Antioxidative Activity and Chemical Constituents

of Edible Terrestrial Alga Nostoc commune Vauch.

Masayuki NINOMIYA,
1
Hitoshi SATOH,
1
Yuji YAMAGUCHI,
2
Hiroyuki TAKENAKA,
2
and Mamoru KOKETSU
1;y
1
Department of Materials Science and Technology, Faculty of Engineering, Gifu University,
1-1 Yanagido, Gifu 501-1193, Japan
2
MAC Gifu Institute, MicroAlgae Corporation, 4-15 Akebono, Gifu 500-8148, Japan
Received June 17, 2011; Accepted August 3, 2011; Online Publication, November 7, 2011
[doi:10.1271/bbb.110466]
The extract of terrestrial alga Nostoc commune
Vauch. has high antioxidative activity. Our study on
N. commune Vauch. resulted in the isolation of two
-ionone derivatives, nostocionone and 3-oxo--ionone,
together with four indole alkaloids, scytonemin, reduced
scytonemin, N-(p-coumaroyl)tryptamine, and N-acetyl-
tryptamine. The structures of the isolated compounds
were determined on the basis of 1D and 2D NMR and
MS analyses. Among these isolates, nostocionone and
reduced scytonemin demonstrated strong antioxidative
activities which were assessed by using a -carotene
oxidation assay.
Key words: Nostoc commune Vauch.; nostocionone;
reduced scytonemin; antioxidative activity
Nostoc commune Vauch. belongs to the heterocystous
blue-green algae capable of forming jelly clumps
consisting of polysaccharides. It is the most common
phycobiont in N
2
-xing lichens and exists as the N
2
-
xing symbiont in a small and diverse group of green
plants.
1)
Some Nostoc species have been traditionally
used in China as health foods or folk medicines for
treating illnesses. N. commune Vauch., commonly
known as ishikurage, is eaten as a vinegared food in
Japan.
2)
N. commune Vauch. is known to be a rich source of
secondary metabolites with a wide variety of biological
activities, including antimicrobial, antimitotic, cytotoxic,
and enzyme inhibitory properties; for example, diterpe-
noids comnostin AE have exhibited strong antibacterial
activities against Bacillus cereus, Staphylococcus epi-
dermidis, and Escherichia coli.
3)
Nostofugicidine, which
is a lipopeptide, has shown potent antifungal activity
against Aspergillus candidus and cytotoxicity toward
NSF-60 cells.
4)
The indole alkaloid, nostodione A, has
disturbed the mitotic spindle formation of sea-urchin
eggs and gave small spindles with low birefringence
density.
5)
It is thus important to study N. commune
Vauch. in detail to identify additional compounds
having chemical and medicinal value.
68)
Antioxidative activity is essential for life and to
counteract the strongly oxidizing environment in which
we live.
9)
Such benecial biological functions as
protection from mutagenesis, carcinogenesis, and aging
are caused by antioxidative eects.
10)
Synthetic antiox-
idants such as BHA and BHT are also widely employed
as preservatives by the pharmaceutical, cosmetic, and
food industries, regardless of their suspected association
with liver damage and carcinogenesis in laboratory
animals.
11)
Considerable attention has therefore been
devoted to the development and utilization of more
eective and non-toxic antioxidants of natural origin.
We found that the extract of N. commune Vauch. had
high antioxidative activity. We isolated six chemical
constituents in order to identify the specic compounds
responsible for this activity. Among these isolates,
nostocionone and reduced scytonemin demonstrated
strong antioxidative activities.
Results and Discussion
We initially evaluated the antioxidative eects of ve
types of algae extract by using a -carotene oxidation
assay. The ve extracts of Nostoc commune Vauch.,
Spirulina platensis, Chlorella pyrenoidosa, and Duna-
liella salina were each prepared by 5-times extraction of
each alga (2 g) with ethanol (20 mL). The results of the
assay showed that the extract of N. commune Vauch.
had the strongest antioxidative activity in comparison
with the other algae (S. platensis, C. pyrenoidosa, and
D. salina). This antioxidative activity was stronger than
that of BHA which was used as a positive control
(Fig. 1). We attempted to isolate and characterize the
constituent compounds as a continuation of our study.
The ethanol extract of N. commune Vauch., which
was harvested in Alxa (Inner Mongolia, China), was
puried by column chromatography (using silica gel,
ODS, and Sephadex LH-20) and PTLC on silica gel to
give six compounds. Among these six compounds, four
were the known alkaloids, scytonemin, reduced scyto-
nemin,
12)
N-(p-coumaroyl)tryptamine,
13)
and N-acetyl-
tryptamine,
14)
and one was found to be the -ionone
derivative, 3-oxo--ionone.
15) 1
H- and
13
C-NMR spec-
tral data for these ve compounds were identical with
those previously reported (Fig. 2). Structural elucidation
of the one new compound is described here.
Compound 1 was isolated as an orange amorphous
powder, and its molecular formula was established as
C
22
H
28
O
4
from HRFABMS for the peak at m=z
y
To whom correspondence should be addressed. Tel/Fax: +81-58-293-2619; E-mail: koketsu@gifu-u.ac.jp
Biosci. Biotechnol. Biochem., 75 (11), 21752177, 2011
357.2091 MH

(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

C for 20 s, and then

powdered. The dried algal powder (600 g) was extracted with ethanol
(60 L), and an ethanol extract (30 g) was obtained by removing the
solvent under reduced pressure. This extract (18 g) was separated by
silica gel vacuum liquid chromatography (VLC), using n-hexane/ethyl
acetate (9/1 to 1/9; v/v) as the eluent, to give 8 fractions (Frs. 1 to 8).
Fr. 3 (567 mg) was further puried by silica gel CC, using n-hexane/
ethyl acetate in a stepwise manner, to give 12 fractions (Frs. 3-1 to
3-12). Fr. 3-4 (150 mg) was puried by using silica gel, Sephadex LH-
20 (GE Healthcare Bio-Sciences), and ODS (Nacalai Tesque) CCs to
yield 3-oxo--ionone (8.1 mg). Repeated silica gel CC of Fr. 5
(909 mg), having a strong antioxidative eect, gave Fr. 5-1. Fr. 5-1
(453 mg) was applied to Sephadex LH-20 CC, eluting with acetone/
methanol/distilled water (0/3/1, 0/10/1, 1/10/0, and 1/1/0), and
divided into 8 fractions (Frs. 5-1-1 to 5-1-8). Fr. 5-1-1 (29 mg) was
puried by using repeated Sephadex LH-20 and ODS CCs to yield
scytonemin (2.5 mg). Further purication of Fr. 5-1-7 (51 mg), using
ODS CC (chloroform/methanol (1/0, 5/1, and 1/1) as the eluent), gave
reduced scytonemin (6.5 mg). The remaining ethanol extract (11 g) was
separated by silica gel VLC, using n-hexane/ethyl acetate as the eluent
in a stepwise manner, and divided into six fractions (Frs. AF). Fr. B
(107 mg), having a strong antioxidative eect, was puried by using
silica gel CC (n-hexane/ethyl acetate (5/1) as the eluent) and PTLC
(chloroform/methanol 100/1 as the solvent) to yield nostocionone 1
(22.3 mg). Fr. D (344 mg) was puried by using silica gel CC
(chloroform/methanol (100/1) as the eluent) to yield N-(p-coumar-
oyl)tryptamine (15.1 mg). Further purication of Fr. E (106 mg), using
silica gel CC (chloroform/methanol (50/1) as the eluent) and PTLC
(chloroform/methanol 50/1 as the solvent), gave N-acetyltryptamine
(11.4 mg).
Nostocionone 1. Orange amorphous powder, IR (lm) cm
1
: 3363,
1600, 1513, 1457, 1114, 672. FABMS: m=z 357 MH

,
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

C in a water bath. The sample

was measured after 1 h, using a UVvis spectrometer at 470 nm. All the
tested compounds were stable during evaluation by this system, and
the activity was therefore determined only by the decomposition of
-carotene. It was also conrmed that nostocionone 1 did not show
absorption at 470 nm. The -carotene survival rate was calculated as
follows:
-Carotene survival rate (%)
A
470 nm 1 h after sample addition
=A
470 nm just after sample addition
100:
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COSY
HMBC
OMe
O
OH
OMe
Fig. 3. Key COSY and HMBC Correlations for Nostocionone 1.
Table 2. Antioxidative Eects of Nostocionone 1 and Reduced
Scytonemin (means SEM, n 3)
-Carotene survival rate (%)
0.5 mM 1.0 mM
Nostocionone 1 54 5 69 4
Reduced scytonemin 39 1 54 3
BHA 41 5 70 4
Control (EtOH) 4 2
Chemical Constituents of Nostoc commune Vauch. 2177