New diarylheptanoids from Alnus japonica and their antioxidative activity

Article abstract of DOI:10.1248/cpb.53.1519

In the course of research on the bioactive constituents of woody plants in the Cyugoku area of Japan, a methanol extract of the leaves of Alnus japonica were found to have strong antioxidative activity. Ethyl acetate soluble and n-buthanol soluble fractio

Full text of DOI:10.1248/cpb.53.1519

December 2005
Chem. Pharm. Bull. 53(12) 1519—1523 (2005)
1519
New Diarylheptanoids from Alnus japonica and Their Antioxidative
Activity
Masanori KUROYANAGI, ,a Mari SHIMOMAE,a Yasuo NAGASHIMA,a Norio MUTO,a Takuro OKUDA,a
*
d
Nobuo KAWAHARA,b Takahisa NAKANE,c and Toshikazu SANO
a School of Bioresources, Hiroshima Prefectural University; 562 Nanatsuka, Shobara 727–0023, Japan: b National Institute
of Health Sciences; 1–18–1 Kamiyoga, Setagaya-ku, Tokyo 158–8501, Japan: c Shouwa Pharmaceutical University;
3–3165 Higashi-Tamagawagakuen, Machida, Tokyo 194–8543, Japan: and d Hiroshima Prefectural Forestry Research
Center; 168–1 Tokaichi, Miyoshi, Hiroshima 728–0015, Japan. Received April 18, 2005; accepted September 27, 2005
In the course of research on the bioactive constituents of woody plants in the Cyugoku area of Japan, a
methanol extract of the leaves of Alnus japonica were found to have strong antioxidative activity. Ethyl acetate
soluble and n-buthanol soluble fractions of the methanol extract had a potent antioxidative effect. Both fractions
were purified by silica gel column chromatography and HPLC using an ODS column to give four new diarylhep-
tanoids along with known diarylheptanoids and flavonoids. These new compounds were elucidated to be
7-(3,4-dihydroxyphenyl)-5-hydroxy-1-(4-hydroxyphenyl)-3-heptanone-5-O-b-D-xylopyranoside (1), 1-(3,4-dihy-
droxyphenyl)-5-hydroxy-7-(4-hydroxyphenyl)-3-heptanone-5-O-b-D-xylopyranoside (2), 1,7-bis-(3,4-dihydroxy-
phenyl)-5-hydroxy-3-heptanone-5-O-[2-(2-methylbutenoyl)]-b-D-xylopyranoside (3) and 1,7-bis-(3,4-dihydroxy-
phenyl)-5-methoxy-3-heptanone (4) using spectral methods and especially 1H-, 13C-NMR and 2D-NMR measure-
ments. The isolated compounds including their main constituent, oregonin (5), were tested for antioxidative ac-
tivity. Some of these compounds having two catechol structures showed potent antioxidative activity. Compounds
having one catechol structure showed moderate antioxidative activity, but a peracetate of 5 having no catechol
structure exhibited no antioxidative activity. Thus the catechol structure of the diarylheptanoids is indispensable
for antioxidative activity.
Key words Alnus japonica; Betulaceae; diarylheptanoid; antioxidant; oregonin
Phytochemical, constitutional and biological studies on (EtOAc) soluble and n-buthanol (n-BuOH) soluble fractions
plant constituents are less popular in woody plants than of the methanol extract were then fractionated by silica gel
herbal plants. But woody plants have a similar potential for column chromatography and HPLC using a reversed phase
bioresources as herbal plants, having already yield useful column according to the biological activity to give five
bioactive compounds such as taxol.1) In a bioassay-guided known diarylheptanoids and four new diarylheptanoids along
study of woody plants in Japan using biological tests for an- with three known flavone derivatives. The structures of these
tioxidative activity, anti-tumor promoting activity, nerve cell compounds were elucidated using spectral methods including
elongation activity and cell differentiation activity, we iso- 1H-, 13C-NMR and 2D-NMR. The antioxidative activity of
lated antibacterial compounds from Abies sachalinensis2) and these compounds is discussed.
Chaenocyparis pisifera,3) and anti-tumor promoting com-
pounds from Chaenomeles sinensis.4) Alnus japonica pro- Results and Discussion
vided one of the most potent antioxidative samples. A variety
Compounds 5—11 isolated from the AcOEt soluble frac-
of pathologies including cancer and cardiovascular disease tion were identified as oregonin (5),19) hirsutanonol-5-O-b-D-
have been linked to the generation of reactive oxygen glucopyranoside (6),19) hirsutanonol (7),19) platyphyllonol-5-
species.5) Antioxidants may prevent oxidative damage by O-b-D-xylopyranoside (8),20) afzelin (9), quercitrin (10) and
scavenging reactive oxygen species. Phenolic compounds are isohyperoside (11), based on comparisons of the NMR data
recognized to have antioxidative properties. Various phenolic of the compounds with reported data. The optical rotation
compounds including flavonoids, lignoids, stylbenoids, ([a]D ꢀ16.9°) of the key compound (5) was the same as that
tannnins and diarylheptanoids are distributed in the plant reported optical rotation ([a]D ꢀ18.6°).7) This indicated that
kingdom. So the phenolic constituents of plants are of imter- 5 was identical in structure including stereochemistry to the
est as potential chemopreventive agents. A. japonica (Betul- authentic oregonin. Oregonin (5) was also isolated from the
aceae, Japanese name; Hannoki) is a common tree in low n-BuOH soluble fraction of the plant.
mountainous areas of Japan. Compounds isolated from Alnus
Compound 1 was found to have the molecular formula
plants include numerous diarylheptanoids along with triter- C24H30O9 by high resolution fast atom bombardment mass
penoids.6—10) Many kinds of diarylheptanoids have also been spectrometry (HR-FAB-MS), m/z 463.1965 [MꢁH]ꢁ. The
isolated from Zingiberaceae11—13) and Betulaceae plants, in- 1H-NMR spectrum showed the presence of a 4-oxyphenyl
cluding some which showed inhibitiory activity toward cy- group [d 6.67 (2H, d, Jꢂ8.5 Hz), 6.99 (2H, d, Jꢂ8.5 Hz)], a
clooxygenase-2,14) and antiemetic activity.15) Curcumin, one 3,4-dioxyphenyl group [d 6.47 (1H, dd, Jꢂ7.5, 2.0 Hz), 6.60
of the best known diarylheptanoids, has various biological (1H, d, Jꢂ2.0 Hz), 6.64 (1H, d, Jꢂ7.5 Hz)], the same C7-
properties including anticancer,16) antiinflammatory17) and moiety [d 2.74 (4H, br s), 2.79 (1H, dd, Jꢂ16.0, 6.5 Hz),
antioxidative activities.18) In the present study, leaves of A. 2.57 (1H, dd, Jꢂ16.0, 5.0 Hz), 4.10 (1H, br qui, Jꢂ5.0 Hz),
japonica were treated with methanol to obtain an extract 1.72 (1H, m), 1.77 (1H, m), 2.50 (2H, m)] as that of oregonin
which showed strong antioxidative activity. Ethyl acetate (5), and a xylopyranosyl moiety [d 4.21 (1H, d, Jꢂ8.0 Hz),
∗ To whom correspondence should be addressed. e-mail: kuroyang@pu-hiroshima.ac.jp
© 2005 Pharmaceutical Society of Japan

1520
Vol. 53, No. 12
Fig. 1. Selected HMBC Correlations of 1, 2, 3
3.12 (1H, dd, Jꢂ9.0, 7.5 Hz), 3.29 (1H, t, Jꢂ9.0 Hz), 3.41
(1H, ddd, Jꢂ10.5, 8.5, 5.5 Hz), 3.86 (1H, dd, Jꢂ11.5,
5.5 Hz), 3.16 (1H, dd, Jꢂ11.5, 10.0 Hz)]. The 13C-NMR data and C-7; H-6 showed a correlation with C-7, C-5 and C-1ꢅ;
also showed the presence of 4-oxyphenyl (d 156.6, 134.3, the anomeric H showed a correlation with C-5; and H-2ꢅ and
130.3ꢃ2, 116.2ꢃ2) and 3,4-dioxyphenyl (d 146.1, 144.2, H-6ꢅ showed a correlation with C-7 and C-4ꢅ. The [a]D and
134.0, 120.7, 116.6, 116.3) moieties, a carbonyl group (d 13C-NMR values around xylopyranosyl and C-5 were identi-
211.8), a secondary carbinyl group (d 76.2), five methylene cal with those of 5. Thus 2 was determined to be (5S)-1-(3,4-
groups (d 48.7, 46.5, 38.6, 31.8, 29.8) and a xylopyranosyl dihydroxyphenyl)-5-hydroxy-7-(4-hydroxyphenyl)-3-hep-
moiety (d 104.3, 77.9, 75.1, 71.3, 67.9) the same as 5. These tanone-5-O-b-D-xylopyranoside. Compound 2 was named al-
results and the FAB-MS data indicated that 1 had the struc- nuside B.
ture of a diarylheptanoid with a 4-hydroxyphenyl, a 3,4-dihy-
1 and 2 were isolated from A. serrulatoides as a mixture
droxyphenyl and the same C7-moiety as oregonin (5). The and then purified as trimethyl ethers. No spectral data of 1
positions of the two phenyl groups were determined in a het- and 2 were obtained. Structures of the trimethyl ethers were
eronuclear multiple bond connectivity (HMBC) experiment determined from the 1D-NMR data and MS fragmentation
(Fig. 1) in which H-1 showed a correlation with C-3, C-2ꢄ without 2D-NMR data.21) Thus the structure of neither 1 nor
and C-6ꢄ; H-2ꢄ and H-6ꢄ showed a correlation with C-1 and 2 was clearly elucidated. We determined the structures of the
C-4ꢄ; H-7 showed a correlation with C-5, C-2ꢅ and C-6ꢅ; compounds separately based on spectral data including 2D-
anomeric H showed a correlation with C-5; H-5 showed a NMR measurements.
correlation with the anomeric carbon and C-3; and H-6ꢅ
Compound 3 was found to have the molecular formula
showed a correlation with C-7, C-2ꢅ and C-4ꢅ. The absolute C29H38O11 by HR-FAB-MS, m/z 563.2490 [MꢁH]ꢁ. The 1H-
configuration at C-5 was taken to be the same with that of 5 NMR spectrum showed the presence of two 3,4-dihydroxy-
from a optical rotation ([a]D ꢀ19.5°), which was identical phenyl groups, the same C7-moiety as that of 5, and a xy-
with the reported data ([a]D ꢀ18.6°)7) of 5, and the 13C- lopyranoside to which an acyl group was linked. The acyl
chemical shifts of the xylopyranoside and carbons around C- part attached to the xylopyranosyl moiety was elucidated
5 were almost the same as those of 5 as shown in Table 1. from the H-, 13C-NMR and HMBC experiments. The H-
Thus the structure of 1 was determined to be (5S)-7-(3,4-di- NMR spectrum of 3 showed an extra ethyl [d 0.93 (3H, t,
hydroxyphenyl)-5-hydroxy-1-(4-hydroxyphenyl)-3-hep- Jꢂ7.5 Hz), 1.48 (1H, m), 1.66 (1H, m)], a doublet methyl
tanone-5-O-b-D-xylopyranoside. Compound 1 was named al- group [d 1.11 (3H, d, Jꢂ7.0 Hz)] and a methine group [d
1
1
nuside A.
2.36 (1H, six, Jꢂ7.0 Hz)]. The 13C-NMR spectrum showed
Compound 2 was found to have the molecular formula the presence of two methyl groups (d 11.8, 16.9), a methyl-
1
C24H30O9 by HR-FAB-MS, m/z 463.1978 [MꢁH]ꢁ. The H- ene group (d 27.8), a methine group (d 42.2) and an ester
NMR spectrum had almost the same signal pattern as that of carbonyl group (d 177.4). The data indicated that the acyl
1. The 13C-NMR spectrum also showed almost the same sig- group should be a 2-methylbutanoyl (MeBu) group. The acyl
nal pattern as that of 1. These findings indicated that 2 was structure and position of the acyl group were confirmed by
an isomer of 1 in terms of the position of the two phenyl performing HMBC experiments, in which the anomeric H of
groups, a 4-hydroxyphenyl and a 3,4-dihydroxyphenyl group. xylopyranosyl moiety showed a correlation with C-5, xyl-2,
Positions of the phenyl groups were determined in a HMBC xyl-3 and xyl-5 carbon; the xyl-2 proton showed the correla-
experiment as shown in Fig. 1. H-1 showed a correlation with tion with the acyl carbonyl carbon; the triplet methyl group
C-2ꢄ, C-6ꢄ and C-3; H-2ꢄ showed a correlation with C-3ꢄ, C- showed a correlation with at C-2 and C-3 of 2-methylbu-
4ꢄ and C-1; H-6ꢄ showed a correlation with C-2ꢄ, C-4ꢄ and tanoyl moiety; the multiplet methin carbon of MeBu moiety
C-1; H-5 showed a correlation with the anomeric carbon, C-3 showed a correlation with C-1, C-3, C-4 and C-5 of MeBu

December 2005
1521
Table 1. 13C-NMR Data for 1—5 Isolated from A. japonica (125 MHz, in
Table 2. Radical Scavenging Activities of Compounds Isolated from
Alnus japonica
CD3OD)a)
Superoxide radical
scavenging activity (%)
Carbon
1
2
3
4
5
DPPH radical scavenging
activity (%) at 6.25 mg/ml
Compound
at 3.125 mg/ml
C-1
2
3
4
5
6
7
1ꢄ
2ꢄ
3ꢄ
4ꢄ
5ꢄ
6ꢄ
1ꢅ
2ꢅ
3ꢅ
4ꢅ
29.8
46.5
211.8
48.7
76.2
38.6
30.1
46.5
211.8
48.8
76.2
38.7
30.7
46.1
210.7
48.8
75.5
38.5
30.5
46.2
211.5
48.2
77.8
36.9
30.1
46.5
211.8
48.8
76.3
38.6
1
2
3
5
6
7
8
9
10
14.0
14.5
19.2
33.0
3.4
36.9
1.4
5.5
29.0
31.1
31.5
41.9
3.9
40.5
2.0
9.4
31.8
31.5
31.6
31.5
31.7
134.3
130.3
116.2
156.6
116.2
130.3
134.0
116.3
146.1
144.2
116.6
120.7
104.3
75.1
134.0
116.5
146.2
144.5
116.4
120.6
134.3
130.3
116.1
156.3
116.1
130.3
104.3
75.1
133.8
116.5
146.2
144.5
116.2
120.5
135.1
116.6
146.0
144.1
116.4
120.6
101.8
74.8
134.7
116.4
146.1
144.4b)
116.3
120.5
133.9
116.4
146.1
144.2b)
116.3
120.5
134.0
116.5
146.1
144.5
116.2
120.5
135.1
116.6
146.0
144.1
116.3
120.6
104.3
75.1
15.6
16.1
11.6
50.0
Curcumina)
Each value represents the mean of triplicate assays. a) Use as a positive control.
Table 3. Radical Scavenging Activities of Oregonin and Its Derivatives
5ꢅ
6ꢅ
Xyl-1
IC50 (mg/ml)
Compound
Superoxide radical
scavenging activity
DPPH radical
scavenging activity
2
3
77.9
77.9
76.2
77.9
4
5
71.3
67.0
71.3
67.0
71.5
66.8
177.4
42.2
27.8
71.2
66.9
4
5
7
12
13
14
15a
15b
2.8
2.2
3.0
1.2
2.9
31.0
2.8
4.2
2.9
4.6
3.1
2.4
MeBu-1
2
3
4
4.3
11.8
16.9
n.d. (at 10 mg/ml)
5
4.1
7.3
OMe
57.0
a) Assignments were made by HMQC and HMBC spectra. b) Signals in same
column may be interchanged. Xyl is b-D-xylopyranosyl group. MeBu is 2-methylbu-
tanoyl group.
n.d.; activity was not detectable at the concentration indicated.
hydrohirsutanonol (12). Oregonin (5) gave 12 and 5-O-ethyl-
hirsutanonol (13) when treated with acidic ethanol. Acetyla-
tion of 5 gave oregonin peracetate (14). Oregonin (5) gave a
mixture of epimer alcohols (15a, 15b) at C-3 following its
reduction with NaBH4.
moiety; the methylene protons showed a correlation with C-
1, C-2, C-4 and C-5 of MeBu moiety; and the doublet methyl
protons showed a correlation with C-1, C-2 and C-3 of MeBu
moiety. The 13C-NMR spectrum of carbons around C-5 ex-
hibited almost the same chemical shifts as those of 5 (shown
in Table 1). Thus 3 was elucidated to be (5S)-1,7-bis-(3,4-di-
hydroxyphenyl)-5-hydroxy-3-heptanone-5-O-[2-(2-methyl-
butenoyl)]-b-D-xylopyranoside, but the absolute configura-
tion at C-2 of MeBu moiety could not be determined. Com-
pound 3 was named alnuside C.
The constituents and some of these derivatives were tested
for anti-oxidative activity in a superoxide radical scavenging
test21) and a DPPH radical scavenging test22) (see Tables 2,
3). Compounds 4, 5, 6, 7, 8, 12, 13 and 15 having two 3,4-di-
hydroxyphenyl moieties showed potent activity. Compounds
1 and 2 having a 3,4-dihydroxyphenyl and a 4-hydroxy-
phenyl moieties showed moderate activity, but 8 having two
4-hydroxyphenyl moieties and 14 having no free catechol
structure showed no activity. Hirusutanonol glucoside (6)
showed weak activity. These results indicated that the cate-
chol structure was important for the potent anti-oxidative ef-
fect of biarrylheptanoids from A. japonica and the type of
sugar moiety at C-5 was important for the activity.
Compound 4 was found to have the molecular formula
1
C20H24O6 by FAB-MS, m/z 361 [MꢁH]ꢁ. The H-NMR
spectrum showed the presence of two 3,4-dihydroxyphenyl
groups, the same C7-moiety as that of hirsutanonol, and an
1
alcoholic methoxyl group [d 3.25 (3H, s) in the H-NMR
spectrum and d 57.0 in the 13C-NMR spectrum]. The data in-
dicated 4 to be 5-O-methylhirustanonol. It was identified as a
product derived from the methanolysis of 5 in an acidic
methanol solution. The optical rotation of 4 ([a]D ꢁ3.8°)
was identical with that the product from 5 ([a]D ꢁ3.6°).
These results suggested that 4 might be an artifact derived
from 5 by extraction of A. japonica with methanol under re-
Experimental
1H- and 13C-NMR data were measured on a JEOL a-500 NMR spectrom-
eter (500 MHz for 1H-NMR and 125 MHz for 13C-NMR). Chemical shifts
are shown as a d-value (ppm) with tetramethyl silane (TMS) as an internal
standard, including NOE, HMQC and HMBC. HR-FAB-MS data were mea-
flux. 4 was obtained as an optically active compound, so the sured on a JEOL HX110 mass spectrometer. [a]D was recorded on a JASCO
P-1010 polarimeter at 20 °C. Analytical and preparative HPLCs were carried
out using a reverse phase column (mightysil RP-18, Kanto Chemical Co.
Ltd.) with a CH3CN–H2O solvent system. TLC was performed using pre-
coated silica gel 60F254 (Merck) 200ꢃ200ꢃ0.25 mm plates for the analysis
configuration at C-5 must be the opposite of that of 5, be-
cause 4 should be transformed from 5 through the SN2 reac-
tion process at C-5.
The main constituent, oregonin (5), was hydrolyzed by
acidic methanol to give 5-O-methylhirsutanonol (4) and de-
and 200ꢃ200ꢃ0.5 mm plates for the preparation of the constituents.
Plant Materials Alnus japonica trees were identified by Mr. T. Sano of

1522
Vol. 53, No. 12
the Hiroshima Prefectural Forestry Research Center. Leaves of A. japonica
were collected in September 2001 in the mountainous northern part of Hi-
roshima prefecture, Japan.
10.6 Hz, xyl-5), 3.28 (1H, t, Jꢂ9.2 Hz, xyl-3), 3.46 (1H, ddd, Jꢂ10.3, 8.9,
5.5 Hz, xyl-4), 3.84 (1H, dd, Jꢂ11.3, 5.5 Hz, xyl-5), 4.09 (1H, qui,
Jꢂ6.6 Hz, H-5), 6.47 (1H, dd, Jꢂ7.9, 2.0 Hz, H-6ꢅ), 6.48 (H, dd, Jꢂ7.9,
2.0 Hz, H-6ꢄ), 6.60 (1H, d, Jꢂ2.0 Hz, H-2ꢅ), 6.61 (1H, d, Jꢂ2.0 Hz, H-2ꢄ),
6.63 (1H, d, Jꢂ7.9 Hz, H-5ꢅ), 6.64 (1H, d, Jꢂ7.9 Hz, H-5ꢄ). 13C-NMR data
are shown in Table 1.
Treatment of 5 with Acidic Methanol Fifty milligrams of 5 was dis-
solved in 5% HCl–MeOH (10 ml) and stood for 1 h. The reaction solution
was poured into 100 ml of ice-water and passed through a HP 20 column,
thoroughly washed with water, and eluted with methanol to give 35 mg of
product. The crude product was purified by preparative HPLC to give two
compounds, 4 (15 mg) and 12 (5 mg).
Extraction and Isolation Powdered leaves (1.0 kg) of A. japonica were
extracted with methanol under reflux to give a methanol extract, which
showed strong antioxidative activity. The MeOH extract was poured into
water and partitioned with ethyl acetate (AcOEt) and then n-butanol (n-
BuOH), to give an AcOEt layer, a n-BuOH layer and a water layer. The
AcOEt layer and the n-BuOH layer showed antioxidative activity. The n-
BuOH layer (35 g) was chromatographed on a SiO2 column using a gradient
CHCl3–MeOH solvent system to give nine fractions, AJMB-1—AJMB-9.
Of these fractions, AJMB-2 (6.1 g), AJMB-4 (3.7 g) and AJMB-4 (1.1 g)
showed potent activity. AJMB-2 and AJMB-3 were successively purified by
silica gel column chromatography and HPLC using a ODS column and 45%
CH3CN solvent to give 5 (2.9 g) and 11 (180 mg).
The AcOEt soluble fraction (90 g) was chromatographed on a silica gel
column using a gradient CHCl3–MeOH solvent system to give seven frac-
tions. Fr. 3 (6.2 g) was purified by repeated HPLC using a 30% CH3CN sol-
vent system to give 4 (180 mg). Fr. 4 (8.4 g) was purified by HPLC using an
ODS column and preparative TLC, successively to give 3 (88 mg), 6
(10 mg), 7 (43 mg), 8 (74 mg) and 9 (7 mg). Fr. 5 (14.7 g) was purified by re-
peated HPLC using an ODS column and preparative TLC, successively to
give 1 (38 mg), 2 (35 mg), 5 (390 mg), 9 (25 mg), 10 (170 mg) and 11
(45 mg). Fr. 6 (16.2 g) was purified by HPLC using an ODS column to give 5
(1.1 g).
Alnuside A (1): Colorless viscous liquid, HR-FAB-MS: m/z 463.1965
[MꢁH]ꢁ (Calcd 463.1968 for C24H31O9. [a]D ꢀ19.5° (cꢂ0.03, MeOH). 1H-
NMR (CD3OD): d 1.72 (1H, m, H-6), 1.77 (1H, m, H-6), 2.50 (2H, m, H-7),
2.57 (1H, dd, Jꢂ17.0, 5.5 Hz, H-4), 2.74 (4H, s, H-1, H-2), 2.79 (1H, dd,
Jꢂ17.0, 7.0 Hz, H-4), 3.12 (1H, dd, Jꢂ9.0, 7.5 Hz, xyl-2), 3.16 (1H, dd,
Jꢂ11.5, 10.0 Hz, xylH-5), 3.29 (1H, t, Jꢂ9.0 Hz, xyl-3), 3.47 (1H, ddd,
Jꢂ10.5, 8.5, 5.5 Hz, xyl-4), 3.86 (1H, dd, Jꢂ11.5, 5.5 Hz, xyl-5), 4.10 (1H,
br qui, Jꢂ5.0 Hz, H-5), 4.21 (1H, d, Jꢂ8.0 Hz, xyl-1), 6.47 (1H, dd, Jꢂ7.5,
2.0 Hz, H-6ꢅ), 6.60 (1H, d, Jꢂ2.0 Hz, H-2ꢅ), 6.64 (1H, d, Jꢂ7.5 Hz, H-5ꢅ),
6.67 (2H, d, Jꢂ8.5 Hz, H-3ꢄ, 5ꢄ), 6.99 (2H, d, Jꢂ8.5 Hz, H-2ꢄ, 6ꢄ). 13C-NMR
data are shown in Table 1.
Alnuside B (2): Colorless viscous liquid. HR-FAB-MS: m/z 463.1978
[MꢁH]ꢁ (Calcd 463.1968 for C24H31O9). [a]D ꢀ15.5° (cꢂ0.04, MeOH).
1H-NMR (CD3OD): d 1.72 (1H, m, H-6), 1.77 (1H, m, H-6), 2.5 (2H, m, H-
7), 2.57 (1H, dd, Jꢂ17.0, 5.5 Hz, H-4), 2.69 (2H, br t, Jꢂ5.5 Hz, H-2), 2.73
(2H, br t, Jꢂ5.5 Hz, H-1), 2.79 (1H, dd, Jꢂ17.0, 7.0 Hz, H-4), 3.12 (1H, dd,
Jꢂ9.0, 8.0 Hz, xyl-2), 3.16 (1H, dd, Jꢂ11.5, 10.0 Hz, xyl-5), 3.29 (1H, t,
Jꢂ9.0 Hz, xyl-3), 3.47 (1H, ddd, Jꢂ10.5, 8.5, 5.5 Hz, xyl-4), 3.84 (1H, dd,
Jꢂ11.5, 5.5 Hz, xyl-5), 4.10 (1H, br qui, Jꢂ5.0 Hz, H-5), 4.21 (1H, d,
Jꢂ8.0 Hz, xyl-1), 6.47 (1H, dd, Jꢂ7.5, 2.0 Hz, H-6ꢄ), 6.60 (1H, d, Jꢂ2.0 Hz,
H-2ꢄ), 6.64 (1H, d, Jꢂ7.5 Hz, H-4ꢄ), 6.67 (2H, d, Jꢂ8.5 Hz, H-3ꢅ, 5ꢅ), 6.99
(2H, d, Jꢂ8.6 Hz, H-2ꢅ, 6ꢅ). 13C-NMR data are shown in Table 1.
12: HR-FAB-MS: m/z 329.1379 [MꢁH]ꢁ (Calcd 329.1389, C19H21O5).
1H-NMR (CD3OD): d 2.46 (2H, br q, Jꢂ7.5 Hz, H-6), 2.61 (2H, t,
Jꢂ7.5 Hz, H-7), 2.71 (2H, t, Jꢂ7.0 Hz, H-2), 2.79 (2H, t, Jꢂ7.0 Hz, H-1),
6.06 (1H, d, Jꢂ15.5 Hz, H-4), 6.48, 6.49 (each 1H, dd, Jꢂ8.0, 1.5 Hz, H-6ꢄ,
6ꢅ), 6.60 (each 2H, d, Jꢂ1.5 Hz, H-2ꢄ, 2ꢅ), 6.65, 6.66 (each 1H, d, Jꢂ8.0 Hz,
H-5ꢄ, 5ꢅ), 6.87 (1H, dt, Jꢂ15.5, 7.5 Hz, H-5). 13C-NMR (CDCl3): d 30.9 (C-
6), 34.8 (C-7), 36.3 (C-2), 45.4 (C-1), 116.3ꢃ2, 116.5ꢃ2 (C-2ꢄ, 5ꢄ, 2ꢅ, 5ꢅ),
120.5, 120.6 (C-6ꢄ, 6ꢅ), 131.5 (C-4), 133.8, 134.0 (C-1ꢄ, 1ꢅ), 144.4, 144.5
(C-3ꢄ, 3ꢅ), 146.1, 146.2 (C-4ꢄ, 4ꢅ), 149.2 (C-5), 202.8 (C-3).
Treatment of 5 with Acidic Ethanol Fifty milligrams of 5 was dis-
olved in 3% HCl–EtOH (10 ml) and stood for 1 h. The reaction solution was
poured into 100 ml of ice-water and passed through a HP 20 column, thor-
oughly washed with water, and eluted with methanol to give 35 mg of prod-
uct. The crude product was purified by preparative HPLC (ODS column,
35% CH3CN) to give 12 (8 mg) and 13 (15 mg).
13: HR-FAB-MS: m/z 375.1777 [MꢁH]ꢁ (Calcd 375.1785, C21H27O6).
1H-NMR (CDCl3): d 1.11 (3H, t, Jꢂ7.0 Hz, CH3 of ethoxyl), 1.67 (2H, m,
H-6), 2.47 (2H, overlap, H-7), 2.49 (1H, overlap, H-4), 2.64 (1H, dd,
Jꢂ16.0, 6.0 Hz, H-4) 2.69 (4H, overlap, H-1, 2), 3.33 (2H, m, CH2 of
ethoxyl), 6.60, 6.61 (1H each, d, Jꢂ1.5 Hz, H-2ꢄ, 2ꢅ), 6.47, 6.48 (1H each,
dd, Jꢂ8.0, 1.5 Hz, H-6ꢄ, 6ꢅ), 6.65, 6.66 (1H each, d, Jꢂ8.0 Hz, H-5ꢄ, 5ꢅ).
13C-NMR (CDCl3): d 15.8 (CH3 of ethoxyl), 30.1 (C-1), 31.8 (C-7), 37.5 (C-
6), 46.8 (C-2), 48.8 (C-4), 65.6 (CH2 of ethoxyl), 76.4 (C-5), 116.3, 116.4
(C-5ꢄ, 5ꢅ), 116.5ꢃ2 (C-2ꢄ, 2ꢅ), 120.6ꢃ2 (C-6ꢄ, 6ꢅ), 134.1, 134.9 (C-1ꢄ, 1ꢅ),
144.3, 144.5 (C-3ꢄ, 3ꢅ), 146.2ꢃ2 (C-4ꢄ, 4ꢅ), 211.7 (C-3).
Acetylation of 5 Fifty-four milligrams of 5 was dissolved in pyridine
(1 ml) and acetic anhydride (1 ml) and stood for 4 h at room temperature.
The reaction solution was poured into ice-water and extracted with CHCl3.
The CHCl3 solution was washed successively with dil-HCl aq. soln, sat-
NaHCO3 aq. soln and water. The CHCl3 solution was evaporated to give
90 mg of crude product. The crude product was purified by preparative TLC
to give a per acetate (14) (60 mg).
1
14: MS: m/z 773 [MꢁH]ꢁ C38H44O17, H-NMR (CDCl3): d 1.83 (2H, m,
H-7), 2.01, 2.02, 2.04 (3H each, s, Ac on xyl), 2.27, 2.28 (6H each, s, Ac on
benzene rings), 2.40 (1H, dd, Jꢂ16.5, 4.0 Hz, H-4), 2.66 (1H, overlap, H-4),
2.67 (4H, overlap, H-1, 2), 3.32 (1H, dd, Jꢂ12.0, 9.5 Hz, xyl-5), 4.08 (1H,
dd, Jꢂ12.0, 5.0 Hz, xyl-5), 4.19 (1H, m, H-5), 4.55 (1H, d, Jꢂ7.0 Hz, xyl-
1), 4.86 (1H, dd, Jꢂ9.5, 7.0 Hz, xyl-2), 4.95 (1H, dt, Jꢂ5.0, 9.5 Hz, xyl-4),
5.17 (1H, t, Jꢂ9.5 Hz, xyl-3), 6.99 (1H, Jꢂ2.0 Hz, H-2ꢄ or 2ꢅ), 7.01 (1H, d,
Jꢂ2.0 Hz, H-2ꢅ or 2ꢄ), 7.02—7.10 (4H, overlap, H-5ꢄ, 6ꢄ, 5ꢅ, 6ꢅ). 13C-NMR
(CDCl3): d 20.6ꢃ3 (Mes of acetyl groups), 20.7ꢃ4 (Mes of acetyl groups),
28.6 (C-1), 30.3 (C-17), 36.4 (C-16), 45.0 (C-2), 47.5 (C-4), 62.2 (xyl-5),
69.1, 71.3, 71.9 (xyl-2, 3, 4), 75.3 (C-5), 168.2, 168.3ꢃ2, 168.4, 168.5,
168.8, 170.0 (COs of acetyl groups), 206.9 (C-3).
Alnuside C (3): Colorless viscous liquid. HR-FAB-MS: m/z 563.2490
[MꢁH]ꢁ (Calcd 563.2493 for C29H39O11). [a]D ꢀ15.6° (cꢂ0.04, MeOH).
1H-NMR (CD3OD): d 0.93 (3H, t, Jꢂ7.5 Hz, MeBu-4), 1.11 (3H, d,
Jꢂ7.0 Hz, MeBu-5), 1.48 (1H, m, MeBu-3), 1.55 (1H, m, H-6), 1.66 (1H,
m, MeBu-3), 1.68 (1H, m, H-6), 2.36 (1H, six, Jꢂ7.0 Hz, MeBu-2), 2.47
(3H, overlap, H-2, 4), 2.5 (2H, m, H-7), 2.64 (3H, overlap, H-1, 4), 3.19
(1H, dd, Jꢂ11.5, 10.0 Hz, xyl-5), 3.40 (1H, t, Jꢂ9.5 Hz, xyl-3), 3.53 (1H,
ddd, Jꢂ10.8, 8.5, 5.5 Hz, xyl-4), 3.88 (1H, dd, Jꢂ11.5, 5.5 Hz, xyl-5), 4.07
(1H, br qui, Jꢂ5.5 Hz, H-5), 4.39 (1H, d, Jꢂ7.2 Hz, xyl-1), 4.63 (1H, dd,
Jꢂ9.5, 7.5 Hz, xyl-2), 6.46 (1H, dd, Jꢂ8.5, 2.5 Hz, H-6ꢅ), 6.48 (1H, dd,
Jꢂ7.5, 2.5 Hz, H-6ꢄ), 6.59 (1H, d, Jꢂ2.5 Hz, H-2ꢅ), 6.60 (1H, d, Jꢂ2.5 Hz,
H-2ꢄ), 6.64 (1H, d, Jꢂ8.5 Hz, H-5ꢅ), 6.66 (1H, d, Jꢂ7.5 Hz, H-5ꢄ). 13C-
NMR data are shown in Table 1.
NaBH4 Reduction of 5 Sixty milligrams of 5 was dissolved in 10 ml of
MeOH, and 20 mg of NaBH4 was added and stirred for 1 h at room tempera-
ture. The reaction solution was poured into water and passed through a HP
20 column, washed with water and eluted with MeOH to give a MeOH elu-
ate (42 mg). The MeOH eluate was purified by HPLC (ODS column, 40%
CH3CN) to give two compounds 15a (9 mg) and 15b (7 mg). 15a and 15b
should be epimers at C-3 each other, but their configurations at C-3 of these
compound have not been determined.
5-O-Methylhirusutanonol (4): Colorless viscous liquid. FAB-MS: m/z 361
[MꢁH]ꢁ C20H25O6. [a]D ꢁ3.8° (cꢂ0.07, MeOH). 1H-NMR (CD3OD): d
1.65 (1H, m, H-6), 1.72 (1H, m, H-6), 2.45 (2H, m, H-7), 2.48 (1H, dd,
Jꢂ16.2, 5.4 Hz, H-4), 2.65 (1H, dd, Jꢂ16.2, 7.2 Hz, H-4), 2.68 (4H, br s, H-
1, 2), 3.25 (3H, s, OMe), 3.62 (1H, qui, Jꢂ6.6 Hz, H-5), 6.46 (1H, dd,
Jꢂ8.4, 1.8 Hz, H-6ꢅ), 6.48 (1H, dd, Jꢂ8.4, 1.8 Hz, H-6ꢄ), 6.59 (1H,
Jꢂ1.8 Hz, H-2ꢅ), 6.60 (1H, Jꢂ1.8 Hz, H-2ꢄ), 6.64 (1H, d, Jꢂ8.4 Hz, H-5ꢄ),
6.65 (1H, d, Jꢂ8.4 Hz, H-5ꢅ). 13C-NMR data are shown in Table 1.
Oregonin (5): Pale yellow viscous liquid. FAB-MS: m/z 479 [MꢁH]ꢁ
15a: FAB-MS: m/z 503 [MꢁH]ꢁ C24H33O10. 1H-NMR (CD3OD): d
1.61—1.82 (5H, m, overlap H-2, H-4, H-6), 2.46—2.61 (4H, m, overlap, H-
1, H-7), 3.15 (1H, dd, Jꢂ9.5, 7.5 Hz, xyl-2), 3.17 (1H, dd, Jꢂ11.5, 10.0 Hz,
xyl-5), 3.29 (1H, t, Jꢂ9.5 Hz, xyl-3), 3.49 (1H, ddd, Jꢂ10.5, 9.5, 5.5 Hz,
xyl-4), 3.67 (1H, m, H-3), 3.80 (1H, quin, Jꢂ6.5 Hz, H-5), 3.86 (1H, dd,
Jꢂ11.5, 5.5 Hz, xyl-5), 4.22 (1H, d, Jꢂ7.5 Hz, xyl-1), 6.48 (1H, dd, Jꢂ8.0,
2.0 Hz, H-6ꢄ or H-6ꢅ), 6.50 (1H, dd, Jꢂ8.0, 2.0 Hz, H-6ꢅ or H-67), 6.61 (1H,
d, Jꢂ2.0 Hz, H-2ꢄ or H-2ꢅ), 6.63 (1H, d, Jꢂ2.0 Hz, H-2ꢅ or H-2ꢄ), 6.64 (1H,
d, Jꢂ8.0 Hz, H-5ꢄ or H-5ꢅ), 6.66 (1H, d, Jꢂ8.0 Hz, H-5ꢅ or H-5ꢄ). 13C-NMR
(CD3OD): d 31.7, 32.2 (C-1, C-7), 38.7 (C-6), 41.2 (C-2), 43.0 (C-4), 67.0
1
C24H31O10. [a]D ꢀ16.9° (cꢂ0.12, MeOH). H-NMR (CD3OD): d 1.71 (1H,
m, H-6), 1.76 (1H, m, H-6ꢄ), 2.46 (1H, ddd, Jꢂ13.8, 9.6, 6.2 Hz, H-7), 2.53
(1H, ddd, Jꢂ13.8, 10.0, 5.3 Hz, H-7), 2.56 (1H, dd, Jꢂ16.8, 5.2 Hz, H-4),
2.70 (2H, overlap, H-1), 2.70 (2H, overlap, H-2), 2.78 (1H, dd, Jꢂ16.8,
6.8 Hz, H-4), 3.11 (1H, dd, Jꢂ9.2, 7.9 Hz, xyl-2), 3.16 (1H, dd, Jꢂ11.3,

December 2005
1523
(xyl-5), 69.9 (C-3), 71.3 (xyl-4), 75.1 (xyl-2), 78.1 (xyl-3), 79.9 (C-5), 104.7
(C-1ꢄ), 116,3, 116.4, 116.6, 116.7 (C-2ꢄ, C-5ꢄ, C-2ꢅ, C-5ꢅ), 120.7ꢃ2 (C-6ꢄ,
C-6ꢅ), 135.2, 135.5 (C-1ꢄ, C-1ꢅ), 114.1, 114.2 (C-4ꢄ, C-4ꢅ), 146.0, 146.1 (C-
3ꢄ, C-3ꢅ).
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Superoxide Radical Scavenging Activity Superoxide radical (Oꢀ2 )
scavenging activity was determined with the hypoxanthine–xanthine oxidase
Oꢀ2 -generating system by the nitrite method modified by Ooyanagui.21) One
hundred microliters of 65 mM KH2PO4–35 mM Borax–0.5 mM EDTA buffer
(pH 8.2), 100 ml of 0.5 mM hypoxanthine, 50 ml of 10 mM hydroxylamine hy-
drochloride–1 mg/ml hydroxylamine-O-sulfonic acid solution, 100 ml of dis-
tilled water, 50 ml of sample solution containing test compounds, and 100 ml
of xanthine oxidase (14.5 mU/ml) were added successively into a 1.5 ml mi-
crotube. After the reaction mixture was incubated at 37 °C for 30 min, 1 ml
of 30 mM N-1-naphthylethylene diamine–3 mM sulfanilic acid–25% acetic
acid mixture was added. The absorbance at 550 nm was determined in a
spectrophotometer (JASCO 550). Compounds tested were dissolved in
methanol and diluted with water at appropriate concentrations. The final 11) Kikuzaki H., Usuguchi J., Nakatani N., Chem. Pharm. Bull., 39, 120—
concentration of methanol in the reaction mixture was less than 1%. Oꢀ2 -
122 (1991).
Scavenging activity was evaluated as the percentage (%) of the reduction of 12) Kuroyanagi M., Noro T., Fukushima S., Aiyama R., Ikuta A., Itokawa
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1,1-Diphenyl-2-picrylhydrazyl (DPPH) Radical Scavenging Activity 13) Ma J., Jin X., Yang L., Liu Z.-L., Phytochemistry, 65, 1137—1143
The free radical, DPPH, was used as an analytical reagent for radical scav-
engers. DPPH radical scavenging activity was determined by the method of
Ahnfelt-Bønne and Nielsen22) with a modification. A test sample (20 ml) was
mixed with 980 ml of an ethanolic solution of 100 mM DPPH. After 20 min,
the absorbance at 516 nm was measured in a spectrophotometer (JASCO
550). All test compounds were dissolved in methanol and diluted at appro-
priate concentrations. DPPH radical scavenging activity was evaluated as the
percentage (%) of the reduction of absorbance.
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Acknowledgements This research was supported by a research grant of
Hiroshima Prefecture. We thank Dr. K. Masuda and Y. Takase of Showa
Pharmaceutical University for FAB-MS and HR-FAB-MS measurements.
18) Balasubramanyam M., Koteswari A. Q. A., Kumar R. S., Monickaraj
S. F., Maheawari J. U., Mohan V., J. Biosci., 28, 15—21 (2003).
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