Article
pubs.acs.org/JAFC
Cytotoxic and Antibiotic Cyclic Pentapeptide from an Endophytic
Aspergillus tamarii of Ficus carica
Yang-Min Ma,*,† Xi-Ai Liang,† Hong-Chi Zhang,†,§ and Rui Liu§
†
Key Laboratory of Auxiliary Chemistry & Technology for Chemical Industry, Ministry of Education. Shaanxi University of Science &
Technology, Xi’an 710021, Shaanxi, China
§
College of Agronomy and Life Science, Shanxi Datong University, Datong 037009, Shanxi, China
S Supporting Information
*
ABSTRACT: A new cyclic pentapeptide, disulfide cyclo-(Leu-Val-Ile-Cys-Cys) (1), named malformin E, together with 13
known cyclic dipeptides, was isolated from the culture broth of endophytic fungus FR02 from the roots of Ficus carica. The strain
FR02 was identified as Aspergillus tamarii on the basis of morphological characteristics and molecular analyses of internal
transcribed spacer (ITS). Their structures were determined by the combination of 1D and 2D NMR spectroscopy, HRMS (ESI),
UV, and Marfey’s analysis. Compound 1 exhibited strong cytotoxic activities against human cancer cell strains MCF-7 and A549
with IC50 values of 0.65 and 2.42 μM, respectively. It also displayed remarkable antimicrobial activities against Bacillus subtilis,
Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Penicillium chrysogenum, Candida albicans, and Fusarium solani with
MIC values of 0.91, 0.45, 1.82, 0.91, 3.62, 7.24, and 7.24 μM, respectively.
KEYWORDS: malformin E, Aspergillus tamarii, cytotoxicity, antimicrobial activity, cyclopeptide
■
chromatography (CC). Analytical thin-layer chromatography (TLC)
was performed on silica gel GF254 plates, and spots were visualized by
UV absorption and staining with Dragendorff’s reagent. HPLC analysis
was run on a Waters C18 column (250 mm × 4.6 mm) on a Waters
2996 instrument (Waters Corp.). The bacteria (P. aeruginosa, E. coli, B.
subtilis, and S. aureus) and the fungi (C. albicans, F. solani, and P.
chrysogenum) were provided from the School of Food & Biological
Engineering, Shaanxi University of Science & Technology (Xi’an,
China). The cancer cell lines MCF-7 (breast cancer cells), HepG2
(liver cancer cells), and A549 (lung cancer cells) were provided from
the College of Life Sciences, Northwest A&F University (Yangling,
China).
Fungal Material. The strain FR02 was isolated from the healthy
roots of F. carica that was collected from Qinling Mountain in China’s
Shaanxi province in September 2014. It was identified as A. tamarii on
the basis of morphological characteristics and molecular analyses of
ITS. The fungus was maintained on potato dextrose agar (PDA) slants
and deposited at the College of Chemistry & Chemical Engineering,
Shaanxi University of Science & Technology.
Fermention, Extraction, and Isolation. Fermentation of
endophytic fungus A. tamarii was carried out in Czapek’s medium
composed of sucrose (30 g/L), MgSO4·7H2O (0.5 g/L), KH2PO4 (1.0
g/L), KCl (0.5 g/L), FeSO4 (0.01 g/L), and NaNO3 (3.0 g/L). Three
pieces of the strain FR02 (diameter = 0.5 cm) from a mature PDA
plate were inoculated into Erlenmeyer flasks (250 mL) containing 100
mL of the above sterile liquid medium, the fermentation flasks were
cultured with an agitation rate of 135 rpm at 28 °C for 4 days to afford
seed culture solution, and then 5 mL of seed culture solution was
added to Erlenmeyer flasks (1000 mL) containing 300 mL of Czapek’s
medium and cultured under the same condition for 15 days. The
culture broth (80 L) was filtered and concentrated and then extracted
with ethyl acetate three times to yield a crude extract (66.7 g). The
INTRODUCTION
Endophytic fungi can produce structurally and biologically
diverse natural products and have become an important source
of novel structures and strong biological compounds.1,2 In
recent years, more and more bioactive compounds from
endophytic fungi of medical plants have been reported.3−5 Ficus
carica is not only an edible plant but also a traditional medical
plant; strain FR02 was isolated from the roots of F. carica,
identified as Aspergillus tamarii. The metabolites displayed wide
antibacterial and antifungal activities in vitro. In the course of
investigating new strong bioactive compounds from the
endophytic fungus A. tamarii, a new cyclic pentapeptide,
disulfide cyclo-(Leu-Val-Ile-Cys-Cys) (1), named malformin E,
together with 13 known cyclic dipeptides (2−14), fumitremorgin B (2), fumitremorgin C (3), cyclotryprostatin B (4),
verruculogen (5), tryprostatin B (6), tryprostatin A (7),
cyclotryprostatin A (8), cyclotryprostatin C (9), cyclo-(ProTrp) (10), cyclo-(N-methyl-Trp-Leu) (11), cyclotryprostatin
D (12), 13-oxofumitremorgin B (13), and cyclo-(N-BenzylTrp-Pro) (14), were isolated. Compound 1 exhibited strong
cytotoxicity to human cancer cell strains A549 and MCF-7. It
also displayed remarkable antimicrobial activities against
Pseudomonas aeruginosa, Escherichia coli, Bacillus subtilis, Staphylococcus aureus, Candida albicans, Fusarium solani, and
Penicillium chrysogenum.
■
MATERIALS AND METHODS
General. 1D and 2D nuclear magnetic resonance (NMR) spectral
data were obtained on a Bruker ADVANCE III 400 MHz
spectrometer with CDCl3 and DMSO as solvents and tetramethylsilane (TMS) as internal standard. HRMS (ESI) was measured using a
Bruker maXis 4G UHR-TOF. Silica gel (200−300 mesh, Qingdao
Marine Chemical Inc., Qingdao, China) and Sephadex LH-20
(Amersham Biosciences Inc., Shanghai, China) were used for column
© 2016 American Chemical Society
Received:
Revised:
Accepted:
Published:
3789
March 4, 2016
April 18, 2016
April 26, 2016
May 5, 2016
DOI: 10.1021/acs.jafc.6b01051
J. Agric. Food Chem. 2016, 64, 3789−3793
Article
Journal of Agricultural and Food Chemistry
Table 1. 1H and 13C NMR Data of Compound 1 (DMSO-d6)
δC
DEPT-135
173.5
50.8
41.3
C
CH
CH2
4
5
6
2-NH
24.9
23.2
22.2
CH
CH3
CH3
7
8
9
10
11
8-NH
171.0
59.3
27.3
20.2
19.2
C
CH
CH
CH3
CH3
12
13
14
15
173.2
58.5
34.4
25.2
C
CH
CH
CH2
10.4
15.4
CH3
CH3
174.5
52.8
46.7
C
CH
CH2
position
Leu
Val
Ile
1
2
3
16
17
13-NH
Cys1
18
19
20
19-NH
Cys2
21
22
23
22-NH
170.2
53.4
45.7
C
CH
CH2
δH (J in Hz)
1
H−1H COSY
HMBC
4.48 (td, J = 9.2, 6.2)
1.57−1.58 (m)
1.44−1.32 (m)
1.63−1.58 (m)
0.84 (s)
0.82 (s)
7.39 (d, J = 9.2)
3,2-NH
2
1, 3
1, 2, 4
3, 5, 6
4
4
2
3, 5, 6
4, 3
4
2, 7
3.94 (d, J = 9.8)
2.12−1.99 (m)
0.90 (d, J = 6.6)
0.87 (d, J = 6.6)
8.00 (d, J = 8.8)
9,8-NH
8
9
9
8
7,
8,
8,
8,
8,
3.89 (dd, J = 10.4, 3.6)
1.71 (dd, J = 15.1, 7.2)
1.46−1.37 (m)
1.23−1.08 (m)
0.82 (s)
0.79 (d, J = 6.8)
8.63 (d, J = 6.7)
14,17,13-NH
13, 15
14, 16
12, 14
13, 15, 17
14, 16, 13, 17
14
13
13
14, 15
13, 14, 15
13, 18
20,19-NH
19
18, 20
18, 19
19
19, 21
23,22-NH
22
22
21, 23
21, 22
22, 1
3.99
3.52
3.15
8.87
(d, J = 3.5)
(dd, J = 14.9, 3.2),
(m)
(d, J = 3.9)
4.72 (td, J = 10.9, 4.4)
3.28−3.10 (m)
7.12 (d, J = 11.0)
9
10, 11
9
9
12
Hz), 0.84 (s), 0.82 (s), 0.79 (d, J = 6.8 Hz); HRMS (ESI) m/z
552.2291 [M + Na]+ (calcd 552.2290).
Biological Assays. Antimicrobial activity was evaluated in vitro by
the serial 2-fold agar dilution method in the 96-well culture plates.6
The bacteria and pathogenic fungi used in this study were S. aureus, B.
subtilis, E. coli, P. aeruginosa, C. albicans, F. solani, and P. chrysogenum,
respectively. Briefly, 100 μL of compound 1 dissolved in DMSO
(concentrations of 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95, and
0.98 μg/mL) was added to each well, respectively, and then 100 μL of
bacteria or fungus suspension at a density of 106 CFU/mL was
inoculated to each well. All test plates were then incubated at 37 °C for
24 h for bacteria and at 28 °C for 48 h for fungi. The tested strains
were incubated in liquid media, Lysogeny broth (LB) medium for
bacteria and Sabouraud dextrose broth (SDB) for fungi. The result of
assay was recorded by the values of the minimal inhibition
concentration (MIC). Gentamicin was used as a positive control
against bacteria and nystatin as a positive control for the antifungal
assay.
The cytotoxic effect of compounds in vitro was preliminarily
assayed on MCF-7, HepG2, and A549 cell lines by the MTT (3-(4,5dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide) method.7
Three cell lines were maintained in a humidified atmosphere
containing 5% CO2 at 37 °C. The cells were subcultured twice a
week in medium, RPMI-1640 medium for MCF-7 cells and Dulbecco’s
modified Eagle medium (DMEM) for HepG2 and A549 cells.
Normally, 200 μL of these cell suspensions (5 × 104 cells mL−1)
was plated in 96-well culture plates at 37 °C. After 12 h, then 20 μL of
crude extract was subjected to chromatography over a silica gel column
followed by a stepwise gradient elution of petroleum ether in ethyl
acetate or ethyl acetate in methanol to afford seven fractions (fractions
1−7). Fraction 2 was further separated by chromatography over a silica
gel column eluting with ethyl acetate/methanol (100:1 to 50:1, v/v),
and a Sephadex LH-20 column eluting with ethyl acetate/methanol
(1:1) afforded compound 2 (37 mg), compound 3 (29 mg),
compound 4 (19 mg), and compound 5 (28 mg). Fraction 3 was
further separated by chromatography over a silica gel column eluting
with ethyl acetate/methanol (50:1 to 25:2, v/v) to afford compound 6
(32 mg), compound 1 (12 mg), compound 7 (11 mg), compound 8
(9 mg), compound 9 (9 mg), and compound 10 (48 mg). Fraction 4
was further separated by chromatography over a silica gel column
eluting with ethyl acetate/methanol (50:1 to 25:3, v/v) to afford
compound 11 (17 mg), compound 12 (7 mg), compound 13 (15 mg),
and compound 14 (23 mg).
Compound 1: white amorphous powder; 13C NMR (DMSO-d6)
(Table 1) δ 174.5, 173.5, 173.2, 170.2, 171.0, 59.3, 58.5, 53.4, 52.8,
50.8, 46.7, 45.7, 41.3, 34.4, 27.3, 25.2, 24.9, 23.2, 22.2, 20.2, 19.2, 15.4,
10.4; 1H NMR (DMSO-d6) δ 8.87 (d, J = 3.9 Hz), 8.63 (d, J = 6.7
Hz), 8.00 (d, J = 8.8 Hz), 7.39 (d, J = 9.2 Hz), 7.12 (d, J = 11.0 Hz),
4.72 (td, J = 10.9, 4.4 Hz), 4.48 (td, J = 9.2, 6.2 Hz), 3.99 (d, J = 3.5
Hz), 3.94 (d, J = 9.8 Hz), 3.89 (dd, J = 10.4, 3.6 Hz), 3.52 (dd, J =
14.9, 3.2 Hz), 3.28−3.10 (m), 3.15 (m), 2.12−1.99 (m), 1.63−1.58
(m), 1.71 (dd, J = 15.1, 7.2 Hz), 1.57−1.58 (m), 1.46−1.37 (m),
1.44−1.32 (m), 1.23−1.08 (m), 0.90 (d, J = 6.6 Hz), 0.87 (d, J = 6.6
3790
DOI: 10.1021/acs.jafc.6b01051
J. Agric. Food Chem. 2016, 64, 3789−3793
Article
Journal of Agricultural and Food Chemistry
Figure 1. Chemical structures of compounds 1−14.
NMR spectra revealed five carbonyl signals (δC 174.5, 173.5,
173.2, 171.0, 170.2) and five amide protons signals (δH 8.87,
8.63, 8.00, 7.39, 7.12). According to the preliminary estimation,
compound 1 may be the peptide class of compounds. The
1
H−1H COSY (Figure 2) experiment showed the five amino
compound 1 dissolved in DMSO (concentrations of 10 mM, 1 mM,
100 μM, 10 μM, and 1 μM) was added to each well respectively and
cultured at 37 °C for 24 h. Then the MTT was dissolved in saline to 5
mg/mL, and 20 μL of this solution was added to each well. After 4 h,
100 μL of DMSO was added to dissolve any formazan crystals formed.
Absorbance was then determined on a Spectra Max Plus plate reader
at 570 nm. Positive controls were either adriamycin or 5-fluorouracil.
Reed and Muench’s method was used to calculated IC50 values.8
■
RESULTS AND DISCUSSION
Structure Elucidation. Marfey’s analysis method was
performed to measured the stereochemistry of the amino acid
residues.9 Briefly, 2 mg of compound 1 dissolved in 2 mL of 6
mol/L HCl was heated in a sealed glass tube at 110 °C; after 24
h, the pH of the mixture was adjusted to 6.0 with NaOH at
room temperature, then concentrated and redissolved in 2 mL
of distilled water, and finally reacted with 1-fluoro-2,4dinitrophenyl-5-L-alaninamide (FDAA). The result was analyzed by reversed-phase HPLC and compared with standard
amino acids. The analysis revealed all of the amino acid residues
were L-configurations.
The molecular formula of compound 1 (Figure 1) was
determined as C23H39N5O5S2 according to the HRMS (ESI) at
m/z 552.2291 [M + Na]+ (calcd 552.2290). The 1H and 13C
Figure 2. Key 1H−1H COSY and HMBC correlations of compound 1.
acid residues as leucine, valine, isoleucine, and two cysteines.
1
H−1H COSY correlations of H-2/2-NH and H-3, H-5, H-6/
H-4, as well as HSQC correlation and δC 173.5, 50.8, 41.3, 24.9,
23.2, and 22.2 revealed the leucine residue. H-8/8-NH and H-9,
H-10, H-11/H-9, as well as HSQC correlation and δC 171.0,
59.3, 27.3, 20.2, and 19.2, revealed the valine residue. H-13/13NH and H-14, H-15, H-17/H-14, H-15/H-16, as well as
3791
DOI: 10.1021/acs.jafc.6b01051
J. Agric. Food Chem. 2016, 64, 3789−3793
Article
Journal of Agricultural and Food Chemistry
Table 2. Antimicrobial Activity of Compound 1
MIC (μM)
Gram-positive bacteria
a
Gram-negative bacteria
fungi
compound
B. subtilis
S. aureus
E. coli
P. aeruginosa
C. albicans
F. solani
P. chrysogenum
1
gentamicin
nystatin
0.91
4.06
−
0.45
2.03
−
0.91
4.06
−
1.82
2.03
−
7.24
−a
6.16
7.24
−
6.72
3.62
−
13.5
“−”, not set test.
■
HSQC correlation and δC 173.2, 58.5, 34.4, 25.2, 10.4, and 15.4,
revealed the isoleucine residue. H-19/19-NH and H-20, H-22/
22-NH and H-23, as well as HSQC correlation and δC 174.5,
52.8, 46.7, 170.2, 53.4, and 45.7, revealed two cysteine residues.
The amino acid sequence was conformed mainly by HMBC
correlations of the carbonyl carbon of one amino acid residue
with the amide protons of the neighboring residue. As shown in
Figure 2, we could see the HMBC correlations of δH 7.39/δC
171.0, δH 8.00/δC 173.2, δH 8.63/δC 174.5, δH 8.87/δC 170.2,
and δH 7.12/δC 173.5. Thus, from the above-mentioned
evidence compound 1 was confirmed to be disulfide cyclo(Leu-Val-Ile-Cys-Cys).
By detailed physicochemical properties and spectroscopic
analyses, as well as by comparisons of the literature data, the
structures of compounds 2−14 (Figure 1) were identified as
fumitremorgin B (2),10,11 fumitremorgin C (3),12 cyclotryprostatin B (4),13 verruculogen (5),14 tryprostatin B (6),12
tryprostatin A (7),12 cyclotryprostatin A (8),13 cyclotryprostatin C (9),13 cyclo-(Pro-Trp) (10),15 cyclo-(N-methyl-Trp-Leu)
(11),16 cyclotryprostatin D (12),13 13-oxofumitremorgin B
(13),17 and cyclo-(N-Benzyl-Trp-Pro) (14),18 respectively.
Biological Activities. The result of antimicrobial activity
showed compound 1 has potent antimicrobial activity. It
displayed significant growth inhibition against B. subtilis, S.
aureus, P. aeruginosa, and E. coli with MIC values of 0.91, 0.45,
1.82, and 0.91 μM, respectively. Meanwhile, it also has strong
antifungal activities against P. chrysogenum, C. albicans, and F.
solani with MIC values of 3.62, 7.24, and 7.24 μM, respectively
(Table 2). In the MTT bioassay, compound 1 exhibited strong
cytotoxicity to human cancer cell strains A549 and MCF-7 with
IC50 values of 2.42 and 0.65 μM, respectively. It also displayed
slight activity with an IC50 value of 36.02 μM on HepG2 cells
(Table 3).
S Supporting Information
*
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.6b01051.
Various spectra of compound 1; strain identification of
FR02; NMR of compounds 2−14 (PDF)
■
*(Y.-M.M.) E-mail: [email protected]. Phone: +86-2986168312. Fax: +86-29-86168312.
Funding
This research was financially supported by the Natural Science
Basic Research Plan in Shaanxi Province of China (2014JZ003).
Notes
The authors declare no competing financial interest.
■
MCF-7
A549
HepG2
0.65
1.24
3.35
2.42
4.28
1.62
36.02
16.78
4.31
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AUTHOR INFORMATION
Corresponding Author
Table 3. In Vitro Cytotoxicity Evaluation of Compound 1
against Three Tumor Cell Lines
1
adriamycin
5-fluorouracil
ASSOCIATED CONTENT
In conclusion, a new cyclic pentapetide, malformin E,
together with 13 known cyclic peptides, was obtained from
the culture broth of endophytic fungus A. tamarii from the
roots of F. carica. The results revealed that endophytic fungus
A. tamarii is a rich source of potent bioactive cyclic peptides.
Malformin E showed significant antimicrobial and cytotoxic
activities in vitro, which indicated malformin E could be a
promising candidate for new antitumor and antimicrobial
agents in the agricultural and medical industries.
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