Evaluation of novel antioxidant triterpenoid saponins from the halophyte Salicornia herbacea

Article abstract of DOI:10.1016/j.bmcl.2012.05.017

As a part of an ongoing search for novel antioxidants from the salt marsh plants, bioactivity-isolation and structure determination of constituents from Salicornia herbacea were performed. One new triterpenoid saponin (4), along with three known saponins (1-3), has been isolated from n-BuOH fraction of S. herbacea. On the basis of the spectroscopic methods, the structure of the new saponin 4 was elucidated as 3β-hydroxy-23-oxo-30-noroleana-12, 20(29)-diene-28-oic acid 3-O-β-d-glucuronopyranosyl-28-O-β-d- glucopyranoside. Scavenging effects of saponins 1-4 were examined on 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical and peroxynitrite. Particularly, saponin 3 exerted significant antioxidant activity on both authentic peroxynitrite and peroxynitrite generated from morpholinosydnonimine (SIN-1).

Full text of DOI:10.1016/j.bmcl.2012.05.017

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4318–4322
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Evaluation of novel antioxidant triterpenoid saponins from the halophyte
Salicornia herbacea
You Ah Kim a, Chang-Suk Kong b, Jung Im Lee c, Hojun Kim c, Hee Yeon Park d, Hyi-Seung Lee e,
Chulhyun Lee f, Youngwan Seo c,
⇑
a Natural Compounds Bank Establishment Department, Korea Promotion Institute for Traditional Medicine Industry, Gyeongbuk 712-260, Republic of Korea
b Department of Food and Nutrition, College of Medical and Life Science, Silla University, Busan 617-736, Republic of Korea
c Division of Marine Environment and Bioscience, Korea Maritime University, Busan 606-791, Republic of Korea
d Food and Safety Research Center, National Fisheries Research and Development Institute, Busan 619-705, Republic of Korea
e Marine Natural Product Chemistry Laboratory, Ocean Research and Development Institute, Ansan 425-600, Republic of Korea
f Magnetic Resonance Team, Korea Basic Science Institute, Eoun-dong 52, Yuseong-gu, Daejeon 305-333, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
As a part of an ongoing search for novel antioxidants from the salt marsh plants, bioactivity-isolation and
structure determination of constituents from Salicornia herbacea were performed. One new triterpenoid
saponin (4), along with three known saponins (1–3), has been isolated from n-BuOH fraction of S. herb-
acea. On the basis of the spectroscopic methods, the structure of the new saponin 4 was elucidated as
Received 10 January 2012
Revised 29 April 2012
Accepted 7 May 2012
Available online 11 May 2012
3b-hydroxy-23-oxo-30-noroleana-12, 20(29)-diene-28-oic acid 3-O-b-D-glucuronopyranosyl-28-O-b-D-
glucopyranoside. Scavenging effects of saponins 1-4 were examined on 1,1-diphenyl-2-picryl-hydrazyl
(DPPH) radical and peroxynitrite. Particularly, saponin 3 exerted significant antioxidant activity on both
authentic peroxynitrite and peroxynitrite generated from morpholinosydnonimine (SIN-1).
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Salicornia herbacea
Saponin
DPPH
Peroxynitrite
In recent years, the role of reactive oxygen/nitrogen species
(ROS/RNS) has attracted great attention due to their oxidation pro-
cesses occurring in various biological systems.1,2 The ROS/RNS
comprehends free radicals and non-radicals. For example, there
are superoxide (O2Åꢀ), hydroxyl (HOÅ), hydroperoxyl (HO2Å), nitric
oxide (ÅNO) and nitrogen dioxide (ÅNO2) radical in free radicals,
and hydrogen peroxide (H2O2) and peroxynitrite anion (ONOOꢀ)
in non-radicals, respectively.1,3 Because oxidation processes of
ROS/RNS usually progress under severe control by several mecha-
nisms in the body, imbalanced oxidative stresses result in several
diseases such as cancer, Alzheimer’s disease, neurodegeneration
and cardiovascular disease.1,4–6 Therefore, there is a growing inter-
est in searching for natural antioxidants due to their lower toxici-
ties than synthetic antioxidants.7–10
Salicornia herbacea L. (also known as glasswort) is annual succu-
lent herb of chenopodiaceae family and one of the most salt toler-
ant plants. It is growing on salt marshes and muddy seashores
along the western coast of Korea11–14 and is widespread in Europe,
Japan and Iran.15–17 It contains large amounts of minerals, essential
amino acid and essential fatty acid.18–21 It has been used as not
only a seasoned vegetable, salad and fermented food in coastal
areas of Europe, Japan and Korea, but also as folk medicine for dis-
orders such as constipation, obesity and diabetes.22,12 Recently, the
consumption of the plants has been extended into the functional
food and medicinal plant due to its beneficial effects.20,21 However,
studies on its secondary metabolite have rarely been re-
ported.9,14,23–26
In our continuing search for bioactive compounds from the salt
marsh plants, we recently reported the isolation and peroxynitrite-
scavenging activities of two flavonoid glycosides from S. herba-
cea.14 In addition to these two compounds, an 1H NMR spectral
analysis of C18 vacuum flash chromatographic fractions for the n-
BuOH layer of crude extracts revealed the presence of a structur-
ally different group of metabolites. Large-scale extraction of organ-
ic materials followed by bioactivity-guided separation using
combined chromatographic techniques yielded four saponin
derivatives. Herein we report the isolation and structure determi-
nation of one new saponin (4), together with three known ones
(1–3) (Fig. 1) and their DPPH radical and peroxynitrite scavenging
activities.
The data were presented as mean SD. Differences between the
means of the individual groups were assessed by one-way ANOVA
with Duncan’s multiple range tests. Differences were considered
significant at p <0.05.
The collected halophyte S. herbacia was processed to yield four
solvent-partitioned fractions including n-hexane, 85% aqueous (aq)
⇑
Corresponding author. Tel.: +82 51 410 4328; fax: +82 51 404 3538.
E-mail address: ywseo@hhu.ac.kr (Y. Seo).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.05.017

Y. A. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4318–4322
4319
150
ONOO-
SIN-1
a
a
a
a
a
COOR2
a
100
50
0
b
b
b
1: R1 = Glucuronic acid, R2 = H,
R3 = CHO
R1O
2: R1 = Glucuronic acid, R2 = Glucose, R3 = CHO
c c
c
R3
L-ascorbic
acid
Penicill
-amine
H2O
n-BuOH 85% aq. n-hexane
MeOH
COOR2
Solvent fractions
Figure 2. Peroxynitrite scavenging activities of each solvent fraction at a concen-
tration of 50
g/mL.14 a–cMeans with the different letters in the same reactive
species are significantly different (p <0.05) by Duncan’s multiple range test.
l
3: R1 = Glucuronic acid, R2 = Glucose, R3 = CH2OH
4: R1 = Glucuronic acid, R2 = Glucose, R3 = CHO
4a: R1 = triacetyl Glucuronic acid, R2 = tetraacetyl Glucose,
R3 = CHO
R1O
R3
Saponin 4 was obtained as an amorphous white powder. Its
molecular formula (C41H60O15) was analyzed by a combination of
negative HR-FABMS and 13C NMR analyses. In the 1H NMR spec-
Figure 1. Chemical structure of saponins 1–4 isolated from Salicornia herbacea.
MeOH, n-BuOH and water.27 Their antioxidant activities were as-
sessed by measuring the DPPH radical and peroxynitrite scaveng-
ing activities.28,29 Among the solvent fractions, 85% aq MeOH and
n-BuOH fractions showed potent DPPHꢁscavenging activity with
trum, signals for two anomeric protons at
d 4.18 (1H, d,
J = 7.7 Hz) and 5.36 (1H, d, J = 8.2 Hz) were correlated with signals
for two carbons at d 104.6 and 95.8 ppm by an HMQC experiment,
and were diagnostic for the presence of two sugar molecules. Care-
ful examination of the 1H and 13C NMR spectroscopic data revealed
that saponin 4 possessed the same A–D rings as saponin 2. How-
ever, there were significant differences in the 13C NMR spectrum.
Signals of two methyl carbons at d 33.5 (C-29) and 24.0 (C-30),
and quaternary carbon at d 31.5 (C-20) in saponin 2 were replaced
by the olefinic signals at d 107.6 (CH2) and 149.5 (C) in saponin 4.
Corresponding differences were found in the 1H NMR spectrum, in
which signals of methyl protons at d 0.90 (s, H-29) and 0.92 (s, H-
30) in saponin 2 were replaced by the exomethylene proton signals
at d 4.62 (1H, s) and 4.61 (1H, s) in saponin 4. These differences
were explained by formation of a double bond between C-20 and
C-29, suggesting the aglycone part of saponin 4 as 3b-hydroxy-
23-oxo-30-noroleana-12,20(29)-diene-28-oic acid, which has pre-
viously been reported.38 This interpretation was also supported
by comparison of the NMR spectral data with E ring moiety of
saponin 3. It was verified by sugar analysis and its NMR spectral
data that the sugar moieties of saponin 4 were the same as those
of 2 and 3.
IC50 values of 15.6 and 40.9 lg/mL, respectively, which is compara-
ble with positive control butylated hydroxytoluene (BHT) (IC50
,
39.8 g/mL) (Table 1). Also, scavenging ratios of 85% aq MeOH
l
and n-BuOH fractions were 70.8% and 71.6% for authentic perox-
ynitrite, and 99.5% and 99.8% for induced peroxynitrite from SIN-
1 at a concentration of 50 lg/mL, respectively, as reported in our
previous study (Fig. 2).14 Induced peroxynitrite is ONOOꢀ formed
Å
by NO and O2Åꢀ, which are generated from SIN-1.
To find the possible scavenging components on ROS/RNS, fur-
ther purification of n-BuOH fraction followed by bioactivity-guided
separation using combined chromatographic techniques yielded
four saponins (1–4),30,31 including one new saponin. The structures
of the isolated saponins were identified on the basis of spectro-
scopic analyses including 1H and 13C NMR spectroscopy, and veri-
fied by comparison with reported spectral data.32 The chemical
structures of the purified compounds are illustrated in Fig. 1.
Saponins 1–3 were identified by a combination of spectroscopic
analysis and comparison with the literature data as gypsogenin 3-
O-b-D-glucuronopyranoside, gypsogenin 3-O-b-D-glucuronopyrano-
Acid hydrolysis of saponin 4 followed by TLC and direct com-
parison with standard sugars indicated the presence of glucose
and glucuronic acid.30 These results were confirmed by GC anal-
ysis of the alditol peracetates and by comparison to the stan-
syl-28-O-b-
D
-glucopyranoside, and 30-norhederagenin 3-O-b-
D-glu-
curonopyranosyl-28-O-b-D
-glucopyranoside, respectively.32–37
dard. In addition, acetylation of saponin
4 resulted in a
Table 1
DPPH radical scavenging activities of solvent fractions from Salicornia herbacea
downfield shift of all sugar protons except those attached to car-
bons involved in the sugar-aglycone, which were then present in
a much less overlapped part of the 1H NMR spectrum. The cyclic
Concentrations (
l
g/mL)
IC50 (lg/mL)
10
50
100
structures of monosaccharides were determined to be
onopyranose and -glucopyranose by 1D TOCSY and HMBC data
on the acetylated product (saponin 4a). A linkage of aglycone be-
tween -glucuronopyranose and -glucopyranose was assigned
to be between C-3 and C-10, and between C-28 and C-100 on
the basis of long-range correlation in HMBC experiment,
respectively. Thus, the structure of saponin 4 was unambigu-
ously determined as 3b-hydroxy-23-oxo-30-noroleana-
12,20(29)-diene-28-oic acid 3-O-b- -glucuronopyranosyl-28-O-b-
-glucopyranoside.
D-glucur-
94.9 0.25a,* 95.1 0.24a 95.0 0.25a 10<<
L
-Ascorbic acid
D
BHA
BHT
63.2 1.84b
39.7 3.01d
16.1 4.20f
33.8 3.31e
79.1 1.05b 86.9 0.66b 10<<
54.9 2.25c 68.0 1.60c 39.8
D
D
H2O fr.
n-BuOH fr.
19.7 4.01d 25.7 3.72d
—
52.4 2.38c 85.2 0.74b 40.9
56.9 2.16c 70.0 1.50c 15.6
85% aq MeOH fr. 49.3 2.53c
a
n-Hexane fr.
15.6 4.22f
19.6 4.02d 27.7 3.62d
—
*
DPPH radical scavenging activities (%).
a–f
Means with the different letters in the same concentration are significantly
different (p <0.05) by Duncan’s multiple range test.
D
D

4320
Table 2
Y. A. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4318–4322
centration of 50
was relatively low (Fig. 3). Especially, saponin 3 exhibited the low-
est value of IC50 (<<1
M) for both authentic ONOOꢀ and induced
lg/mL, but that of monodesmosidic saponin (1)
DPPH radical and peroxynitrite scavenging activities of saponins 1–4 isolated from
Salicornia herbacea
l
ONOOꢀ from SIN-1. The activity differences between these deriva-
tives were likely due to the number of hydroxy group present in
each of them. Although it’s difficult to explain clearly a scavenging
effect of saponins on peroxynitrite, it’s apparent that all of sapo-
nins 1–4 have much more scavenging effect on peroxynitrite than
DPPH radical. Thus, the present study demonstrated that the per-
oxynitrite-scavenging effect of S. herbacea is at least partly attrib-
uted to saponins 1–4. Furthermore, from 85% aq MeOH,
purification of peroxynitrite-scavenging compounds is in progress.
In conclusion, it was found that S. herbacea is capable of scav-
enging the DPPH radical and peroxynitrite. In addition, four sapo-
nins (1–4), including one new saponin 4 were isolated from n-
BuOH fraction by bioactivity-guided separation with chromato-
graphic techniques. Their structures were determined by extensive
2D NMR experiments and by comparison with known compounds,
and their antioxidant activities were evaluated on peroxyntrite.
Saponin 3 showed the strongest scavenging effect on peroxynitrite.
Therefore, this study suggests that saponins 1–4 have potential for
their uses as natural antioxidants, which might be useful in the
prevention of ROS/RNS and in the treatment of pathological pro-
cesses such as aging, cancer and cardiovascular disease.
DPPH
Authentic ONOOꢀ
SIN-1
IC50 (mM)
IC50
(
l
M)
IC50
(lM)
<<0.02
1.5
<<1
L
-Ascorbic acid
BHA
BHT
<<0.1
<<0.2
Penicillamine
Saponin 1
Saponin 2
Saponin 3
Saponin 4
<<1
21.9
7.1
<<1
4.9
<<1
20.4
1.4
<<1
6.6
>>0.5
>>0.5
0.39
>>0.5
150
A
50 µg/mL 25 µg/mL 5 µg/mL
a
a
ab
b
ab
bc
100
50
cd
d
a
c
b
c
d
e
e
d
d
e
Acknowledgments
This research was financially supported by
a Grant (No.
0
L-ascorbic Penicill
20100293) from Technology Development Program for Fisheries,
Ministry for Food, Agriculture, Forestry and Fisheries, Republic of
Korea and is the outcome of a Manpower Development Program
for Marine Energy by the Ministry of Land, Transport and Maritime
Affairs (MLTM).
1
2
3
4
acid
-amine
1
3
Saponins
150
B
50 µg/mL
25 µg/mL
5 µg/mL
a
a
Supplementary data
b
a
a
a
Supplementary data associated with this article can be found, in
100
50
0
b
d
the
online
version,
at
http://dx.doi.org/10.1016/j.bmcl.
b
c
b
c
bc
d
2012.05.017.
d
c
c
References and notes
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d
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acid -amine
1
2
3
4
4
L-
1
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Bioeng. 2004, 19, 57.
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Figure 3. Peroxynitrite scavenging activities of saponins 1–4 on (A) authentic
ONOOꢀ and (B) induced ONOOꢀ from SIN-1.a–eMeans with the different letters in
the same concentration are significantly different (p <0.05) by Duncan’s multiple 10. Pokorny, J. Eur. J. Lipid Sci. Technol. 2007, 109, 629.
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Environ. Sci. 2010, 8, 79.
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19. Kim, M. W. Korean J. Nutr. 2007, 40, 403.
Potent antioxidant activity of four saponins isolated from the n-
BuOH fraction was evaluated by measuring DPPH radical and per-
oxynitrite scavenging activities (Table 2). To the best of our knowl-
edge, this is the first report on the DPPHÅ and ONOOꢀ scavenging
activities of these compounds. Saponins 1–4 showed relatively
good activity on authentic ONOOꢀ and induced ONOOꢀ from mor-
20. Cho, Y. S.; Kim, S. I.; Han, Y. S. Korean J. Food Sci. Technol. 2008, 40, 326.
21. Rhee, M. H.; Park, H. J.; Cho, J. Y. J. Med. Plant. Res. 2009, 3, 548.
22. Greenway, H.; Munns, R. Annu. Rev. Plant Physiol. 1980, 31, 149.
23. Han, S. K.; Kim, S. M.; Pyo, B. S. Korean J. Food Sci. Anim. Res. 2003, 23, 46.
pholinosydnonimine (SIN-1), comparable with those of L-ascorbic
acid and penicillamine, while they showed no significant effect
on DPPH radical. Among saponins, activities of bidesmosidic sapo-
nins 2–4 on peroxynitrites were similar to each other at the con-

Y. A. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4318–4322
4321
24. Min, J. G.; Lee, D. S.; Kim, T. J.; Park, J. H.; Cho, T. Y.; Park, D. I. J. Food Sci. Nutr.
Norhederagenin 3-O-b-
D
-glucuronopyranosyl-28-O-b-
D
-glucopyranoside (3):
2002, 7, 105.
white amorphous solid, mp 220–240 °C. ½a D24
ꢃ
+3.7o (c = 1.14, MeOH). IR (KBr)
25. Im, S. A.; Kim, G. W.; Lee, C. K. Nat. Prod. Sci. 2003, 9, 273.
tmax cmꢀ1: 3336 (br), 2943, 2834, 1640, 1450, 1415, 1021 and 610 cmꢀ1. H-
NMR (600 MHz, CD3OD): 5.37 (1H, d, J = 8.2 Hz, H-100), 5.32 (1H, t, J = 3.1 Hz, H-
12), 4.62 (1H, s, H-29), 4.61 (1H, s, H-29), 4.44 (1H, d, J = 7.8 Hz, H-10), 3.80 (1H,
dd, J = 11.8, 1.5 Hz, H-600), 3.68 (1H, m, H-3), 3.67 (1H, dd, J = 11.5, 4.8 Hz, H-600),
3.64 (1H, d, J = 11.5 Hz, H-23), 3.46–3.29 (7H, m, H-30, -40, -50, -200, -300, -400, -500),
3.27 (1H, d, J = 11.5 Hz, H-23), 3.26 (1H, dd, J = 9.2, 7.8 Hz, H-20), 2.73 (1H, dd,
J = 13.5, 4.1 Hz, H-18), 2.56 (1H, J = 14.5, 13.1, H-19), 2.26–2.11 (3H, m, H-16, -
21), 2.08 (1H, dd, J = 13.5, 4.1, H-19), 1.98–1.87 (3H, m, H-2, -11, -22), 1.87–
1.81 (2H, m, H-15, -16), 1.76 (1H, m, H-2), 1.66–1.46 (4H, m, H-6, -7, -9, -22),
1.40–1.21 (3H, m, H-5, -6, -7), 1.13 (1H, m, H-15), 0.98 (1H, m, H-6), 1.20 (3H, s,
H-27), 0.98 (3H, s, H-25), 0.80 (3H, s, H-26), 0.70 (3H, s, H-24); 13C NMR
spectroscopic data29; HMBC correlations: H-3/C-4, C-23, C-24, C-10; H-12/C-9,
C-11, C-14, C-18; H-18/C-12, -13, -16, -17, -19, -28; H-19/C-18, -21, -20, -29;
H-23/C-3, -4, -5, -24; H-24/C-3, -4, -5, -23; H-25/C-1, C-5, C-9, C-10; H-26/C-7,
C-8, C-9, C-14; H-27/C-8, C-14, C-15; H-29/C-19, C-21; H-10/C-3; H-20/C-10; H-
100/C-28; H-600/C-400, C-500. Negative HR-FABMS m/z: 793.4013 (MꢀH)ꢀ (Calcd
26. Park, S. H.; Kim, K. S. J. Korean Soc. Appl. Biol. Chem. 2004, 47, 120.
27. Extraction and isolation of bioactive compounds: The Halophyte Salicornia
herbacea was collected in Daebudo, Ansan, Korea in July 2004. The collected
samples were briefly dried under shade and kept at ꢀ25 °C until use. The air-
dried sample of S. herbacea (480 g) was chopped into small pieces and soaked
for 2 days with MeOH (3 L ꢂ 2) and CH2Cl2 (3 L ꢂ 2), sequentially. The
combined crude extracts (164 g) were evaporated under reduced pressure
and partitioned between CH2Cl2 and water. The organic layer was further
partitioned between 85% aq MeOH and n-hexane, and then the aqueous layer
was fractionated with n-BuOH and H2O, successively, to afford the n-hexane
(10.7 g), 85% aq MeOH (39.6 g), n-BuOH (53.7 g) and water (50.8 g) fractions. A
portion of n-BuOH (50 g) fraction was separated into 6 subfractions by C18
(YMC–GEL ODS-A, 12 nm, S-75 lm) reversed-phase vacuum flash
chromatography eluting with stepwise gradient mixtures of MeOH and H2O
(50%, 60%, 70%, 80%, 90% aq MeOH and 100% MeOH). The fourth fraction was
further separated on
a
silica gel column chromatography with gradient
for C41H61O15: 793.4010). 3b-Hydroxy-23-oxo-30-noroleana-12,20(29)-diene-
mixtures of MeOH and CHCl3. The fraction eluted with 20% MeOH in CHCl3
(1.078 g) was further separated by reversed-phase HPLC (YMC ODS-A, 80% aq
MeOH, 1 cm x 25 cm, 2 mL/min) to yield saponin 1 (9.8 mg). The fraction
eluted with 50% MeOH in CHCl3 (0.548 g) was separated by reversed-phase
HPLC (YMC ODS-A, 50% aq MeOH, 1 ꢂ 25 cm, 2 mL/min) to give saponins 3
(26.0 mg), 4 (13.5 mg), and 2 (9.8 mg) in order of elution.
28-oic acid 3-O-b-
D
-glucuronopyranosyl-28-O-b-
D
-glucopyranoside (4): white
+16.4o (c = 1.35, MeOH). IR (KBr) tmax
: .
cmꢀ1 3343 (br), 2930, 2850, 1670, 1560, 1210 and 670 cmꢀ1 1H NMR
amorphous solid, mp 235–245 °C. ½a D24
ꢃ
(600 MHz, CD3OD): 9.42 (1H, s, H-23), 5.36 (1H, d, J = 8.2 Hz, H-100), 5.33 (1H, t,
J = 3.3 Hz, H-12), 4.62 (1H, s, H-29), 4.61 (1H, s, H-29), 4.18 (1H, d, J = 7.7 Hz, H-
10), 3.95 (1H, dd, J = 11.6, 4.5 Hz, H-3), 3.80 (1H, br d, J = 11.5 Hz, H-600), 3.67
(1H, dd, J = 11.5, 4.8 Hz, H-600), 3.51 (1H, d, J = 9.8 Hz, H-500), 3.43–3.37 (2H, m,
H-30, -300), 3.36–3.29 (4H, m, H-30, -200, -400, -500), 3.12 (1H, dd, J = 9.2, 7.7 Hz, H-
20), 2.74 (1H, dd, J = 13.1, 4.6 Hz, H-18), 2.56 (1H, J = 13.5, 13.5 Hz, H-19), 2.26–
2.11 (3, m, H-16, -21), 2.09 (1H, dd, J = 14.5, 4.6, H-19), 2.04 (1H, m, H-2), 1.96–
1.88 (3H, m, H-11, -22), 1.86–1.73 (3H, m, H-2, -15, -16), 1.72–1.66 (2H, m, H-1,
-9), 1.58–1.46 (3H, m, H-6, -7, -22), 1.35 (1H, br d, J = 10.2 Hz, H-5), 1.24 (1H, m,
H-7), 1.12 (2H, m, H-1, -15), 0.91 (1H, m, H-6), 1.21 (3H, s, H-27), 1.10 (3H, s, H-
24), 1.00 (3H, s, H-25), 0.81 (3H, s, H-26); 13C NMR spectroscopic data29; HMBC
correlations: H-3/C-2, C-4, C-23, C-24, C-10; H-5/C-4, C-6, C-7, C-10, C-23, C-24;
H-12/C-9, C-11, C-14, C-18; H-18/C-12, C-13, C-16, C-17, C-19, C-28; H-19/C-
13, C-18, C-20, C-21, C-29; H-23/C-5, C-24; H-24/C-3, C-4, C-5, C-23; H-25/C-1,
C-5, C-9, C-10; H-26/C-7, C-8, C-9, C-14; H-27/C-8, C-13, C-14, C-15; H-29/C-
19, C-20, C-21; H-10/C-3, C-30, C-50; H-20/C-10, C-30; H-50/C-30, C-40, C-60; H-100/
C-28, C-300; H-600/C-400, C-500. Negative HR-FABMS m/z: 791.3819 (M-H)- (Calcd
for C41H59O15: 791.3854). Peracetylation of saponin 4: Saponin 4 (5 mg) in
pyridine (0.3 mL) was treated separately with Ac2O (0.2 mL) for 24 h at room
temperature. After removing the pyridine and excess acetic anhydride under
vacuum, the residue was purified by reversed-phase HPLC (100% MeOH) to
yield 4a (3.5 mg): a colorless gum; 1H NMR (600 MHz, CD3OD), d 9.32 (1H, s, H-
23), 5.68 (1H, d, J = 8.3 Hz, H-100), 5.35 (1H, t, J = 3.0 Hz, H-12), 5.34 (1H, dd,
J = 10.3, 8.3 Hz, H-300), 5.17 (1H, dd, J = 9.8, 9.3 Hz, H-30), 5.11 (1H, dd, J = 9.8,
9.3 Hz, H-40), 5.06 (1H, dd, J = 8.3, 8.3 Hz, H-200), 5.05 (1H, dd, J = 10.3, 10.3 Hz,
H-400), 4.81 (1H, dd, J = 9.3, 8.4 Hz, H-20), 4.62 (1H, s, H-29), 4.61 (1H, d,
J = 8.4 Hz, H-10), 4.59 (1H, s, H-29), 4.27 (1H, dd, J = 12.5, 4.1 Hz, H-600), 4.04
(1H, dd, J = 12.5, 2.2 Hz, H-600), 3.98 (1H, dd, J = 10.3, 4.1, 2.2 Hz, H-500), 3.89 (1H,
d, J = 9.3 Hz, H-500), 3.86 (1H, dd, J = 11.7, 4.4 Hz, H-3), 2.69 (1H, dd, J = 13.2,
4.4 Hz, H-18), 2.54 (1H, t, J = 13.2 Hz, H-19), 2.26–1.91 (6H, m, H-2, -11, -16, -
19, -21), 2.03 (3H, s, OAc), 2.01 (3H, s, OAc), 2.00 (3H, s, OAc), 1.99 (3H, s, OAc),
1.98 (3H, s, OAc), 1.97 (3H, s, OAc), 1.94 (3H, s, OAc), 1.86 (1H, m, H-22), 1.81–
1.64 (4H, m, H-1, -2, -9, -16), 1.62–1.46 (3H, m, H-6, -7, -15), 1.43 (1H, m, H-
22), 1.34 (1H, br d, J = 10.2 Hz, H-5), 1.21 (3H, s, H-27), 1.19 (3H, m, H-1, -7, -
15), 0.97 (3H, s, H-25), 0.95 (3H, s, H-24), 0.88 (1H, m, H-6), 0.76 (3H, s, H-26).
31. 13C NMR spectral assignments for saponins 1–4 from Salicornia herbacea.
28. DPPH radical scavenging effect: Aliquots of extracts or pure compounds in
MeOH solution were added to DPPHꢁ methanol solution (1.5 ꢂ 10ꢀ1 M). After
gentle mixing and holding for 30 min at room temperature, the optical density
was measured at 520 nm.
L-ascorbic acid and penicillamine, BHA and BHT were
used as a positive control.
29. ONOOꢀ scavenging ability by monitoring the ONOOꢀ-induced oxidation of
dihydrorhodamine 123 (DHR 123) to rhodamine 123: A stock solution of DHR
123 (5 mM) was purged with nitrogen and stored at ꢀ80 °C.
A working
solution of DHR 123 (final conc. 5 M) diluted from the stock solution was
l
placed on ice in the dark immediately prior to the determinations. The buffer
containing 90 mM sodium chloride, 50 mM sodium phosphate (pH 7.4) and
5 mM potassium chloride was purged with nitrogen and placed on ice before
use. At the beginning step of the experiments, diethylenetriaminepentaacetic
acid (DTPA) (final conc. 100 l
M) was added to the buffer. The ONOOꢀ
scavenging activity was determined at room temperature with a microplate
fluorescence spectrophotometer FL 500 (Bio-Tek instruments, USA) using
excitation and emission wavelengths of 485 and 530 nm. The background and
final fluorescent intensities were measured 5 min after treatment with or
without SIN-1 (final conc. 10 l lM) in
M) or authentic ONOOꢀ (final conc. 10
0.3 M sodium hydroxide. Oxidation of DHR 123 by decomposition of SIN-1
gradually increased whereas authentic ONOOꢀ rapidly oxidized DHR 123 with
its final fluorescent intensity being stable over time.
penicillamine were used as a positive control.
L-ascorbic acid and
30. Acid hydrolysis of saponins 2–4: each compound (3 mg) in MeOH (2 mL) was
individually treated with an equal volume of 1 N HCl under reflux for 2 h and
then extracted with CH2Cl2 (3 ꢂ 20 mL). The aqueous was dried under a stream
of nitrogen at 40 °C. Aliquots of 1 M NH4OH and 5% NaBH4 (each 1 mL) were
added to each sample and the solution incubated at 40 °C for 90 min. After
addition of AcOH (0.1 mL) to eliminate excess NaBH4, the mixture was
concentrated to dryness and the AcOH codistilled with MeOH (2 ꢂ 10 mL). The
resulting alditols were acetylated by refluxing for 6 h with Ac2O/pyridine (1:1,
5 mL). The reaction mixture was quenched with water and extracted with
CH2Cl2 and identified by GC-MS as those of glucose and glucuronic acid.
Sapogenin of each compound was separated from each organic layer by
preparative TLC on silica gel (CHCl3–MeOH, 9:1) and confirmed by comparison
with spectral data in the literature. Gypsogenin 3-O-b-
D
-glucuronopyranoside
(1): white amorphous solid, mp 180–195 °C. ½a D24
ꢃ
+16.7o (c = 0.72, MeOH). IR
No
1a
2b
3a
4a
(KBr) tmax cmꢀ1
: 3340, 2938, 2810, 1690, 1470, 1300, 1215, 1020 and
1
2
3
4
5
6
7
8
39.2t
25.5t
83.0d
56.3s
48.9d
21.4t
33.2t
41.0s
48.9d
36.9s
23.9t
123.5d
144.8s
43.0s
28.8t
24.5t
39.2t
25.8t
83.5d
56.1s
49.0d
21.4t
33.6t
40.9s
48.8d
37.0s
23.9t
123.3d
145.0s
43.0s
28.8t
24.5t
39.6t
26.3t
82.2d
43.9s
48.2d
24.2t
33.4t
40.7s
49.0d
37.7s
24.6t
124.3d
144.3s
43.0s
28.9t
24.2t
39.4t
25.7t
82.8d
56.5s
48.8d
21.5t
33.3t
41.2s
48.9d
37.1s
24.6t
124.5d
144.4s
43.1s
29.0t
24.2t
680 cmꢀ1 1H NMR (300 MHz, CD3OD): 9.39 (1H, s, H-23), 5.24 (1H, t,
.
J = 3.3 Hz, H-12), 4.24 (1H, d, J = 7.2 Hz, H-10), 3.86 (1H, dd, J = 11.4, 4.3 Hz,
H-3), 3.74 (1H, d, J = 9.1 Hz, H-50), 3.46 (1H, dd, J = 9.6, 9.1 Hz, H-40), 3.30 (1H,
dd, J = 9.6, 9.1 Hz, H-30), 3.11 (1H, dd, J = 9.1, 7.7 Hz, H-20), 2.83 (1H, dd, J = 13.8,
3.9 Hz, H-18), 2.60–1.86 (4H, m, H-2, -11, -16), 1.84–1.63 (6H, m, H-1, -2, -9, -
15, -19, -22), 1.63–1.42 (4H, m, H-6, -7, -16, -22), 1.42–1.26 (2H, m, H-5, -21,),
1.26–1.02 (5H, m, H-1, -7, -15, -19, -21), 1.18 (3H, s, H-27), 1.12 (3H, s, H-24),
1.00 (3H, s, H-25), 0.94 (3H, s, H-30), 0.91 (3H, s, H-29), 0.91 (1H, m, H-6), 0.82
(3H, s, H-26); 13C NMR spectroscopic data29
.
Negative HR-FABMS m/z:
645.3637 (MꢀH)ꢀ (Calcd for C36H53O10
:
645.3639). Gypsogenin 3-O-b-D-
glucuronopyranosyl-28-O-b-D-glucopyranoside (2): white amorphous solid, mp
9
213–230 °C. ½a 2D4
ꢃ
ꢀ6.6o (c = 1.07, MeOH). IR (KBr)
t
max cmꢀ1: 3308, 2975, 2832,
.
1H NMR (600 MHz, CD3OD):
10
11
12
13
14
15
16
1660, 1450, 1415, 1090, 1025, 880 and 630 cmꢀ1
9.41 (1H, s, H-23), 5.36 (1H, d, J = 8.3 Hz, H-100), 5.25 (1H, t, J = 3.2 Hz, H-12),
4.20 (1H, d, J = 7.8 Hz, H-10), 3.92 (1H, dd, J = 11.5, 4.1 Hz, H-3), 3.81 (1H, br d,
J = 11.2 Hz, H-600), 3.67 (1H, dd, J = 11.2, 3.9 Hz, H-600), 3.57 (1H, m, H-500), 3.46–
3.38 (3H, m, H-40, -50, -300), 3.38–3.29 (3H, m, H-30, -200, -400), 3.12 (1H, dd, J = 8.3,
7.8 Hz, H-20), 2.84 (1H, dd, J = 13.2, 3.9 Hz, H-18), 2.08–1.85 (4H, m, H-2, -11, -
16), 1.81–1.56 (7H, m, H-1, -2, -15, -16, -19, -22), 1.54–1.43 (2H, m, H-6, -7),
1.43–1.25 (2H, m, H-5, -21), 1.25–1.01 (4H, m, H-1, -7, -15, -21), 1.16 (3H, s, H-
27), 1.09 (3H, s, H-24), 0.98 (3H, s, H-25), 0.92 (3H, s, H-30), 0.90 (3H, s, H-29),
0.83 (1H, m, H-6), 0.78 (3H, s, H-26); 13C NMR spectroscopic data29. Negative
(continued on next page)
HR-FABMS m/z: 807.4167 (M-H)- (Calcd for C42H63O15
: 807.4170). 30-

4322
Y. A. Kim et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4318–4322
32. Borel, C.; Gupta, M. P.; Hostettmann, K. Phytochemistry 1987, 26, 2685.
(continued)
33. Yoshikawa, K.; Arihara, S.; Wang, J.-D.; Narui, T.; Okuyama, T. Chem. Pharm.
Bull. 1991, 39, 1185.
No
1a
2b
3a
4a
34. Jia, Z.; Koike, K.; Kudo, M.; Li, H.; Nikaido, T. Phytochemistry 1998, 48, 529.
35. Acebes, B.; Diaz-Lanza, A. M.; Bernabe, M. Phytochemistry 1998, 49, 2077.
36. Ikuta, A.; Itokawa, H. Phytochemistry 1989, 28, 2663.
37. Espada, A.; Riguera, R. J. Nat. Prod. 1997, 60, 17.
38. Park, S.-Y.; Chang, S.-Y.; Oh, O.-J.; Yook, C.-S.; Nohara, Y. Phytochemistry 2002,
59, 379.
17
18
19
20
21
22
23
24
25
26
27
28
29
30
10
48.0s
42.6d
47.1t
31.5s
34.8t
47.6s
42.7d
47.2t
31.6s
34.9t
33.3t
208.7d
10.5q
16.1q
17.7q
26.5q
181.5s
33.8q
24.0q
105.0q
74.8d
77.4d
73.0d
76.5d
172.5s
—
48.4s
48.6d
42.6t
149.4s
30.9t
38.4t
64.8t
13.4q
16.5q
17.8q
26.4q
177.4s
107.4t
—
48.3s
48.6d
42.7t
149.5s
31.0t
38.5t
209.5d
10.5q
16.3q
17.9q
26.5q
177.5s
107.6t
—
33.1t
209.6d
10.4q
16.2q
17.7q
26.4q
178.3s
33.5q
24.0q
104.5d
75.0d
77.6d
73.5d
76.6d
178.3s
95.7d
73.8d
78.1d
71.0d
78.6d
62.3t
105.0d
75.0d
78.1d
73.6d
76.5d
177.0s
95.8d
73.9d
78.2d
71.1d
78.6d
62.4t
104.6d
75.3d
77.9d
73.7d
76.5d
177.0s
95.8d
74.0d
78.3d
71.2d
78.8d
62.6t
20
30
40
500
600
100
200
300
4
—
—
—
—
500
600
—
a,bMeasured in CD3OD at 150 and 75 MHz, respectively.