Secondary Metabolites from Marine Bacterium Nocardiopsis Sp. (G057)

Journal of Science & Technology 118 (2017) 009-013
9
Secondary Metabolites from Marine Bacterium Nocardiopsis Sp. (G057)
Tran Van Hieu1, Doan Thi Mai Huong1,*, Vu Thi Quyen1, Nguyen Quang Tung2,
Chau Van Minh1, Pham Van Cuong1,*
1Institute of Marine Biochemistry-VAST, 18, Hoang Quoc Viet, Cau Giay, Hanoi, Viet Nam
2Hanoi University of Industry - Minh Khai Ward, Bac Tu Liem District, Hanoi, Vietnam
Received: September 30, 2016; accepted: June 9, 2017
Abstract
Seven compounds were isolated and characterized from the culture broth of the marine bacteria
Nocardiopsis sp. (strain G057), which was isolated from sediment collecting at Cô Tô – Quảng Ninh. Their
structures were determined by spectroscopic analysis including MS and 2D NMR, as well as by comparison
with reported data in the literature. All compounds were evaluated for their antimicrobial activity against a
panel of clinically significant microorganisms. Compounds 1, 2 and 7 selectively inhibited Escherichia coli
with a MIC value of 32, 64, 8 μg/mL, respectively. Compound 3 exhibited antimicrobial activity against
several strains of both gram-positive and gram-negative bacteria.
Keywords: Nocardiopsis, marine microorganisms, antimicrobial activity, Cylo-(Leu-Pro), xanthone.
1. Introduction
Marin* microorganisms have been the important
study in recent years because of production of novel
metabolites which represent various biological
properties such as antiviral, antitumor or
antimicrobial activities [1-2]. These secondary
metabolites serve as model systems in discovery of
new drugs [3]. In search of bioactive metabolites
from marine bacteria, we examined the extract of the
culture broth of the marine bacterium Nocardiopsis
sp. (G057 strain). During our screening program, the
EtOAc extract of this strain exhibited antimicrobial
activity against both gram-positive (Enteroccocus
faecalis - ATCC13124) and gram-negative
(Escherichia coli - ATCC25922 and Salmonella
enterica ATCC12228) bacteria strains, and the
fungus strain (Candida albicans - ATCC1023).
Herein, we described the isolation and structural
determination of seven compounds (1 - 7) from the
culture broth of Nocardiopsis sp. (G057) (Figure 1).
Compound 1, 2 and 7 selectively inhibited
Escherichia coli with a MIC value of 32, 64, 8
μg/mL, 3 exhibited antimicrobial activity against
several strains of gram-positive and gram-negative
bacteria.
2. Materials and Methods
2.1. General Experimental procedures
ESI-MS were recorded on an Agilent 1100 LC-
MSD Trap spectrometer. NMR spectra were recorded
* Corresponding author: Tel: (+84) 4.37564995
Email:doanhuong7@yahoo.com
on a Bruker 500.13 MHz spectrometer operating at
125.76 MHz for 13C NMR, and at 500.13 MHz for 1H
NMR. 1H chemical shifts were referenced to CDCl3,
DMSO-d6 and CD3OD at δ 7.27, 2.50 and 3.31 ppm,
respectively, while the 13C chemical shifts were
referenced to the central peak of at δ 77.1 (CDCl3),
39.5 (DMSO-d6), and 49.0 (CD3OD). For HMBC
experiments the delay (1/2J) was 70 ms. TLC silica
gel Merk 60 F254 was used as Thin-layer
chromatography. Column chromatography (CC) was
carried out using silica gel 40-63 µm or Sephadex
LH-20.
2.2. Bacteria isolation and fermentation
The marine sediment was collected from the
coast of Cô Tô – Quảng Ninh in Vietnam in June of
2014. The sediment sample (1 g) was added to 10 mL
of sterile sea water in a conical flask. The flask was
agitated for about one hour. The marine sediment was
filtered and the filtrate was serially diluted to obtain
10-1 to 10-7 dilutions using the sterilized sea water. An
aliquot of 100 μL of each dilution was spread on the
media. Different media like Starch Casein Agar
(SCA), Glycerol Asparagine Agar (GA Agar), humic
acid-B vitamin agar (HV Agar) and Glucose yeast
malt extract agar (GYM) were used for isolation of
actinomycetes. The media containing 50% of sterile
sea water were supplemented with rifampicin (5
μg/mL) and nystatin (25 μg/mL) (Himedia Mumbai)
to inhibit bacterial and fungal contamination,
respectively. The petriplates were incubated for up to
3 weeks at 28 °C. The isolated discrete colonies were
observed and used for identification. The
fermentation was carried out in a 5 L flask using a
modification of the published method [4].
Journal of Science & Technology 118 (2017)
10
2.3. Extraction and isolation
The culture broth (50 L) of Nocardiopsis sp.
(G057 strain) was filtered, and then extracted with
ethyl acetate (30 L x 5 times). The extract was
concentrated in vacuo to give 3.5 g of ethyl acetate
extract. The ethyl acetate extract (3.5 g) was
fractionated by column chromatography (CC) on
silica gel, eluted with CH2Cl2/MeOH gradient to give
five fractions. Fraction F1 was subjected to Sephadex
LH-20 CC (MeOH) to afford four subfractions,
subfraction F1.2 was subjected to CC on silica gel,
eluted with mixtures of CH2Cl2/acetone (9/1) to
afford 7 (6 mg). Fraction F2 (700 mg) was
chromatographed by CC on Sephadex LH-20
(MeOH/CH2Cl2: 9/1), providing 3 subfractions.
Subfraction F2.2 (200 mg) was subjected to CC on
Sephadex LH-20 (MeOH/CH2Cl2: 9.5/0.5) affording
4 (10 mg). Subfraction F2.3 (170 mg) was
chromatographed on Sephadex LH-20
(MeOH/CH2Cl2: 9/1), giving 6 (3.5 mg). Fraction F3
(1.3 g) was separated by CC on Sephadex LH-20
(MeOH/CH2Cl2: 9/1), leading to five subfractions.
Subfraction 2 (200 mg) was purified by CC on
Sephadex LH-20 (MeOH/CH2Cl2: 9/1) followed by
preparative TLC (CH2Cl2/EtOAc: 7/1) to furnish
compounds 5 (3.0 mg). Subfraction F3.5 (250 mg)
was separated by Sephadex LH-20 CC
(MeOH/CH2Cl2: 9/1) to provide 1 (4.5 mg). Fraction
F4 (500 mg) was subjected to Sephadex LH-20 CC,
eluting with a mixture of MeOH/CH2Cl2: 7/3, to yield
4 subfractions. Subfraction F4.2 (250 mg) was
chromatographed on silica gel column, eluted with a
solvent gradient of CH2Cl2/MeOH to afford 2 (4 mg)
and 3 (15 mg).
1-hydroxy-4-methoxy-2-naphthoic acid (1): White
amorphous solid, ESI-MS: m/z 241.04 [M+Na]+; 1H-
NMR (500 MHz, MeOD): δH (ppm) 3.98 (3H, s,
CH3O-4), 7.29 (1H, s, H-3), 7.51 (1H, dt, J=1.5, 8.0
Hz, H-7), 7.56 (1H, dt, J=1.5, 8.0 Hz, H-6), 8.15 (1H,
br d, J= 8.0 Hz, H-5), 8.29 (1H, br d, J= 8.0 Hz, H-8).
13C- NMR (125 MHz, MeOD): δC (ppm) 56.1 (4-
OCH3), 104.5 (C-3), 110.2 (C-2), 122.6 (C-5), 124.2
(C-8), 126,4 (C-7), 127.0 (C-8a), 128.3 (C-6), 130.1
(C-4a), 148.1 (C-4), 155.0 (C-1), 176.3 (C=O).
Scopoletin (2): White amorphous solid, ESI-MS: m/z
193 [M+H]+; 1H- NMR (500 MHz, CDCl3): δH (ppm)
3,96 (3H, s, O-CH3), 6.27 (1H, d, J=9.5 Hz, H-3),
6.85 (1H, s, H-5), 6.92 (1H, s, H-8), 7.60 (1H, d,
J=9.5 Hz, H-4). 13C- NMR (125 MHz, CDCl3): δC
(ppm) 56.4 (O-CH3), 103.2 (C-8), 107.5 (C-5), 111.5
(C-4a), 113.5 (C-3), 143.2 (C-4), 144.0 (C-6), 149.7
(C-7), 150.3 (C-8a), 161.4 (C-2).
Xanthone (3): Yellow solid, ESI-MS: m/z 194.9 [M-
H]-; 1H- NMR (500 MHz, CDCl3): δH (ppm) 6.67
(2H, m, H-2, H-4); 7.30 (1H, t, J=7.5 Hz, H-3), 7.92
(1H, d, J=7.5 Hz, H-1). 13C- NMR (125 MHz,
CDCl3): δC (ppm) 109.8 (C-8a), 116.5 (C-2), 116.8
(C-4), 132.1 (C-1), 135.0 (C-3), 151.1 (C-4a), 173.0
(C=O).
Cylo-(Leu-Pro) (4): White amorphous solid, m.p 147-
148oC, ESI-MS: m/z 249 [M+K]+. 1H NMR (500
MHz, CD3OD): 0.98 (3H, d, J= 6.5 Hz, CH3-13), 0.99
(3H, d, J= 6.5 Hz, CH3-12), 1.54 (1H, m, Ha-10), 1.91
(1H, m, H-11), 1.93 (1H, m, Ha-4), 1.98 (1H, m, Hb-
4), 2.07 (1H, m, Hb-10), 2.08 (1H, m, Ha-5), 2.34
(1H, m, Hb-5), 3.55 (2H, m, CH2-3), 4.14 (1H, m, H-
9), 4.28 (1H, t, J= 7.5 Hz, H-6). 13C NMR (125 MHz,
CD3OD): 21.2 (C-13), 22.7 (C-4), 23.3 (C-12), 24.7
(C-11), 28.1 (C-5), 38.7 (C-10), 45.5 (C-3), 53.4 (C-
9), 59.0 (C-6), 166.2 (C-1), 170.2 (C-7).
Cyclo-(Pro-Tyr) (5): White amorphous solid, m.p
156-158oC, ESI-MS: m/z 261 [M+H]+. 1H NMR (500
MHz, CDCl3): 1.87 (2H, m, CH2-10), 1.99 (1H, m, H-
5b), 2.31 (1H, m, H-5a), 2.79 (1H, dd, J = 9.5, 14.5
Hz, Hb-10), 3.44 (1H, dd, J = 9.5, 14.5 Hz, Ha-10),
3.53 (1H, m, Hb-3), 3.64 (1H, m, Ha-3), 4.06 (1H, dd,
J = 1.5, 7.5 Hz, H-6), 4.22 (1H, dd, J = 2.5, 9.5 Hz),
6.07 (1H, s, NH), 6.77 (2H, d, J = 8.5 Hz, H-3’), 7.03
(2H, d, J = 8.5 Hz, H-2’).
Cyclo-(Pro-Phe) (6): White solid, HRESI-MS: m/z
245.1316 [M+H]+ (Calcd. 245.1290 for C14H17N2O2);
1H-NMR (400 MHz, CD3OD): δH (ppm) 1.60 (2H, m,
Ha-4, Ha-5), 1.91-2.07 (3H, m, Hb-4, Hb- 5, Ha-10),
2.64 (1H, m, Hb-10), 3.02 (1H, dd, J = 4.8, 13.6 Hz,
Ha-3), 3.22 (1H, dd, J = 4.4, 13.6 Hz, Hb-3), 3.59 (1H,
m, H-6), 4.22 (1H, t, J = 4.8 Hz, H-9), 7.20-7.33 (5H,
aromatic); 13C-NMR (100 MHz, CD3OD): δC (ppm)
21.1 (C-4), 28.4 (C-5), 39.6 (C-10), 44.7 (C-3), 57.7
(C-9), 58.4 (C-6), 127.1-129.9 (CH-aromatic), 135.3
(C-1’), 166.0 (C=O), 170.0 (C=O).
4-hydroxybenzaldehyde (7): Amorphous solid, 1H-
NMR (500 MHz, CDCl3): δH (ppm) 6.95 (2H, d, J=
8.5 Hz, H-3, H-5), 7.80 (2H, d, J= 8.5 Hz, H-2, H-6),
9.87 (1H, s, -CHO).
3. Results and discussion
Compound 1 was isolated as white amorphous
solid. The 1H NMR spectrum of 1 showed signals of a
1,2-disubstituted benzene ring [δH 87.51 (1H, dt,
J=1.5, 8.0 Hz, H-7), 7.56 (1H, dt, J=1.5, 8.0 Hz, H-
6), 8.15 (1H, br d, J= 8.0 Hz, H-5), 8.29 (1H, br d, J=
8.0 Hz, H-8)], and a singlet aromatic proton at δH
7.29 (1H, s, H-3). Signal of a singlet methoxy at δH
3.98 (3H, s, CH3O-4) was also noted. Analysis of the
13C NMR and DEPT spectra of 1 revealed the
presence of 12 carbons, including one methoxy group
at δC 56.1 (4-OCH3), ten aromatic carbons (five
methines and five quaternary carbons), and one
carbonyl at δC 176.3 (C=O). The carbon chemical
Journal of Science & Technology 118 (2017)
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shifts of C-4 (δC 148.1), and C-1(δC 155.0) suggested
its connection to oxygen. Analysis of the HMBC
spectrum confirmed the 1,2-disubstituted benzene
ring by cross-peaks of C-4a with H-8, and those of C-
8a with H-5 (Figure 2). Furthermore, the methoxy
protons correlated to the carbonyl C-4, indicating the
linkage of methoxy group to C-4 (Figure 2). Detailed
analysis of NMR spectra and comparison with
reported values in the literature [5], the structure of 1
was determined to be 1-hydroxy-4-methoxy-2-
naphthoic acid.
Compound 2 was obtained as white amorphous
solid. The ESI-MS indicated the pseudo-molecular
ion peak at m/z 193 [M+H]+. The 13C NMR spectrum
established the presence of 10 carbons corresponding
to 4 aromatic methines, one aromatic methoxy, one
lactone carbonyl (C 161.4, C-2), and 4 other
quaternary carbons. The 1H NMR spectrum
displayed a pair of doublet (J=10.0 Hz) at δH 6.27 and
7.60, which are typical for a coumarin unsubstituted
in the pyrone ring, whereas a two singlet at δH 6.85
and 6.82 were consistent with the presence of two
subtituents in the aromatic ring. At higher field,
typical signals accounted for one aromatic methoxy at
δH 3.96 (3H, s, O-CH3). The long-range coupling
observed in HMBC spectrum between the methoxy
protons with C-6 permitted to locate the aromatic
methoxy subtituent at C-7. The full analysis of the 1D
and 2 D NMR together with literature data [6] clealy
indicated the structure of 2 as scopoletin.
Fig. 1. Isolated compounds from the broth culture of
Nocardiopsis sp. (G057 strain)
Compound 3 was isolated as yellow solid. The
ESI mass spectrum (negative) of 3 showed a
pseudomolecular ion peak at m/z 194.9 [M-H]-. The
1H-NMR spectrum of 3 displayed signals of a 1,2-
disubstituted benzene ring [δH 6.67 (2H, m, H-2, H-
4), 7.30 (1H, t, J=7.5 Hz, H-3), 7.92 (1H, d, J=7.5
Hz, H-1)]. Analysis of the 13C NMR and DEPT
spectra of 3 revealed the presence of one carbonyl at
δC 173.0 (C=O), four methine carbons at δC 116.5 (C-
2), 116.8 (C-4), 132.1 (C-1), 135.0 (C-3), and two
quaternary carbons at δC 109.8 (C-9a), 151.1 (C-4a).
The carbon chemical shifts of C-4a suggested its
connection to oxygen. This observation indicated that
compound 3 had a symmetric structure. The structure
of 3 was then confirmed by analyses of 2D-NMR
spectra which allowed establishing as xanthone [7].
Compound 4 was isolated as white amorphous
solid. The ESI-MS indicated the pseudomolecular ion
peak at m/z 249 [M+K]+. The 1H NMR spectrum of 4
displayed signals of 2 methyl groups as doublet of
doublet at δH 0.98 (d, J = 6.5 Hz, CH3-13), 0.99 (d, J
= 6.5 Hz, CH3-12) and signals of ten aliphatic
protons. Analysis of the 13C-NMR and DEPT spectra
of 4 revealed the presence of 11 carbons, including
two carbonyl at δC 166.2 (C-1) and 170.2 (C-7), two
methyl groups at δC 21.2 (C-13) and 23.3 (C-12),
three methines at δC 24.7 (C-11), 53.4 (C-9) and 59.0
(C-6), and four methylenes at δC 22.7 (C-4), 28.1 (C-
5), 38.7 (C-10), 45.5 (C-3). The chemical shifts of
CH2-3, CH-6 and CH-9 suggested their linkage to
nitrogen atoms. This data suggested the presence of
two amino acid units, proline and leucine in the
structure of 4. Based on detailed analysis of NMR
spectra and comparison with reported values in the
literature [8-9], the structure of 4 was determined to
be Cylo-(Pro-Leu). This compound inhibited against
Gram positive bacteria B. subtilis and S. aureus with
a MIC value of 16, 32 μg/mL, respectively [10].
Compound 5 was isolated as a white amorphous
solid. The ESI-MS indicated the pseudo-molecular
ion peak at m/z 261 [M+H]+. The 1D NMR spectrum
of 5 displayed signals of the proline unit as 4.
However, in comparison with 4, the presence of an
A2B2 aromatic system [δH 6.77 (2H, d, J = 8.5 Hz, H-
3’) and 7.03 (2H, d, J = 8.5 Hz, H-2’)] instead of
signals of the 2-propyl group was noted for 4. This
observation strongly suggested that the leucine unit of
4 was replaced by the 4-hydroxyphenylanaline
moiety in 5. Comparison with the literature [11],
compound 5 was identified as Cyclo-(Pro-Tyr). This
cyclodipeptide had antibacterial activity against both
Gram-positive and Gram-negative bacteria and
antifungal property [10].
Compound 6 was isolated as a white solid. In its
positive HRESI mass spectrum, the pseudo-molecular
ion was observed at m/z 245.1316 [M+H]+, consistent
with the molecular formula C14H16N2O2. The 1D-
NMR spectra (1H and 13C) of compound 6 were close
to those of 5, except for the presence of a phenyl ring
instead of the A2B2 system. This data strongly
suggested that the 4-hydroxy-phenylalanine fragment
in 5 was replaced by a phenylalanine moiety in 6.
Comparison of NMR data revealed the structure of 6
which was identical to Cyclo-(Pro-Phe) [12].
Compound 7 was obtained as a white amorphous
solid and determined to be 4-hydroxybenzaldehyde.
Its NMR data were consistent with those reported in
the literature [13]
Journal of Science & Technology 118 (2017) 009-013
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Table 1. Antibacterial and antifungal activities of compounds 1-7 (MIC: μg/mL).
Compounds Gram (+) Gram (-) Fungal
E. faecalis S. aureus B. cereus E.coli P. aeruginosa S. enterica C. albicans
1 >256 >256 >256 32 >256 >256 >256
2 >256 >256 >256 64 >256 >256 >256
3 128 256 256 64 256 128 >256
4 >256 >256 >256 >256 >256 >256 >256
5 >256 >256 >256 >256 >256 >256 >256
6 >256 >256 >256 >256 >256 >256 >256
7 >256 >256 >256 8 >256 >256 >256
S 256 256 128 32 256 128 -
C - - - - - - 32
(S= Streptomycin, C= Cyclohexamide)
Fig. 2. Selected COSY ( ) and HMBC ( ) correlations of 1-3 and 5
All the isolated compounds were evaluated for
their antibacterial activity against Escherichia coli
(ATCC25922), Pseudomonas aeruginosa
(ATCC27853), Salmonella enterica (ATCC12228),
Enterococcus faecalis (ATCC13124), Staphylococcus
aureus (ATCC25923), Bacillus cereus
(ATCC13245), and antifungal activity against
Candida albicans (ATCC1023). Compound 1, 2 and
7 selectively inhibited Escherichia coli with a MIC
value of 32, 64, 8 μg/mL, respectively. Compounds 3
exhibited antimicrobial activity against several strains
of both gram-positive and gram-negative bacteria
(table 1).
4. Conclusion
Seven secondary metabolites 1-hydroxy-4-
methoxy-2-naphthoic acid (1), scopoletin (2),
xanthone (3), Cylo-(Pro-Leu) (4), Cyclo-(Pro-Tyr)
(5), Cyclo-(Pro-Phe) (6), and 4-hydroxybenzaldehyde
(7) were isolated from the cultures broth of
Nocardiopsis sp. (G057). Compound 1, 2 and 7
selectively inhibited Escherichia coli with a MIC
value of 32, 64, 8 μg/mL, respectively. Compounds 3
exhibited antimicrobial activity against several strains
of gram-positive and gram-negative bacteria.
Acknowledgements
The authors thank Prof. Do Cong Thung (VAST
- Vietnam) for marine sample collection. The
Vietnam Academy of Science and Technology
(VAST) are gratefully acknowledged for financial
support (Grant No: VAST.TĐ.ĐAB.04/13-15).
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