Two new holostan-type triterpene glycosides from the sea cucumber Bohadschia marmorata Jaeger

Article abstract of DOI:10.1248/cpb.56.1207

Two new holostan-type triterpene glycosides, 17-hydroxy fuscocineroside B (1) and 25-hydroxy fuscocineroside B (2), together with a known triterpene glycoside, fuscocineroside B (3) were isolated from the sea cucumber Bohadschia marmorata JAEGER. The structures of the new triterpene glycosides were elucidated on the basis of spectroscopic analyses and chemical reactions. Compounds 1 and 3 showed considerable antifungal activities against six strains.

Full text of DOI:10.1248/cpb.56.1207

August 2008
Notes
Chem. Pharm. Bull. 56(8) 1207—1211 (2008)
1207
Two New Holostan-Type Triterpene Glycosides from the Sea Cucumber
Bohadschia marmorata JAEGER
a
Weihua YUAN, Yanghua YI, ,a Haifeng TANG,b Mei XUE,a Zenglei WANG,a Guoquan SUN,a
*
Wen ZHANG,a Baoshu LIU,a Ling LI,a and Peng SUN
a
a Research Center for Marine Drugs, School of Pharmacy, Second Military Medical University; 325 Guohe Road,
Shanghai 200433, P.R. China: and b Department of Pharmacy, Xijing Hospital, Fourth Military Medical University; Xi’an
710032, P. R. China. Received April 18, 2008; accepted May 23, 2008; published online May 27, 2008
Two new holostan-type triterpene glycosides, 17-hydroxy fuscocineroside B (1) and 25-hydroxy fuscocinero-
side B (2), together with a known triterpene glycoside, fuscocineroside B (3) were isolated from the sea cucumber
Bohadschia marmorata JAEGER. The structures of the new triterpene glycosides were elucidated on the basis of
spectroscopic analyses and chemical reactions. Compounds 1 and 3 showed considerable antifungal activities
against six strains.
Key words sea cucumber; Bohadschia marmorata; triterpene glycoside, antifungal active
In order to find new biologically active substances from (MS and 2D-NMR) with literature values.5)
marine organisms, we have been studying kinds of sea cu-
17-Hydroxy fuscocineroside B (1), colorless amorphous
cumbers.1—6) Triterpene glycosides are the predominant sec- powder, was positive to Liebermann–Burchard and Molish
ondary metabolites of the sea cucumber, and exhibit wide tests. The molecular formula was established as
spectra of biological activity, such as antifungal, cytotoxic ef- C54H85O27NaS by the pseudomolecular ion [MꢀNa]ꢀ at m/z
fects,4,7) and they are usually of the lanosterol-type with a 1243.4805 in the HR-ESI-MS (positive-ion mode). A frag-
18(20)-lactone and the sugar chain of up to six monosaccha- ment ion peak at m/z 1123 [MꢀNaꢁNaHSO4]ꢀ in the posi-
ride units is generally linked to the C-3 of the aglycone, tive ion mode ESI-MS indicated the presence of a sulfate
which are composed by D-xylose, D-quinovose, D-glucose, group in 1, which was confirmed by the IR spectrum with ab-
and 3-O-methyl-D-glucose.8) In this paper, we report the iso- sorption bands at 1265 and 1205 cmꢁ1. The IR spectrum also
lation and the antifungal activity of the three glycosides from showed the presence of hydroxyl (3421 cmꢁ1), carbonyl
the sea cucumber Bohadschia marmorata JAEGER (family Bo- (1775, 1717 cmꢁ1), olefinic (1651 cmꢁ1).
1
hadschia) [distribute abundantly in the South China Sea],
The H-, 13C-NMR, and DEPT spectra displayed reso-
nances due to seven tertiary methyl groups, one olefinic bond
(dH 5.69; dC 154.3, 115.5), one lactone carbonyl group (dC
173.8), and one doublet (dH 5.06; dC 71.1) that was ascribed
and structural elucidation of the new triterpene glycosides.
Results and Discussion
The 60% EtOH extracts of B. marmorata JAEGER (3.5 kg, to a methine proton linked to a carbon bearing a hydroxy
dried) were suspended in H2O, partitioned with n-BuOH. The group and suggested that the aglycone of 1 had a holostane
n-BuOH extract was sequentially subjected to column chro- triterpenoid skeleton with a 9(11)-en-12-ol moiety.9)
matography on Si gel and ODS, and followed by HPLC on a
The location of the ketone carbonyl group at C-22 was de-
Silasorb C-18 column to afford the pure 17-hydroxy fus- duced from the chemical shift of the C-22 signal, which
cocineroside B (1), 25-hydroxy fuscocineroside B (2) and showed long-range correlation with the protons at dH 2.00 (s,
fuscocineroside B (3).
CH3-21) in the HMBC spectrum. The aglycone of 1 is simi-
The known compound 3 was identified as fuscocineroside lar to that of fuscocineroside B in spite of a hydroxyl instead
B, originally isolated from the sea cucumber Holothuria fus- of a a-H at C-17.5) The signal of C-17 was shifted downfield
cocinerea, by comparison of physical and spectroscopic data from 46.9 to 87.6 ppm. The carbon C-17 (dC 87.6) showed
long-rang correlation with the protons at dH 2.00 (s, CH3-21)
and dH 5.06 (12-CH) in the HMBC spectrum.
The relative stereochemistry of all chiral centers of the
aglycone was established with the aid of a NOESY experi-
ment. Thus, as is depicted in Fig. 2, H-3 showed correlations
with H-1 (Xyl), H-1a (1.47 ppm), H-5a and H-28 confirm-
ing the b configuration at C-3. The 12a configuration of the
hydroxyl group was confirmed by a cross-peak at dH 5.06
(H-12)/2.00 (H-21) in the NOESY spectrum and from the
coupling constant for H-12 with H-11 (4.0 Hz).7) A correla-
tion between H-12 and H-21 in the NOESY spectrum, evi-
denced the a configuration of the hydroxyl group at C-17,10)
see Fig. 2. Complete assignments were achieved by studying
the results of HMQC, HMBC, COSY, TOCSY and NOESY
experiment (see Table 1, Fig. 2). Thus, the structure of the
Fig. 1. The Structures of Compounds 1—3
aglycone part of 1 was identified as a 22-oxo-9(11)-
∗ To whom correspondence should be addressed. e-mail: yiyanghua@126.com
© 2008 Pharmaceutical Society of Japan

1208
Vol. 56, No. 8
Fig. 2. The Key NOESY Correlations and Relative Configuration of 1
Table 1. 1H- and 13C-NMR Chemical Shifts and Selected HMBC Correlations for the Aglycone Moiety of 1 and 2 (in Pyridine-d5, 400/100 MHz)
1
2
C
d
H mult. (J in Hz)
dC
HMBC
d
H mult. (J in Hz)
dC
HMBC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
1.47, 1.90 m
2.02, 2.19 m
3.22 (overlap)
—
1.07 m
1.56, 1.80 m
1.58, 1.81 m
3.37 m
—
36.5
27.2
88.9
40.1
52.8
21.3
28.5
41.1
154.3
39.9
115.5
71.1
58.7
46.4
36.9
39.2
87.6
173.8
22.7
92.3
21.6
208.7
37.2
32.6
27.9
22.6
22.6
28.2
16.9
20.0
1.51, 1.93 m
2.20 m
3.22 (overlap)
—
1.07 m
1.78 m
1.78 m
3.39 m
—
36.6
27.2
88.9
40.2
52.9
21.4
29.0
40.3
153.3
39.8
116.1
67.9
64.0
46.7
37.2
27.4
47.0
176.9
22.7
90.6
25.1
211.6
35.3
37.4
69.0
30.2
29.9
28.2
16.9
22.0
4,28, 29, 1ꢃ
4,28, 29, 1ꢃ
3, 4, 6, 10, 19, 28
3, 4, 6, 10, 19, 28
7, 9, 11, 14, 30
7, 9, 11, 14, 30
—
—
5.69 d (4.0)
5.06 d (4.0)
—
8, 10, 12, 13
9, 11, 14, 18
5.79 d (4.1)
4.63 d (4.1)
—
8, 10, 12, 13
9, 11, 14, 18
—
—
1.47, 1.89 m
2.24, 2.51 m
—
—
1.45 s
1.47, 1.64 m
2.25 m
3.73 m
—
1.47 s
—
1.87 s
—
3.24 m
2.20 m
—
1.48 s
1.48 s
1.32 s
1.13 s
1.30 s
1, 5, 9, 10
17, 20, 22
1, 5, 9, 10
17, 20, 22
—
2.00 s
—
2.87, 2.98 m
1.70 m
1.62 m
0.95 d (6.5)
0.95 d (6.5)
1.32 s
24, 25, 27
24, 25, 26
3, 4, 5, 29
3, 4, 5, 28
8, 13, 14, 15
24, 25, 27
24, 25, 26
3, 4, 5, 29
3, 4, 5, 28
8, 13, 14, 15
1.12 s
1.72 s
holostene-3b,12a,17a-triol.
for four anomeric protons and their corresponding carbons,
The sugar moieties of 1 were determined to be D-xylose, dH 4.76 (dC 105.4), dH 5.12 (dC 105.6), dH 5.06 (dC 105.0)
D-glucose, D-quinovose and 3-O-methyl-D-glucose in a and dH 5.42 (dC 106.0). All anomeric protons were doublet
3
1 : 1 : 1 : 1 ratio by acidic hydrolysis with aqueous 2 M trifluo- with JH-1/H-2ꢂ6.4—7.6 Hz, the large vicinal coupling con-
roacetic acid and preparation of the corresponding aldononi- stants of each anomeric proton indicated a trans-diaxial ori-
trile peracetates, which were analyzed by GC-MS using stan- entation with respect to their coupling partners (b-configura-
dard aldononitrile peracetates as reference samples. The tion).
common D-configuration for the four carbohydrate units was
Complete assignment was achieved by studying the results
assumed also according to those most often encountered of HMQC, HMBC, COSY and TOCSY experiment (Table
among the sea cucumber glycosides.11,12)
2). The HMQC experiment correlated all proton resonances
1
The H-, 13C-NMR and DEPT spectra displayed signals with those of their corresponding carbons. The COSY exper-

August 2008
1209
Table 2. 1H- and 13C-NMR Chemical Shifts and Selected HMBC Correlations for the Sugar Moiety of 1 and 2 (in Pyridine-d5, 400/100 MHz)
1
2
C
d
H mult. (J in Hz)
dC
HMBC
d
H mult. (J in Hz)
dC
HMBC
Xyl
1
2
3
4
4.76 d (6.4)
4.12 m
4.36 m
5.20 m
3.84, 4.79 m
105.4
83.5
75.4
76.0
64.4
C-3 (Aglycone)
4.76 d (6.7)
4.12 m
4.38 m
5.20 m
3.81, 4.83 m
105.4
83.5
75.4
76.0
64.4
C-3 (Aglycone)
5
4-O-SO3Na
Qui
1
2
3
4
5
6
Glc
1
2
3
4
5
5.12 d (6.8)
4.22 m
4.07 m
3.76 m
3.82 m
105.6
75.5
76.5
86.8
72.1
18.2
C-2 (Xyl)
5.12 d (7.1)
4.19 m
4.06 m
3.73 m
3.82 m
105.5
75.5
76.4
86.8
72.1
18.2
C-2 (Xyl)
1.79 d (4.9)
C-4, 5 (Qui)
C-4 (Qui)
1.79 d (5.2)
C-4, 5 (Qui)
C-4 (Qui)
5.06 d (7.0)
4.20 m
4.37 m
4.23 m
4.12 m
105.0
74.2
88.1
69.6
77.8
61.9
5.06 d (7.3)
4.16 m
4.36 m
4.16 m
4.12 m
105.0
74.2
88.1
69.7
77.8
62.0
6
4.33, 4.53 m
4.31, 4.55 m
3OMeGlc
1
2
3
4
5
5.42 d (7.6)
4.13 m
3.78 m
4.16 m
4.08 m
106.0
75.1
88.1
70.7
78.4
62.2
61.0
C-3 (Glc)
5.41 d (7.7)
4.12 m
3.78 m
4.16 m
4.07 m
106.0
75.2
88.1
70.8
78.4
62.3
61.0
C-3 (Glc)
6
4.34, 4.55 m
3.94 s
4.33, 4.56 m
3.94 s
OMe
iment allowed the sequential assignment of most of the reso- group in 2, which was confirmed by the IR spectrum with ab-
nances for each sugar ring, staring from the easily distin- sorption bands at 1262 and 1208 cmꢁ1. The IR spectrum also
guished signals due to anomeric protons. By the HMBC ex- showed the presence of hydroxyl (3421 cmꢁ1), carbonyl
periment, the location of the interglycosidic linkages was de- (1773, 1716 cmꢁ1), olefinic (1651 cmꢁ1).
duced, the signals of them were downfield relative to shifts
The aglycone of 2 differs from 3 by the presence of a hy-
expected for the corresponding methyl glycopyranosides: dC droxyl at C-25,5) the signals of C-24 (CH2), C-25 (C), C-26
83.5 (Xyl C-2), dC 86.8 (Qui C-4), dC 88.1 (Glc C-3),13) and (CH3) and C-27 (CH3) were shifted downfield to 37.4, 69.0,
a cross-peak between the H-1 of Xyl and C-3 of the aglycone 30.2 and 29.9 ppm. The HMBC spectrum showed cross-
indicated that the Xyl was connected to C-3 of the aglycone, peaks H3-26/C-25, H3-26/C-24, H3-27/C-25, H3-27/C-24, H3-
then the interglycosidic linkages could be deduced by the 26/C-27 and H3-27/C-26, together with the chemical shift of
same way. The site of linkage of the sulfate group in the C-25 supported the presence of the two methyl groups at-
sugar units was determined by studying the NMR chemical tached to C-25 bearing the hydroxyl group. Complete assign-
shifts of the sugar moiety, C-4 (Xyl) shifted downfield to ments were achieved by studying the results of HMQC,
76.0 ppm, indicating that the sulfate group was located at C-4 HMBC, COSY, and TOCSY experiment (Table 1). The a
of the xylose unit.5) The structure of the carbohydrate chain configuration of the H at C-17 was confirmed by cross-peaks
of 1 was corroborated by HMBC, COSY and NOESY corre- at dH 3.19 (H-17)/1.57 (H-21) and dH 3.19 (H-17)/1.28 (H-
lations, see Fig. 2.
30) in the NOESY spectrum10) (see Fig. 3). Thus, the struc-
Combined with NMR data, it was showed that 1 and 3 ture of the aglycone part of 2 was identified as a 22-oxo-
have the same sugar sequence.5) Thus, the structure of 1 9(11)-holostene-3b,12a, 25-triol.
was established to be 3-O-[3-O-methyl-b-D-glucopyranosyl-
Combined with acid hydrolysis and NMR (see Table 2), it
(1→3)-b-D-glucopyranosyl-(1→4)-b-D-quinovopyranosyl- was found that the compounds 1, 2 and 3 all have the same
(1→2)-4-O-sodiumsulfato-b-D-xylopyranosyl]-22-oxo- sugar sequence.5) Thus, the structure of 2 was established
9(11)-holostene-3b,12a,17a-triol.
to be 3-O-[3-O-methyl-b-D-glucopyranosyl-(1→3)-b-D-glu-
25-Hydroxy fuscocineroside B (2), colorless amorphous copyranosyl-(1→4)-b-D-quinovopyranosyl-(1→2)-4-O-so-
powder, was positive to Liebermann–Burchard and Molish diumsulfato-b-D-xylopyranosyl]-22-oxo-9(11)-holostene-
tests. The molecular formula was established as 3b,12a, 25-triol.
C54H85O27NaS by the pseudomolecular ion [MꢀNa]ꢀ at m/z
The Antifungal Activities Some triterpene glycosides
1243.4790 in the HR-ESI-MS (positive-ion mode). A frag- hitherto isolated from sea cucumber exhibited significant an-
ment ion peak at m/z 1123 [MꢀNaꢁNaHSO4]ꢀ in the posi- tifungal activity.7) In this paper, we report the antifungal ac-
tive ion mode ESI-MS indicated the presence of a sulfate tivities of three triterpene glycosides isolated from B. mar-

1210
Vol. 56, No. 8
Fig. 3. The Key NOESY Correlations and Relative Configuration of 2
Table 3. Antifungal Activity of the Compounds 1—3 (MIC80,a) mg/ml)
Candida albicans
Cryptococcus neoformans
Aspergillus fumigatus
Trichophyton rubrum
Candida tropicalis
Candida krusei
1
2
3
64
ꢅ64
64
16
ꢅ64
16
16
ꢅ64
16
16
ꢅ64
16
64
ꢅ64
64
64
ꢅ64
64
TBNF
KCZ
AMB
FCZ
4
1
0.0625
1
4
0.0625
0.0625
4
0.0625
0.0156
0.25
ꢅ64
0.25
2
0.0625
0.25
0.25
0.0625
0.25
0.25
ꢅ64
4
0.25
4
a) MIC80: the minimum concentration to inhibit ꢆ80% growth for 1—3 against six fungi strains.
heating Si gel plates sprayed with 10% H2SO4 in EtOH.
morata against six strains: Candida albicans, Cryptococcus
neoformans, Aspergillus fumigatus, Trichophyton rubrum,
Candida tropicalis and Candida krusei. Terbinafine (TBNF),
Ketoconazole (KCZ), Amphotericin B (AMB), Fluconazole
(FCZ) were used as positive controls, and the results are
shown in Table 3.
Earlier studies on the triterpene glycosides isolated from
B. bivittata revealed that the presence of the 12a-OH func-
tion seemed to be essential for exhibiting antifungal activi-
ties.7) As we see in Table 3, 1 differs from 3 by the presence
of a hydroxyl instead of a a-H at C-17, the antifungal activi-
ties were at the same level, but when the hydroxyl attaches to
C-25 in the side chain, the antifungal activities decreased re-
markably. It was envisioned that the better for the hydrophilic
property of the side chain, the lower for the antifungal activi-
ties.
Six strains: Candida albicans (ATCC76615), Cryptococcus neoformans
(ATCC32609), Aspergillus fumigates (Clinic Strains), Trichophyton rubrum
(Clinic Strains), Candida tropicalis (Clinic Strains) and Candida krusei
(Clinic Strains). Positive controls: Terbinafine (TBNF), Ketoconazole
(KCZ), Amphotericin B (AMB), Fluconazole (FCZ).
Animal Material Specimens of B. marmorata were collected from off-
shore waters of Hainan island in the South China Sea in March 2006, and
authenticated by Mr. Yu-Lin Liao (Institute of Oceanology, Chinese Acad-
emy of Science, P. R. China). A voucher specimen (No. BM-2006-3) was
deposited at the Research Center for Marine Drugs, School of Pharmacy,
Second Military Medical University, Shanghai.
Extraction and Isolation The dried sea cucumbers (3.5 kg) were
chopped and refluxed with 60% EtOH (3 lꢄ6, 3 h/time), and evaporated
under reduced pressure, and then suspended in 6 l water, partitioned with n-
BuOH for three times. The n-BuOH extract (90 g) was subjected to column
chromatography on Si gel eluting with a CHCl3 : MeOH : H2O (10 : 1 : 0.1;
9 : 1 : 0.1; 8 : 2 : 0.2; 7 : 3 : 0.5; 6 : 4 : 0.8) gradient to give 12 fractions. Frac-
tion 10 (11 g) was further purified by reversed-phase silica MPLC eluting
with an aq. CH3OH (40—80%) gradient to give 8 fractions. And then the
fraction 7 (0.5 g) was purified by HPLC (Zorbax 300 SB-C18; 70% aq.
MeOH, 1.5 ml/min) to afford the compounds 1 (16 mg), 2 (15 mg) and 3
(35 mg).
Experimental
General Melting points were determined on an XT5-XMT apparatus.
Optical rotations were measured with a Perkin-Elmer 341 polarimeter. IR
spectra were recorded on a Bruker Vector 22 infrared spectrometer. NMR
spectra were recorded in C5D5N on a Varian Inova-400 spectrometer, and the
2D NMR spectra were obtained using standard pulse sequences. ESI-MS
and HR-ESI-MS were recorded on a Micromass Quattro mass spectrometer.
GC/MS were performed on a Finnigan Voyager apparatus using a DB-5 col-
umn (30 mꢄ0.25 mm i.d., 0.25 mm) with an initial temperature of 150 °C for
2 min and then temperature programming to 300 °C at a rate of 15 °C /min.
Semipreparative HPLC was carried out on an Agilent 1100 liquid chromato-
graph equipped with a refractive index detector using a Zorbax 300 SB-C18
column (25 cmꢄ9.4 mm i.d.).
17-Hydroxy Fuscocineroside B (1): Colorless amorphous powder; mp
224—226 °C; [a]D20 ꢁ0.2 (cꢂ0.675, pyridine); H- and 13C-NMR data, see
1
Tables 1 and 2; ESI-MS (positive-ion mode) m/z 1243 [MꢀNa]ꢀ, 1123
[MꢀNaꢁNaHSO4]ꢀ; ESI-MS (negative-ion mode) m/z 1197 [MꢁNa]ꢁ. IR
(KBr) nmax: 3421, 1775, 1717, 1651, 1265, 1205, 1076 cmꢁ1. HR-ESI-MS
(positive ion mode) m/z 1243.4805 [MꢀNa]ꢀ, (Calcd for C54H85O27Na2S,
1243.4794).
25-Hydroxy Fuscocineroside B (2): Colorless amorphous powder; mp
219—221 °C; [a]D20 ꢀ8.3 (cꢂ0.41, pyridine); H- and 13C-NMR data, see
1
Tables 1 and 2; ESI-MS (positive-ion mode) m/z 1243 [MꢀNa]ꢀ, 1123
[MꢀNaꢁNaHSO4]ꢀ; ESI-MS (negative-ion mode) m/z 1197 [MꢁNa]ꢁ. IR
(KBr) nmax: 3421, 1773, 1716, 1651, 1262, 1208, 1074 cmꢁ1. HR-ESI-MS
(positive ion mode) m/z 1243.4790 [MꢀNa]ꢀ, (Calcd for C54H85O27Na2S,
1243.4794).
Column chromatographies were performed on silica gel (200—300 mesh,
10—40 mm; Yantai, P. R. China) and ODS (40—63 mm; Merck) and
Sephadex LH-20 (Pharmacia). Fractions were monitored by TLC (precoated
silica-gel GF254 plates (10—40 mm; Yantai), and spots were visualized by

August 2008
1211
Acid Hydrolysis of 1—3 Each glycoside (5 mg) was heated in an am- References
pule with 5 ml of aqueous 2
M
CF3COOH at 120 °C for 2 h. The agly-
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cone was extracted with dichloromethane, and the aqueous residue was
evaporated under reduced pressure. Then 1 ml of pyridine and 2 mg of
NH2OH·HCl were added to the dry residue, and the mixtures were heated at
90 °C for 1 h. After the reaction mixtures were cooled, 1.5 ml of Ac2O was
added and the mixtures were heated at 90 °C for 1 h. The reaction mixtures
were evaporated under reduced pressure, and the resulting aldononitrile per-
acetates were analyzed by GC-MS using standard aldononitrile peracetates
as reference samples. D-Xylose, D-quinovose, D-glucose, and 3-O-methyl-D-
glucose were identified for each of 1 and 2 in a 1 : 1 : 1 : 1 ratio (D-quinovose:
tRꢂ5.44 min; D-xylose: tRꢂ5.53 min; 3-O-methyl-D-glucose: tRꢂ6.57 min;
D-glucose: tRꢂ6.75 min).
Bioassays The antifungal activities of the compounds 1—3 were tested
against six strains: Candida albicans (ATCC76615), Cryptococcus neofor-
mans (ATCC32609), Aspergillus fumigates (Clinic Strains), Trichophyton
rubrum (Clinic Strains), Candida tropicalis (Clinic Strains) and Candida
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Acknowledgment This work was financially supported by National
High-Tech Research and Development Project (863 Project, 2006AA09Z417
and 2006AA09Z423) and Chinese National Natural Science Foundation
(Grant number: 20502035).