H, d, Jꢃ8.1 Hz, 6-H), 6.91 (1H, d, Jꢃ8.1 Hz, 5-
H). 13C-NMR (125 MHz, CD3OD) dC: given in Table 1. Positive-ion FAB-
MS: m/z 857 (2MꢀH)ꢀ, 451 (MꢀNa)ꢀ. Negative-ion FAB-MS m/z: 855
(2MꢁH)ꢁ, 427 (MꢁH)ꢁ, 265 (MꢁC6H11O5)ꢁ.
nosyl part [d 4.21 (1H, d, Jꢃ7.6 Hz, Glc-1-H)]}. By various
2D-NMR experiments, the planar structure of 3 was eluci-
dated to be the same structure of 2. Treatment of 3 with b-
glucosidase furnished its aglycon (3a),11) and the stereo-
structure of 3 was determined to be the 7,8-threo-form.
Hyuganoside IIIa (2): A white powder, [a]D25 ꢁ6.1° (cꢃ0.22, MeOH).
High-resolution positive-ion FAB-MS: Calcd for C26H34O12Na (MꢀNa)ꢀ:
561.1948. Found: 561.1953. UV (MeOH, nm, log e): 267 (4.16). IR (KBr):
Experimental
3410, 1607, 1508, 1076, 1032 cmꢁ1 1H-NMR (500 MHz, DMSO-d6) d:
.
The following instruments were used to obtain physical data: specific ro-
tations, Horiba SEPA-300 digital polarimeter (lꢃ5 cm); UV spectra, Shi-
madzu UV-1600 spectrometer; IR spectra, Shimadzu FTIR-8100 spectrome-
ter; EI-MS and high-resolution MS, JEOL JMS-GCMATE mass spectrome-
ter; FAB-MS and high-resolution MS, JEOL JMS-SX 102A mass spectrom-
eter; 1H-NMR spectra, JNM-LA500 (500 MHz) spectrometer; 13C-NMR
spectra, JNM-LA500 (125 MHz) spectrometer with tetramethylsilane as an
internal standard; and HPLC detector, Shimadzu RID-6A refractive index,
Shimadzu SPD-10Avp UV-VIS, and Shodex OR-2 optical rotation detectors.
The following experimental conditions were used for chromatography: or-
dinary-phase silica gel column chromatography, Silica gel BW-200 (Fuji
Silysia Chemical, Ltd., 150—350 mesh); reversed-phase silica gel column
chromatography, Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd.,
100—200 mesh); TLC, precoated TLC plates with Silica gel 60F254 (Merck,
0.25 mm) (ordinary phase) and Silica gel RP-18 F254S (Merck, 0.25 mm) (re-
verse phase); reverse-phase HPTLC, precoated TLC plates with Silica gel
RP-18 WF254S (Merck, 0.25 mm); and detection was achieved by spraying
with 1% Ce(SO4)2–10% aqueous H2SO4, followed by heating.
Extraction and Isolation The extraction and isolation from the fresh
roots of A. furcijuga KITAGAWA (cultivated in Miyazaki prefecture, Japan)
were described in our previous paper.1,2) The methanolic extract from the
fresh roots of A. furcijuga was subjected to Diaion HP-20 column chro-
matography to afford H2O-eluted fraction and three fractions (Fr. 1—3).
Fraction 2 was separated by ordinary- and reversed-phase column chro-
matographies to give five fractions (Fr. 2-7-1—2-7-5).1,2) Fraction 2-7-3
(554 mg) was purified by HPLC [detection: RI, column: YMC-pack R&D-
ODS-5-A, 20ꢄ250 mm i.d. (YMC Co., Ltd., Japan), mobile phase:
MeOH–H2O (30 : 70, v/v) and 2-PrOH–H2O (10 : 90, v/v)] to give hyugano-
sides II (1, 93 mg, 0.003% from the fresh roots), IIIa (2, 25 mg, 0.0008%),
and IIIb (3, 44 mg, 0.001%) together with 4-[b-D-apiofuranosyl-(1→6)-b-D-
glucopyranosyloxy]-3-methoxypropiophenone (4, 30 mg, 0.0009%), hymex-
elsin (5, 18 mg, 0.0006%), and (R)-peucedanol 7-O-b-D-glucopyranoside (6,
120 mg, 0.004%).
3.46, 3.69 (1H each, both br d, Jꢃca. 12 Hz, Glc-6-H2), 3.60 (2H, br s, 9-
H2), 3.72, 3.73 (3H each, both s, 3, 3ꢂ-OCH3), 4.18, 4.40 (1H each, both
br dd, Jꢃca. 6, 13 Hz, 9ꢂ-H2), 4.21 (1H, d, Jꢃ7.6 Hz, Glc-1-H), 4.31 (1H,
br q, Jꢃca. 5 Hz, 8-H), 4.70 (1H, br s, 7-H), 6.21 (1H, ddd, Jꢃ5.8, 6.1,
15.9 Hz, 8ꢂ-H), 6.54 (1H, br d, Jꢃca. 16 Hz, 7ꢂ-H), 6.67 (1H, d, Jꢃ7.9 Hz, 5-
H), 6.77 (1H, dd, Jꢃ1.5, 7.9 Hz, 6-H), 6.86 (1H, dd, Jꢃ1.5, 8.5 Hz, 6ꢂ-H),
6.92 (1H, d, Jꢃ8.5 Hz, 5ꢂ-H), 6.99 (1H, d, Jꢃ1.5 Hz, 2-H), 7.00 (1H, d,
Jꢃ1.5 Hz, 2ꢂ-H). 13C-NMR (125 MHz, DMSO-d6) dC: given in Table 1. Pos-
itive-ion FAB-MS: m/z 561 (MꢀNa)ꢀ Negative-ion FAB-MS m/z: 537
(MꢁH)ꢁ.
Hyuganoside IIIb (3): A white powder, [a]D23 ꢁ16.9° (cꢃ0.51, MeOH).
High-resolution positive-ion FAB-MS: Calcd for C26H34O12Na (MꢀNa)ꢀ:
561.1948. Found: 561.1961. UV (MeOH, nm, log e): 270 (3.96). IR (KBr):
3432, 1603, 1508, 1076, 1030 cmꢁ1 1H-NMR (500 MHz, DMSO-d6) d:
.
3.25, 3.58 (1H each, both m, 9-H2), [3.45 (1H, dd, Jꢃ6.3, 10.6 Hz), 3.69
(1H, br d, Jꢃca. 11 Hz), Glc-6-H2], 3.72, 3.80 (3H each, both s, 3,3ꢂ-OCH3),
4.18, 4.42 (1H each, both br dd, Jꢃca. 6, 13 Hz, 9ꢂ-H2), 4.21 (1H, d,
Jꢃ7.6 Hz, Glc-1-H), 4.28 (1H, br q, Jꢃca. 5 Hz, 8-H), 4.71 (1H, br s, 7-H),
6.24 (1H, ddd, Jꢃ5.6, 6.1, 15.9 Hz, 8ꢂ-H), 6.56 (1H, br d, Jꢃca. 16 Hz, 7ꢂ-
H), 6.68 (1H, d, Jꢃ8.1 Hz, 5-H), 6.76 (1H, dd, Jꢃ1.2, 8.1 Hz, 6-H), 6.89
(1H, br d, Jꢃca. 8 Hz, 6ꢂ-H), 6.98 (1H, d, Jꢃ8.2 Hz, 5ꢂ-H), 6.97 (1H, br s, 2-
H), 7.06 (1H, d, Jꢃ1.2 Hz, 2ꢂ-H). 13C-NMR (125 MHz, DMSO-d6) dC: given
in Table 1. Positive-ion FAB-MS: m/z 561 (MꢀNa)ꢀ. Negative-ion FAB-MS
m/z: 537 (MꢁH)ꢁ.
Acid Hydrolysis of 1—3 A solution of 1—3 (2.0 mg each) in 1.0 M HCl
(0.1 ml) were heated under reflux for 1 h. After cooling, the reaction mixture
was extracted with AcOEt (0.1 ml). The H2O layer was analyzed by HPLC
under the following conditions [detection: optical rotation, column: Ka-
seisorb LC NH2-60-5, 4.6ꢄ250 mm i.d., 5 mm (Tokyo Kasei Kogyo Co.,
Ltd., Japan), mobile-phase: CH3CN–H2O (3 : 1, v/v), flow rate: 0.8 ml/min,
injection volume: 10 ml, column temperature: room temperature]. Identifica-
tion of D-glucose present in the H2O layer was carried out by comparison of
its retention time and optical rotation with that of authentic sample. tR:
12.3 min (D-glucose, positive optical rotation).
Compounds 4—6 were identified by comparison of its physical data
([a]D, MS, 1H-, and 13C-NMR) with reported values.1,7)
Hyuganoside II (1): A white powder, [a]D25 ꢀ8.7° (cꢃ1.11, MeOH). High-
resolution positive-ion FAB-MS: Calcd for C20H28O10Na (MꢀNa)ꢀ:
451.1580. Found: 451.1576. UV (MeOH, nm, log e): 230 (sh, 3.82), 285
(3.43), 323 (3.24). IR (KBr): 3410, 1719, 1708, 1613, 1560, 1509, 1075,
Enzymatic Hydrolysis of 1 A solution of 1 (12.6 mg, 0.029 mmol) in
0.2 M acetate buffer (pH 3.8, 1.5 ml) was treated with naringinase (15.0 mg,
Sigma), and the solution was stood at 38 °C for 24 h. After treatment of the
reaction mixture with EtOH, the mixture was evaporated to dryness under
reduced pressure and the residue was purified by ordinary-phase silica gel
column chromatography [1.0 g, CHCl3–MeOH–H2O (10 : 3 : 1, v/v/v, lower
layer)] to give hyuganol II (1a, 7.8 mg, quant.).
1
812 cmꢁ1. H-NMR (500 MHz, CD3OD) d: 1.22, 1.24 (3H each, both s, 3ꢂ-
gem-CH3), [2.55 (1H, br dd, Jꢃca. 7, 16 Hz), 2.60 (1H, br dd, Jꢃca. 8,
16 Hz), 8-H2], [2.89 (1H, ddd, Jꢃ7.6, 7.9, 14.4 Hz), 2.97 (1H, ddd, Jꢃ7.0,
7.3, 14.4 Hz), 7-H2], [3.29 (1H, dd, Jꢃ8.8, 16.2 Hz), 3.35 (1H, dd, Jꢃ9.5,
16.2 Hz), 1ꢂ-H2], [3.70 (1H, dd, Jꢃ5.2, 11.9 Hz), 3.85 (1H, dd, Jꢃ2.4,
Hyuganol II (1a): A white powder, [a]D25 ꢀ47.3° (cꢃ0.41, MeOH). High-
resolution EI-MS: Calcd for C14H18O5 (Mꢀ): 266.1154. Found: 266.1156.