Triterpene Saponins from the Sea Cucumber Stichopus chloronotus

Abstract

Sea cucumbers have been used as a dietary delicacy and importantingredient in Asian traditional medicine and functional foods over many centuries. Using combined chromatographic methods, six triterpene saponins (1-6), including a new compound, stichloroside F (1), were isolated from a methanol extract of the sea cucumber Stichopus chloronotusBrandt. Their structures were determined on the basis of spectroscopic (1H and 13C NMR, HSQC, HMBC, 1H-1H COSY, ROESY) and FTICR-MS data and by comparison with literature values.

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Department of Organic Chemistry,
University of Granada,
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Dipartimento di Chimica Bioorganicae Biofarmacia,
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State Key Laboratory of Natural and Biomimetic Drugs,
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School of Pharmacy,
Tokyo University of Pharmacy and Life Sciences,
Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan
mimakiy@ps.toyaku.ac.jp
PROFESSOR STEPHEN G. PYNE
Department of Chemistry
University of Wollongong
Wollongong, New South Wales, 2522, Australia
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Department of Chemistry,
Texas Christian University,
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Department of Chemistry
The University of Alabama in Huntsville
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Faculty of Pharmaceutical Sciences
Hokuriku University
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PROFESSOR DAVID E. THURSTON
Department of Pharmaceutical and Biological Chemistry,
The School of Pharmacy,
University of London, 29-39 Brunswick Square,
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@p
harmac
y
.ac.uk
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Triterpene Saponins from the Sea Cucumber Stichopus chloronotus
Nguyen Phuong Thao
a,b
, Bui Thi Thuy Luyen
a,b
, Le Thi Vien
a
, Bui Huu Tai
a
, Le Duc Dat
a
,
Nguyen Xuan Cuong
a
, Nguyen Hoai Nam
a
, Phan Van Kiem
a
, Chau Van Minh
a,*
and Young Ho Kim
b,*
a
Institute of Marine Biochemistry (IMBC), Vietnam Academy of Science and Technology (VAST),
18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam
b
College of Pharmacy, Chungnam National University, Daejeon 305–764, Republic of Korea
cvminh@vast.ac.vn (Minh, C.V.); yhk@cnu.ac.kr (Kim, Y. H.)
Received: January 6
th
, 2014; Accepted: February 17
th
, 2014
Sea cucumbers have been used as a dietary delicacy and important ingredient in Asian traditional medicine and functional foods over many centuries. Using
combined chromatographic methods, six triterpene saponins (16), including a new compound, stichloroside F (1), were isolated from a methanol extract of
the sea cucumber Stichopus chloronotus Brandt. Their structures were determined on the basis of spectroscopic (
1
H and
13
C NMR, HSQC, HMBC,
1
H-
1
H
COSY, ROESY) and FTICR-MS data and by comparison with literature values.
Keywords: Stichopus chloronotus, Stichopodidae, Sea cucumber, Stichloroside F.
Sea cucumbers belonging to the family Stichopodidae (phylum
Echinodermata, class Holothurioidea, order Aspidochirotida) are
usually served as a culinary delicacy and traditional tonic. Among
the members of this family, Stichopus chloronotus Brandt is a
marine invertebrates found in benthic areas and deep seas in the
Pacific, Indo-Pacific, and Atlantic oceans [1]. Sea cucumbers
represent one of several marine organisms used as food, particularly
among the Asian population [2-3]. They have also been popular as a
traditional tonic in Japan, Vietnam, China, Taiwan, and Korea [4-7].
Chemical investigation of S. chloronotus dates back to the 1980s
when its fatty acid [8], glycosphingolipid [9], and triterpene
oligoglycoside (saponin) [10-12] components were reported to have
antifungal [11], antitumor, cytotoxic [13], anticoagulant,
antioxidation, antithrombotic, and anticancer activities [3-14]. Most
of the known sea cucumber glycosides have a lanostane aglycone
(so called holostane aglycone) with an 18(20)-lactone [15] and a
sugar chain composed of up to six monosaccharide units linked to
C-3 of the aglycone [12].
During our ongoing investigations to catalog the chemical
constituents of Vietnamese echinoderms [16-19], we studied the
Figure sea cucumber S. chloronotus. The present study addresses
the isolation and structure elucidation of six holostane glycosides
(16, Figure 1), including a new compound, stichloroside F (1),
from this sea cucumber.
A methanol extract (10.45 g) of S. chloronotus was suspended in
water and partitioned successively with n-butanol. Six holostane
glycosides (16) were isolated from the n-butanol residue (2.42 g)
by using combined chromatographic methods. Detailed analysis of
the spectroscopic data (1D, 2D NMR, and MS) and comparison
with previously reported values led to the elucidation of the known
compounds as stichoposide D (2) [20-21], stichloroside A
2
(3) [20],
stichoposide E (4) [20], neothyonidioside (5) [22], and holothurin B
(6) [23]. Notably, compound 5 was isolated for the first time from a
Stichopus species, and from all other representatives of the family
Stichopodidae studied so far.
Figure 1: Structures of compounds 16 from the sea cucumber S. chloronotus.

NPC Natural Product Communications
2014
Vol. 9
No. 5
615 - 618

616 Natural Product Communications Vol. 9 (5) 2014 Thao et al.
Table 1:
1
H and
13
C NMR spectroscopic data of compound 1 in pyridine-d
5
.
Position δ
C
a
δ
H
b
mult. (J in Hz)
Position δ
C
a
δ
H
b
mult. (J in Hz)
Aglycon Xyl I
1 36.3 1.48 m
1
107.3 4.78 d (8.0)
2 27.2 1.95 m/2.20 m
2
75.2 4.00
c
3 89.1 3.39 dd (4.0, 11.5)
3
76.0 4.16
c
4 39.5 -
4
76.8 4.27
c
5 48.1 1.08 m
5
64.5 3.71
c
/4.46
c
6 23.3 2.02 m Xyl II
7 119.9 5.68 m
1
103.3 4.91 d (8.0)
8 146.9 -
2
72.4 4.03
c
9 47.5 3.52 br d (14.0)
3
86.8 4.14
c
10 36.6 -
4
69.3 4.04
c
11 23.0 1.52 m/1.78 m
5
66.6 3.60 dd (3.0, 11.0)
12 30.5 1.95 m/2.10 m 4.29
c
13 58.6 - MeGlc
14 51.4 -
1
105.3 5.28 d (7.5)
15 34.3 1.70 m/1.80 m
2
75.1 3.99
c
16 25.2 1.93 m/2.06 m
3
88.0 3.85
c
17 53.7 2.44 dd (4.5, 10.5)
4
70.5 4.15
c
18 180.2 -
5
78.3 3.95 m
19 24.6 1.20 s
6
62.1 4.26
c
/4.45
c
20 84.9 -
3-OMe
60.8 3.85 s
21 28.1 1.82 s
22 47.6 2.00 m/2.13 m
23 65.4 4.00 m
24 49.2 1.30 m/1.65 m
25 24.0 2.04 m
26 23.8 0.95 d (6.5)
27 22.1 0.98 d (6.5)
28 17.6 1.07 s
29 28.9 1.33 s
30 31.0 1.11 s
a
125 MHz;
b
500 MHz;
c
Overlapped signals; All assignments were made by HSQC,
COSY, HMBC, and ROESY experiments.
Figure 2: Important
1
H-
1
H COSY and HMBC correlations of compound 1.
Stichloroside F (1) was obtained as a white amorphous powder. Its
molecular formula, C
47
H
76
O
17
(contained ten degrees of
unsaturation), was identified from a pseudo-molecular ion peak at
m/z 935.49804 [M+Na]
+
in the Fourier transform ion cyclotron
resonance mass spectrum (FTICR-MS). The IR spectrum showed an
absorption band due to a γ-lactone moiety (1762 cm
1
) and strong
broad absorptions (3384 and 1074 cm
1
) reminiscent of a glycosidic
structure. Acid hydrolysis of 1 with 10% HCl produced
stichlorogenol [10] and two
D-xylose and one 3-O-methyl-D-
glucose moieties, characterized by GC-MS analysis of their
persilylated derivatives (see Experimental). The NMR features
indicated a triterpene saponin, one of the main constituents of sea
cucumbers [14]. The
13
C NMR spectrum exhibited 47 carbon
signals, including 30 aglycone carbon signals similar to those of
stichlorogenol [10]. The presence was detected of a trisubstituted
double bond [δ
C
119.9 (CH, C-7)/δ
H
5.68 (1H, m, H-7) and 146.9
(C, C-8)], one γ-lactone carbonyl group [δ
C
180.2 (C, C-18)], seven
methyls [δ
C
24.6 (CH
3
, C-19)/δ
H
1.20 (3H, s, H-19); δ
C
28.1 (CH
3
,
C-21)/δ
H
1.82 (3H, s, H-21); δ
C
23.8 (CH
3
, C-26)/δ
H
0.95 (3H, d,
J = 6.5 Hz, H-26); δ
C
22.1 (CH
3
, C-27)/δ
H
0.98 (3H, d, J = 6.5
Hz,H-27); δ
C
17.6 (CH
3
, C-28)/δ
H
1.07 (3H, s, H-28); δ
C
28.9 (CH
3
,
Figure 3: Key ROESY correlations of the aglycone portion of compound 1.
C-29)/δ
H
1.33 (3H, s, H-29); δ
C
31.0 (CH
3
, C-30)/δ
H
1.11 (3H, s,
H-30)], two oxymethines [δ
C
89.1 (CH, C-3)/δ
H
3.39 (1H, dd, J =
4.0, 11.5 Hz, H-3); δ
C
65.4 (CH, C-23)/δ
H
4.00 (1H, m, H-23)], and
an oligosaccharide chain composed of three sugar units (Table 1).
Most sea cucumber triterpene glycosides have a 18(20)-lactone
structure and either a Δ
7
- or Δ
9(11)
-double bond in holostane
aglycones [24-25]. Assignment of the NMR data of 1 was further
confirmed by the HMBC analysis. The HMBC cross-peaks between
H-6 (δ
H
2.02) and C-7 (δ
C
119.9)/C-8 (δ
C
146.9) and H-30 (δ
H
1.11)
and C-8 (δ
C
146.9), clearly indicated the position of the double bond
at C-7/C-8. The γ-lactone moiety was assigned at C-18 due to the
obvious HMBC correlations of H-12 (δ
H
1.95/2.10) and H-17 (δ
H
2.44) with C-18 (δ
C
180.2). Detailed analysis of the other HMBC
and
1
H-
1
H COSY peaks (Figure 2) unambiguously determined the
planar structure of the aglycone.
In the ROESY spectrum, the correlation of H-3 (δ
H
3.39) with H-5
(δ
H
1.08) and that of H-17 (δ
H
2.44) with H-21 (δ
H
1.82) and H-30
(δ
H
1.11) suggested an α-orientation for both H-3 and H-17 (Fig. 3).
The S-configuration at C-23 was suggested by the coexistence of
compounds 14 in S. chloronotus, which was further supported by
an agreement of the
13
C NMR chemical shift at C-23 (δ
C
65.4) of 1
with that of variegatuside B (from S. variegatus having 23S
configuration) at δ
C
65.6 (C-23) [26].
In addition, analysis of the NMR spectra of 1 revealed three
anomeric carbon signals at δ
C
107.3 (C-1), 103.3 (C-1), and 105.3
(C-1), which correlated with corresponding anomeric protons at
δ
H
4.78 (1H, d, J = 8.0 Hz, H-1), 4.91 (1H, d, J = 8.0 Hz, H-1),
and 5.28 (1H, d, J = 7.5 Hz, H-1) in the HSQC spectrum,
confirming the presence of three sugar moieties. The large coupling
constants of the anomeric protons (J = 7.5 or 8.0 Hz) suggested the
presence of β-glycosidic linkages. The positions of attachment of
the sugar units were determined by HSQC, COSY, HMBC, and
ROESY experiments. Detailed analysis of the correlations in the
COSY spectrum allowed the assignment of proton positions for
all of the sugar moieties (Figure 2) with their configurations
identified by acid hydrolysis of 1 followed by GC-MS analysis.
These NMR data and the HMBC correlations of H-1 (δ
H
4.78) with
C-3 (δ
C
89.1), H-1 (δ
H
4.91) with C-4 (δ
C
76.8), and H-1
(δ
H
5.28) with C-3 (δ
C
86.8) clearly indicated the attachments of
the first
D-xylose at C-3, the second D-xylose at C-4, and 3-O-
methyl-
D-glucose at C-3. Thus, the structure of stichloroside F (1)
was elucidated as 3β,23(S)-dihydroxyholost-7-ene 3-O-[3-O-
methyl-β-D-glucopyranosyl-(13)-β-D-xylopyranosyl-(14)-β-D-
xylopyranoside].

Triterpene saponins from the sea cucumber Stichopus chloronotus Natural Product Communications Vol. 9 (5) 2014 617
Experimental
General: Optical rotations were determined on a JASCO P-2000
polarimeter (Hachioji, Tokyo, Japan). IR spectra were obtained on a
Bruker TENSOR 37 FT-IR spectrometer (Bruker Optics, Ettlingen,
Germany). The high resolution mass spectra were gained using a
Varian 910 FT-ICR mass spectrometer (Varian, CA, USA). The
NMR spectra were recorded on Bruker AM500 (Billerica, MA,
USA) and JEOL ECA 600 (Tokyo, Japan) FT-NMR spectrometers.
TMS was used as an internal standard. GC-MS was carried out on a
Shimadzu-2010 spectrometer: detector, FID; detection temperature,
300
o
C; capillary column SPB
TM
-1 (0.25 mm i.d.×30 m); column
temperature, 230
o
C; carrier gas, He (2 mL/min), injection
temperature, 250
o
C; injection volume, 0.5 μL. Column
chromatography (CC) was performed on silica gel (Kieselgel 60,
70–230 mesh and 230–400 mesh, Merck, Darmstadt, Germany) and
YMC RP-18 resins (30–50 μm, Fuji Silysia Chemical Ltd., Kasugai,
Aichi, Japan). TLC used pre-coated silica gel 60 F
254
(1.05554.0001, Merck, Darmstadt, Germany) and RP-18 F
254S
plates
(1.15685.0001, Merck, Darmstadt, Germany), and compounds were
visualized by spraying with aqueous 10% H
2
SO
4
and heating for
35 min.
Biological material: The sample of the sea cucumber S. chloronotus
Brandt was collected at Cat Ba, Haiphong, Vietnam, in November
2011, and identified by Professor Do Cong Thung (Institute of
Marine Environment and Resources, VAST). A voucher specimen
(SC-11-2011_01) was deposited at the Institute of Marine
Biochemistry and Institute of Marine Environment and Resources,
VAST, Vietnam.
Extraction and isolation: The fresh body walls of S. chloronotus
(0.6 kg) were cut into small pieces and immersed in hot methanol (3
times for 6 h each) to afford a MeOH extract (10.45 g, A) after
removal of the solvent under reduced pressure. This extract was
partitioned between H
2
O and n-butanol, 3 times (0.7 L each). The n-
butanol soluble portion (2.42 g, B) was subjected to CC over silica
gel (230‒400 mesh) eluting with a gradient (dichloromethane‒
methanol 10:1, 3:1, 1:1, v/v.). Combination of similar fractions on
the basis of TLC analysis afforded 3 fractions (Fr. B1‒B3). Fraction
B3 (0.45 g) was further separated by reverse-phase silica (75 μm)
MPLC eluting with a H
2
O‒CH
3
OH (35–65%) gradient into two
fractions (Fr. B3.1–B3.2). Subfraction B3.2 (0.27 g) was gel-filtered
on Sephadex LH-20 (CH
3
OH‒H
2
O, 4.5:1) followed by silica gel CC
(CH
2
Cl
2
‒CH
3
OH‒H
2
O, 1.8:1:0.2) to yield glycosides 2 (34.11 mg,
5.68×10
-3
% of fresh weight) and 4 (27.56 mg, 4.59×10
-3
% of fresh
weight). Subfraction B3.1 (0.18 g) was subjected to silica gel CC
with CH
2
Cl
2
–CH
3
OH–H
2
O (2.5:1:0.15) and further separated by
YMC RP-18 CC using CH
3
OH–H
2
O (3.5:1, v/v) as the eluent to
afford 3 (18.81 mg, 3.13×10
-3
% of fresh weight) and 5 (12.25 mg,
2.04×10
-3
% of fresh weight) as a white solid. Next, fraction B2
(0.62 g) was further subjected to silica gel CC with a CH
2
Cl
2
–
MeOH–H
2
O (65:15:2–10:10:2) gradient to obtain 3 subfractions
(Fr. B2.1–B2.3). Subfraction B2.1 (0.33 g) was further separated by
YMC RP-18 CC using acetonewater (2:1, v/v) as eluent to give 6
(15.82 mg, 2.64×10
-3
% of fresh weight). Finally, compound 1
(15.78 mg, 2.63×10
-3
% of fresh weight) was purified as a white
amorphous powder from subfraction B2.2 (0.29 g) following a two-
stage separation beginning with silica gel CC eluting with
CH
2
Cl
2
MeOHH
2
O (4:1:0.1, v/v), followed by YMC CC eluting
with MeOHwater (3:1, v/v).
Acid hydrolysis and determination of absolute configuration of
monosaccharides: Compound 1 (3 mg) was heated in 3 mL of 10%
HCl‒dioxane (1:1) at 80
o
C for 3 h. After the solvent was removed
in vacuo, the residue was partitioned between EtOAc and H
2
O to
give the aglycone and the sugar, respectively. The sugar
components in the aqueous layer were analyzed by silica gel TLC
by comparison with standard sugars. The solvent system was
CH
2
Cl
2
‒CH
3
OH‒H
2
O (1.5:1:0.2), and compounds were visualized
by spraying with 95% EtOH‒H
2
SO
4
‒anisaldehyde (7:0.5:0.5, v/v),
and heating at 180
o
C for 5 min. The R
f
values of D-xylose and 3-O-
methyl-
D-glucose by TLC were 0.30 and 0.65, respectively. The
results were confirmed by GC-MS analysis. The aqueous layer was
evaporated to dryness to give a residue, which was dissolved in
anhydrous pyridine (100 μL), and then mixed with a pyridine
solution of 0.1 M
L-cysteine methyl ester hydrochloride (100 μL).
After warming at 60
o
C for 2 h, trimethylsilylimidazole solution was
added, and the reaction solution was warmed at 60
o
C for 2 h. The
mixture was evaporated in vacuo to give a dried product, which was
partitioned between n-hexane and H
2
O. The n-hexane layer was
filtered and analyzed by GC. The retention times of the persilylated
monosaccharide derivatives were as follows: D-xylose (t
R
, 5.78),
and 3-O-methyl-
D-glucose (t
R
, 8.19 min). Their identities were
confirmed by comparison with authentic standards.
Stichloroside F (1)
White amorphous powder


24
D
α
: –18.9 (c 0.26, MeOH);
IR (KBr): ν
max
3384, 2954, 1762, 1074, 972 cm
1
;
FTICR-MS: m/z 935.49804 [M+Na]
+
(Calcd. for C
47
H
76
O
17
Na:
935.49802);
1
H (Pyridine-d
5
, 500 MHz) and
13
C NMR (Pyridine-d
5
, 125 MHz):
Table 1.
Acknowledgment - This study was supported by Vietnam Ministry
of Science and Technology (MOST), The World Academy of
Science (RGA No: 12-066 RG/CHE/AS_G-UNESCO FR:
3240271332), and the Priority Research Center Program through the
National Research Foundation of Korea (NRF) funded by the
Ministry of Education, Science and Technology (2009-0093815),
Republic of Korea. The authors are grateful to the Institute of
Chemistry, VAST for the provision of the spectroscopic instrument.
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Natural Product Communications Vol. 9 (5) 2014
Published online (www.naturalproduct.us)
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Antimicrobial Activity of Ammodaucus leucotrichus Fruit Oil from Algerian Sahara
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In Vitro Cytotoxic Activity Guided Essential Oil Composition of Flowering Twigs of Stevia rebaudiana
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Natural Product Communications
2014
Volume 9, Number 5
Contents
Gerald Blunden Award (2013) Page
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Prediction of Anticancer Activity of Diterpenes Isolated from the Paraiban Flora through a PLS Model and Molecular Surfaces
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Cladieunicellin J, a New Hydroperoxyeunicellin from Cladiella sp.
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Wei-Hsien Wang, Yang-Chang Wu and Ping-Jyun Sung 613
Triterpene Saponins from the Sea Cucumber Stichopus chloronotus
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Ferulaldehyde and Lupeol as Direct and Indirect Antimicrobial Compounds from Cordia gilletii (Boraginaceae) Root Barks
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Continued inside backcover