Secondary metabolites from marine Bacterium nocardiopsis Sp. (G057)

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Journal of Science & Technology 118 (2017) 009-013

Secondary Metabolites from Marine Bacterium Nocardiopsis Sp. (G057)

Tran Van Hieu¹, Doan Thi Mai Huong^1,*, Vu Thi Quyen¹, Nguyen Quang Tung²,

Chau Van Minh¹, Pham Van Cuong^1,*

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 – Qu ng 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 g/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

on a Bruker 500.13 MHz spectrometer operating at

125.76 MHz for ¹³C NMR, and at 500.13 MHz for ¹H

NMR. H chemical shifts were referenced to CDCl₃,

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

DMSO-d₆and 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 F₂₅₄was 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

faecalis

ATCC13124) and gram-negative

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^-1to 10^-7dilutions 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].

(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,

and

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

Journal of Science & Technology 118 (2017)

(1H, d, J=7.5 Hz, H-1). ¹³C- NMR (125 MHz,

2.3. Extraction and isolation

CDCl₃): δ_C(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).

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

Cylo-(Leu-Pro) (4): White amorphous solid, m.p 147-

148^oC, ESI-MS: m/z 249 [M+K]⁺. H 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, H_a-10), 1.91

(1H, m, H-11), 1.93 (1H, m, H_a-4), 1.98 (1H, m, H_b-

4), 2.07 (1H, m, H_b-10), 2.08 (1H, m, H_a-5), 2.34

(1H, m, H_b-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

156-158^oC, ESI-MS: m/z 261 [M+H]⁺. H NMR (500

(10 mg). Subfraction F2.3 (170 mg) was

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, H_b-10), 3.44 (1H, dd, J = 9.5, 14.5 Hz, H_a-10),

3.53 (1H, m, H_b-3), 3.64 (1H, m, H_a-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’).

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)

Cyclo-(Pro-Phe) (6): White solid, HRESI-MS: m/z

245.1316 [M+H]⁺(Calcd. 245.1290 for C₁₄H₁₇N₂O₂);

¹H-NMR (400 MHz, CD₃OD): δ_H(ppm) 1.60 (2H, m,

H_a-4, H_a-5), 1.91-2.07 (3H, m, H_b-4, H_b- 5, H_a-10),

2.64 (1H, m, H_b-10), 3.02 (1H, dd, J = 4.8, 13.6 Hz,

H_a-3), 3.22 (1H, dd, J = 4.4, 13.6 Hz, H_b-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): δ_C(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).

was

separated

by Sephadex LH-20

(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]⁺; H-

Amorphous solid, ¹H-

NMR (500 MHz, MeOD): δ_H(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): δ_C(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).

4-hydroxybenzaldehyde (7):

NMR (500 MHz, CDCl₃): δ_H(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 [δ_H87.51 (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 δ_H

7.29 (1H, s, H-3). Signal of a singlet methoxy at δ_H

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 δ_C56.1 (4-OCH₃), ten aromatic carbons (five

methines and five quaternary carbons), and one

carbonyl at δ_C176.3 (C=O). The carbon chemical

Scopoletin (2): White amorphous solid, ESI-MS: m/z

193 [M+H]⁺; ¹H- NMR (500 MHz, CDCl₃): δ_H(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): δ_C

(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]^-; H- NMR (500 MHz, CDCl₃): δ_H(ppm) 6.67

(2H, m, H-2, H-4); 7.30 (1H, t, J=7.5 Hz, H-3), 7.92

Journal of Science & Technology 118 (2017)

shifts of C-4 (δ_C148.1), and C-1(δ_C155.0) suggested

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].

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 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 δ_H0.98 (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 δ_C166.2 (C-1) and 170.2 (C-7), two

methyl groups at δ_C21.2 (C-13) and 23.3 (C-12),

three methines at δ_C24.7 (C-11), 53.4 (C-9) and 59.0

(C-6), and four methylenes at δ_C22.7 (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 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 (_C161.4, C-2), and 4 other

quaternary carbons.

The ¹H NMR spectrum

displayed a pair of doublet (J=10.0 Hz) at δ_H6.27 and

7.60, which are typical for a coumarin unsubstituted

in the pyrone ring, whereas a two singlet at δ_H6.85

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

δ_H3.96 (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.

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

A₂B₂aromatic system [δ_H6.77 (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 A₂B₂system. 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].

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 [δ_H6.67 (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

δ_C173.0 (C=O), four methine carbons at δ_C116.5 (C-

2), 116.8 (C-4), 132.1 (C-1), 135.0 (C-3), and two

quaternary carbons at δ_C109.8 (C-9a), 151.1 (C-4a).

The carbon chemical shifts of C-4a suggested its

connection to oxygen. This observation indicated that

Compound 7 was obtained as a white amorphous

4-hydroxybenzaldehyde

solid and determined to be

Its NMR data were consistent with those reported in

the literature [13]

Journal of Science & Technology 118 (2017) 009-013

Table 1. Antibacterial and antifungal activities of compounds 1-7 (MIC: μg/mL).

Compounds

Gram (+)

Gram (-)

Fungal

E. faecalis

>256

128

S. aureus

>256

256

>256

256

B. cereus

>256

256

>256

128

E.coli

P. aeruginosa S. enterica

C. albicans

>256

256

>256

128

>256

256

>256

256

>256

128

(S= Streptomycin, C= Cyclohexamide)

Fig. 2. Selected COSY (

) and HMBC (

) correlations of 1-3 and 5

Acknowledgements

All the isolated compounds were evaluated for

their antibacterial activity against Escherichia coli

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).

(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).

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4. Conclusion

Seven secondary metabolites 1-hydroxy-4-

methoxy-2-naphthoic acid (1), scopoletin (2),

xanthone (3), Cylo-(Pro-Leu) (4), Cyclo-(Pro-Tyr)

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Cong Vinh Le, Hong Minh Le, Brian T. Murphy, Van

Minh Chau and Van Cuong Pham - Secondary

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Sea, Natural Product communication 11 (2016), 401-

404.

4-hydroxybenzaldehyde

(5), Cyclo-(Pro-Phe) (6), and

(7) were isolated from the cultures broth of

Nocardiopsis sp. (G057). Compound 1, 2 and 7

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