Cytotoxic holostane-type triterpene glycosides from the sea cucumber pentacta quadrangularis

Article abstract of DOI:10.1055/s-0030-1249854

Two new holostane-type triterpene glycosides, named pentactasides I (1) and II (2), and a new natural product, pentactaside III (3), together with two known glycosides, philinopsides A (4) and B (5), were isolated from the sea cucumber Pentacta quadrangularis. Their structures were elucidated by extensive spectroscopic analysis and chemical evidences. Compounds 1 and 2 possess the same trisaccharide moiety which is a rare structural feature among naturally occurring sea cucumber glycosides and has been infrequently reported, while 3 is a sulfated diglycoside. All the glycosides showed significant in vitro cytotoxicities against six tumor cell lines (P-388, A-549, MCF-7, MKN-28, HCT-116, and U87MG) with IC₅₀ in the range of 0.603.95μM.

Full text of DOI:10.1055/s-0030-1249854

1900 Letters
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Cytotoxic Holostane-Type Triterpene
Glycosides from the Sea Cucumber
Pentacta quadrangularis
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1976; 24: 1961–1964
Hua Han1, Qiang-Zhi Xu2*, Hai-Feng Tang3, Yang-Hua Yi4,
Wei Gong4
1
College of Animal Sciences, Zhejiang University, Hangzhou,
P.R. China
2
received June 28, 2009
Department of Biochemistry and Molecular Biology, Second Mili-
revised
accepted April 15, 2010
March 24, 2010
tary Medical University, Shanghai, P.R. China
Department of Pharmacy, Xijing Hospital, Fourth Military Medical
3
University, Xiʼan, P.R. China
Research Center for Marine Drugs, School of Pharmacy, Second Mili-
tary Medical University, Shanghai, P.R. China
Bibliography
4
DOI http://dx.doi.org/10.1055/s-0030-1249934
Published online May 19, 2010
Planta Med 2010; 76: 1896–1900
© Georg Thieme Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
Abstract
!
Two new holostane-type triterpene glycosides, named pentacta-
sides I (1) and II (2), and a new natural product, pentactaside III
(3), together with two known glycosides, philinopsides A (4)
and B (5), were isolated from the sea cucumber Pentacta quad-
rangularis. Their structures were elucidated by extensive spec-
troscopic analysis and chemical evidences. Compounds 1 and 2
possess the same trisaccharide moiety which is a rare structural
feature among naturally occurring sea cucumber glycosides and
has been infrequently reported, while 3 is a sulfated diglycoside.
All the glycosides showed significant in vitro cytotoxicities
against six tumor cell lines (P-388, A-549, MCF-7, MKN-28, HCT-
116, and U87MG) with IC50 in the range of 0.60–3.95 µM.
Correspondence
Prof. Pei-Ming Yang
Department of Chinese Traditional Medicine
Shanghai Institute of Pharmaceutical Industry
1320 West Beijing Road
Shanghai 200040
Peopleʼs Republic of China
Phone: + 862162479808 ext. 463
Fax: + 862162790148
yangpeiming@yahoo.cn
Key words
sea cucumber · Cucumariidae · Pentacta quadrangularis · triter-
pene glycosides · cytotoxicity
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/plantamedica
Triterpene glycosides are the predominant secondary metabo-
lites of sea cucumbers and exhibit a wide spectrum of biological
effects [1]. Most of them are lanosterol type triterpenes with an
18(20) lactone and a sugar chain of up to six monosaccharide
units linked to the C-3 of the aglycone [2,3]. The sea cucumber
Pentacta quadrangularis (family Cucumariidae) is distributed
abundantly in the South China Sea especially the offshore waters
of Guangdong Province, P.R. China [4]. To date, four triterpene
glycosides, philinopsides A, B, E, and F have been reported from
the species [5,6]. Philinopsides A and E were reported possessing
dual antiangiogenic and antitumor effects [7–9]. As a part of our
ongoing investigation on bioactive glycosides from sea cucum-
bers [5,6,10,11], a reinvestigation on this species led to the isola-
tion of two new triterpene glycosides named pentactasides I (1)
and II (2), and a new natural product, pentactaside III (3), along
with the two known glycosides, philinopsides A (4) and B (5)
"
(l Fig. 1). We described herein the isolation, structural elucida-
tion, and cytotoxicity of these glycosides.
* First co-author.
Han H et al. Cytotoxic Holostane-Type Triterpene… Planta Med 2010; 76: 1900–1904

Letters 1901
Fig. 1S, Supporting Information. The site of the sulfate linkage
was demonstrated by comparing the 13C‑NMR data of 1 with
those of 4 and 5 [5]. The signal of Xyl I C-4 also shifted downfield
to δ 75.5, indicating that the sulfate group was located at C-4 of
the inner xylose unit. Hence, the structure of 1 was determined
as 16β-acetoxy-3-O-[β-D-xylopyranosyl-(1 → 4)-β-D-quinovo-
pyranosyl-(1 → 2)-4-O-sulfate-β-D-xylopyranosyl]-holosta-7,24-
diene-3β-ol sodium salt. The presence of a trisaccharide moiety is
a rare structural feature among naturally occurring sea cucumber
glycosides and has been infrequently reported [14]. We pre-
sumed that 1 is a natural product in view of the mild conditions
in the course of extraction and isolation of 1. However, more ex-
tensive probative studies are needed before a clear conclusion
can be reached.
The molecular formula of pentactaside II (2) was determined as
C48H73O20SNa by the positive ion mode HR‑ESI‑MS, identical to
that of 1. The presence of D-xylose and D-quinovose in a 2:1 ratio
was established by acid hydrolysis and preparation of the corre-
sponding aldononitrile peracetates which were analyzed by
"
GC‑MS. The NMR spectral features of 2 (l Tables 1 and 2) showed
a close resemblance to those of 1, except for signals due to the side
chain, and thus indicated that 2 and 1 possess the same trisacchar-
ide chain and holostane skeleton. The 1H- and 13C‑NMR spectra of
2 showed signals ascribable to one olefinic methyl group [δH 1.66
(3H, s) and δC 25.4, CH3-27] and one disubstituted terminal dou-
ble bond [δH 4.76 (2H, s, H2-26); δC 145.3 (C-25) and 110.7 (C-26)],
rather than those due to two olefinic methyl groups and one tri-
substituted double bond as shown in the spectra of 1. This sug-
gested the presence of a double bond at Δ25(26) in the side chain of
2, which was confirmed by the HMBC correlations H3-27/C-26,
H3-27/C‑24, H2-26/C‑24, and H2-23/C-25. Therefore, the structure
of 2 was deduced as 16β-acetoxy-3-O-[β-D-xylopyranosyl-(1 →
4)-β-D-quinovopyranosyl-(1 → 2)-4-O-sulfate-β-D-xylopyrano-
syl]-holosta-7,25-diene-3β-ol sodium salt.
Fig. 1 Structures of glycosides 1–5.
Pentactaside I (1) was positive to Liebermann-Burchard and Mol-
ish tests. Its molecular formula was determined as C48H73O20SNa
by the positive ion mode HR‑ESI‑MS. A fragment ion peak at m/z
927 [M + Na − NaHSO4]+ indicated the presence of a sulfate group
in 1 which was confirmed by the IR spectrum with absorption
bands at 1240 and 1205 cm−1. An examination of the 1H-,
13C‑NMR, and DEPT spectra of 1 suggested the presence of a tri-
terpenoid aglycone with seven methyls, two olefinic bonds, one
acetoxyl, and one lactone carbonyl group, identical to those re-
ported for 16β-acetoxy-holosta-7,24-diene-3β-ol, the aglycone
of philinopsides A and F from P. quadrangularis [5,6]. The assign-
ments of the NMR signals associated with the aglycone moiety
were derived from the DQFCOSY, TOCSY, HMQC, and HMBC ex-
periments. The sugar moieties of 1 were determined as D-xylose
(Xyl) and D-quinovose (Qui) in the ratio 2:1 by acidic hydrolysis
with aqueous 2 M trifluoroacetic acid and preparation of the cor-
responding aldononitrile peracetates which were analyzed by
GC‑MS using standard aldononitrile peracetates as reference
samples. The common D-configuration for the three sugar units
were assumed according to those most often encountered among
the sea cucumber glycosides [12,13]. In the 1H‑NMR spectrum,
three doublets ascribable to the anomeric protons were observed
at δ 4.77, 5.14, and 4.87, with 3JH-1/H‑2 = 7.2–7.6 Hz, indicating the
β-configurations. The 1H- and 13C‑NMR signals attributable to the
three sugar residues were completely assigned by DQFCOSY,
The molecular formula of pentactaside III (3) was determined as
1
C
"
43H65O16SNa by HR‑ESI‑MS. The H- and 13C‑NMR spectra of 3
(l Table 1) revealed the signals due to aglycone protons and car-
bons identical with those observed in 1, and suggested that 3 and
1 possess the same aglycone. The NMR spectrum of 3 indicated
the presence of two β-sugar units and suggested that they were
D-xylose and D-quinovose. This was confirmed by acid hydrolysis
followed by GC‑MS analysis of the corresponding aldononitrile
peracetates. The NMR signals due to the sugar moieties were fully
"
assigned by 2D‑NMR experiments (l Table 2). The sequence of
the sugar residues and their attachment points were determined
by analysis of HMBC correlations: δH 4.70 (Xyl H-1)/δC 89.2 (C-3)
and δH 5.09 (Qui H-1)/δC 83.5 (Xyl C-2). By comparison of the 13
C
NMR data of 3 with those of 1 and 2, an esterification shift was
also observed for the signal of Xyl C-4 (shifted downfield to δ
75.8), indicating that the sulfate group was located at C-4 of the
xylose unit. Thus, 3 was defined as 16β-acetoxy-3-O-[β-D-quino-
vopyranosyl-(1 → 2)-4-O-sulfate-β-D-xylopyranosyl]-holosta-
7,24-diene-3β-ol sodium salt. 3 has been obtained as a progenin
of cucumarioside G1 by enzymatic hydrolysis with cellulose [15,
16], but reported as a natural product for the first time.
Glycosides 4 and 5 were identified as philinopsides A and B by
comparison of their physical and spectroscopic data with litera-
ture values and the tR values (HPLC) with those of the authentic
samples isolated from this species before [5].
"
TOCSYand HMQC experiments and the data (l Table 2) indicated
that the sugar units are in their pyranose forms. The structure of
the carbohydrate chain of 1 was indicated by HMBC spectrum,
which showed a cross-peak between C-3 of the aglycone and H-
1 of the inner xylose (Xyl I), indicating that Xyl I was connected to
C-3 of the aglycone, and the cross-peaks Qui H-1/Xyl I C-2 and Xyl
II H-1/Qui C-4 which indicated the linkages of the terminal xylose
(Xyl II) at the C-4 of Qui in turn linked to C-2 of Xyl I. This conclu-
sion was also confirmed by the NOESY correlations as shown in
The cytotoxicities of 1–5 against six tumor cell lines were eval-
uated by sulforhodamine B (SRB) protein assay [11,17]. The IC50
value of each glycoside tested was measured on the basis of cell
Han H et al. Cytotoxic Holostane-Type Triterpene… Planta Med 2010; 76: 1900–1904

1902 Letters
Table 1 1H- (600 MHz) and 13C‑NMR (150 MHz) data for the aglycone moieties of 1–3 in C5D5 N‑D2O (4:1).
Position
δ
H mult. (J in Hz)
δC
1
2
3
1
2
3
1
2
1.38 m, 1.89 m
1.35 m, 1.86 m
1.40 m, 1.87 m
36.1
35.4
36.2
1.87 m, 2.05 m
1.85 m, 2.04 m
1.92 m, 2.07 m
27.4
89.2
39.6
48.3
23.6
120.3
145.7
47.3
35.8
22.6
31.7
59.2
47.5
43.7
26.9
88.2
39.3
47.8
23.1
120.2
145.2
47.0
35.9
23.0
31.2
59.2
47.3
43.5
27.1
89.2
39.6
48.1
23.3
120.4
145.7
47.2
35.6
22.6
31.4
59.3
47.5
43.7
3
3.32 dd (11.0, 4.4)
3.20 dd (12.0, 4.2)
3.25 brd (8.4)
4
–
–
–
5
1.08 m
0.97 m
1.01 m
6
1.69 m, 2.01 m
1.65 m, 1.96 m
1.68 m, 2.04 m
7
5.65 brs
5.64 brs
5.65 brs
8
–
–
–
9
3.56 brd (11.4)
3.40 brd (11.5)
3.46 brd (13.2)
10
11
12
13
14
15
–
–
–
1.69 m, 1.89 m
1.52 m, 1.66 m
1.51 m,1.79 m
2.05 m, 2.20 m
2.04 m, 2.11 m
2.02 m, 2.14 m
–
–
–
–
–
–
1.86 dd (12.0, 7.6)
1.66 dd (12.0, 7.8)
1.71 dd (12.0, 7.8)
2.68 dd (12.0, 7.8)
2.59 dd (12.0, 8.0)
2.58 dd (12.0, 8.2)
16
6.02 ddd (8.4, 7.8, 7.6)
5.92 ddd (9.0, 8.0, 7.8)
5.93 ddd (9.6, 8.2, 7.8)
75.2
54.5
179.7
24.3
85.6
28.3
38.7
28.9
124.5
131.9
25.6
17.6
17.3
28.8
32.6
169.9
21.6
75.1
54.3
179.7
23.8
85.1
28.0
38.3
23.5
38.2
145.3
110.7
25.4
28.5
17.1
32.1
169.7
21.3
75.2
54.6
179.5
24.0
85.0
28.3
38.6
23.2
124.2
131.9
25.6
18.4
17.4
28.7
32.3
169.7
21.3
17
2.71 d (8.4)
2.65 d (9.0)
2.65 d (9.6)
18
–
–
–
19
1.31 s
1.16 s
1.22 s
–
20
–
–
21
1.65 s
1.49 s
1.51 s
1.88 m, 2.50 m
1.98 m, 2.03 m
5.09 m
–
22
2.50 m, 2.59 m
1.98 m, 2.05 m
5.18 s
1.18 m, 2.32 m
1.43 m, 1.61 m
1.95 t (7.8)
–
23
24
25
–
26
1.75 s
4.76 s
1.65 s
1.58 s
1.15 s
1.28 s
1.15 s
–
27
1.66 s
1.66 s
30
1.20 s
1.22 s
31
1.06 s
1.06 s
32
1.23 s
1.15 s
CH3CO
CH3CO
–
–
1.96 s
1.98 s
1.98 s
Table 2 1H- (600 MHz) and 13C‑NMR (150 MHz) data for the sugar moieties of 1–3 in C5D5 N‑D2O (4:1).
Position
δ
H mult. (J in Hz)
δC
1
2
3
1
2
3
Xyl I
1
4.77 d (7.2)
4.13 m
4.69 d (7.2)
4.04 m
4.70 d (7.2)
3.99 m
105.2
83.5
76.3
75.5
64.3
105.0
105.2
83.5
75.1
75.8
64.1
2
82.6
75.9
75.4
64.2
3
4.08 m
4.30 m
4.27 m
4
5.20 m
5.10 m
5.09 m
5
3.72 m, 4.86 m
3.74 m, 4.83 m
3.69 m, 4.69 m
Qui
1
5.14 d (7.6)
4.03 m
5.05 d (7.8)
3.97 m
5.09 d (7.8)
3.99 m
105.1
75.3
75.8
86.2
71.9
18.2
105.0
76.2
75.5
85.7
71.5
17.9
105.7
76.6
77.4
76.5
73.4
18.4
2
3
4.35 m
4.04 m
4.12 m
4
3.69 t (9.0)
4.08 m
3.58 m
3.65 m
5
3.68 m
3.75 m
6
1.82 d (6.2)
1.64 d (6.4)
1.61 d (5.4)
Xyl II
1
4.87 d (7.6)
3.81 m
4.76 d (7.2)
3.93 m
105.2
73.7
87.3
69.2
66.8
105.3
74.4
77.7
70.4
66.8
2
3
4.26 m
4.11 m
4
4.23 m
4.11 m
5
3.76 m, 4.67 m
3.65 m, 4.20 m
"
viability, after 72 h treatment. The results (l Table 3) showed
that all the glycosides exhibited significant cytotoxicities against
the six cell lines, while the diglycoside 3 was less active than the
others. There were not significant differences between the cyto-
toxic effects of 1 and 2 against cancer cells, except for MKN-28
cells. This suggested that the cytotoxicity of the sea cucumber
Han H et al. Cytotoxic Holostane-Type Triterpene… Planta Med 2010; 76: 1900–1904

Letters 1903
Table 3 Cytotoxicity of glycosides 1–5 against six tumor cell lines in vitro (IC50, µM).
Cell line
P-388
A-549
MKN-28
HCT-116
MCF-7
U87MG
1
0.99 0.15b
1.02 0.22
1.31 0.19
0.86 0.14
0.62 0.09
0.45 0.10
1.95 0.22
1.72 0.27
2.14 0.30
1.53 0.16
0.60 0.11
0.92 0.08
1.53 0.26
1.05 0.17
2.46 0.31
1.48 0.20
1.25 0.15
0.83 0.15
1.75 0.21
1.82 0.16
2.28 0.34
1.52 0.14
0.79 0.08
1.36 0.12
1.92 0.20
1.89 0.18
2.67 0.36
1.33 0.15
1.09 0.14
1.05 0.11
3.57 0.35
3.44 0.40
3.95 0.53
2.74 0.32
1.90 0.21
1.39 0.17
2
3
4
5
HCPa
a HCP (10-hydroxycamptothecine) as the positive control; b The data represent the mean plus SD of three independent experiments in which each compound concentration was
tested in three replicate wells
glycosides is not sensitive to the positions of the C=C bond, i.e.,
C(24)=C(25) in 1 and C(25)=C(26) in 2. The increasing trend of
the cytotoxicity against cancer cells, except for MKN-28 cells,
from diglycoside 3 to triglycosides 1, 2 and tetraglycosides 4, 5,
may be ascribed to the difference of their sugar moieties. Howev-
er, more extensive studies are needed before a clear structure-ac-
tivity relationship can be reached.
tR 29.3 min), respectively. Fr 6 [1.5 g, eluted with CHCl3-
MeOH‑H2O (6.5:3.5:1, lower phase), 20–22 L] was subjected to
size exclusion chromatography on a 100 g Sephadex LH-20 col-
umn (2 × 120 cm), eluted with MeOH‑H2O (2:1) 800 mL to re-
move the pigments and carbohydrates. Frs 38–42 (380–420 mL,
410 mg) were purified by HPLC (55% aq. MeOH, 1.5 mL/min) to
yield glycosides 4 (20 mg, tR 19.6 min) and 5 (16 mg, tR 21.5 min).
Pentactaside I (1): colorless amorphous powder; m.p. 232–234°C;
[α]2D0 − 17.4 (c 0.64, pyridine); IR (KBr) νmax 3444, 2939, 1747,
Materials and Methods
!
1647, 1240, 1205, 1067 cm−1; 1H- and 13C‑NMR data, see l Tables
"
Melting points were determined on an XT5-XMT apparatus. Op-
tical rotations were measured with a Perkin-Elmer 341 polarim-
eter. IR spectra were recorded on a Bruker Vector 22 infrared
spectrometer. NMR spectra were recorded on a Varian Inova-
600 spectrometer, and the 2D NMR spectra were obtained using
standard pulse sequences. ESI‑MS and HR‑ESI‑MS were acquired
using a Micromass Quattro mass spectrometer. HPLC was carried
out on an Agilent 1100 liquid chromatograph equipped with a re-
fractive index detector using a Zorbax 300 SB‑C18 column
(250 × 9.4 mm i.d.). Column chromatographic separations were
performed on DA-101 resin (Nankai University, Tianjin, China),
silica gel H (200–300 mesh; Qingdao Marine Chemical, Inc.), Lo-
bar Lichroprep RP‑C18 (40–63 µm; Merck), and Sephadex LH-20
(Pharmacia, Inc.). Fractions were monitored by TLC on precoated
silica gel GF254 plates (10–40 µm; Yantai) or RP‑C18 plates (Qing-
dao Marine Chemical, Inc.), and spots were visualized by heating
plates sprayed with 15% H2SO4 in EtOH.
1 and 2; ESI‑MS (pos. ion mode) m/z 1047 [M + Na]+, 927 [M +
Na – NaHSO4]+; ESI‑MS (neg. ion mode) m/z 1001 [M – Na]−;
HR‑ESI‑MS (pos. ion mode) m/z 1047.4216 [M + Na]+ (calcd. for
C48H73O20Na2S 1047.4211).
Pentactaside II (2): colorless amorphous powder; m.p. 230–
233°C; [α]2D0 − 17.6 (c 0.64, pyridine); IR (KBr) νmax 3445, 1747,
1649, 1240, 1206, 1069 cm−1; 1H- and 13C‑NMR data, see l Tables
"
1 and 2; ESI‑MS (pos. ion mode) m/z 1047 [M + Na]+; ESI‑MS (neg.
ion mode) m/z 1001 [M – Na]−, 899 [M – Na – SO3Na + H]−; HR‑E-
SI‑MS (pos. ion mode) m/z 1047.4213 [M + Na]+ (calcd. for
C48H73O20Na2S 1047.4211).
Pentactaside III (3): colorless amorphous powder; m.p. 228–
230°C; [α]2D0 − 36.1 (c 0.9, pyridine); IR (KBr) υmax 3510, 1743,
1647, 1244, 1208, 1068 cm−1; 1H- and 13C‑NMR data, see l Tables
"
1 and 2; ESI‑MS (pos. ion mode) m/z 915 [M + Na]+; ESI‑MS (neg.
ion mode) m/z 869 [M – Na]−; HR‑ESI‑MS (pos. ion mode) m/z
915.3788 [M + Na]+ (calcd. for C43H65O16Na2S 915.3789).
Specimens of P. quadrangularis were collected from offshore
waters of Zhanjiang in the South China Sea in May 2007, and
identified by Prof. J.R. Fang (Fujian Institute of Oceanic Research,
P.R. China). A voucher specimen (No. SA-2007-04) was reserved
at the Research Center for Marine Drugs, School of Pharmacy, Sec-
ond Military Medical University, Shanghai, P.R. China.
Supporting information
Key HMBC correlations of glycosides 1–3, key NOESY correlations
and relative configuration of 1, details for the acid hydrolysis of
1–5, and the description of the SRB assay are available as Sup-
porting Information.
The sea cucumbers (3.4 kg, dry weight) were cut into pieces and
refluxed with 70% EtOH (4 L × 5, 1 h/time). The combined EtOH
extracts were evaporated. The residue was dissolved in H2O
(5 L), and then passed through a DA-101 resin column (2 kg,
15 × 120 cm), stepwise eluted with H2O (5 L), 50% EtOH (10 L)
and 95% EtOH (5 L). The 50% EtOH eluate was evaporated (crude
glycoside-containing mixture, 71 g) and subjected to CC on silica
gel (2.1 kg, 15 × 90 cm) eluted with a CHCl3-MeOH‑H2O (8:2:1,
7:3:1, 6.5:3.5:1, lower phase, 10 L each) gradient. Fractions
were examined by TLC and combined to afford 8 pooled fractions
(Fr 1–8). Fr 4 [4.8 g, eluted with CHCl3-MeOH‑H2O (7:3:1, lower
phase), 10–13 L] was subjected to reversed-phase silica MPLC
(3 × 100 cm) eluted with an aq. MeOH (50–80%) gradient to give
5 subfractions 4.1–4.5 (400 mL eluate each). Fr 4.3 (430 mg) was
purified by HPLC (65% aq. MeOH, 1.5 mL/min) to yield glycosides
1 (18 mg, tR 21.3 min), 2 (17 mg, tR 23.5 min), and 3 (23 mg,
Acknowledgements
!
This work was financially supported by the National High-Tech
Research and Development Project (863 Project, 2006AA
09Z423) and the National Natural Science Foundation of China
(No. 20502035 and No. 20772155).
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Biotransformation of 3-Oxo-Oleanolic
Acid by Absidia glauca
Na Guo1,2,3, Ying Zhao1,2,3, Wei-Shuo Fang1,2,3
1
Key Laboratory of Bioactive Substances and Resources Utilization
of Chinese Herbal Medicine (Peking Union Medical College),
Ministry of Education, Beijing, P.R. China
Key Laboratory of Biosynthesis of Natural Products,
2
Ministry of Health, Beijing, P.R. China
Institute of Materia Medica, Chinese Academy
3
of Medical Sciences, Beijing, P.R. China
Abstract
!
3-Oxo-oleanolic acid (1) was biotransformed in growing cultures
of the fungus Absidia glauca, resulting in three novel hydrox-
ylated metabolites, identified as 1β-hydroxy-3-oxo-olean-11-
en-28,13-lactone (2), 1β,11α-dihydroxy-3-oxo-olean-12-en-28-
oic acid (3), and 1β,11α,21β-trihydroxy-3-oxo-olean-12-en-28-
oic acid (4).
Key words
Absidia glauca Hagem · hydroxylation · triterpenoids · β‑cyclo-
dextrin · anticancer
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solving taxonomic problems in the sea cucumber genus Cucumaria
(Holothurioidea, Echinodermata). Biochem Syst Ecol 2004; 32: 715–
733
Supporting information available online at
http://www.thieme-connect.de/ejournals/toc/plantamedica
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3-Oxo-oleanolic acid (1) has been recognized as an anticancer
and antiangiogenic agent [1,2]. However, both the activity and
the water solubility of 1 are unsatisfying. Therefore, microbial hy-
droxylation of 1 was attempted [3] to increase its water solubility
and to introduce functional groups for further modifications.
Here, we report the biotransformation of 1 by the fungus Absidia
glauca Hagem (3.67) [4], whichafforded three novel triterpenoids:
1β-hydroxy-3-oxo-olean-11-en-28,13-lactone (2), 1β,11α-dihy-
droxy-3-oxo-olean-12-en-28-oic acid (3), and 1β,11α,21β-trihy-
"
droxy-3-oxo-olean-12-en-28-oic acid (4) (l Fig. 1).
To enhance the efficiency of this biotransformation, the maxi-
mum concentration of substrate was increased from 0.1 g/L to
1 g/L by forming inclusion of 1 with β-cyclodextrin [5]. The triter-
pene 1 was incubated for 9 days with the cells of A. glauca, afford-
ing compounds 2–4 in yields of 0.74%, 2.3%, and 0.23%, respec-
tively.
received December 25, 2009
revised
–
accepted April 1, 2010
Bibliography
DOI http://dx.doi.org/10.1055/s-0030-1249854
Published online April 27, 2010
Planta Med 2010; 76: 1900–1904
© Georg Thieme Verlag KG Stuttgart · New York ·
ISSN 0032‑0943
Compound 2 possessed the molecular formula C30H44O4 as deter-
mined by HR‑ESI‑MS at m/z = 469.32950 [M + H]+ (calcd. for
C30H45O4: 469.33178), suggesting the addition of one oxygen
atom and the loss of two hydrogen atoms to 1. 1H- and 13C‑NMR
of 2 indicated that one methylene group in 1 was hydroxylated
(δ = 3.87, t, H-1; δ = 79.9, d, C-1), which was supported by the
long-range correlations of H-1 to C-3 and C-9 and of H-2 and H-9
to C-1. The 13C‑NMR of 2 indicated that the signal of C-28 in 1 [6]
shifted from δ = 184.4 to 179.9, possibly due to forming a lactone
[7,8] between 28-COOH and C-13 (δ = 89.4), which was con-
firmed by HMQC and HMBC (from H-12, H-18, and H-27 to C-
13). 1H‑NMR of 2 showed that the signal at δ = 5.30 (1H, br s, H-
12) in 1 disappeared, and two new methine signals were finally
assigned to cis double-bond protons (δ = 5.41, H-11 and δ = 6.89,
Correspondence
Prof. Hai-Feng Tang
Department of Pharmacy
Xijing Hospital, Fourth Military Medical University
15 Changle West Rd.
Xiʼan 710032
P.R. China
Phone: + 862984775471
Fax: + 862984775471
tanghaifeng71@163.com
"
H-12) because of the coupling constant (10.4 Hz) (l Table 1). The
Guo N et al. Biotransformation of 3-Oxo-Oleanolic… Planta Med 2010; 76: 1904–1907