Unusual Cytotoxic Steroidal Saponins from the Gorgonian Astrogorgia dumbea

Article abstract of DOI:10.1055/s-0042-106168

Three steroidal saponins, including astrogorgiosides A (1) and B (2) bearing acetamido-glucose moieties, and astrogorgioside C (3) with a 19-nor and bearing an aromatized B ring steroid aglycone, together with a known major saponin dimorphoside A (4), were obtained from the gorgonian Astrogorgia dumbea collected near Dongshan Island in East China Sea. Structures of these compounds were elucidated by in-depth spectral and chemical methods, including 2D-NMR, HR-ESI-MS spectra, and acidic hydrolysis. For the first time, acetamido-glucose moiety is being reported from a gorgonian. The B-ring aromatized steroid aglycone of compound 3 is also rare in marine natural products. Compounds 1-3 exhibited moderate cytotoxic activity with IC₅₀ values of 26.8-45.6 μM against human tumor cells Bel-7402 and K562.

Full text of DOI:10.1055/s-0042-106168

8
82 Original Papers
Unusual Cytotoxic Steroidal Saponins from the
Gorgonian Astrogorgia dumbea
1
2
1
3
1
Authors
Ya-Nan Lu , Ping Cui , Xiao-Qing Tian , Li-Guang Lou , Cheng-Qi Fan
1
Affiliations
Key Laboratory of East China Sea & Oceanic Fishery Resources Exploitation and Utilization, Ministry of Agriculture, East China
Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai, P.R. China
Changchun University of Chinese Medicine, Changchun, P.R. China
2
3
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, P.R. China
Key words
Abstract
MS spectra, and acidic hydrolysis. For the first
time, acetamido-glucose moiety is being reported
from a gorgonian. The B-ring aromatized steroid
aglycone of compound 3 is also rare in marine
natural products. Compounds 1–3 exhibited
moderate cytotoxic activity with IC50 values of
26.8–45.6 µM against human tumor cells Bel-
7402 and K562.
"
l Astrogorgia dumbea
!
"
l Plexauridae
Three steroidal saponins, including astrogorgio-
sides A (1) and B (2) bearing acetamido-glucose
moieties, and astrogorgioside C (3) with a 19-nor
and bearing an aromatized B ring steroid agly-
cone, together with a known major saponin di-
morphoside A (4), were obtained from the gorgo-
nian Astrogorgia dumbea collected near Dongshan
Island in East China Sea. Structures of these com-
pounds were elucidated by in-depth spectral and
chemical methods, including 2D‑NMR, HR‑ESI‑
"
l steroidal saponin
"
l astrogorgiosides A–C
"
l cytotoxic activity
Supporting information available online at
http://www.thieme-connect.de/products
Introduction
Linʼs group also reported fourteen 9,10-secoste-
roids and eight analogues from Astrogorgia sp.
collected off the coast of Beibuwan Bay, South
China Sea [8]. Five of them showed significant in-
hibitory activities against human tumor-related
protein kinases. Herein, the isolation, structure
elucidation, and bioactivity of three steroidal sa-
ponins, astrogorgiosides A–C (1–3), from the gor-
gonian A. dumbea are reported.
!
As one of the hot topics in the natural products
research field, many structural types of com-
pounds, including terpenoids, steroids, alkaloids
and lipids, have been reported from gorgonians.
These compounds exhibited many kinds of activ-
ities, such as anti-TB, anti-inflamation, antitumor,
etc. [1–4]. Dongshan Island (23°42′N, 117°26′E, on
the south of Fujian province in China) has a rich
diversity of gorgonians where the species were
estimated to be over 20 [5]. To find more bioactive
constituents from these gorgonians, samples of
the genera Astrogorgia, Echinogorgia, Guaiagor-
gia, and Melithaea were collected from the Dong-
shan coastal waters. Among them, Astrogorgia
dumbea Grasshoff (Plexauridae) was the first re-
corded species of this genus in this district [5].
Gorgonians of the Astrogorgia genus are distrib-
uted widely in Indonesia, New Caledonia, and Ja-
pan, but there are only a few reports about their
bioactive secondary metabolites. A secosteroid
and a diterpene have been reported by Fusetani
et al. from Astrogorgia sp. collected from Shikoku,
which inhibited cell division of fertilized starfish
eggs [6]. The secosteroid astrogorgiadiol also has
the potential to treat osteoporosis and autoim-
mune diseases as an analogue of vitamin D [7].
received
revised
accepted
Sep. 9, 2015
March 11, 2016
March 22, 2016
Results and Discussion
!
Bibliography
After percolation with methanol and extraction
DOI http://dx.doi.org/
0.1055/s-0042-106168
Planta Med 2016; 82: 882–887
Georg Thieme Verlag KG
1
with petroleum ether and CHCl , the aqueous
3
phase of the gorgonian A. dumbea was subjected
to column chromatography on MCI gel and silica
gel to give three novel steroidal saponins, astro-
gorgiosides A–C (1–3), with a known one (4;
©
Stuttgart · New York ·
ISSN 0032‑0943
Correspondence
Prof. Cheng-Qi Fan
East China Sea Fisheries
Research Institute
Chinese Academy of Fishery
Sciences
"
l Fig. 1).
Compound 1, namely astrogorgioside A, was
obtained as a colorless powder, and exhibited a
quasi-molecular ion peak at m/z 790.4343 ([M +
+
Na] , calcd. 790.4354) in its HR‑ESI MS, compati-
3
00 Jungong Road
Shanghai 200090
P.R. China
Phone: + 862165807718
Fax: + 862165807718
fancq@ecsf.ac.cn
ble with the molecular formula C40
H
65
−1
NO13. The
IR absorptions at 3425 and 1639 cm indicated
the presence of OH, NH, and carbonyl groups. By
1
13
interpretation of the H-, C‑NMR (with DEPT, in
Lu YN et al. Unusual Cytotoxic Steroidal… Planta Med 2016; 82: 882–887

Original Papers 883
Table 1 1H‑NMR data of compounds 1–3 (400 MHz, δ in ppm, J in Hz)a.
Atom
1
2
3
1
2.52, m
2.79, m
1.00, m
2.21, m
2.75, m
0
.97, m
1.57, br d, 14.3
.00, br d, 4.4
2.59, m
2
2.16, m
2
1.85, m
3
4
3.62, m
3.94, m
4.12, m
1.80, br d, 1.8
2.41, t, 11.0
2.89, d, 10.4
5.63, m
3.07, dd, 4.0, 16.0
2.80, m
2
.52, m
6
7
5.51, d, 5.5
6.83, d, 8.7
6.76, d, 8.1
3.73, t like, 5.5
2.11, m
1.60, m
8
9
2.04, d, 3.7
1.15, m
1.84, m
1.10, m
1.85, m
Fig. 1 Structures of compounds 1–4.
1
1
1.71, br d, 10.7
2.75, m
2.59, m
1.63, m
2.24, m
2.63, m
1.42, m
1.28, m
2.07, m
1.48, m
1.32, m
0.56, s
1
.65, m
1.92, m
.08, m
1
2
1.13, m
Table 2 13C‑NMR data of compounds 1–4 (100 MHz, δ in ppm).a
1
1
1
4
5
1.11, m
1.85, m
0.97, m
1.50, m
1.01, m
1.67, m
C
1
2
2*
34.3
3
26.3b
4
1
.49, m
1
35.3
35.5
35.3
1
6
1.30, m
2
3
32.7
79.5
42.0
138.0
131.1
73.6
41.7
48.9
52.3
24.8
41.5
44.7
58.0
27.9
30.3
57.4
13.0
177.5
37.6
19.9
37.9
25.5
41.2
29.6
23.5
23.7
100.9
57.3
85.3
71.0
77.8
62.4
104.6
79.2
74.9
84.1
63.2
174.6
23.5
32.7
79.7
42.5
136.9
126.0
32.7
42.0
50.6
48.9
25.0
41.4
44.0
58.5
25.6
30.3
57.7
13.1
177.9
33.8
21.9
139.9
128.0
43.6
30.3
23.5
23.2
100.9
57.3
85.5
71.0
77.9
62.4
104.6
79.2
75.0
84.2
63.2
174.6
23.2
32.0
78.0
41.8
136.1
124.3
31.5
42.1
49.1
51.2
23.8
39.7
42.5
56.7
24.4
29.0
55.9
12.2
176.1
40.4
21.0
138.5
126.4
42.1
28.7
22.3
22.4
99.7
57.2
83.9
70.3
78.0
62.3
103.8
79.1
73.8
83.9
61.2
171.4
23.4
31.6
76.5
37.2
134.9
128.2
125.1
135.3
132.9
139.2
26.3 b
38.9
43.5
53.5
25.5
30.5
57.2
11.9
–
32.7
79.4
41.7
138.0
131.2
73.6
41.7
48.9
52.3
24.8
41.5
44.7
58.0
27.9
30.2
57.4
13.0
177.5
37.6
19.8
37.9
25.4
41.2
29.6
23.4
23.7
102.9
75.2
86.1
78.0
70.5
62.8
104.1
79.3
75.0
84.2
63.2
1.21, m
1
1
2
2
2
7
8
0
1
2
1.09, m
1.07, m
0.66, s
4
0.69, s
5
1.36 t like, 7.7b
0.93, d, 6.6
1.02, dd, 2.6, 6.2
1.95, m
0.99, d, 6.6
5.24, m
1.08, m
1.02, d, 6.6
1.08, m
6
7
8
1
.45, m
2.07, m
.48, m
9
2
3
1.36, t like, 7.7b
5.28, m
1.85, m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
1
2
2
2
2
4
5
6
7
1
2
3
4
5
6
1.13, m
1.17, m
1.52, m
1.54, m
0.87, d, 6.6
0.87, d, 6.6
4.6, d, 7.3
3.63, m
0.86, d, 6.6
0.86, d, 6.6
5.35, d, 8.4
4.20, m
0.89, d, 6.6
0.89, d, 6.6
4.50, d, 7.7
3.34, m
′
′
′
′
′
′
3.62, m
4.58, m
3.45, dd, 8.8, 19.0
3.31, m
3.4, dd, 9.5, 8.0
3.32, m
4.08, t, 9.0
3.94, m
3.40, m
38.0
19.8
37.9
25.8
41.2
29.7
23.5
23.7
103.5
75.0
86.3
78.1
70.5
62.8
104.2
79.4
75.3
84.2
63.2
3.68, d, 5.5
4.24, m
3.63, dd, 4.4, 12.8
3.74, m
3
.65, m
4.44, d like, 9.9
5.64, d, 4.0
4.60, m
1
2
3
4
5
′′
′′
′′
′′
′′
4.93b
5.24, d, 4.8
3.99, dd, 8.4, 4.7
4.18, t like, 8.0
3.72, m
3.91, dd, 4.8, 8.1
4.11, dd, 7.7, 8.1
3.72, m
5.05, t, 8.1
4.33, d like, 7.4
4.14, m
3.86, br d, 11.4
3.87, dd, 1.8,11.7
3.67, m
3
.73, m
1′
2′
COCH3
1.96, s
2.08, s
3
4
5
6
1
′
aData of 1 and 3 were recorded in CD3OD, and the data of 2 were recorded in C5D5N.
bProton signals were overlapped
′
′
′
′′
"
2′′
CD OD; l Tables 1 and 2) and HSQC spectra, the presence of a
3
3
4
5
′′
′′
′′
steroid skeleton and two saccharide moieties were observed,
which contained five methyls, ten methylenes, six methines,
two sp3 quaternary carbons, two oxygenated methylenes, ten
COCH3
COCH3
oxygenated methines, one N-linked methine at δ 57.3, two ole-
C
finic carbons (a quaternary carbon at δ 138.0 and a methine at δC
C
a
Data of 1–4 were recorded in CD3OD, and the data of 2* were recorded in C5D5N.
1
31.1), and two carbonyl carbons (δ 177.5 and 174.6). These da-
C
b
Carbon signals were overlapped
ta, especially those of the steroid aglycone, showed a high simi-
larity with those of a known steroidal saponin, dimorphoside A
"
(
4; l Table 2), which was the major metabolite of A. dumbea in
our study and has been reported as 3-[(3-O-β-D-arabinofurano-
syl-β-D-glucopyranosyl)oxy]-7β-hydroxycholest-5-en-19-oic ac-
Lu YN et al. Unusual Cytotoxic Steroidal… Planta Med 2016; 82: 882–887

8
84 Original Papers
id from another gorgonian, Anthoplexaura dimorpha [9]. Com-
pared with the molecular formula C38H62O13 of dimorphoside A,
compound 1 not only had two additional carbons, including a
methyl at δH 1.96 (3H, s) and δ 23.5, and the carbonyl group at
C
δC 174.6, which might indicate an acetyl group, but it also con-
tained one N-linked methine at δ 57.3 instead of one oxygenated
C
methine in the glycoside moiety of dimorphoside A. Therefore,
compound 1 should be a dimorphoside A analogue bearing an
acetyl and amino-glucose.
1
13
1
1
Combined analysis of H- and C‑NMR data, H- H COSY, HSQC,
"
and HMBC spectra (l Fig. 2) indicated that compound 1 should
1
1
have the same steroid aglycone of 3β,7β-dihydroxycholest-5-en-
Fig. 2 Key H- H COSY (—) and HMBC (H→C) correlations of compounds 1
and 2.
19-oic acid and an arabinose moiety as dimorphoside A. Anome-
ric protons of the arabinose and another hexose were observed at
δH 4.93 (1H, overlapped) and 4.61 (1H, d, J = 7.3 Hz), and corre-
sponded with carbon signals at δ 104.6 and 100.9, respectively.
C
The linkage from C-1′ to C-6′ of the undetermined hexose could
1
1
also be lined out by H- H COSY and HSQC spectra. In the HMBC
spectra, both the methyl at δH 1.96 and H-2′ at δH 3.63 correlated
with the carbonyl at δ 174.6, which confirmed the linkage of the
C
CH CONH- group to the methine at δ 57.3 (C-2′). By direct com-
3
C
parison of the two structures, the OH at C-2′ in dimorphoside A
was replaced by the acetamido group in compound 1, so an acet-
amido-glucose should exist in the structure of compound 1. In
addition, key correlation signals from δH 4.61 (H-1′) to δC 79.5
(C-3), and from δH 4.93 (H-1′′) to δC 85.3 (C-3′) confirmed that
the C-1′ of acetamido-glucose linked to C-3 of the aglycone, and
C-1′′ of arabinose linked to C-3′ of acetamido-glucose. The couple
constant of the anomeric proton (7.3 Hz) of glucose indicated that
its relative configuration was β-positioned [10]. The cross-peak
between the anomeric proton of acetamido-glucose at δH 4.60
and H-3′ at δH 3.62 in the ROESY spectra confirmed the indica-
tion. The couple constant of the arabinose could not be read be-
1
cause the signal in H‑NMR was overlapped by D O. But the data
2
1
13
of H- and C‑NMR of arabinose were similar to those of com-
pound 4. So the relative configuration of C-1′′ was evaluated to
be β-positioned, the same as compound 4. The cross-peak be-
tween the anomeric proton of arabinose at δ 4.93 and H-3′ of
H
"
acetamido-glucose at δH 3.62 (l Fig. 3) in the ROESY spectra sup-
ported our evaluation. Thus, the structure of astrogorgioside A
(
1) was firstly elucidated to be 3-[(2-acetamido-3-O-β-arabino-
furanosyl-β-glucopyranosyl)-oxy]-7β-hydroxycholest-5-en-19-
"
oic acid as shown in l Fig. 1, with the absolute configurations of
Fig. 3 Key ROESY correlations of compounds 1–3.
arabinose and acetamido-glucose determined later by compari-
son with compound 2.
Compound 2, namely astrogorgioside B, was obtained as colorless
plates. Its molecular formula was deduced as C40H63NO12 by
1
1 or 4, the aglycone of compound 2 showed a few different H-
+
13
HR‑ESI MS at m/z 772.4254 ([M + Na] , calcd. 772.4248). The IR
or C‑NMR signals. There were four olefinic carbons at δ 136.1
C
−1
absorptions at 3405, 1708, and 1652 cm indicated the presence
(C), 124.3 (CH), 138.5 (CH), and 126.4 (CH) with three multiple-
coupled protons at δH 5.63 (1H, m), 5.24 (1H, m), and 5.28 (1H,
m) in compound 2, together with the absence of one methylene
and one oxygenated methine. These data indicated the presence
of an additional –CH=CH– group and one methylene in com-
pound 2, taking the place of two methylenes and OH linked 7-
methine in compound 1, respectively. In the HMBC spectra
1
13
of OH, NH, and carbonyl groups. The H- and C‑NMR data (in
"
pyridine-d ; l Tables 1 and 2) of 2 also exhibited the acetamido-
5
glucose and arabinose moieties. The acetamido-glucose showed
an anomeric proton at δH 5.35 (1H, d, J = 8.4 Hz), C-1′ at δ 99.7,
C
and a -CH(NHCOCH )- group at δ 4.20 (1H, m), 2.08 (3 H, s), δC
3
H
5
7.2, 23.4, and 171.4. The arabinose had the anomeric proton at
1
3
"
δH 5.64 (1H, d, J = 4.0 Hz) and C-1′′ at δ 103.8. The C‑NMR data
(l Fig. 2), the key correlations from δ 0.99 (H-21) to δC 138.5
C
H
of acetamido-glucose and arabinose moieties of compound 2
and from δ 5.28 to δ 23.4 (C-25) confirmed the presence of the
H
C
2
2, 23
both in C D N and CD OD showed a high similarity with those
Δ
, and the correlation between the olefinic proton at δH 5.63
5
5
3
"
of compound 1 in CD OD (l Table 2), which indicated the same
(1H, m, H-6) and a methylene at δ 32.0 (C-7) was also observed.
3
C
structure unit as the 2-acetamido-3-O-β-arabinofuranosyl-β-
glucopyranosyl moiety in 1. However, compared with the agly-
cone of 3β,7β-dihydroxycholest-5-en-19-oic acid in compounds
The coupling constants of glucose (8.4 Hz) and arabinose (4.0 Hz)
implied that the relative configuration of these two saccharides
were all β-positioned [10,11]. In the ROESY spectra, the cross-
Lu YN et al. Unusual Cytotoxic Steroidal… Planta Med 2016; 82: 882–887

Original Papers 885
peak between the H-1′ at δH 5.35 and H-3′ also showed the β con-
figuration at C-1′ of the acetamido-glucose (l Fig. 3). On the basis
1
1
Fig. 4 Key H- H COSY
—) and HMBC (H→C)
"
(
1
13
of above evidences and by combined analysis of H- and C‑NMR
correlations of com-
1
1
data, H- H COSY, HSQC, and HMBC spectra, the aglycone of com-
pound 2 was determined as 3β-hydroxycholesta-5,22-dien-19-
oic acid.
pound 3.
To determine the absolute configurations of arabinose and acet-
amido-glucose as the same in compounds 1 and 2, acidic hydro-
lysis of compound 2 was carried out. By comparisons of the re-
tention time (t ) in HPLC with a polyamine column and [α] val-
R
D
ues with those of authentic D-arabinose and 2-N-acetylamino-D-
glucose standards, the structure of astrogorgioside B (2) was fi-
nally elucidated as 3-[(2-acetamido-3-O-β-D-arabinofuranosyl-
β-D-glucopyranosyl)oxy]-cholesta-5,22-dien-19-oic acid, and
that of astrogorgioside A (1) was 3-[(2-acetamido-3-O-β-D-ara-
binofuranosyl-β-D-glucopyranosyl)oxy]-7β-hydroxycholesta-5-
Table 3 Cytotoxic activity (IC50 value, µM) of compounds.
"
en-19-oic acid. Their NMR data were assigned in l Tables 1 and 2
1
1
by total analysis of H- H COSY, HSQC, and HMBC spectra. The
BEL-7402
K562
1
3
—a
C‑NMR data of 2 in CD OD were also obtained for comparison
1
34.8
26.8
45.6
0.08
3
with the other compounds.
2
41.1
28.9
0.3
3
Compound 3, namely astrogorgioside C, was a colorless powder.
Its molecular formula C37H58O10 was deduced by HR‑ESI MS at
ADRb
+
m/z 685.3920 ([M + Na] , calcd. 685.3928). The IR absorptions at
a
The activity of 1 was not evaluated against K562. b Positive control
−
1
3
415 and 1637 cm indicated the presence of OH and carbonyl
1
13
groups. By interpretation of the H-, C‑NMR (with DEPT, in
CD OD; l Tables 1 and 2), and HSQC spectra and by comparison
"
and 2, the 2′-OH of glucose was replaced by the acetamino group,
and it is the first report of the acetamido-glucose moieties in the
steroidal saponin structures from gorgonian. Compound 3 has an
aromatized B-ring. Although the steroids bearing an aromatized
B-ring are common in synthesized structures and also have been
found as natural products of a fungus [12] and sponge [13], they
are found in gorgonians for the first time and are seldom reported
in other marine organisms. The structures from gorgonians have
been reported mainly as terpenoids, especially diterpenoids. Ste-
roid saponins were seldom founded in gorgonians except for di-
morphoside A from A. dimorpha [9]. Dimorphoside A and its
three analogues from A. dumbea showed that these steroid sapo-
nins should be one of the chemical characteristics of this genus.
Cytotoxicity of the compounds against two human tumor cell
lines, hepatoma Bel-7402 and erythroleukemia K562, was eval-
uated by means of MTT methods, with ADR as a positive control.
3
with the NMR data of dimorphoside A (4), the anomeric protons
at δH 4.50 (1H, d, J = 7.7 Hz) and 5.24 (1H, d, J = 4.8 Hz) with corre-
sponded carbon signals at δC 103.5 and 104.2, respectively, to-
gether with seven oxygenated methines and two oxygenated
methylenes, indicated the presence of glucose, arabinose, and
the same linkage as the 3-O-β-arabinofuranosyl-β-glucopyrano-
syl moiety in compound 4. The correlations between δH 4.50 (H-
1
′) and δC 76.5 (C-3) fixed the linkage site at C-3 of the steroid
aglycone.
The signals of the steroid aglycone were observed as four meth-
yls, ten methylenes, five sp3 methines (one oxygenated), and a
quaternary carbon, together with six olefinic carbons (four qua-
2
ternary carbons at δC 139.2, 134.9, 135.3, 132.9, and two sp
methines at δC 128.2 and 125.1), which implied that one of the
methyls in the cholesterol skeleton disappeared. In addition, ole-
finic proton signals at δH 6.83 (1H, d, J = 8.7 Hz) and 6.76 (1H, d,
"
All compounds exhibited moderate cytotoxic activity (l Table 3).
1
1
J = 8.7 Hz) showed a correlation in the H- H COSY spectra, indi-
Compound 2 exhibited the best activity on Bel-7402 with an IC50
value of 26.8 µM, while 3 exhibited better activity on K562 (IC50
value of 28.9 µM).
cating the existence of a –CH=CH– moiety. In the HMBC spectra
"
(
l Fig. 4), the proton signals at δ 6.83 correlated with δC 139.2
H
and 135.3, and the proton signals at δH 6.76 correlated with δC
34.9 and 132.9, which revealed that the six aromatic carbons
1
should be an aromatic system. The other HMBC correlations, in-
Materials and Methods
!
cluding δH 6.83 to δ 37.2 (C-4), δ 6.76 to δ 53.5(C-14), H-3 to δC
C
H
C
1
34.9, H-11 to δ 132.9, and H-14 to δ 135.3, confirmed that the
Instruments and chemicals
C
C
B-ring of the aglycone was aromatized, and its C-19 was cut from
C-10 to give the aglycone 19-norcholesta-5,7,9-trien-3-β-ol.
Acidic hydrolysis of compound 3 yielded D-arabinose and D-glu-
Optical rotations were measured on a Perkin-Elmer 341 polarim-
eter and Rudolph Autopol VI. IR spectra were obtained on a Per-
−1
kin-Elmer 577 spectrometer with KBr discs in cm . 1D and 2D
NMR spectra were recorded on a Bruker AM-400 spectrometer;
cose by comparisons of t in HPLC and [α]D values with those of
R
authentic standards, thus the structure of astrogorgioside C (3)
was elucidated as 19-norcholesta-5,7,9-trien-3-β-ol 3-O-(3-O-β-
D-arabinofuranosyl)-β-D-glucopyranoside, and confirmed by
δ in ppm rel. to Me Si, J in Hz. ESI MS was carried out on a
4
Finnigan LCQ-DECA instrument in m/z (rel. %). HPLC analysis and
preparation were carried on a system including a Shimadazu LC-
20AT pump, SIL-20A autosampler, SPD‑M20A PDA and RI (Buchi)
detectors, and a YMC-Pack ODS‑AQ column (AQ12S05-2510WT,
250 × 10 mm, S-5 µm, 12 nm) or YMC-Pack polyamine II column
(PB12S05-2510WT, 250 × 10 mm, S-5 µm, 12 nm). TLC was car-
ried out with precoated silica gel GF254 plates (Qingdao Haiyang
1
1
further analysis of H- H COSY, HSQC, and HMBC spectra.
This is the first time reporting the chemical compounds of
A. dumbea. Three new steroidal saponins were isolated with a
cholesterol skeleton bearing a 3-O-β-arabinofuranosyl- β-gluco-
pyranosyl moiety at C-3. It is interesting that in compounds 1
Lu YN et al. Unusual Cytotoxic Steroidal… Planta Med 2016; 82: 882–887

8
86 Original Papers
Chemical Co. Ltd.). Spots were visualized under UV light and by
spraying with 10% H SO in EtOH, followed by heating. CC: re-
tracted with CHCl (1 mL × 3). The pH value of the water solution
3
was adjusted to 6–7 with NaHCO , and the mixture was evapo-
2
4
3
versed-phase silica gel (ODS‑A, 150–200 mesh; YMC), silica gel
H (200–300 mesh, Qingdao Haiyang Chemical Co. Ltd.), and Se-
phadex LH-20 gel (Amersham Biosciences). All solvents used
were of analytical grade (Shanghai Chemical Plant). ADR came
from Sigma with a purity ≥ 98% (TLC).
rated to give a residue. The sugars were finally purified by HPLC
equipped with a polyamine column and refractive index detector,
eluted by 2.50 mL/min 75% CH CN in H O to yield D-arabinose
3
2
and 2-N-acetylamino-D-glucose, which were identified by com-
parison of t and with authentic samples. D-arabinose: tR 12.4,
R
20
[
[α]
α]
D
2 R
123.6 (c = 0.055, H O); 2-N-acetylamino-D-glucose: t 15.2,
20
Gorgonian material
D
2
118.2 (c = 0.065, H O). Then astrogorgioside C (3, 4.7 mg)
The gorgonian A. dumbea was collected from Dongshan Island of
P.R. China and kept frozen at − 20°C until used. The specimen was
identified by Dr. Xiu-Bao Li (South China Sea Institute of Ocean-
ology, Chinese Academy of Sciences). A voucher specimen has
been deposited at the East China Sea Fisheries Research Institute,
Chinese Academy of Fishery Sciences, P.R. China (Accession num-
ber LAD-200804-DS).
was also hydrolyzed to afford D-arabinose and D-glucose: t
R
20
D
2
13.3, [α] 44.4(c = 0.045, H O).
Cytotoxicity assay
Cytotoxicity of the compounds against two human tumor cell
lines, hepatoma Bel-7402, and erythroleukemia K562 was eval-
uated by means of the MTT methods as described [14]. Bel-7402
was obtained from Shanghai Institutes for Biological Sciences,
CAS, and K562 was obtained from the American Type Culture
Collection (ATCC). ADR (Adriamycin) was used as a positive con-
trol.
Extraction and isolation
The whole samples (2.25 kg) were cut into pieces and were per-
colated with methanol (4 L × 4). After evaporation of the solvent,
the aqueous solution was extracted successively with petroleum
ether (PE, 4 × 500 mL) and CHCl3 (4 × 500 mL) to give the PE ex-
tracts (16.5 g), CHCl3 extracts (46.5 g), and the aquatic phase.
The aquatic solution was evaporated and subjected to MCI gel
column chromatography eluted with methanol (MeOH) in water
Supporting information
IR, MS, and NMR spectra of compounds 1–3 are available as Sup-
porting Information.
(75 µm, 3 × 50 cm, 20%, 40%, 60%, 100%, v/v, 1000 mL each) to af-
ford four solutions (LAD I–IV).
LAD IV (10.8 g) was subjected to column chromatography on sili-
Acknowledgements
!
ca gel (3.5 × 50 cm, 300 mesh) eluted with a gradient of CH Cl/
MeOH (17:1 → 1:1, each 1000 mL) to give 13 fractions (Fr. 1–
This project was supported by the Natural Science Foundation of
Shanghai (Grant No. 11ZR1449800), the foundation of Key Labo-
ratory of Marine Bio-resources Sustainable Utilization (No.
LMB101003), and a special research fund for the national non-
profit institutes (East China Sea Fisheries Research Institute,
No. 2007M18) of P.R. China. We thank Dr. Xiu-Bao Li from the
Key Laboratory of Marine Bio-resources Sustainable Utilization
(South China Sea Institute of Oceanology, Chinese Academy of
Sciences) for the identification of gorgonian material.
3
13). Fr. 11 (142 mg) was subjected on a Sephadex LH-20 gel col-
umn (1.5 × 80 cm) and eluted with 70% ethanol (EtOH) in water
to afford compounds 1 (18 mg, ≥ 90% by TLC) and 4 (87 mg). Com-
pound 4 was also a major constituent of Fr. 9–10. Fr. 8 (66 mg)
was separated by a reversed-phase ODS‑A column eluted with
MeOH/H O (150 µm, 3 × 40 cm, 60% → 100%) to give compound
2
2
(25 mg, ≥ 90% by TLC). Fr. 6 (242 mg) was separated by the same
reversed-phase ODS‑A column eluted with MeOH/H O (75%
2
→
100%) to afford 11 fractions (Fr. 601–611). A major constituent
in Fr. 605–607 was too difficult to purify. Fr. 609 (32 mg) was sub-
jected to column chromatography on silica gel (1.5 × 30 cm, 300
Conflict of Interest
!
mesh) and eluted with a gradient of CH Cl/MeOH (15:1 → 8:1,
The authors declare no competing financial interest.
3
v/v, 200 mL each) to give compound 3 (8 mg, ≥ 90% by TLC).
2
0
Astrogorgioside A (1): Colorless powder. [α]
D
− 42 (c = 0.125,
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1
"
13
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"
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h, and then after evaporation of MeOH, the solution was ex-
6
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