Anthraquinone glycosides from marine Streptomyces sp. strain

Article abstract of DOI:10.1016/j.phytol.2012.04.005

Two new anthraquinone glycosides Strepnoneside A (1) and Strepnoneside B (2), together with Chromomycin A₃ (3), were isolated from cultures of the marine Streptomyces sp. strain. The structures were elucidated on the basis of NMR spectroscopic and mass spectrometry data. Compound 3 exhibited cytotoxic activities against HCT 116 cell lines (IC₅₀ = 300 ± 11 pM).

Full text of DOI:10.1016/j.phytol.2012.04.005

Phytochemistry Letters 5 (2012) 459–462
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Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Anthraquinone glycosides from marine Streptomyces sp. strain
Yuanyuan Lu a,b,1, Yingying Xing b,1, Chen Chen b, Jiansheng Lu b, Yihua Ma b, Tao Xi b,
*
a State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing, Jiangsu 210009, China
b Department of Marine Pharmacy, China Pharmaceutical University, Nanjing, Jiangsu 210009, China
A R T I C L E I N F O
A B S T R A C T
Article history:
Two new anthraquinone glycosides Strepnoneside A (1) and Strepnoneside B (2), together with
Chromomycin A3 (3), were isolated from cultures of the marine Streptomyces sp. strain. The structures
were elucidated on the basis of NMR spectroscopic and mass spectrometry data. Compound 3 exhibited
cytotoxic activities against HCT 116 cell lines (IC50 = 300 ꢁ 11 pM).
Received 6 February 2012
Received in revised form 2 April 2012
Accepted 6 April 2012
Available online 20 April 2012
ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Key words:
Streptomyces
Metabolites
Anthraquinone glycosides
Cytotoxic activity
1. Introduction
2. Results and discussion
Development of multiple drug resistant in microbes and tumor
cells has become a major problem and has revealed the need to
search for new and novel anticancer antibiotics (Wise, 2008;
Demain and Sanchez, 2009). In previously study, the class
Actinobacteria, and especially Streptomyces sp. showed to be a
prominent reservoir for bioactive compounds and continues to be a
source of new bioactive compounds (Berdy, 2005; Kavitha et al.,
2010a). However, the percent of discovery of new bioactive
compounds produced by Streptomyces sp. from common and
ubiquitous sources (such as soil, river, etc.) has been declined
(Berdy, 2005; Kavitha et al., 2010b). Hence, in present era,
metabolites from marine Streptomyces sp. have becoming a new
urgent focus in the research of anticancer antibiotics (Hughes et al.,
2010; Motohashi et al., 2010; Raju et al., 2010). In a previous study,
a marine Streptomyces sp. strain was isolated from sediments of
Weihai, and its taxonomic study indicated that it may belong to
Streptomyces coelicolor (Meng et al., 2008). Since its fermentation
show cytotoxic activities against human colon carcinoma cell line
(HCT116), the potential medicinal importance and our interest in
new active products prompted us to investigate metabolites of the
strain. This paper reports on the isolation and biological evaluation
of two new anthraquinone glycosides (Strepnonesides A (1) and B
(2)) and Chromomycin A3 (3) produced by the marine Streptomyces
sp. (Fig. 1).
Compound 1 (Strepnoneside A) was obtained as a yellow
amorphous powder (CHCl3), showing positive reaction with
Molish and Borntra¨ger reagent. It was assigned the molecular
formula C32H36O14 from its negative-ion HRESI-MS (m/z 643.2022
[MꢀH]ꢀ, calcd for C32H35O14ꢀ [MꢀH]ꢀ: 643.2027). Acid hydrolysis
of 1 yielded
D-oliose (identified by co-TLC with an authentic
sample) and an aglycon.
The 1H NMR spectrum of compound 1 (see Table 1) showed
two phenolic hydroxy protons (
at 7.91 and 8.38 (each 1H, d, J = 1.5 Hz) assigned to meta
coupled aromatic protons H-2 and H-4, respectively; one singlet
aromatic proton at 7.54 due to H-5, showed the presence of
third substituent on ring A (the aglycon); two methoxyl groups
were observed at 3.98 and 3.61, and were assigned to H-13 and
H-700, respectively; aromatic methyl proton signal at
2.24; one
methyl proton at
2.18 assigned to H-80; two anomeric protons
at
5.42 (1H, d, J1 = 9.5 Hz, J2 = 2.0 Hz, H-10) and 5.13 (1H, br s, H-
100), respectively; two methyl protons of 2, 6-deoxyhexopyranose
d 12.12 and 12.33); two doublets
d
d
d
d
d
d
at
d
1.30 (3H, d, J = 7.0 Hz, Me-60) and 1.31 (3H, d, J = 7.0 Hz, Me-
600), respectively. The 13C NMR spectrum data of 1 also revealed
the presence of anthraquinone moiety: two conjugated carbonyl
moieties (
oxygen substitution (
signals (three methines
carbon signals 137.3, 122.3, 133.9, 111.1, 118.4, 132.3). The A
d
180.9 and 191.4), three aromatic carbons with
161.5, 162.3, 162.7), nine low-field carbon
106.6, 120.1, 125.4 and six quaternary
d
d
d
ring’s substitutes position of the aglycon were suggested by
HMBC correlations observed from H-5 to C-6, C-7, C-10, C-8a, and
C-10a, and from H-11 to C-6 and C-8. H-13, H-2 and H-4 signals
* Corresponding author. Tel.: +86 25 83271022; fax: +86 25 83271249.
E-mail address: taoxi18@hotmail.com (T. Xi).
1
were detected the HMBC correlations to C-12
(d 164.9),
Both authors contributed equally to this work.
1874-3900/$ – see front matter ß 2012 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.phytol.2012.04.005

460
Y. Lu et al. / Phytochemistry Letters 5 (2012) 459–462
Fig. 1. Structures of Strepnoneside A (1) and Strepnoneside B (2), and Chromomycin A3 (3) isolated from cultures of the marine Streptomyces sp. strain.
Table 1
1H NMR (500 MHz, CDCl3), 13C NMR (125 MHz, CDCl3) and HMBC spectroscopic data for 1–2 with J values (in Hz) in parentheses.
No.
1
2
dH
dC
HMBC
dH
dC
HMBC
1
–
162.3
125.4
137.3
120.1
106.6
161.5
122.3
162.7
191.4
180.9
8.5
–
154.7
131.2
123.5
155.7
106.1
161.3
122.0
162.2
191.5
183.4
8.6
2
7.91 d (1.5)
C-1, 4, 9a, 12
7.81 s
C-1, 4, 9a, 12
–
3
–
–
–
4
8.38 d (1.5)
C-9a, 10, 12
–
5
7.54 s
C-6, 7, 8a, 10, 10a
7.60 s
C-6, 7, 8a, 10, 10a
6
–
–
–
–
7
–
–
–
–
8
–
–
–
–
9
–
–
–
–
10
–
–
–
–
11
2.24 s
C-6, 8
2.27 s
C-6, 8
12
–
164.9
52.8
–
–
164.4
52.8
–
13
3.98 s
C-12
–
4.00 s
C-12
4a
–
133.8
111.1
118.4
132.3
–
117.8
111.8
117.4
132.4
–
8a
–
–
–
–
9a
–
–
–
–
10a
–
–
–
–
OH at C1
12.12 s,
–
C-2
12.17 s
13.80 s
12.44 s
5.44 dd (9.7,2.1)
2.24 m, 2.13 m
4.04 m
5.23 d (2.6)
3.96 br q (6.7)
1.34 d (6.7)
–
C-2
OH at C4
C-3, 4a
C-7, 8a
C-6
OH at C8
12.33 s
C-7, 8a
C-6
10
5.42 dd (9.5, 2.0)
2.22 m, 2.11 m
4.04 m
95.4
32.8
70.0
67.2
70.1
16.6
170.7
20.8
96.9
33.5
65.9
81.5
67.1
17.2
62.3
95.4
32.8
70.0
67.2
70.2
16.7
170.6
20.8
97.0
33.5
65.9
81.5
67.1
17.3
62.4
20
30
40
5.20 br d (3.0)
3.92 br q (7.0)
1.30 d (7.0)
–
C-30, 70
C-40
C-30, 70
C-40
50
60
70
–
C-70
C-40, 300, 500
–
C-70
C-40, 300, 500
80
2.18 s
2.21 s
100
200
300
400
500
600
700
5.13 br s
1.79 m
5.13 br s
1.80 m
4.04 m
3.25 d (2.7)
3.90 q (6.9)
1.32 d (6.9)
3.64 s
3.98 m
3.23 br d (3.0)
3.87 br q (7.0)
1.31 d (7.0)
3.61 s
C-400
C-400
C-400
C-400
indicating that C-3 was substituted by a carboxylic methoxyl
ester. Those NMR date indicated that the aglycon was identified
to 1, 6, 8-trihydroxy-7-methyl-3-methoxycarbonyl anthraqui-
none. NMR data of sugar moieties were identical to a disaccha-
was elucidated as 6-O-[3-O-
D-4-O-acetyl-olioside] 1, 6, 8-trihydroxy-7-methyl-3-methoxy-
carbonyl-anthraquinone.
a
-D-4-O-methyl-oliosyl-(1 ! 3)-
b-
Compound 2 (Strepnoneside B) was obtained as a yellow
amorphous powder (CHCl3), showing positive reaction with
Molish and Borntra¨ger reagent. It was assigned the molecular
formula C32H36O15 from its negative-ion HRESI-MS (m/z 659.6107
[MꢀH]ꢀ, calcd for C32H35O14ꢀ [MꢀH]ꢀ: 659.6113). The 1D and 2D
NMR data of 2 were in good coincident with those of 1 except for
noticeable differences in ring C of its aglycon (see Table 1). A new
phenolic hydroxyl of 2 was further evidenced by 1H NMR
ride
Chromomycin A3 (Yoshimura et al., 1988). Also, the key HMBC
correlations (see Fig. 2) from -oliose)
5.42 (H0-1 of 4-O-acetyl-
to 161.5 (C-6 of aglycon), from
5.13 (H00-1 of 4-O-methyl-
oliose) to -oliose) indicated that the
70.0 (C-30 of 4-O-acetyl-
sugar chain was linked to C-6 of the aglycon and the 4-O-methyl-
-oliose was attached to C-30 of the 4-O-acetyl-
-oliose. The
anomeric proton coupling constants suggested 4-O-acetyl-
oliose (3J1,2 > 7 Hz) and 4-O-methyl- -oliose (3J1,2 < 2 Hz) have
and -configuration, respectively. In conclusion, compound 1
(4-O-acetyl-
D
-oliose
and
4-O-methyl-
D
-oliose)
of
d
D
d
d
D-
d
D
D
D
D
-
spectroscopic (a phenolic hydroxy hydrogens
HRESI-MS data. Its substituted position was suggested by HMBC
correlations (see Fig. 2) from H-2 ( 7.81) to C-1 (154.7), C-4
d 13.80) and the
D
b
a
d

Y. Lu et al. / Phytochemistry Letters 5 (2012) 459–462
461
Fig. 2. Key HMBC correlations of compounds 1 and 2.
(155.7), C-9a (117.4) and C-12 (168.4). Also, position of the sugar
moieties was unambiguously deduced by the HMBC experiment.
using CDCl3 as solvent with tetramethylsilane (TMS) as internal
reference, The chemical shifts were given in
constants in Hz.
d and coupling
Structure 2 was therefore identified to 6-O-[3-O-a-D-4-O-methyl-
oliosyl-(1 ! 3)-
b
-D-4-O-acetyl-olioside] 1, 4, 6, 8-tetrahydroxy-7-
TLC was performed on silica gel GF254 (Qingdao Haiyang
Chemical Co. Ltd.) plates. D101 macroporous adsorption resin
(pore size B 13–14 mm, Cangzhou Bonchem Co. Ltd., China),
methyl-3-methoxy-carbonyl-anthraquinone.
Additionally, compound 3 was identified to Chromomycin A3 by
comparing its NMR spectroscopic data with those in the literature
(Yoshimura et al., 1988).
Three isolated compounds were tested for cytotoxic activity
against HCT116 cell lines. Strepnoneside A (1), Strepnoneside B (2)
and Chromomycin A3 (3) were found to exhibit potent activities
with IC50 values of 30.2 ꢁ 3.2 mM, 40.2 ꢁ 2.0 mM, and 300 ꢁ 11 pM.
Whereas the positive control Cisplatin showed an IC50 value of
18.5 ꢁ 2.3 mM.
Sephadex LH-20 (20–100
silica gel (ODS) (40–63
chromatography. -Oliose was purchased from Hangzhou Wisdom
m
m, Pharmacia), and octadecylsilanized
m
m, YMC, Japan) were used for column
D
Network Technology Co. Ltd.
3.3. Fermentation, extraction, purification and structural elucidation
of bioactive metabolites
In order to isolate and characterize the metabolites from the
strain, a loopful culture of Streptomyces sp. WBF-16 was cultivated
in seed broth (1% glucose, 1.2% tyrosine, and 0.2% yeast extract
dissolving in artificial seawater, pH 7.2) and incubated on a rotary
shaker (200 rpm) at 28 8C. After 96 h of incubation, the seed culture
(5%, v/v) was transferred to the optimized fermentation medium
(2% soluble starch, 2% soybean meal, and 1% glucose dissolving in
artificial seawater, pH 7.0) (Chen et al., 2010).
Culture filtrates (20 L) were obtained by cultivation of the
strain in fermentation tank Biostat C20-3 (B. Brawn Biotech
International, USA) for 128 h under the following conditions:
rotation speed: 200 rpm, gas flow: 4.5 L/min, pH: 7.0, and
temperature: 28 8C. The filtrates were subjected to D101
macroporous adsorption resin using the gradient solvent system
of H2O:EtOH (H2O, 70:30, v/v, EtOH 100%) affording three
fractions (Fr. 1–3). Each fraction was evaluated for its antitumor
activities in vitro. Fraction 3 (2 g) shows the strongest activity of
all the fractions, therefore was chromatographed on a ODS column
3. Materials and methods
3.1. Microorganism isolation
The actinomycetes strain WBF-16 was isolated from the
sediments of soil sample of Wei-hai Sea in china (378320N,
1228030E) employing soil dilution technique on Gause-agar
medium mixing with artificial seawater. It was assigned to the
S. coelicolor cluster and 16S rRNA sequence was submitted to the
NCBI Genbank with accession number HQ848084.
3.2. General methods
UV-vis absorption spectra were obtained using a Shimadzu UV-
1650PC spectrophotometer; ESI-MS and HRESI-MS experiments
were performed on an Agilent 1100 Series MSD Trap mass
spectrometer and Mariner ESI-TOF spectrometer, respectively;
NMR spectra were recorded on a Bruker DRX-500 spectrometers
(
K 3 cm ꢂ 50 cm) using gradient solvent system of H2O:MeOH

462
Y. Lu et al. / Phytochemistry Letters 5 (2012) 459–462
(90:10–10:90, v/v). Three fractions (Fr. 3.1–3.3) were obtained at
different eluent conditions, Fr. 3.3 (1 g, 30:70, v/v, H2O:MeOH)
showed antitumor activities in vitro. Fr. 3.3 was further purified by
the column chromatography on sephadex LH-20 to give com-
pound 1 (7 mg), 2 (4 mg) and 3 (32 mg), the structure of the
compounds was elucidated mainly by spectroscopic methods
(HRESIMS, 1D and 2D NMR).
3.7. Strepnoneside B (2)
Yellow amorph powder (CHCl3); ½a D25
ꢀ52.18 (c 0.11, MeOH);
ꢃ
UV (MeOH)
l max (e): 233 (36,800), 280 (22,100), 302 (7810), 492
(11,400), 513 (7700) nm; 1H NMR (500 MHz, CDCl3), Table 1; 13C
NMR (125 MHz, CDCl3), Taꢀble 1; HR-ESI-MS: m/z 659.6107
[MꢀH]ꢀ (calcd for C32H35O14 [MꢀH]ꢀ: 659.6113).
3.4. Acid hydrolysis
Acknowledgments
Compounds 1 and 2 (1 mg each) in 5% H2SO4-dioxane (1:1,
1 mL) were each heated at 85 8C for 4 h in a water bath. The
reaction mixtures were neutralized with Ba(OH)2, filtered, and
then extracted with CHCl3 (1 mL ꢂ 3). After concentration, each
H2O layer (monosaccharide portion) was examined by TLC with
solvent Me2CO–BuOH–H2O (8:2:1) and compared with authentic
samples.
This study was supported by the Project Program of State Key
Laboratory of Natural Medicines, China Pharmaceutical University
(No. JKGQ201102) and National Nature Science Foundation of
China for Young Scientists (No. 81001395).
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3.5. Antitumor assays
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3.6. Strepnoneside A (1)
Raju, R., Piggott, A.M., Barrientos Diaz, L.X., Khalil, Z., Capon, R.J., 2010. Herona-
pyrroles A-C: farnesylated 2-nitropyrroles from an Australian marine-derived
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Yellow amorph powder (CHCl3); ½a D25
ꢀ50.78 (c 0.10, MeOH);
ꢃ
UV (MeOH)
l max (e): 231 (38,000), 270 (23,000), 310 (7100), 440
(12,300) nm; 1H NMR (500 MHz, CDCl3), Table 1; 13C NMR
(125 MHz, CDCl3), Table 1; HR-ESI-MS: m/z 643.2022 [MꢀH]ꢀ
Yoshimura, Y., Koenuma, M., Matsumoto, K., Tori, K., Terui, Y., 1988. NMR studies of
Chromomycins, Olivomycins, and their derivatives. J. Antibiot. 41, 53–67.
(calcd for C32H35O14 [MꢀH]ꢀ: 643.2027).
ꢀ