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Isolation and Identification of Antioxidative Compounds and Their Activities

from Suaeda japonica

Article  in  Food science and biotechnology · December 2013

DOI: 10.1007/s10068-013-0250-2

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Food Sci. Biotechnol. 22(6): 1547-1557 (2013)
DOI 10.1007/s10068-013-0250-2
RESEARCH ARTICLE
Isolation and Identification of Antioxidative Compounds and
Their Activities from Suaeda japonica
Jeong-Yong Cho, Xing Yang, Kyung-Hee Park, Hye Jin Park, Sun-Young Park, Jae-Hak Moon, and
Kyung-Sik Ham
Received: 2 April 2013 / Revised: 23 May 2013 / Accepted: 18 June 2013 / Published Online: 31 December 2013
© KoSFoST and Springer 2013
Abstract The ethyl acetate (EtOAc) and chloroform
(CHCl3) layers obtained after solvent fractionation of a
H2O suspension of powdered Suaeda japonica juice showed
higher 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-
scavenging activity than other layers. Eighteen compounds
were purified and isolated from the EtOAc and CHCl3
layers using chromatography following DPPH radical-
scavenging assay. These compounds were identified as
dihydroferulic acid methyl ester (1), pyrocatechol (2), syringic
acid (3), apigenin (4), isorhamnetin (5), kaempferol (6),
dihydroferulic acid (7), vanillic acid (8), 4-hydroxybenzoic
acid (9), acetophlorglucine (10), homoeriodictyol (11),
naringenin (12), quercetin (13), luteolin (14), 9-epi-
blumenol C (15), scopoletin (16), dihydroisorhamnetin
(17), and chrysoeriol (18). The structures of these compounds
were determined by mass spectrometry and nuclear magnetic
resonance analyses. The isolated compounds were newly
identified from this plant. Compounds 13 and 14 exhibited
higher DPPH radical-scavenging activity and an inhibition
effect against ferric ion-induced lipid oxidation of rat liver
when compared to α-tocopherol and other compounds.
Keywords: Suaeda japonica, antioxidant, flavonoid,
phenolics, DPPH radical-scavenging activity
Jeong-Yong Cho, Xing Yang, Kyung-Hee Park, Hye Jin Park, Sun-Young
Park, Kyung-Sik Ham (
)
Department of Food Science & Biotechnology, and Solar Salt
Biotechnology Research Center, Mokpo National University, Muan,
Jeonnam 534-729, Korea
Tel: +82-61-450-2425; Fax: +82-61-454-1521
E-mail: ksham@mokpo.ac.kr
Jae-Hak Moon
Department of Food Science & Technology, and Functional Food
Research Center, Chonnam National University, Gwangju 500-757, Korea
Introduction
Suaeda japonica Makino belongs to the family of
Chenopodiacea and is an annual halophytic herb which
grows on seaside tidal flats in Korea and Japan (1,2). The
leaf color of this plant, which is called ‘chilmyeoncho’ in
Korean, changes from green to red at different growth
stages. S. japonica has been used as a traditional medicine
to alleviate fever in Korea (3). The young aerial parts of S.
japonica are eaten as a seasoned vegetable and herb on the
western and southern coasts of Korea. Recently, the young
leaves of this plant were approved as a food material by
Korea Food and Drug Administration (4). This plant is rich
in minerals such as sodium, magnesium, calcium, potassium,
and iron (5), and has also been used as an organic salt
material in Korea.
Several studies have reported that S. japonica showed
strong antioxidative effects, as determined by various in
vitro assays using 1,1-diphenyl-2-picrylhydrazyl (DPPH)
radical, hydroxyl radical, ferric-reducing antioxidant potential,
and β-carotene bleaching (3,6-8). This plant also suppressed
tyrosinase activity and melanin synthesis in B16BL6 cells
(6). A few chemical constituents, such as glycinebetaine
(betaine), 2'-hydroxy-6,7-methylenedioxyisoflavone, loliolide,
dehydrovomifoliol, and uridine have been isolated and
identified in S. japonica (9,10). In addition, betacyanins are
known to be an important red pigment in S. japonica and
have also been identified as antioxidative compounds (11).
Among halophytes grown on saline environments such
as coastal sand, salt marshes, and tidal flats in Korea,
Salicornia herbacea (glasswort) is well known as a useful
crop resource and food material. Many researchers have
reported that glasswort exerts various biological effects
(anti-hyperglycemic, anti-diabetic, antioxidant, and anticancer,
etc.) (12-15) and contains various biological compounds
1548
(sterols, polysaccharides, flavonol glycosides, and caffeoyl
quinic acid derivatives) (16-20). It is likely that glasswort
contains large quantities of secondary metabolites, such as
phenolics and flavonoids, due to salt stress induced by its
environment.
S. japonica and glasswort are members of the family
Chenopodiacea and are succulent halophytes. Like the
glasswort, S. japonica may synthesize and accumulate
various secondary metabolites for multiple biochemical
defenses, such as the retention and acquisition of water, the
maintenance of ion homeostasis, and protection of cell
functions to survive under severe saline environments. S.
japonica is also considered to be a potentially useful
halophyte for food material, like the glasswort. In addition,
growth environment and morphological characteristics of
S. japonica are very similar to those of glasswort. However,
as described above, studies on the chemical constituents of
S. japonica are not yet complete. Understanding the
chemical constituents of S. japonica is very important in
acquiring basic information on health beneficial activity
and value of this plant as a food material. Therefore,
investigation for antioxidative compounds in the aerial
parts of S. japonica was carried out. In this study, the
isolation and identification of 18 antioxidative compounds
from S. japonica were described. In addition, the
antioxidative activities of the isolated compounds were
evaluated by DPPH radical and ferric ion-induced lipid
oxidation system in rat liver.
Materials and Methods
Materials and chemicals The powdered S. japonica juice
was obtained from Hanuri Company (Yesan, Chungnam,
Korea). The aerial parts of this plant were collected in
October 2010 at Taean County, located on the western
coast of Korea. After the aerial parts were washed, juiced,
and filtered, the filtrates were mixed with dextran and were
then spray-dried. The final product of powdered S. japonica
juice contained 37%(w/v) dextran. Thiobarbituric acid
(TBA), DPPH, L-ascorbic acid, and (±)-α-tocopherol were
purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Isorhamnetin [3'-methoxy-3,4',5,7-tetrahydroxyflavanone],
kaempferol [3,4',5,7-tetrahydroxyflavanone], vanillic acid
(4-hydroxy-3-methoxybenzoic acid), and naringenin [5,7-
dihydroxy-2-(4-hydroxyphenyl)chroman-4-one] were also
obtained from Sigma Chemical Co. Methanol-d4 (CD3OD)
and dimethyl sulfoxide-d6 (DMSO-d6) were obtained from
Merck Co. (Darmstadt, Germany).
Partition Powdered S. japonica juice (1,000 g, equivalent
to about 15 kg fresh weight) was suspended in 3.0 L of
H2O and successively partitioned with n-hexane (3 L, 3
Cho et al.
times), chloroform (CHCl3, 3 L, 3 times), ethyl acetate
(EtOAc, 3 L, 3 times), and water-saturated n-butanol
(BuOH, 3 L, 3 times). Each layer was evaporated in vacuo
at 38oC.
Purification of EtOAc and CHCl3 layers The EtOAc
layer (13.2 g) was fractionated on silica gel (Kiesel gel 60,
70-230 mesh; Merck Co.) column (396 g, 6.0×30 cm) and
eluted with solvent mixtures of CHCl3/MeOH (9:1, 7:3,
5:5, 3:7, 0:10, v/v, each 3 L). Fractions EA and EB were
re-fractionated by the chromatography of silica gel (70-230
mesh; Merck Co.) column (90 g, 2.5×40 cm) that had been
eluted with a series of solvent mixtures of n-hexane/EtOAc
(6:4, 4:6, 2:8, and 0:10, v/v, each 400 mL). Fraction EA1a
was subjected to chromatography using a silica gel column
(20 g, 2.5×40 cm) and was eluted with an isocratic system
of n-hexane/EtOAc=7:3 (v/v). Fractions EA1a2, EA1c,
and EA2b were purified by chromatography of Sephadex
LH-20 (25-100 mesh; GE Healthcare Bio-Sciences AB,
Uppsala, Sweden) column (1.5×56 cm) eluted with 100%
MeOH. Fraction EB1 was purified using ODS (170 mesh;
YMC, Kyoto, Japan) column (2.0×18 cm) chromatography
eluted with 50% MeOH.
The CHCl3 layer (7.2 g) was separated on a silica gel
column (200 g, 6.0×30 cm) and was eluted with solvent
mixtures of n-hexane/EtOAc (8:2, 6:4, 4:6, 2:8, and 0:10,
v/v, each 400 mL). Fraction CD (355 mg) was dissolved in
MeOH (10 mL) at room temperature. After filtration (No.
2 filter paper; Whatman International, Maidstone, UK), the
MeOH-soluble fraction (CD) was concentrated in vacuo at
38. Fractions CD1, CD1d, and CG were purified using a
Sephadex LH-20 column (1.5×56 cm) eluted with 100%
MeOH.
Structural analysis Nuclear magnetic resonance (NMR)
spectra were obtained from Digital Avance 400 (Bruker
Analytik GmbH, Karlsruhe, Germany) and unitINOVA 500
(Varian, Walnut Creek, CA, USA) spectrometers using
tetramethylsilane as an internal standard in CD3OD and
DMSO. All MS spectra were obtained using electrospray
ionization tandem mass spectrometers (LC-ESI-MS/MS,
Synapt HDMS; Waters, Milford, MA, USA, EsqireHCT;
Bruker Daltonics, Bremen, Germany).
Assay of DPPH radical-scavenging activity The free
radical-scavenging activities of the identified compounds,
the solvent-fractionated fractions, and a standard (ascorbic
acid) were evaluated using the DPPH radical according to
modified modification of the method described by Abe et
al. (21). In brief, MeOH solutions (100 µL) of solvent-
fractionated layers at different concentrations and the
isolated compounds (final concentration, 20 mM) were
added to the DPPH radical ethanol solution (900 µL, final
Chemical Constituents in Suaeda japonica
concentration, 100 mM). The solution was gently mixed
and allowed to stand for 30 min in darkness. The DPPH
free radical-scavenging activities of solvent-fractionated
layers and the isolated compounds were then quantified by
measuring the absorbance of DPPH at 517 nm using
spectrophotometer (8452A; Hewlett Packard, Palo Alto,
CA, USA). The DPPH radical-scavenging activities of
solvent-fractionated layers and the isolated compounds
were also determined as the percentage decrease in
absorbance compared to the absorbance shown by a
control test. The value of 50% free radical-scavenging
concentration (SC50) for the solvent fractionated fractions
was determined from the dose-response curve.
An assay for the purification of antioxidative compounds
was performed by spraying DPPH reagent on thin-layer
chromatography (TLC) (22). Briefly, each fraction was
purified by chromatography on a series of columns, and
was then spotted onto a silica gel TLC plate (silica gel 60
F254, 0.25-mm thickness; Merck) and developed using a
mixture of CHCl3/EtOAc (1:1, v/v) or n-hexane/EtOAc
(7:3, v/v). After spraying the plate with 100 mM DPPH
EtOH solution, the fractions were visualized as a reflection
of antioxidative activity.
Determination of the inhibitory effect of the isolated
compounds against ferric ion-induced oxidation of rat
liver The antioxidative activities of the identified
compounds and α-tocopherol (Toc) as a positive control
were evaluated by measuring thiobarbituric acid reactive
substance (TBARS) for ferric ion-induced rat liver
oxidation, following the method described elsewhere (23)
with slight modifications. Briefly, Sprague-Dawley rats
(male, 6 week age, 180-200 g; Damool Science, Daejeon,
Korea) were kept at 25oC under a 12 h dark/light cycle and
were fasted for 12-15 h. After anesthesia with diethyl ether,
the abdomen wall was opened and the liver was collected
and immediately frozen in liquid nitrogen, and stored at
−70oC for no longer than 1 week. The rat liver (1 g) was
homogenized in 0.02 M Tris-HCl buffer (pH 7.4) and
centrifuged at 12,000×g and 4oC for 15 min. The supernatant
(0.35 mL) was mixed with the isolated compound solubilized
in MeOH (0.05 mL, final concentration 25 mM). After pre-
incubation at 37oC for 5 min, the mixture was oxidized by
the addition of 100 µM ascorbic acid (0.05 mL) and
aqueous FeSO4 solution (0.05 mL, final concentration,
100 µM). Each mixture was incubated at 37oC for 1 h with
continuous shaking. This solution was added to 0.5 mL of
0.6 M HCl solution containing 28% trichloroacetic acid
and 0.75 mL of 1 M NaOH solution containing 1%
thiobarbituric acid. After standing at 95oC for 15 min, the
reaction solution was partitioned with water-saturated n-
butanol and was then centrifuged at 3,000 rpm for 10 min.
The absorbance of the upper layer (water-saturated n-
1549
butanol layer) was measured at 532 nm. The inhibition
effects of the isolated compounds against ferric ion-
induced oxidation in rat liver were determined as the
percentage decrease in absorbance compared to that of
control test.
Statistical analysis The data for DPPH radical-scavenging
activities of the isolated compounds were expressed as the
mean±standard deviation (n=3) using the Statistical
package for Social Sciences (SPSS, Chicago, IL, USA)
18.0 package programs. The significant differences were
evaluated by one-way ANOVA, followed by Duncan’s
multiple-range test, where significance was determined by
a value of p<0.05.
Results and Discussion
Antioxidative activities of solvent-fractionated layers
The powdered S. japonica juice (1,000 g) was solvent-
fractionated with various solvents to obtain n-hexane
(4.6 g), CHCl3 (7.2 g), EtOAc (13.2 g), and BuOH (90.0 g)
layers. The antioxidative activities of these layers were
evaluated using a DPPH radical. DPPH radical-scavenging
activity of all these layers increased in a dose-dependent
manner (Fig. 1). The SC50 values were determined from
the results of dose-response curves. The antioxidative
activity (SC50) decreased in the following order: EtOAc
(SC50, 37.1 µg/mL)>CHCl3 (57.4 µg/mL)>BuOH (182.8 µg/
mL) >n-hexane (220.6 µg/mL) layers (Fig. 1). The EtOAc
and CHCl3 layers showed relatively higher antioxidative
activity when compared to other layers. Therefore,
purification of antioxidative compounds from the EtOAc
and BuOH layers was performed.
Isolation of antioxidative compounds from the EtOAc
and CHCl3 layers The EtOAc layer (13.2 g) was
separated on a silica gel column eluted with a solvent
system of CHCl3/MeOH. Each fraction was developed on
a silica gel TLC with n-hexane/EtOAc (1:1, v/v) solution.
After spraying with 100 µM DPPH free radical EtOH
solution, 18 active fractions (EA-ER) were divided from
the EtOAc layer. Two active fractions (EA and EB) showed
significantly higher antioxidative activity than other
fractions. Fraction EA1 (CHCl3/MeOH=9:1, v/v, 2.6 g)
was re-fractionated by silica gel column chromatography
using a step-wise elution system of n-hexane/EtOAc as a
mobile phase to give 10 active fractions (EA1a-EA1j).
Fraction EA1a (n-hexane/EtOAc=6:4, v/v, 285.1 mg) was
separated on a silica gel column (n-hexane/EtOAc=7:3,
v/v) to give three active fractions (EA1a1-EA1a3). Fraction
EA1a2 (30.0 mg) was separated by Sephadex LH-20 column
chromatography (100% MeOH) to obtain compounds 1
1550
Fig. 1. DPPH radical-scavenging activities of fractions
obtained after solvent-fractionation of H2O suspension of
powdered S. japonica juice. ●-●, n-Hexane layer; ■-■,
CHCl3 layer; ▲-▲, EtOAc layer; ▼-▼, BuOH layer. Each value
is the mean±SD of three experiments. a-d, results with a different
letter at the same concentration differ significantly (p<0.05).
[elution volume/total volume (Ve/Vt) 0.82-0.91, 4.3 mg]
and 2 (Ve/Vt 1.02-1.10, 8.9 mg). In addition, Fraction
EA1c (n-hexane/EtOAc=6:4, v/v, 130.5 mg), obtained after
silica gel column chromatography of EA1 (2.6 g), was
purified by Sephadex LH-20 column chromatography
(100% MeOH) to obtain compounds 3 (Ve/Vt 0.81-0.90,
20.9 mg), 4 (Ve/Vt 1.01-1.10, 6.4 mg), and a mixture of 5
and 6 (Ve/Vt 1.10-1.23, 1.9 mg). Fraction EA2 (n-hexane/
EtOAc=6:4, v/v, 891.9 mg) obtained after silica gel
column chromatography of EA (2.6 g) and was fractionated
by silica gel column chromatography (n-hexane/EtOAc=
7:3, v/v) to give nine fractions (EA2a-EA2i). Fraction
EA2b (502.5 mg) was purified on a Sephadex LH-20
column (100% MeOH) to give eight compounds: a mixture
of 7 and 8 (Ve/Vt=0.82-0.87, 85.1 mg), 9 (Ve/Vt 0.89-0.92,
2.6 mg), 10 (Ve/Vt 0.95-1.00, 3.9 mg), a mixture of 11 and
12 (Ve/Vt 1.00-1.08, 3.4 mg), and a mixture of 4 and 5 (Ve/
Vt 1.12-1.30, 2.5 mg). Fraction EB (CHCl3/MeOH=9:1,
v/v, 3.2 g), obtained after silica gel column chromatography
of the EtOAc layer (13.2 g), was fractionated by
chromatography using a silica gel column (n-hexane/
EtOAc, 7:3, v/v) to give ten fractions (EB1-EB9). A
portion (200 mg) of EB1 (1.27 g) was further purified by
chromatography using a ODS column (50% MeOH) to
give compounds 13 (16.6 mg), a mixture of 13 and 14
(132.5 mg), and 14 (8.4 mg). The purification procedure
for the active compounds (1-14) isolated in the EtOAc
layer was shown in Fig. 2.
The CHCl3 layer (7.2 g) was separated using silica gel
column chromatography (n-hexane/EtOAc). All fractions
were spotted on a silica gel TLC plate and then developed
Cho et al.
with n-hexane/EtOAc (7:3, v/v) solution. After spraying
with 100 µM DPPH free radical EtOH solution, 8 active
fractions (CA-CH) were separated from the CHCl3 layer.
All of the fractions showed DPPH free radical-scavenging
activity. In particular, fractions CD (n-hexane/EtOAc, 4:6,
v/v, 355 mg) and CG (n-hexane/EtOAc, 0:10, v/v, 83 mg)
exhibited higher DPPH free radical-scavenging activity
than other fractions. Therefore, fraction CD (n-hexane/
EtOAc, 4:6, v/v, 355 mg) was dissolved with MeOH and
divided into dissolved (CD1, 188.3 mg) and undissolved
(CD2, 132.8 mg) fractions. Fraction CD1 (188.3 mg) was
purified on the chromatography of Sephadex LH-20 column
(100% MeOH) to give two fractions (CD1b, CD1d) and
four compounds (Ve/Vt=0.75-0.81, 13.6 mg, 15; Ve/Vt
0.84-0.95, 11.7 mg, 7; Ve/Vt 1.12-1.20, 22.0 mg, 17).
Fraction CD1d (30 mg) was further purified on Sephadex
LH-20 column (100% MeOH) to give two compounds
(Ve/Vt 0.82-0.88, 8.6 mg, 7; Ve/Vt 0.91-0.97, 6.8 mg, 16).
Fraction CG (n-hexane/EtOAc, 0:10, v/v, 83 mg) obtained
after silica gel column chromatography of the CHCl3 layer
was subjected to Sephadex LH-20 column (100% MeOH)
chromatography to give two subfractions (CG1 and CG2)
and 18 (Ve/Vt 0.93-1.05, 9.7 mg). The purification procedure
for the active compounds (7, 15-18) isolated in the CHCl3
layer is indicated in Fig. 3.
Identification of the isolated 17 compounds Eighteen
antioxidative compounds (1-18) were isolated from the
EtOAc and CHCl3 layers of powdered S. japonica juice.
The structures of the isolated compounds were determined
by NMR and ESI-MS analyses.
Compound 1: 1H-NMR (400 MHz, CD3OD) δ 6.77 (1H,
d, J=2.0 Hz, H-2), 6.68 (1H, d, J=8.0 Hz, H-5), 6.62 (1H,
dd, J=8.0, 2.0 Hz, H-6), 2.59 (2H, t, J=15.3 Hz, H-7), 2.82
(2H, t, J=15.3 Hz, H-8), 3.84 (3H, s, -OCH3 of C-3), 3.63
(3H, s, -OCH3 of C-9); 13C-NMR (100 MHz, CD3OD) δ
133.9 (C-1), 113.3 (C-2), 148.3 (C-3), 146.3 (C-4), 116.5
(C-5), 122.0 (C-6), 30.9 (C-7), 37.4 (C-8), 175.7 (C-9),
56.7 (-OCH3 of C-4), 52.4 (-OCH3 of C-9); ESI-MS
(positive) m/z 210.7 [M+H]+ and 232.7 [M+Na]+.
Compound 2: 1H-NMR (400 MHz, CD3OD) δ 6.74 (2H,
br. d, J=6.0 Hz, H-4, 5), 6.64 (2H, br. d, J=6.0 Hz, H-3, 6);
13
C-NMR (100 MHz, CD3OD) δ 116.8 (C-4, 5), 121.3 (C-
3, 6), 146.8 (C-1, 2); ESI-MS (negative) m/z 108.8 [M-H]−.
Compound 3: 1H-NMR (400 MHz, CD3OD) δ 7.33 (2H, s,
H-2, 6), 3.88 (6H, s, -OCH3×2); 13C-NMR (100 MHz,
CD3OD) δ 170.8 (C=O), 149.1 (C-3, 5), 141.3 (C-4), 124.3
(C-1), 108.5 (C-2, 6), 57.1 (-OCH3×2); ESI-MS (negative)
m/z 196.6 [M-H]−.
Compound 4: 1H-NMR (400 MHz, CD3OD) δ 6.59 (1H, s,
H-3), 6.20 (1H, d, J=2.0 Hz, H-6), 6.45 (1H, d, J=2.0 Hz,
H-8), 7.84 (2H, br. d, J=8.8 Hz, H-2', 6'), 6.89 (2H, br. d,
J=8.8 Hz, H-3', 5'); ESI-MS (positive) m/z 270.8 [M+H]+
 Chemical Constituents in Suaeda japonica

Fig. 2. Isolation procedure of antioxidants from EtOAc layer of powdered S. japonica juice.
1551
1552
Fig. 3. Isolation procedure of antioxidants from CHCl3 layer of powdered S. japonica juice.
and 292.8 [M+Na]+.
Compound 5: 1H-NMR (400 MHz, DMSO-d6) δ 6.19 (1H,
d, J=2.0 Hz, H-6), 6.48 (1H. d, J=2.0 Hz, H-8), 7.75 (1H,
d, J=2.0 Hz, H-2'), 6.94 (1H, d, J=8.5 Hz, H-5'), 7.69 (1H,
dd, J=8.5, 2.0 Hz, H-6'), 3.84 (3H, s, -OCH3); ESI-MS
(negative) m/z 314.8 [M-H]−.
Compound 6: 1H-NMR (400 MHz, DMSO-d6) δ 6.20 (1H,
d, J=2.0 Hz, H-6), 6.44 (1H, d, J=2.0 Hz, H-8), 8.04 (2H,
br. d, J=9.0 Hz, H-2', 6'), 6.91 (2H, br. d, J=9.0 Hz, H-3',
5'); ESI-MS (negative) m/z 284.7 [M-H]−.
Compound 7: 1H-NMR (500 MHz, CD3OD) δ 6.79 (1H,
d, J=2.0 Hz, H-2), 6.69 (1H, d, J=8.0 Hz, H-5), 6.64 (1H,
dd, J=8.0, 2.0 Hz, H-6), 2.55 (2H, t, J=15.3 Hz, H-7), 2.82
(2H, t, J=15.3 Hz, H-8), 3.89 (3H, s, -OCH3); 13C-NMR
(125 MHz, CD3OD) δ 133.9 (C-1), 113.1 (C-2), 148.9 (C-
3), 146.0 (C-4), 115.9 (C-5), 121.8 (C-6), 31.8 (C-7), 37.4
(C-8), 177.1 (C-9), 56.5 (-OCH3); ESI-MS (negative) m/z
194.7 [M-H]−.
Compound 8: 1H-NMR (500 MHz, CD3OD) δ 7.57 (1H,
d, J=2.0 Hz, H-2), 6.84 (1H, d, J=9.0 Hz, H-5), 7.55 (1H,
dd, J=9.0, 2.0 Hz, H-6), 3.88 (3H, s, -OCH3); 13C-NMR
(125 MHz, CD3OD) δ 123.2 (C-1), 113.9 (C-2), 148.8 (C-
3), 152.8 (C-4), 116.3 (C-5), 125.4 (C-6), 170.2 (C-7), 56.5
(-OCH3); ESI-MS (negative) m/z 166.7 [M-H]−.
Cho et al.
Compound 9: 1H-NMR (400 MHz, CD3OD) δ 7.85 (2H,
br. d, J=8.6 Hz, H-3, 5), 6.88 (2H, d, J=8.6 Hz, H-2, 6);
ESI-MS (negative) m/z 136.7 [M-H]−.
Compound 10: 1H-NMR (500 MHz, CD3OD) δ 5.79 (2H,
s, H-3, 5), 2.59 (3H, s, H-2'); 13C-NMR (125 MHz,
CD3OD) δ 105.6 (C-1), 166.3 (C-2), 95.6 (C-3), 165.9 (C-
4), 95.4 (C-5), 166.0 (C-6), 204.0 (C-1'), 32.9 (C-2'); ESI-
MS (negative) m/z 166.6 [M-H]−.
Compound 11: 1H-NMR (500 MHz, CD3OD) δ 5.34 (1H,
dd, J=6.5, 3.5 Hz, H-2), 3.16 (1H, dd, J=17.0, 12.5 Hz, H-
3a), 2.72 (1H, dd, J=17.0, 3.5 Hz, H-3b), 5.89 (1H, d,
J=2.0 Hz, H-6), 5.90 (1H, d, J=2.0 Hz, H-8), 7.07 (1H, d,
J=2.0 Hz, H-2'), 6.82 (1H, d, J=8.0 Hz, H-5'), 6.92 (1H, dd,
J=8.0, 2.0 Hz, H-6'), 3.88 (3H, s, -OCH3); 13C-NMR (125
MHz, CD3OD) δ 80.9 (C-2), 48.6 (C-3), 198.0 (C-4), 165.1
(C-5), 96.3 (C-6), 168.5 (C-7), 97.2 (C-8), 165.7 (C-9),
103.5 (C-10), 131.9 (C-1'), 120.7 (C-2'), 149.3 (C-3'),
148.3 (C-4'), 116.5 (C-5'), 111.4 (C-6'), 56.6 (-OCH3); ESI-
MS (negative) m/z 300.8 [M-H]−.
Compound 12: 1H-NMR (500 MHz, CD3OD) δ 5.33 (1H,
dd, J=6.5, 3.5 Hz, H-2), 3.09 (1H, dd, J=15.5, 6.5 Hz, H-
3a), 2.68 (1H, dd, J=15.5, 3.50 Hz, H-3b), 5.88 (2H, d,
J=2.0 Hz, H-6, overlapped with H-8 signal), 5.88 (1H, d,
J=2.0 Hz, H-8, overlapped with H-6 signal), 7.31 (2H, br.
Chemical Constituents in Suaeda japonica
d, J=8.0 Hz, H-2', 6'), 6.81 (2H, br. d, J=8.0 Hz, H-3', 5');
13
C-NMR (125 MHz, CD3OD) δ 80.7 (C-2), 44.2 (C-3),
198.0 (C-4), 165.1 (C-5), 96.3 (C-6), 168.5 (C-7), 97.2 (C-
8), 165.7 (C-9), 103.5 (C-10), 131.2 (C-1'), 129.2 (C-2'),
116.3 (C-3'), 159.2 (C-4'), 116.3 (C-5'), 129.2 (C-6'); ESI-
MS (negative) m/z 270.8 [M-H]−.
Compound 13: 1H-NMR (400 MHz,CD3OD) 6.16 (1H, d,
J=2.0 Hz, H-6), 6.37 (1H, d, J=2.0 Hz, H-8), 7.73 (1H, d,
J=1.6 Hz, H-2'), 6.87 (1H, d, J=8.5 Hz, H-5'), 7.63 (1H, dd,
J=8.5, 1.6 Hz, H-6'); ESI-MS (negative) m/z 300.7 [M-H]−.
Compound 14: 1H-NMR (400 MHz,CD3OD) δ 6.52 (1H,
s, H-3), 6.18 (1H, d, J=1.8 Hz, H-6), 6.40 (1H, d, J=1.8 Hz,
H-8), 7.37 (1H, br. s, H-2'), 6.89 (1H, d, J=7.8 Hz, H-5'),
7.38 (1H, dd, J=7.8, 1.8 Hz, H-6'); ESI-MS (negative) m/
z 284.7 [M-H]−.
Compound 15: 1H-NMR (400 MHz, CD3OD) δ 2.00 (1H,
d, J=17.0 Hz, H-2a), 2.44 (1H, d, J=17.0 Hz, H-2b), 5.81
(1H, s, H-4), 1.99 (1H, m, H-6), 1.61 (1H, m, H-7a), 1.76
(1H, m, H-7b), 1.51-1.56 (2H, m, H-8), 3.69 (1H, m, H-9),
1.16 (3H, d, J=6.0 Hz, H-10), 1.02 (3H, s, H-11), 1.09 (3H,
s, H-12), 2.04 (3H, d, J=1.0 Hz, H-13); 13C-NMR (100
MHz, CD3OD) δ 37.7 (C-1), 48.4 (C-2), 202.6 (C-3), 125.8
(C-4), 170.2 (C-5), 52.7 (C-6), 27.9 (C-7), 40.1 (C-8), 68.9
(C-9), 24.0 (C-10), 29.4 (C-11), 27.7 (C-12), 25.3 (C-13);
ESI-MS (positive) m/z 210.9 [M+H]+ and 232.9 [M+Na]+.
Compound 16: 1H-NMR (400 MHz, CD3OD) δ 7.86 (1H,
d, J=9.6 Hz, H-2), 6.20 (1H, d, J=9.6 Hz, H-3), 7.11 (1H,
s, H-4), 6.76 (1H, s, H-8), 3.90 (3H, s, -OCH3); ESI-MS
(positive) m/z 192.7 [M+H]+ and 214.7 [M+Na]+.
Compound 17: 1H-NMR (400 MHz, CD3OD) δ 5.00 (1H,
d, J=11.6 Hz, H-2), 4.58 (1H, d, J=11.6 Hz, H-3), 5.87
(1H, br. s, H-6), 5.90 (1H, br. s, H-8), 7.11 (1H, br. s, H-
2'), 6.97 (1H, br. d, J=8.2 Hz, H-6'), 6.83 (1H, d, J=8.2 Hz,
H-5'), 3.88 (3H, s, -OCH3); 13C-NMR (150 MHz, CD3OD)
δ 85.6 (C-2), 74.0 (C-3), 198.0 (C-4), 165.1 (C-5), 96.9 (C-
6), 165.8 (C-7), 97.9 (C-8), 164.1 (C-9), 112.7 (C-10),
130.3 (C-1'), 121.8 (C-2'), 148.7 (C-3'), 149.3 (C-4'), 116.3
(C-5'), 122.6 (C-6'), 56.8 (-OCH3); ESI-MS (positive) m/z
340.9 [M+Na]+.
Compound 18: 1H-NMR (400 MHz, CD3OD) δ 6.63 (1H,
s, H-3), 6.19 (1H, br. s, H-6), 6.46 (1H, br. s, H-8), 7.60
(1H, br. s, H-2'), 6.93 (1H, d, J=8.0 Hz, H-5), 7.51 (1H, br.
d, J=8.0 Hz, H-6), 3.94 (3H, s, -OCH3); ESI-MS (negative)
m/z 298.7 [M-H]−.
Seventeen known compounds (all except for 15) were
identified as dihydroferulic acid methyl ester (1) (24),
pyrocatechol (2) (25), syringic acid (3) (26), apigenin (4)
(27), isorhamnetin (5) (28), kaempferol (6) (29),
dihydroferulic acid (7) (30), vanillic acid (8) (31), 4-
hydroxybenzoic acid (9) (32), acetophlorglucine (10) (33),
homoeriodictyol (11) (34), naringenin (12) (35), quercetin
(13) (36), luteolin (14) (37), scopoletin (16) (38),
dihydroisorhamnetin (17) (39), and chrysoeriol (18) (40)
1553
(Fig. 4). These compounds were identified via comparison
of NMR and MS spectroscopic data reported previously in
the relevant literature.
Structural determination of compound 15 Compound
15 was obtained as a colorless amorphous powder. The
molecular weight of this compound was determined to be
210 from the pseudomolecular ion peaks of m/z 210.9
[M+H]+ and 232.9 [M+Na]+ detected in the ESI-MS
(positive ion) spectrum. Thirteen carbon signals were
observed in the 13C NMR (100 MHz, CD3OD) and HSQC
spectra, including a carbonyl carbon (δ 202.6, C-3), two
olefinic double bond carbons [δ 170.2 (C-5) and 125.8 (C-
4)], and 10 sp3 carbons (δ 68.9-24.0). From 13C-NMR and
ESI-MS spectroscopic data, the molecular formula of 15
was suggested to be C13H22O2. The 1H-NMR spectrum
(400 MHz, CD3OD) of 15 showed the presence of an
olefinic double bond proton signal at δ 5.81 (1H, s, H-4),
3 methylene proton signals at δ 2.00 (1H, d, J=17.0 Hz, H-
2a), 2.44 (1H, d, J=17.0 Hz, H-2b), 1.61 (1H, m, H-7a),
1.76 (1H, m, H-7b), and 1.51-1.56 (2H, m, H-8), and 4
methyl proton signals at δ 2.04 (3H, d, J=1.0 Hz, H-13),
1.16 (3H, d, J=6.0 Hz, H-10), 1.09 (3H, s, H-12), 1.02 (3H,
s, H-11)]. In addition, a methine proton signal at δ 1.99
(1H, m, H-6) and an oxygenated methine proton signal at
δ 3.69 (1H, m, H-9) were observed. Therefore, compound
15 was proposed to be a derivative of C13 cyclohexenone
derivative (blumenin) coupled to a hydroxyl group. The
partial structure of 5 contiguous protonated carbons (C-
6~C-10, pentan-4-ol) was assigned, based on cross peaks
between the protons detected in the 1H-1H COSY spectrum
(Fig. 4, bold lines). The important long-range correlations
between protons and carbons were determined by the
HMBC spectrum (Fig. 4, arrows). The partial structures of
15 were confirmed as isophorone (3,5,5-trimethyl-2-
cyclohexene-1-one) and butan-3-ol. In particular, the
presence of the cross peaks from the proton signals of H-
7 [δ 1.61 (1H, m, H-7a) and 1.76 (1H, m, H-7b)] to the
carbon signals of C-1 (δ 37.7) and C-5 (δ 170.2) and from
the proton signal of H-8 (δ 1.51-1.56) to the carbon signal
of C-6 (δ 52.7) indicated that butanol was harbored at the
C-6 position of trimethylcyclohexenone. Therefore, the
structure of 15 was determined to be 9-hydroxymegastigman-
4-en-3-one (blumenol C). The absolute stereochemical
structure of 15 was confirmed as (6R,9S)-9-hydroxy-
megastigman-4-en-3-one (9-epi-blumenol C) by comparing
its NMR spectroscopic data with that reported previously
(41). Therefore, compound 15 was unambiguously identified
as 9-epi-blumenol C (Fig. 4).
DPPH radical-scavenging activity of the isolated
compounds The free radical- scavenging activities of the
identified compounds (1-10 and 12-18) except for
 1554

Fig. 4. Structures of the isolated compounds and important 1H-1H COSY (bold line) and HMBC (arrows) correlations for 15.
Cho et al.
Chemical Constituents in Suaeda japonica
Fig. 5. DPPH radical-scavenging activity of the isolated
compounds. Each reaction mixture contained 100 µM DPPH and
20 µM antioxidant in EtOH solution. After reaction at room
temperature for 30 min in the dark, the absorbance of each
reaction mixture was monitored at 517 nm. 1, Dihydroferulic acid
methyl ester; 2, pyrocatechol; 3, syringic acid; 4, apigenin; 5,
isorhamnetin; 6, kaempferol; 7, dihydroferulic acid; 8, vanillic
acid; 9, 4-hydroxybenzoic acid; 10, acetophlorglucine; 12,
naringenin; 13, quercetin; 14, luteolin; 15, 9-epi-blumenol C; 16,
scopoletin; 17, dihydroisorhamnetin; 18, chrysoeriol. AsA
(ascorbic acid) was used as a positive control. Each value is the
mean±SD of three experiments. a-j, results with a different letter
differ significantly (p<0.05)
homoeriodictyol (11) at the same concentration (20 µM)
were evaluated using the DPPH radical (final concentration,
100 µM). In the antioxidative activity evaluation of
commercially available products, isorhamnetin (5),
kaempferol (6), vanillic acid (8), and naringenin (12) were
used, because these compounds were identified as a
mixture from powdered S. japonica juice in this study.
Homoeriodictyol (11) was also identified as a mixture from
this plant and and its commercial products is not available,
and therefore, the antioxidative activitiy of 11 could not be
compared with other compounds. The DPPH radical-
scavenging activities of the identified compounds are
shown in Fig. 5. Quercetin (13) among the identified
compounds showed the highest DPPH radical-scavenging
activity. Luteolin (14) also displayed relatively higher
radical-scavenging activity compared to other compounds
and ascorbic acid (AsA) as a positive control, although its
activity was slightly lower in comparison to that of quercetin
(13). In addition, isorhamnetin (5) and kaempferol (6),
which contain a double bond between C-2 and C-3 and a
hydroxyl group in the C-3 position of the C ring like a
quercetin, showed relatively higher DPPH radical-
scavenging activities than other flavonoids (4, 12, 17, 18)
without containing either a C-2 double bond or C-3
hydroxyl group in the C ring. It was already reported that
the catechol structure of the B ring, C-2 double bond, and
1555
Fig. 6. Inhibition effects of the isolated compounds against
ferric ion-induced rat liver lipid oxidation. Rat liver
homogenate in 0.02 M Tris-HCl buffer (pH 7.4) was incubated
with 100 µM FeSO4 and 100 µM ascorbic acid at 37oC for 1 h. 1,
Dihydroferulic acid methyl ester; 2, pyrocatechol; 3, syringic acid;
4, apigenin; 5, isorhamnetin; 6, kaempferol; 7, dihydroferulic acid;
8, vanillic acid; 9, 4-hydroxybenzoic acid; 10, acetophlorglucine;
12, naringenin; 13, quercetin; 14, luteolin; 15, 9-epi-blumenol C;
16, scopoletin; 17, dihydroisorhamnetin; 18, chrysoeriol. Toc (α-
tocopherol) was used as a positive control. a-g, results with a
different letter differ significantly (p<0.05)
C-3 hydroxyl group of the C ring are important active sites
in the antioxidant activity of flavonoids (42,43). This study
confirms that the catechol structure of the B ring is a very
important factor for the excellent DPPH radical-scavenging
activities of quercetin (13) and luteolin (14). However,
compounds 8, 9, 12, and 15-17 did not scavenge DPPH
radicals at the same concentration (20 µM) under the
reaction conditions used.
Inhibition effects of the isolated compounds against
ferric ion-induced lipid oxidation in rat liver The
inhibition effects of the identified compounds (1-10 and
12-18) and α-tocopherol as a positive control at 25 µM
against ferric ion-induced rat liver lipid oxidation were
evaluated by measuring contents of TBARS produced (Fig.
6). The results showed a similar pattern with those in the
DPPH radical-scavenging experiment. That is, quercetin
(13) and luteolin (14) showed higher inhibition than α-
tocopherol (Toc) as a positive control. Quercetin (13)
exhibited higher antioxidative activity than luteolin (14).
Pyrocatechol (2), isorhamnetin (5), and kaempferol (6)
showed slightly lower inhibition than α-tocopherol (Toc),
but their effects were relatively higher in comparison with
those of other compounds (1, 3, 4, 7-10, 12, and 15-18).
In this study, 18 antioxidative compounds were isolated
and identified from the aerial part of S. japonica. To the
best of our knowledge, these compounds are the first
identified from this plant. It is well known that phenolic
1556
compounds, including flavonoids, are widely distributed in
various plants. 9-epi-Blumenol C (15) and its glycosides
have been isolated from plants including barley, wheat, rye,
and oat (41,44). From the results of antioxidative evaluation
for 18 antioxidative compounds identified from powdered
S. japonica juice, quercetin (13) and luteolin (14)
significantly scavenged DPPH radicals and inhibited lipid
oxidation in rat liver homogenates induced by ferric ions.
They are well known as excellent free radical-scavengers
and metal chelators (20,42,44,45). These results indicate
that quercetin (13) and luteolin (14) mainly contribute to
the antioxidative activity of S. japonica. Although other
phenolic compounds (1-11, 16-18) showed low or no
antioxidative activities in the model systems used in this
study, they exerted high antioxidative activities in other
assay systems (46,47). Therefore, these phenolic compounds
may be partially responsible for the antioxidative activity
of S. japonica. In addition, the results obtained in this study
may warrant further studies to understand the chemical
constituents and biological effects of S. japonica, and to
provide very useful information related to the utilization of
this plant as a crop resource and food material.
Antioxidative phenolic compounds are well known to
exert various biological effects such as anti-inflammatory,
anticancer, cardiovascular protection, and anti-aging effects
in human (48). Phenolic compounds in plants have also
been reported to be synthesized and accumulated as
biochemical defense molecules in response to biotic and
abiotic stresses such as salinity (49). The phenolic
compounds (1-14, 16-18) identified as antioxidative
compounds in this study may play a role in the adaptation
of S. japonica to severe saline environments. In our
previous study, flavonol glycosides and dicaffeoylquinic
acid derivatives have been isolated as antioxidative
compounds from glasswort (20), which has similarities in
morphological and saline growth characteristics with S.
japonica. Interestingly, antioxidative compounds isolated
from S. japonica and glassworts were found to be very
different, although investigations of the chemical constituents
of these plants have not yet been fully performed until now.
These plants may synthesize and accumulate antioxidative
phenolic compounds by their different defense mechanisms
in response to salt triggered-oxidative stress. To further
understand the chemical constituents of S. japonica and
glasswort as succulent halophytes and potent health
beneficial materials, investigation of chemical profile using
isolated compounds as external standards is in progress.
Acknowledgments This study was carried out with the
support of “Cooperative Research Program for Agricultural
Science & Technology Development (Project No.
PJ009366022013)”, Rural Development Administration,
Republic of Korea. NMR spectroscopy was performed by
Cho et al.
the Korea Basic Science Institute, Gwangju, Korea.
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