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(calcd. for C
22
H
29
O
4
, 357.2066).
The IR spectrum displayed intense absorption bands for
hydroxy (3363 cm
1
) and olen (1600 cm
1
) function-
alities. The
1
H-NMR and
13
C-NMR spectra showed
signals for two tertiary methyl protons at
H
1.09, a vinyl
methyl proton at
H
1.81, and their respective carbons
at
C
29.0 and 22.0. Three methylene protons were
observed at
H
1.471.49, 1.601.65, and 2.07, and their
respective carbon signals were found at
C
39.9, 19.0,
and 33.8. A set of two doublets having an
1
H
1
H
coupling constant of 16.0 Hz was observed in the
aromatic region at
H
6.48 and 7.48, a result character-
istic of an E-olen. These olenic protons were
correlated to
C
188.9 (carbonyl carbon) in the HMBC
spectrum. Three quaternary carbons were observed at
C
34.3, 136.5, and 136.7. This NMR data suggested that
compound 1 had the -ionone moiety. In addition, the
presence of equivalent methoxy substituents was con-
rmed by a singlet at
H
3.91 (six protons). A singlet and
two doublets, which were correlated to
C
188.9
(carbonyl carbon), appeared in the downeld region of
the
1
H-NMR spectrum. These belong to the E-sinapinic
moiety, the singlet at
H
6.82 represents the aromatic
protons, and the doublets at
H
6.82 and 7.56 represent
the E-olenic protons. Since the olenic protons in the
-ionone and E-sinapinic moieties were correlated to the
same carbon (
C
188.9), it is clear that this carbonyl
carbon bridged the two fragments (Table 1).
16)
The
ultraviolet absorption maximum was observed at 372
nm. These spectral studies lead us to propose that the
structure of compound 1 was (1E,4E)-1-(4-hydroxy-3,5-
dimethoxyphenyl)-5-(2,6,6-trimethylcyclohex-1-enyl)-
penta-1,4-dien-3-one, nostocionone (Fig. 3).
The antioxidative eects of the isolated compounds
were evaluated by using the -carotene oxidation
system. Nostocionone 1 and reduced scytonemine
exhibited strong antioxidative eects among the iso-
lates. Nostocionone 1 at 1.00 mM showed a dintinct
antioxidative eect with a carotene survival rate of 69%;
in contrast, BHA gave a carotene survival rate of 70%.
Compound 1 at 0.5 mM gave a survival rate of 54%, this
eect being stronger than that of BHA (41%). Nosto-
cionone 1 was therefore conrmed to be a good
antioxidant (Table 2).
In conclusion, we performed a chemical study on
N. commune Vauch. and isolated two -ionone deriva-
tives, nostocionone 1 and 3-oxo--ionone, together with
four indole alkaloids, scytomenin, reduced scytonemin,
N-(p-coumaroyl)tryptamine, and N-acetyltryptamine.
Among these compounds, nostocionone 1, which is
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60
C
a
r
o
t
e
n
e
s
u
r
v
i
v
a
l
r
a
t
e
(
%
)
Incubation time (min)
Fig. 1. Antioxidative Activities of Several Algal Extracts.
Ethanol extract solutions (1 mg/mL) of Nostoc commune Vauch.
( ), Spirulina platensis ( ), Chlorella pyrenoidosa ( ), Dunaliella
salina (), 10 mg/mL of BHA ( ) as a positive control, and EtOH
alone ( ) were each added to the prepared -carotene solution. UV
absorption (470 nm) was measured 10, 20, 30, 40, 50, and 60 min
after adding the sample.
N
HO
O
N
O
OH
O
OMe
OH
OMe
N
H
N
H
O
OH
N
H
N
H
O
O
O
HN
HO
O
NH
O
OH
Scytonemin Reduced scytonemin
Nostocionone 1
N-Acetyltryptamine N-(p-Coumaroyl)-tryptamine 3-Oxo--ionone
1
2
3
4
5 6
7
8
9
10
11
12
13
14
15
16
17
18 19
20
Fig. 2. Chemical Structures of the Isolated Compounds.
Table 1. NMR Data for Nostocionone 1 (400 MHz, CDCl
3
)
Position
Nostocionone 1
H
mult (J, Hz)
C
1 136.7
2 136.5
3 2.07, t (6.4) 33.8
4 1.601.65, m 19.0
5 1.471.49, m 39.9
6 34.3
7 7.48, d (16.0) 142.9
8 6.48, d (16.0) 129.3
9 188.9
10 6.82, d (16.0) 124.3
11 7.56, d (16.0) 143.2
12 126.5
13 and 17 6.82, s 105.4
14 and 16 147.4
15 137.4
18 and 19 1.09, s 29.0
20 1.81, s 22.0
14- and 16-OMe 3.91, s 56.5
15-OH 5.87, br s
2176 M. NINOMIYA et al.
newly reported, was found to be an eective antioxidant.
Compounds with antioxidative activity, especially those
which are naturally present in food, are of great interest
to consumers because of their possible benecial eects
on human health. Our ndings provide evidence for the
antioxidative activity of N. commune Vauch. and iden-
tify the potential compounds responsible for these
eects.
Experimental
General experimental procedures. All solvents were purchased
from the suppliers and used without further purication. IR spectra
were recorded with a Jasco FT/IR-460 Plus spectrophotometer.
1
H-
(400 MHz) and
13
C-(100 MHz)NMR spectra were recorded with a Jeol
ECX 400 spectrometer, using tetramethylsilane as an internal standard.
MS data were obtained with a Jeol JMS-700/GI spectrometer, and the
UV spectrum was measured with a Hitachi U-4500 spectrophotometer.
Silica gel column chromatography (CC) was performed on silica gel
N-60 (4050 mm), and thin-layer chromatography (TLC) spots on
plates pre-coated with silica gel 60 F
254
were detected with a UV lamp
(254 nm). Silica gel and TLC plates were respectively purchased from
Kanto Chemical Co. and Merck. Fractionation for all CC operations
was based on TLC analyses.
Extraction and isolation from N. commune Vauch. The terrestrial
blue-green alga, N. commune Vauch., was harvested in Alxa (Inner
Mongolia, China) in the summer of 2001. After washing with water, the
alga was dried in the sun, sterilized at 130
,
HRFABMS: m=z 357.2091 MH
(calcd. for C
22
H
29
O
4
,
357.2066). UV (MeOH)
max
(log "): 372 (4.3) nm. See Table 1 for
the
1
H- and
13
C-NMR data.
Antioxidative activity. The evaluation procedures for antioxidative
activity were performed as previously described.
17)
-Carotene
(1.0 mg/mL) in a chloroform solution (3.0 mL), linoleic acid
(0.1 mg/mL) in a chloroform solution (0.4 mL), and polyoxyethylene
(20) sorbitan monopalmitate (0.2 mg/mL) in a chloroform solution
(2.0 mL) were mixed in a ask and the resulting solution was dried in
an N
2
atmosphere. Distilled water (200 mL) was subsequently added to
the ask. This -carotene solution (18.6 mL), a pH 7.2 phosphate
buer (1.6 mL), and the sample (1.00 mM or 0.50 mM in ethanol,
0.4 mL) were mixed and warmed at 55