A new diterpenoid glucoside and two new diterpenoids from the fruit of Vitex agnus-castus

Article abstract of DOI:10.1248/cpb.59.392

A new labdane-type diterpenoid glucoside and two new labdane-type diterpenoids were isolated from the fruit (chasteberry) of Vitex agnus-castus L. (Verbenaceae) along with 14 known compounds comprising seven labdane-type diterpenoids, one halimane-type diterpenoid, two oleanane-type triterpenoids, two ursane-type triterpenoids, one aromadendrane-type sesquiterpenoid, and one flavonoid. Their structures were characterized on the basis of spectroscopic data as well as chemical evidence. Furthermore, the antioxidative activities of the flavonoid were evaluated using five different analyses.

Full text of DOI:10.1248/cpb.59.392

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
392
Note
Chem. Pharm. Bull. 59(3) 392—396 (2011)
Vol. 59, No. 3
A New Diterpenoid Glucoside and Two New Diterpenoids from the Fruit
of Vitex agnus-castus
Masateru ONO, ,a Keisuke EGUCHI,a Masatarou KONOSHITA,a Chisato FURUSAWA,a Junich SAKAMOTO,a
*
Shin YASUDA,a Tsuyoshi IKEDA,b Masafumi OKAWA,c Junei KINJO,c Hitoshi YOSHIMITSU,d and
d
Toshihiro NOHARA
a School of Agriculture, Tokai University; 5435 Minamiaso, Aso, Kumamoto 869–1404, Japan: b Faculty of Medical and
Pharmaceutical Sciences, Kumamoto University; 5–1 Oe-honmachi, Kumamoto 862–0973, Japan: c Faculty of
Pharmaceutical Sciences, Fukuoka University; 8–19–1 Nanakuma, Jonan-ku, Fukuoka 814–0180, Japan: and d Faculty of
Pharmaceutical Sciences, Sojo University; 4–22–2 Ikeda, Kumamoto 860–0082, Japan.
Received November 3, 2010; accepted December 18, 2010; published online December 22, 2010
A new labdane-type diterpenoid glucoside and two new labdane-type diterpenoids were isolated from the
fruit (chasteberry) of Vitex agnus-castus L. (Verbenaceae) along with 14 known compounds comprising seven
labdane-type diterpenoids, one halimane-type diterpenoid, two oleanane-type triterpenoids, two ursane-type
triterpenoids, one aromadendrane-type sesquiterpenoid, and one flavonoid. Their structures were characterized
on the basis of spectroscopic data as well as chemical evidence. Furthermore, the antioxidative activities of the
flavonoid were evaluated using five different analyses.
Key words Vitex agnus-castus; Verbenaceae; chasteberry; diterpenoid; glucoside; antioxidative activity
Vitex agnus-castus L. (Verbenaceae) is widely distributed 2a-hydroxyursolic acid (13),16) 3-epimaslinic acid (14),17)
in Central Asia, the Mediterranean region, and Southern Eu- maslinic acid (15),16) and 4a,10a-dihydroxyaromadendrane
rope. The fruit (chasteberry) of this plant is used as a dietary (16),18) and casticin (17),19) respectively, based on compari-
supplement for treating the hormone-imbalance syndrome in son of their physical and spectral data with authentic samples
women.1) This fruit has been reported to contain essential or those already reported (Fig. 1).
oils, iridoids, flavonoids, and diterpenoids.1—9) Further,
Compound 1, tentatively named viteagnuside A, was ob-
linoleic acid isolated from this fruit was identified as an es- tained as an amorphous powder, and gave an [MꢀNa]ꢀ ion
trogenic compound.10) In the course of our study on the con- peak at m/z 521 in the positive FAB-MS. The molecular for-
stituents of Verbenaceae plants, we previously reported the mula of 1 was determined to be C26H42O9 by high-resolution
isolation and structural elucidation of 14 diterpenoids, one (HR)-positive FAB-MS. The 1H-NMR spectrum of 1 showed
each of norlabdane-type diterpenoid, aromadendrane-type
sesquiterpenoid, and flavonoid from the fruit of V. agnus-cas-
tus.11,12) As part of our continuing study on the constituents
of this fruit, the present paper deals with the isolation and
structural elucidation of a new labdane-type diterpenoid glu-
coside and two new labdane-type diterpenoids along with 14
known compounds comprising seven labdane-type diter-
penoids, one halimane-type diterpenoid, two oleanane-type
triterpenoids, two ursane-type triterpenoids, one aromaden-
drane-type sesquiterpenoid, and one flavonoid.
The fruit of V. agnus-castus was successively percolated
with hexane, acetone, and methanol (MeOH) to give hexane
extract, acetone extract, and MeOH extract. The acetone ex-
tract was subjected to silica gel, Chromatorex octadecyl sil-
ica (ODS), and Sephadex LH-20 column chromatography as
well as HPLC on ODS to yield 16 compounds (2—17). The
successive chromatography of the MeOH extract over Diaion
HP20, Sephadex LH-20, Chromatrex ODS, and silica gel
column yielded 1.
Compounds 4—17 were identified as (rel 5S,6R,8R,9R,
10S,13S,16S)-6-acetoxy-9,13-epoxy-16-methoxy-labdan-
15,16-olide (4),13) (rel 5S,6R,8R,9R,10S,13R,16S)-6-acetoxy-
9,13-epoxy-16-methoxy-labdan-15,16-olide (5),13) (rel 5S,
6R,8R,9R,10S,13S)-6-acetoxy-9,13-epoxy-15-methoxy-lab-
dan-16,15-olide (6),13) (rel 5S,6R,8R,9R,10S,13R)-6-acetoxy-
9,13-epoxy-15-methoxy-labdan-16,15-olide (7),13) vitexilac-
tone (8),14) viteagnusin C (9),11) 8-epi-sclareol (10),11) vitetri-
folin D (11),14) 2,3-dihydroxy-12-ursen-28-oic acid (12),15)
Fig. 1. Structures of 1—19 and 1a
∗ To whom correspondence should be addressed. e-mail: mono@agri.u-tokai.ac.jp
© 2011 Pharmaceutical Society of Japan

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
March 2011
393
Table 1. 1H-NMR Spectral Data for 1 and 1a (in C5D5N, 500 MHz)
Table 2. 13C-NMR Spectral Data for 1, 1a, 2, and 3 (125 MHz)
1
1a
1a)
1aa)
2b)
3b)
1a 2.12 ddd (3.5, 10.5, 10.5)
1b ca. 1.53
2a 2.37 dddd (3.5, 3.5, 4.0, 13.0)
ca. 1.95
ca 1.66
2.18 m
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
30.6
26.8
89.1
39.6
45.8
21.7
31.6
37.0
76.7
43.2
33.1
23.1
134.8
145.0
70.7
174.8
16.5
28.6
17.0
16.7
30.7
28.4
78.1
39.5
45.7
22.0
31.7
37.1
76.8
43.6
33.2
23.1
134.9
144.9
70.6
—
32.5
19.0
44.0
34.2
48.8
70.5
36.3
31.6
95.0
42.8
28.9
37.0
86.0
39.1
173.6
108.0
16.9
32.9
23.7
19.7
170.4
21.9
56.4
34.1
18.7
43.8
34.1
48.4
70.4
36.6
31.0
94.9
43.1
28.3
37.6
85.8
39.9
173.6
109.2
16.5
33.0
23.8
19.9
170.4
21.9
56.8
2b 1.90 dddd (3.5, 10.5, 11.5, 13.0) ca. 1.95
3
5
3.48 dd (4.0, 11.5)
2.00 d (10.0)
3.52 dd (6.0, 10.0)
2.00 dd (3.0, 12.5)
ca. 1.66
6a ca. 1.59
6b 1.38 dddd (4.0, 10.0, 12.5, 12.5) 1.45 dddd (4.0, 12.5, 12.5, 12.5)
7a 1.69 dddd (4.0, 12.5, 12.5, 12.5) 1.73 dddd (4.0, 12.5, 12.5, 12.5)
7b ca. 1.48
ca. 1.75
1.52 dddd like (2.5, 2.5, 2.5, 12.5)
ca. 1.80
2.09 m
8
11a ca. 2.00
11b ca. 1.76
12a 2.63 br t like (8.0)
12b 2.63 br t like (8.0)
14 7.22 s
15a 4.76 d (1.5)
15b 4.76 d (1.5)
17 1.11 d (6.0)
18 1.32 s
ca. 1.82
2.68 ddd (1.5, 8.5, 8.5)
2.68 ddd (1.5, 8.5, 8.5)
7.20 dd (1.5, 1.5)
4.74 d (1.5)
4.74 d (1.5)
1.13 d (6.5)
1.23 s
1.11 s
1.03 s
16.6
29.1
16.7
16.5
19 1.07 s
20 0.95 s
Glc-1 4.85 d (8.0)
1ꢃ
2ꢃ
OCH3
2
3
4
5
4.02 dd (8.0, 9.0)
4.20 dd (9.0, 9.0)
4.23 dd (9.0, 9.0)
3.91 m
Glc-1
106.9
75.7
78.7
71.7
78.1
62.9
2
3
4
5
6
6a 4.52 dd (2.5, 11.5)
6b 4.40 dd (5.0, 11.5)
d in ppm from tetramethylsilane (TMS) (coupling constants (J) in Hz are given in
parentheses). Glc, glucopyranosyl.
d in ppm from TMS. a) In C5D5N. b) In CDCl3. Glc, glucopyranosyl. —, signal
not recorded.
signals due to three tertiary methyl groups (d 1.32, 1.07,
0.95), one secondary methyl group [d 1.11 (d, Jꢁ6.0 Hz)],
one olefinic proton [d 7.22 (s)], and one anomeric proton [d
4.85 (d, Jꢁ8.0 Hz)]. The 13C-NMR spectrum of 1 gave 26
carbon signals including one each of carbonyl carbon (d
174.8), oxygenated quaternary carbon (d 76.7), oxygenated
methine carbon (d 89.1), oxygenated methylene carbon (d
70.7), and glucopyranosyl group (d 106.9, 75.7, 78.7, 71.7,
78.1, 62.9), and two olefinic carbons (d 145.0, 134.8). These
1H- and 13C-NMR spectral signals were assigned with the aid
1
of H–1H correlation spectroscopy (COSY), heteronuclear
multiple-quantum coherence (HMQC), and heteronuclear
multiple-bond correlation (HMBC) techniques as shown in
Tables 1 and 2, and the planar structure of 1, which was a
labdane-type diterpenoid glucoside possessing an a-substi-
tuted butenolide ring, could be characterized as illustrated in
Fig. 2. HMBC Correlations Observed for 1
Fig. 2. In the nuclear Overhauser and exchange spectroscopy secondary methyl group [d 1.13 (d, Jꢁ6.5 Hz)], one olefinic
(NOESY) spectrum of 1, the key nuclear Overhauser effects proton [d 7.20 (dd, Jꢁ1.5, 1.5 Hz)], one oxygenated methyl-
(NOEs) were observed between H-3 and H3-18; H-5 and H3- ene group [d 4.74 (d, Jꢁ1.5 Hz)], and one oxygenated me-
18; H-8 and H3-20; and Ha-11 and H3-20 (Fig. 3). Thus, rela- thine proton [d 3.52 (dd, Jꢁ6.0, 10.0 Hz)]. Thus, 1a was de-
tive configurations at C-3, C-5, C-8, C-9, and C-10 were con- fined to be a genuine aglycone of 1. In order to determine the
cluded to be S*, S*, R*, R*, and S*, respectively.
absolute configuration of the aglycone, the 13C-NMR spectral
On acidic hydrolysis, 1 afforded D-glucose, which was data of 1 were compared with those of 1a. The glycosylation
identified by using optical rotation chiral detection in the shifts were observed at C-2, C-3, and C-4 of the aglycone
HPLC analysis, along with several unidentified artificial moiety of 1 with magnitudes ꢂ1.6, ꢀ11.0, and ꢀ0.1 ppm,
aglycones. The glycosdic linkage was shown to be b by the respectively, indicating the absolute configuration at C-3 of
1
coupling constant of the H-NMR spectral signal due to the aglycone to be S according to Kasai et al.20) and Seo et
anomeric proton. Enzymatic hydrolysis of 1 with b-glucosi- al.21) Consequently, the structure of 1 was concluded to be
1
dase gave 1a, whose H-NMR spectrum exhibited signals (3S,5S,8R,9R,10S)-3,9-dihydroxy-13(14)-labdaen-16,15-
due to three tertiary methyl groups (d 1.23, 1.11, 1.03), one olide 3-O-b-D-glucopyranoside.

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
394
Vol. 59, No. 3
Table 3. 1H-NMR Spectral Data for 2 and 3 (in C5D5N, 500 MHz)
2
3
1a
1b
2a
2b
3a
3b
5
6
ca. 1.62
ca. 1.25
ca. 1.67
ca. 1.46
ca. 1.33
ca. 1.20
1.58 d (3.0)
5.40 ddd (3.0, 3.0, 3.0)
ca. 1.72
ca. 1.50
ca. 2.06
ca. 2.17
1.76 m
ca. 1.34
ca. 1.27
ca. 1.64
ca. 1.49
ca. 1.31
1.15 m
1.58 d (3.0)
5.39 ddd (3.0, 3.0, 3.0)
ca. 1.70
ca. 1.54
ca. 2.06
2.17 m
1.82 m
ca. 2.09
1.88 m
2.97 d (16.5)
2.41 d (16.5)
4.93 s
0.82 d (6.5)
0.95 s
7a
7b
8
11a
11b
12a
12b
14a
14b
16
17
18
19
20
2ꢃ
ca. 2.06
1.97 m
3.10 d (16.5)
2.38 d (16.5)
4.80 s
0.82 d (6.5)
0.97 s
0.99 s
1.22 s
2.04 s
0.98 s
1.23 s
2.04 s
OCH3
3.52 s
3.55 s
d in ppm from TMS (coupling constants (J) in Hz are given in parentheses).
one methoxy group (d 3.52). The 13C-NMR spectrum of 2,
which could be superimposed on that of 4, gave 23 carbon
signals, including two carbonyl carbons (d 173.6, 170.4), one
acetal carbon (d 108.0), one methoxy carbon (d 56.4), one
oxygenated methine carbon (d 70.5), and two oxygenated
1
quaternary carbons (d 95.0, 86.0). These H- and 13C-NMR
spectral signals were examined in detail, and the planar
structure of 2, a labdane-type diterpenoid possessing one
each of spiro-tetrahydrofuran ring, g-spiro-lactone ring,
methoxy group, and acetyl group, could be determined to be
the same as that of 4. The relative configurations were de-
fined on the basis of the NOESY spectrum, in which correla-
tions were observed between H-5 and H3-18; H-6 and H3-18;
H-8 and H3-20; Ha-11 and H3-20; Ha-12 and H-16; and Hb-
14 and H3-17 (Fig. 3). Accordingly, 2 was defined as (rel
5S,6R,8R,9R,10S,13S,16R)-6-acetoxy-9,13-epoxy-16-
methoxy-labdan-15,16-olide.
Compound 3, tentatively named viteagnusin J, was ob-
tained as a colorless syrup, and the molecular formula of 3
was concluded to be the same as that of 2 by using HR-posi-
tive FAB-MS. The 1H- and 13C-NMR spectra of 3 were anal-
ogous to those of 2, and the planar structure of 3 was deter-
mined to be the same as that of 2 by using 2D-NMR tech-
niques. In the NOESY spectrum of 3, key NOEs were ob-
served between H-5 and H3-18; H-6 and H3-18; H-8 and H3-
20; Ha-11 and H3-20; and H-16 and H3-17 (Fig. 3). Further-
more, the 1H- and 13C-NMR spectra of 3 were different from
Fig. 3. Key NOE Correlations Observed for 1, 2, and 3
Compound 2, tentatively named viteagnusin I, was ob- those of 5.13) Therefore, 3 was defined as the 16-epimer of 5.
tained as a colorless syrup. The positive FAB-MS of 2 indi-
Although, the absolute configurations of 2 and 3 have not
cated an [MꢀNa]ꢀ ion peak at m/z 431; the molecular for- been confirmed, they are probably the same as that of 1 from
mula of 2 was concluded to be C23H36O6 by HR-positive a biogenetic point of view. However, 2—7 might be artifacts
1
FAB-MS. The H-NMR spectrum of 2, which was closely produced from aldehydes during the isolation procedures, be-
analogous to that of 4, exhibited signals due to three tertiary cause of co-occurrence of epimers at C-16. Since Henderson
methyl groups (d 1.22, 0.99, 0.97), one secondary methyl and McCrindle reported that premarrubiin (18) was con-
group [d 0.82 (d, Jꢁ6.5 Hz)], one acetyl group (d 2.04), and verted into marrubiin (19) on distillation in vacuo, heating

Evaluation Only. Created with Aspose.PDF. Copyright 2002-2021 Aspose Pty Ltd.
March 2011
395
Table 4. Antioxidative Activity (%) of 17 on the Five Different Antioxi-
dant Analyses
(4 mg) and 12 (13 mg) from fr. 5.7 and 15 (41 mg) and 13 (13 mg) from fr.
5.8. Fr. 6 (7.57 g) was recrystallized from MeOH to give 17 (734 mg) and fr.
6.1. Fr. 6.1 (6.82 g) was chromatographed over silica gel column [Merck,
Art. 1.09385, hexane–acetone (7 : 1, 5 : 1, 3 : 1, 0 : 1), MeOH] to yield frs.
6.1.1—6.1.8. Fr. 6.1.6 (1331 mg) was subjected to Sephadex LH-20 column
(Pharmacia Fine Chemicals, Uppsala, Sweden, MeOH) to give frs. 6.1.6.1—
6.1.6.2 and 17 (776 mg). The MeOH extract was subjected to Diaion HP20
column chromatography (Mitsubishi Chemical Industries Co., Ltd., Tokyo,
Japan, H2O, MeOH, acetone) to yield MeOH eluate (25.5 g) and acetone elu-
ate (1.4 g). The MeOH eluate was chromatographed over Sephadex LH-20
column (MeOH) to yield frs.10—14. The chromatography of fr. 12
(10881 mg) over silica gel column [Merck, Art. 1.09385, CHCl3–MeOH–
H2O (14 : 2 : 0.1, 10 : 2 : 0.1, 8 : 2 : 0.2, 7 : 3 : 0.5, 6 : 4 : 1)] gave frs. 12.1—
12.15. Fr. 12.4 (1227 mg) was subjected to Chromatorex ODS column chro-
matography (50% MeOH, 60% MeOH, 70% MeOH, 80% MeOH, 90%
MeOH, MeOH) to yield frs. 12.4.1—12.4.14 and 1 (37 mg).
17
Trolox
EDTA
Oꢂ2 radical scavenging activity
NO scavenging activity
14.53ꢅ3.25 12.47ꢅ3.80
26.79ꢅ2.25 35.40ꢅ2.51
DPPH radical scavenging activity 36.60ꢅ4.48 92.99ꢅ0.65
H2O2 scavenging activity
Metal chelating activity
9.88ꢅ2.36 99.37ꢅ0.17
29.19ꢅ4.02
100.1ꢅ1.21
Data shown represent meanꢅS.D. derived from four determinations. The final con-
centration of each sample tested was 0.50 mM.
for 3 h in refluxing ethanol, or dissolution in CHCl3,22)
might be also artifact.
1
Viteagnuside A (1): Amorphous powder, [a]D15 ꢀ2.8 (cꢁ2.5, MeOH).
During the course of our studies on natural antioxi- Positive FAB-MS m/z: 521 [MꢀNa]ꢀ. HR-FAB-MS m/z: 521.2762 (Calcd
1
for C26H42O9Na: 521.2727). H-NMR spectral data: see Table 1. 13C-NMR
dants,23,24) the antioxidatve properties of 17, which was iso-
spectral data: see Table 2.
lated as a major compound in this study, was evaluated using
Viteagnusin I (2): Colorless syrup, [a]D19 ꢂ11.0 (cꢁ0.7, CHCl3). Positive
several measurements, i.e., superoxide anion (Oꢂ2 ) radical
FAB-MS m/z: 431 [MꢀNa]ꢀ. HR-FAB-MS m/z: 431.2415 (Calcd for
C23H36O6Na: 431.2410). 1H-NMR spectral data: see Table 3. 13C-NMR spec-
tral data: see Table 2.
scavenging assay, nitrogen oxide (NO) scavenging assay, 1,1-
diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay,
hydrogen peroxide (H2O2) scavenging assay, and metal
chelating assay on ferrous ion. The scavenging activities on
Oꢂ2 radical, NO, DPPH radical, and H2O2 were ca. 1.2-fold,
ca. 0.8-fold, ca. 0.4-fold, and ca. 0.1-fold of those of a stan-
dard sample, 6-hydroxy-2,5,7,8-tetramethylchroman-2-car-
boxylic acid (trolox), respectively, at a concentration of
0.50 mM and the activity of metal chelating effect was ca.
0.3-fold of that of a standard sample, ethylenediaminetete-
traacetic acid (EDTA) at a concentration of 0.50 mM (Table
4).
Viteagnusin J (3): Colorless syrup, [a]D19 ꢂ8.5 (cꢁ0.5, CHCl3). Positive
FAB-MS m/z: 431 [MꢀNa]ꢀ. HR-FAB-MS m/z: 431.2408 (Calcd for
C23H36O6Na: 431.2410). 1H-NMR spectral data: see Table 3. 13C-NMR spec-
tral data: see Table 2.
Acidic Hydrolysis of 1 Compound 1 (5 mg) was heated in 2 M HCl
(1 ml) at a temperature of 95 °C for 1 h. The reaction mixture was extracted
with ethyl acetate (1 ml). The aqueous layer was neutralized with Amberlite
MB-3 (Organo Co., Tokyo, Japan) and then evaporated under reduced pres-
sure to give a monosaccharide fr. This fr. was analyzed by HPLC under the
following conditions: column, Shodex RS-Pac DC-613, Showa Denko,
Tokyo, Japan, 150 mmꢄ6.0 mm; solvent, CH3CN–H2O (3 : 1); flow rate,
1.0 ml/min; column temperature, 70 °C; detector, JASCO OR-2090 plus,
JASCO, Tokyo, Japan; pump, JASCO PU-2080; and column oven, JASCO
CO-2060. The retention time and optical activity of the sample were identi-
cal with those [tR (min): 7.1; optical activity: positive] of D-glucose. How-
ever, the ethyl acetate extract exhibited several spots by TLC, and the agly-
cone of 1 could not be obtained.
Experimental
All instruments and materials used were the same as cited in a previous
report24) unless otherwise specified.
Plant Material The fruit of Vitex agnus-castus L. was purchased in
May 2006 from Charis Seijo Co., Ltd., a commercial supplier of herbs in
Tokyo, Japan and identified by one of authors (T. Nohara). A voucher speci-
Enzymatic Hydrolysis of 1 Compound 1 (10 mg) was dissolved in
CH3COOH–CH3COONa buffer solution (pH 5.5, 1 ml), and b-glucosidase
men has been deposited at the laboratory of Natural Products Chemistry, (from Almonds Lot. 124H40281, Sigma Chemical Co., St. Louis, U.S.A.,
School of Agriculture, Tokai University.
30 mg) was added. The mixture was left to stand at 37 °C for 16 d. After re-
moval of the solvent under reduced pressure, the residue was extracted with
MeOH, and the MeOH extract was chromatographed over silica gel [Merck
Art. 1.09385, CHCl3–MeOH–H2O (14 : 2 : 0.1, 10 : 2 : 0.1, 8 : 2 : 0.2, 7 : 3 : 0.5,
6 : 4 : 1)] to give 1a (0.2 mg) and 1 (8.5 mg).
Extraction and Isolation The powdered fruit of V. agnus-castus
(1994 g) was percolated with hexane, acetone, and MeOH at room tempera-
ture, and each solvent was removed under reduced pressure to yield hexane
extract (188.4 g), acetone extract (36.9 g), and MeOH extract (113.4 g), re-
spectively. The acetone extract was subjected to silica gel column [Merck
(Darmstadt, Germany), Art. 1.07734, hexane–acetone (20 : 1, 10 : 1, 5 : 1,
3 : 1, 1 : 1), CHCl3–MeOH–H2O (14 : 2 : 0.1, 7 : 3 : 0.5, 6 : 4 : 1, 0 : 1 : 0)] to
give fractions (frs.) 1—9. The chromatography of fraction (fr.) 3 (6.0 g) over
Chromatorex ODS column (Fuji Silysia Chemical Ltd., Aichi, Japan, 60%
MeOH, 70% MeOH, 80% MeOH, 90% MeOH, and MeOH) gave frs. 3.1—
3.11. Fr. 3.3 was chromatographed over silica gel column [Merck, Art.
1a: Amorphous powder, [a]D17 ꢀ22.3 (cꢁ0.03, MeOH). 1H-NMR spectral
data: see Table 1. 13C-NMR spectral data: see Table 2.
Assay of Scavenging Effect on Oꢀ2 Radical The Oꢂ2 radical scavenging
effect was measured by using the phenazine methosulfate (PMS) b-nicotin-
amide adenine dinucleotide (reduced form) (NADH) system according to
previously described methods.25,26) Briefly, the reaction was started by the
addition of NADH into the assay mixture containing nitroblue tetrazolium
1.09385, hexane–acetone (20 : 1, 15 : 1, 10 : 1, 5 : 1, 3 : 1, 1 : 1, 0 : 1), MeOH] (NBT) plus test sample, then allowed to proceed for 10 min at room temper-
to yield frs. 3.3.1—3.3.12. Fr. 3.3.4 (196 mg) was subjected to HPLC (col- ature. The absorbance of the resulting solution was measured at 570 nm.
umn, COSMOSIL 5C18 AR-II, Nacalai Tesque, Inc., Kyoto, Japan,
20 mmꢄ250 mm; solvent, 80% MeOH) to afford 5 (9 mg), 4 (5 mg), 6
(33 mg), 7 (32 mg), 2 (8 mg), and 3 (16 mg). The chromatography of fr. 3.3.5
(574 mg) over silica gel column [Merck, Art. 1.09385, hexane–acetone
(20 : 1, 10 : 1, 5 : 1, 3 : 1, 1 : 1, 0 : 1)] gave frs. 3.3.5.1—3.3.5.7. Frs. 3.3.5.1
(64 mg) and 3.3.5.2 (90 mg) were each subjected to HPLC (75% MeOH)
under the conditions similar to those used for fr. 3.3.4 to afford 11 (8 mg)
and 10 (3 mg) from fr. 3.3.5.1 and 9 (7 mg) and 10 (3 mg) from fr. 3.3.5.2.
The chromatography of fr. 5 (7.6 g) over Chromatorex ODS column (60%
MeOH, 70% MeOH, 80% MeOH, 90% MeOH, MeOH) produced frs. 5.1—
5.10. Fr. 5.2 (44 mg) was chromatographed over silica gel column [Merck,
Art. 1.09385, hexane–acetone (20 : 1, 15 : 1, 10 : 1, 5 : 1, 1 : 1, 0 : 1)] to give
16 (11 mg). The chromatography of fr. 5.4 (565 mg) over silica gel column
[Merck, Art. 1.09385, hexane–acetone (20 : 1, 10 : 1, 7 : 1, 5 : 1, 1 : 1)]
yielded 8 (4 mg). Frs. 5.7 (363 mg) and 5.8 (404 mg) were each subjected to
HPLC under the same conditions as those used for fr. 3.3.4 to afford 14
Trolox was used as a standard sample.
Assay of Scavenging Effect on NO The NO scavenging effect was
measured by using the Griess method based on the spontaneous NO genera-
tion from sodium nitroprruside (SNP).26,27) Briefly, the reaction was started
by the addition of SNP freshly prepared into the assay mixture containing
test sample, allowed for 150 min incubation at room temperature. Thereafter,
Griess reagent was added and the resulting solution was measured at
550 nm. Trolox was used as a standard sample.
Assay of Scavenging Effect on DPPH Radical The DPPH radical
scavenging effect was measured based on the following method.28) Briefly,
the reaction was started by the addition of DPPH into the assay mixture con-
taining test sample, then allowed to proceed for 30 min at room temperature.
The absorbance of the resulting solution was measured at 517 nm. Trolox
was used as a standard sample.
Assay of Scavenging Effect on H2O2 The H2O2 scavenging effect was
measured using the H2O2-dependent, horseradish peroxidase (HRP)-medi-