Three new compounds from Aspergillus terreus PT06-2 grown in a high salt medium

Article abstract of DOI:10.3390/md9081368

To investigate the structurally novel and bioactive natural compounds from marine-derived microorganisms under high salinity, the fungus Aspergillus terreus PT06-2 was isolated from the sediment of the Putian Sea Saltern, Fujian, China. Three new compound

Full text of DOI:10.3390/md9081368

Mar. Drugs 2011, 9, 1368-1378; doi:10.3390/md9081368
OPEN ACCESS
Marine Drugs
ISSN 1660-3397
www.mdpi.com/journal/marinedrugs
Communication
Three New Compounds from Aspergillus terreus PT06-2 Grown
in a High Salt Medium
Yi Wang †, Jinkai Zheng †, Peipei Liu, Wei Wang and Weiming Zhu *
Key Laboratory of Marine Drugs, Chinese Ministry of Education, School of Medicine and Pharmacy,
Ocean University of China, Qingdao 266003, China; E-Mails: wangyi@hotmail.com (Y.W.);
zhjk8212@yahoo.cn (J.Z.); liupeipei@ouc.edu.cn (P.L.); wwwakin@ouc.edu.cn (W.W.)
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: weimingzhu@ouc.edu.cn;
Tel./Fax: +86-532-8203-1268.
Received: 1 June 2011; in revised form: 15 July 2011 / Accepted: 19 July 2011 /
Published: 12 August 2011
Abstract: To investigate the structurally novel and bioactive natural compounds from
marine-derived microorganisms under high salinity, the fungus Aspergillus terreus PT06-2
was isolated from the sediment of the Putian Sea Saltern, Fujian, China. Three new
compounds, terremides A (1) and B (2) and terrelactone A (3), along with twelve known
compounds (4–15) were isolated and identified from the fermentation broth of A. terreus
PT06-2 at 10% salinity. Among these metabolites, compounds 4 and 15 only produced in
the 10% salinity culture, were identified as methyl 3,4,5-trimethoxy-2-(2-(nicotinamido)
benzamido) benzoate, and (+)-terrein, respectively. The new compounds 1 and 2 exhibited
antibacterial activity against Pseudomonas aeruginosa and Enterobacter aerogenes
with MIC values of 63.9 and 33.5 ꢀM, respectively. Compounds 5 showed moderate
anti-H1N1 activity and lower cytotoxicity with IC50 and CC50 values of and 143.1 and
976.4 ꢀM, respectively.
Keywords: Aspergillus terreus; high salinity metabolites; terremides A and B; terrelactone A
1. Introduction
Solar salterns matured a surprisingly rich diversity and abundance of halophilic and halotolerant
fungi [1]. Furthermore, some genes could be activated at high salt concentrations; some new secondary

Mar. Drugs 2011, 9
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metabolites were probably produced by halotolerant fungi. During pursing the halotolerant microbes
from hypersaline ecological niches [2–6], Aspergillus terreus PT06-2 was isolated and identified from
the sediment collected in the Putian Salterns, Fujian, China. A. terreus is commonly isolated from
cultivated or non-cultivated soils with worldwide distribution and was first published in 1918 [7]. The
famous statins drug lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase
(HMG-CoA reductase), for lowering cholesterol to prevent cardiovascular disease, is mainly produced
by A. terreus [8,9]. A series of compounds such as terreineol [10], terreulactone A [11], terrain [12],
terreic acid [13] and aspulvinones [14] were also isolated from this fungus. Chemical screening for
A. terreus PT06-2 showed that the chemical diversity of the secondary metabolites is the richest at
10% salinity compared with those at 0% and 3% salt. Chemical investigation in 10% salinity resulted
in isolation and identification of three new compounds, terremides A and B (1, 2), terrelactone (3),
along with twelve known compounds (4–15) [15–26]. Antibacterial activity of the new compound 2
against Enterobacter aerogenes, and the new compound 1 and 4 against Pseudomonas aeruginosa
were observed. Compound 5 showed moderate anti-H1N1 activity and lower cytotoxicity.
2. Results and Discussion
2.1. The Identification of New Metabolites from Aspergillus terreus PT06-2 at 10% Salinity
Fungus A. terreus PT06-2 was incubated in a high-salt medium containing 10% salt and extracted
with EtOAc to afford a crude extract. The crude extract (31.2 g) was separated by extensive
chromatography using silica gel, Sephadex LH-20 and HPLC to give compounds 1–15 (Figure 1),
including three new compounds: terremides A and B (1, 2), terrelactone (3), and twelve known ones:
methyl 3,4,5-trimethoxy-2-(2-(nicotinamido)benzamido)benzoate (4) [15], (+)-butyrolactones I–III
(5–7) [16–19], 3-hydroxy-5-[[4-hydroxy-3-(3-methyl-2-buten-1-yl)phenyl]methyl]-4-(4-hydroxyphenyl)-
2(5H)-furanone (8) [20], aspernolide A (9) [16], 5-[(3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-6-yl)-
methyl]-3-hydroxy-4-(4-hydroxyphenyl)-2(5H)-furanone (10) [18], territrem B (11) [22], (−)-(1R,4R)-
1,4-(2,3)-indomethane-1-methyl-2,4-dihydro-1H-pyrazino[2,1-b]quinazoline-3,6-dione (12) [23],
R(−)-6-hydroxymellein (13) [24], trans-4,6-dihydroxymellein (14) [25], and (+)-terrein (15) [26].
Terremide A (1) gave an HRESIMS peak at m/z 392.1257 [M + H]+ (calcd for C21H18N3O5,
392.1246), corresponding to the molecular formula C21H17N3O5. The UV spectrum displayed
absorption at ꢁmax 205 and 262 nm, similar to that of compound 4 [15]. The 1D NMR spectra were
also similar to those of 4, indicating the same molecular skeleton [15]. The differences were a
1,2,3-trisubstituted benzene nucleus instead of the 1,2,3,4,5-pentasubstituted one, and the
corresponding signals of three methoxy groups did not display in the 1D NMR spectra of 1. Besides,
the downfield shifts for C-1ꢂ–C-3ꢂ were observed, suggesting 1 as the derivative of 4 by
demethoxylation at C-4ꢂ and C-5ꢂ, and demethylation at 3ꢂ-MeO. This deduction was further
1
supported by the key H-1H COSY correlations of H-4ꢂ/H-5ꢂ/H-6ꢂ, and the key HMBC correlations
(Figure 2) form HO-3ꢂ (ꢃH 12.22) to C-3ꢂ (ꢃC 149.8), from H-4ꢂ (ꢃH 7.70) and H-6ꢂ (ꢃH 7.36) to C-2ꢂ
(ꢃC 128.2), and from CH3O- (ꢃH 3.95) to carbonyl carbon (ꢃC 169.2). Thus, structure of 1 was
determined to be methyl 3-hydroxy-2-(2-(nicotinamido)benzamido)benzoate.

Mar. Drugs 2011, 9
Figure 1. Chemical structures of the metabolites (1–15) from A. terreus PT06-2.
1370
Terremide B (2) gave an HRESIMS peak at m/z 374.1160 [M + H]+ (calcd for C21H16N3O4,
374.1141), corresponding to the molecular formula C21H15N3O4 that required 16 degrees of
unsaturation. Compared with compound 1, the molecular formula of 2 is one H2O less and one more
degree of unsaturation than that of 1. Both 1 and 2 showed similar 1H and 13C NMR spectra, except for
the disappearance of two exchangeable proton signals at ꢃH 11.96/9.08, and the replacement of the
amido carbon signal at ꢃC 163.7 by a quaternary carbon signal at ꢃC 147.2. Furthermore, another amido
carbon signal shifted upfield from 169.1 ppm to 160.7 ppm, revealing existence of a quinazolinone
nucleus [27]. Thus, compound 2 was deduced as the dehydrated and cyclized product of 1. When
reacted in MeOH, compound 1 formed 2 in 96% yields. Therefore, the structure of terremide B (2) was
assigned as methyl 3-hydroxy-2-(4-oxo-2-(pyridin-3-yl)quinazolin-3(4H)-yl)benzoate that was further
determined by X-ray single-crystal diffraction analysis (Figure 2).
Terrelactone (3) gave an HRESI-MS peak at m/z 465.1529 [M + Na]+ (calcd for C24H26O8Na,
465.1525), corresponding to the molecular formula C24H26O8. Its UV spectrum showed characteristic
absorption of butyrolactones at 231 and 310 nm [21]. 1D NMR showed signals of a 1,2-disubstituted
benzene nucleus at ꢃH 7.62 (2H, d, 8.2)/6.97 (2H, d, 8.2), a 1,2,4-trisubstituted benzene nucleus at
ꢃH 6.55 (d, 8.2)/6.51 (brs)/6.52 (d, 8.2), a carbomethoxy at ꢃH/C 3.78/53.7/170.9, and an unsaturated
butyrolactone at ꢃC 86.0/132.6/139.0/168.7, suggesting the same skeleton as butyrolactone I (5) [18,19].
The NMR differences between 3 and 5 were that the signals belonging to the CH=C double band at
ꢃH/C 5.06/122.4/131.4 in 5 were replaced by a methylene signal at ꢃH/C 1.55/44.4 and a quaternary
carbon signal at ꢃC 70.2 (Table 1). These data suggested that compound 3 was the hydrated derivative
of 5 at CH=C double band. This assignment was further supported by 1H-1H COSY correlation between

Mar. Drugs 2011, 9
1371
H-1′′′ and H-2′′′, and HMBC correlations from H-1′′′ to C-2′, C-4′ and C-3′′′, from H-4′′′/5′′′ to C-2′′′
and C-3′′′ (Figure 2). The dextral specific rotation ([ꢄ]25 +71) indicated R-configuration at C-4 [17].
D
Thus, the structure of terrelactone (3) was determined as (R)-methyl 4-hydroxy-2-(4-hydroxy-3-
(3-hydroxy-3-methylbutyl)benzyl)-3-(4-hydroxyphenyl)-5-oxo-2,5-dihydrofuran-2-carboxylate.
Figure 2. The key COSY and HMBC correlations for compounds 1 and 3, and the final
X-ray drawing of compound 2.
OH
OH
HO
O
N
H
O
O
NH
O
O
O
O
N
O
HO
1
1H-1H COSY
HMBC
3
OH
Table 1. 1H- and 13C-NMR (600 and 150 MHz) data for compounds 1–3 in CDCl3.
1 a 3 b
δH (J in Hz) δH (J in Hz)
2
Position
δC
δC
δH (J in Hz)
δC
1
2
3
4
5
6
7
8
9
10
1′
2′
3′
4′
5′
6′
7′
1ꢂ
2ꢂ
3ꢂ
4ꢂ
5ꢂ
6ꢂ
7ꢂ
119.1, qC
140.3, qC
122.0, CH
134.2, CH
124.0, CH
128.1, CH
169.1, qC
160.7, qC
153.4, qC
147.2, qC
127.4, CH
134.8, CH
127.3, CH
126.4, CH
120.6, qC
131.3, qC
147.9, CH
150.1, CH
122.5, CH
168.7, qC
139.0, qC
132.6, qC
86.0, qC
170.9, qC
39.1, CH2
8.87, d (8.3)
7.67, t (7.8)
7.32, t (7.8)
7.99, d (7.8)
7.79, d (7.9)
7.92, t (7.9)
7.61, t (7.9)
8.16, d (7.9)
3.44, s
130.3, qC
148.8, CH
124.8, qC
132.6, CH
128.2, qC
154.8, qC
9.26, s
8.57, s
6.51, br s
152.6, CH
8.78, d (4.2)
135.0, CH
8.48, d (4.5)
123.6, CH 7.45, t “like” (6.0) 160.7, qC 7.29, dd (4.5, 7.9) 115.1, CH 6.52, br d (8.2)
134.9, CH
163.7, qC
120.1, qC
128.2, qC
149.8, qC
123.5, CH
126.7, CH
126.3, CH
169.2, qC
8.27, d (6.0)
153.4, qC
7.75, d (7.9)
129.4, CH
6.55, d (8.2)
124.4, qC
128.7, qC
153.5, qC
121.2, CH
130.0, CH
120.0, CH
165.2, qC
122.8, qC
130.1, CH 7.62, br d (8.2)
116.5, CH 6.97, br d (8.2)
158.7, qC
116.5, CH 6.97, br d (8.2)
130.1, CH 7.62, br d (8.2)
7.70, d (7.8)
7.26, t (7.7)
7.36, d (8.2)
7.03, d (7.8)
7.28, t (7.8)
7.33, d (7.8)

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Table 1. Cont.
7ꢂ-OMe 53.0, CH3
2-NH
2ꢂ-NH
3.95, s
11.96, s
9.08, s
52.2, CH3
3.65, s
3ꢂ-OH
12.22, s
a
The NMR data for 2-NH, 2ꢂ-NH and 3ꢂ-OH are ꢃH 11.96 (s), 9.08 (s) and 12.22 (s), respectively;
Recorded in acetone-d6. The NMR data for isoprenyl moiety and 5-OCH3 are ꢃH 2.55 (m)/2.45 (m),
b
1.55 (2H, m), 1.18 (6H, s), and 3.78 (3H, s), and ꢃC 25.3 (CH2), 44.4 (CH2), 70.2 (CH), 29.8 × 2 (CH3), and
53.7 (CH3), respectively.
2.2. The Effects of Salt Stress on Production of Secondary Metabolites from Aspergillus terreus PT06-2
Salt-tolerant fungi belong to extremophiles which can survive under the conditions of zero to high
salinity. However, using salt-tolerant fungi to produce new secondary metabolites at high salinity was
rarely reported [2–6]. In order to investigate the effect of high salt stress on fungal secondary
metabolites, A. terreus PT06-2 was cultured at 0%, 3% and 10% salt. The amount of EtOAc extracts of
metabolites in 10% salinity was the largest (222 mg vs. 165 and 200 mg of 0% and 3% salinity). The
chemical diversities of the metabolites in 10% salinity were increased (Figure 3). Compounds 4 and 15
were not produced by A. terreus PT06-2 when cultivated in 0% and 3% salinity media. The other sole
secondary metabolites were a kind of red pigments (Figure 3) whose structures were not identified.
Figure 3. HPLC profiles of secondary metabolites from A. terreus PT06-2 cultured in
different salt conditions (0%, 3% and 10%, respectively). Peaks circled were red pigments
(HPLC eluent: 0–60 min, 5–100% CH3OH; flow rate: 1 mL/min).

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2.3. The Bioactivities of Metabolites from Aspergillus terreus PT06-2 in 10% Salinity
The new compounds (1–3), sole metabolites (4, 15) produced at 10% salinity, and typical
butyrolactones (5–10) of A. terreus were evaluated for their cytotoxicity against HL-60 and BEL-7402
cell lines, antimicrobial activity against Enterobacter aerogenes, Pseudomonas aeruginosa,
Staphylococcus aureus and Candida albicans (Table 2), and antiviral activity against influenza virus
(H1N1) by MTT [28], agar dilution method [29], and CPE inhibition assay [30,31], respectively.
Compound 5 showed weak cytotoxicity against HL-60 with an IC50 (half maximal inhibitory
concentration) value of 57.5 ꢀM. Compounds 1, 4 and 2 showed weak antibacterial activity against
S. aureus and E. aerogenes with MIC (minimum inhibitory concentration) of 63.9, 52.4 and 33.5 ꢀM,
respectively (Table 2). Other compounds did not show cytotoxicity and antibacterial activity (IC50 or
MIC > 100 ꢀM). Butyrolactone I (5) showed anti-H1N1 activity with IC50 and CC50 (50% cytotoxicity
concentration) values of 143.1 and 976.4 ꢀM, respectively (positive control: ribavirin, IC50 100.8 ꢀM).
In addition, it was reported that butyrolactone I (5) showed kinase inhibition with high selectivity
towards CDK1 and CDK2 [32].
Table 2. Antimicrobial activities of compounds 1–4 (“−” indicates not measured).
MIC (µM)
Compound
Enterobacter
aerogenes
>100
Pseudomonas
aeruginosa
>100
Staphylococcus
Candida
albicans
>100
>100
>100
>100
>100
−
aureus
63.9
>100
>100
52.4
>100
1
1
2
33.5
>100
>100
>100
1
−
>100
>100
>100
>100
30
−
3
4
15
Ciprofloxacin lactate
Ketoconazole
−
5
3. Experimental Section
3.1. General Experimental Procedures
Optical rotations were obtained on a JASCO P-1020 digital polarimeter. UV spectra were recorded
on a Beckman DU 640 spectrophotometer. IR spectra were obtained on a Nicolet NEXUS 470
spectrophotometer as KBr disks. 1H NMR, 13C NMR, and DEPT spectra and 2D-NMR were recorded
on a JEOL JNMECP 600 spectrometer using TMS as internal standard, and chemical shifts were
recorded as ꢃ values. ESIMS was measured on a Q-TOF Ultima Global GAA076 LC mass
spectrometer. Semipreparative HPLC was performed using an ODS column (YMC-pack ODS-A,
10 × 250 mm, 5 ꢀm, 4 mL/min). HPLC was performed using an ODS column (YMC-pack C18,
4.6 × 250 mm, 5 ꢀm, 2 mL/min). TLC and column chromatography (CC) were performed on plates
precoated with silica gel GF254 (10–40 ꢀm) and over silica gel (200–300 mesh, Qingdao Marine
Chemical Factory, Qingdao, China) and Sephadex LH-20 (Amersham Biosciences, Sweden),
respectively. All the materials for the culture medium of A. terreus PT06-2 was purchased from
Qingdao Marine Chemical Factory, Qingdao, China.

Mar. Drugs 2011, 9
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3.2. Fungal Material
The fungus A. terreus PT06-2 was isolated from sediment (saline 20%), Putian Saltern of Fujian
Province of China. It was identified according to its morphological characteristics and analyses of its
18S rRNA sequence (Genbank JN006059) by Prof. C. X. Fang, China Center for Type Culture
Collection. A voucher specimen is deposited in our laboratory at −80 °C. The working strain was
prepared on potato dextrose agar slants and stored at 4 °C.
3.3. Fermentation and Extraction
The fungus A. terreus PT06-2 was grown under static conditions at 28 °C for 35 days in 100 1000-mL
conical flasks containing liquid medium (300 mL/flask, pH 7.0) composed of glucose (10 g/L),
maltose (20 g/L), mannitol (20 g/L), monosodium glutamate (10 g/L), yeast extract (3 g/L), corn steep
liquor (1 g/L), and 10% salt (NaCl 8%, MgSO4 0.5%, KH2PO4 0.5%, NH4Cl 0.5% and KCl 0.5%). The
fermented whole broth (30 L) was filtered through cheesecloth to separate the supernatant from the
mycelia. The supernatant was concentrated under reduced pressure to about 5 L and then extracted
three times with EtOAc to give an EtOAc solution, while the mycelia were extracted three times with
acetone. The acetone was removed under reduced pressure to afford a residual aqueous solution. This
aqueous solution was extracted three times with EtOAc to give a further EtOAc crude extract. Both
EtOAc solutions were combined and concentrated under reduced pressure to give an extract (31.2 g).
The fungus A. terreus PT06-2 was incubated under the same conditions in 0%, 3% and 10% salt.
The chemical diversities of the secondary metabolites of EtOAc extract were investigated with HPLC.
3.4. Purification
The extract (31.2 g) from A. terreus PT06-2 was separated into ten fractions (Fraction 1 to 10) on a
silica gel column using a step gradient elution with CHCl3–petroleum ether (0–100%) and then with
MeOH–CHCl3 (0–100%). Fraction 4 was separated on Sephadex LH-20 with MeOH–CHCl3 (1:1) and
100% MeOH to obtain Fraction 4-2 and 4-3. Fraction 4-2 was further purified on semipreparative
HPLC (60% MeOH) to give compound 15 (106 mg, tR 12 min); Fraction 4-3 was separated on a silica
gel column using a step gradient elution with Me2CO–petroleum (0–100%) to obtain Fraction 4-3-1
and 4-3-2. Fraction 4-3-1 was purified on semipreparative HPLC (60% MeOH) to give compound 13
(7 mg, tR 8 min). Fraction 5 was separated on silica gel column using a step gradient elution with
Me2CO–petroleum (0–100%) to obtain fraction 5-1 and fraction 5-5. Fraction 5-1 was rechromatographed
on silica gel column (MeOH–CHCl3, 1:50), Sephadex LH-20 (MeOH–CHCl3, 1:1), and then
semipreparative HPLC (70% MeOH) to give compound 4 (87 mg, tR 8 min), 11 (21 mg, tR 12 min) and
12 (7 mg, tR 14 min). Fraction 5-5 was chromatographed on Sephadex LH-20 (MeOH-CHCl3, 1:1),
and then semipreparative HPLC (40% MeOH) to give compound 14 (2 mg, tR 9 min). Fraction 8 was
separated on Sephadex LH-20 (MeOH-CHCl3, 1:1) twice to obtain Fraction 8-2 and 8-4. Fraction 8-2
was chromatographed on Sephadex LH-20 (100% MeOH), and then semipreparative HPLC
(60% MeOH) to give compound 5 (200 mg, tR 23 min), 7 (10 mg, tR 12 min), and 8 (5 mg, tR 18 min).
Fraction 9 was separated on silica gel column (MeOH–CHCl3, 1:10) to obtain Fraction 9-1–9-4.
Fraction 9-1 was further purified on semipreparative HPLC (70% MeOH) to give compound 1 (6 mg,

Mar. Drugs 2011, 9
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tR 10 min), 9 (70 mg, tR 12 min), and 10 (4 mg, tR 10 min). Fraction 9-3 was chromatographed on ODS
column (50% MeOH), and then semipreparative HPLC (60% MeOH) to give compound 2 (3 mg,
tR 6 min), 6 (3 mg, tR 15 min). Fraction 9-5 was chromatographed on ODS column (40% MeOH), and
then semipreparative HPLC (60% MeOH) to give compound 3 (45 mg, tR 10 min).
Terremide A (1): white amorphous powder; UV (MeOH) ꢁmax (log ꢅ) 205 (4.4), 262 (3.2) nm;
1H and 13C NMR data, see Table 1; HRESIMS m/z 392.1257 [M + H]+ (calcd for C21H18N3O5, 392.1246).
1
Terremide B (2): colorless crystal; UV (MeOH) ꢁmax (log ꢅ) 212 (4.4), 283 (3.3) nm; H and
13C NMR data, see Table 1; HRESIMS m/z 374.1160 [M + H]+ (calcd for C21H16N3O4, 374.1174).
Terrelactone (3): pale yellow oil; [ꢄ]25 +71 (c 0.5, CHCl3); UV (MeOH) ꢁmax (log ꢅ) 231 (4.0),
D
310 (4.4) nm; IR (KBr) ꢆmax 3275, 2932, 1744, 1703, 1604, 1527, 1433, 1380, 1225, 1146, 839 cm−1; 1H
and 13C NMR data, see Table 1; HRESIMS m/z 465.1529 [M + Na]+ (calcd for C24H26O8Na, 465.1525).
3.5. Chemical Transformation of 1 into 2
Compound 1 (2 mg, 5 ꢀmol) was dissolved in MeOH (0.5 mL) and stirred for 24 h at room
temperature. The reaction mixture was purified by semi-preparative HPLC eluting with 70% CH3OH
to give compound 2 (1.8 mg, 96% yield) as colorless crystals.
3.6. X-ray Crystal Structure of 2
Compound 2 was obtained as colorless monoclinic crystals with molecular formula C21H15N3O4.
Space group C2/c, a = 27.130 (3) Å, b = 8.9484 (13) Å, c = 14.6147 (17) Å, ꢄ = 90.00°, ꢇ = 91.248 (2)°,
ꢈ = 90.00°, V = 3547.2 (8) Å3, Z = 8, crystal size 0.45 × 0.40 × 0.29 mm3. A total of 3131 unique
reflections (2ꢉ < 50°) were collected on a CCD area detector diffractometer with graphite
monochromated MoKa radiation (ꢁ = 0.71073 Å). The structure was solved by direct methods
(SHELXS-97) and expanded using Fourier techniques (SHELXL-97). The final cycle of full-matrix
least squares refinement was based on 3131 unique reflections (2ꢉ < 50°) and 254 variable parameters
and converged with unweighted and weighted agreement factors of R1 = 0.0679, Rw = 0.1074 and
R = 0.0366 for I > 2sigma(I) data. Crystallographic data (excluding structure factors) for structure 2 in
this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary
publication number CCDC 819714. Copies of the data can be obtained, free of charge, on
application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (Fax: +44-(0)-1223-336033 or
E-Mail: deposit@ccdc.cam.ac.uk).
3.7. Bioassay
Antimicrobial activity against E. aerogenes, P. aeruginosa, S. aureus and C. albicans was evaluated
using an agar dilution method [29]. The tested strains were cultivated in LB agar plates for bacteria
and in YPD agar plates for C. albicans, at 37 °C. Compounds and positive controls were dissolved in
5% DMSO-H2O at different concentrations from 1000 to 62.5 ꢀg/mL and then from 50 to 0.78 ꢀg/mL,
using continuous 2-fold dilution. The test solutions (5 ꢀL) were absorbed onto paper disks (5 mm
diameter) and placed on the assay plates. After 24 h incubation, zones of inhibition (mm in diameter)

Mar. Drugs 2011, 9
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were recorded. The minimum inhibitory concentrations were defined as the lowest concentration at
which an inhibition zone could be observed.
Cytotoxicity against HL-60 and BEL-7402 cancer cell lines and confluent MDCK cells was assayed
by the MTT method [28]. Cell lines were grown in RPMI-1640 supplemented with 10% FBS under a
humidified atmosphere of 5% CO2 and 95% air at 37 °C. Cell suspensions, 200 ꢀL, at a density of
5 × 104 cell mL−1 were plated in 96-well microtiter plates and incubated for 24 h. Then, 2 ꢀL of the test
solutions (in MeOH) were added to each well and further incubated for 72 h. The MTT solution (20 ꢀL,
5 mg/mL in IPMI-1640 medium) was then added to each well and incubated for 4 h. Old medium
containing MTT (150 ꢀL) was then gently replaced by DMSO and pipetted to dissolve any formazan
crystals formed. Absorbance was then determined on a SPECTRA MAX PLUS plate reader at 540 nm.
The CC50 was calculated as the compound concentration necessary to reduce cell viability by 50%.
Vp-16 (Etoposide) was used as the positive control with IC50 values of 0.042 and 0.83 ꢀM, respectively.
The antiviral activity against H1N1 was evaluated by the CPE inhibition assay [30,31]. Confluent
MDCK cell monolayers were firstly incubated with influenza virus (A/Puerto Rico/8/34 (H1N1),
PR/8) at 37 °C for 1 h. After removing the virus dilution, cells were maintained in infecting media
(RPMI 1640, 4 ꢀg/mL of trypsin) containing different concentrations of test compounds at 37 °C.
After 48 h incubation at 37 °C, cells were fixed with 100 ꢀL of 4% formaldehyde for 20 min at room
temperature. After removal of the formaldehyde, the cells were stained with 0.1% crystal violet for
30 min. The plates were washed and dried, and the intensity of crystal violet staining for each well was
measured in a microplate reader (Bio-Rad, USA) at 570 nm. The IC50 was calculated as the compound
concentration required inhibiting influenza virus yield at 48 h post-infection by 50%. Ribavirin was
used as the positive control with the IC50 values of 100.8 ꢀM.
4. Conclusions
In summary, fifteen metabolites including three new compounds were isolated and identified from
the fermentation broth of A. terreus PT06-2 under 10% salinity conditions. High salt stress affected the
quantity and profile of secondary metabolites. The new compounds 1 and 2, and the sole metabolite 4
produced under high salt stress conditions showed antibacterial activity. The typical metabolite of
A. terreus, butyrolactone I (5), showed anti-H1N1 activity with low cytotoxicity, indicating that 5
might be a promising drug candidate for anti-influenza virus.
Acknowledgements
This work was supported by grants from the Special Fund for Marine Scientific Research in the
Public Interest of China (No. 2010418022-3), the National Basic Research Program of China
(No. 2010CB833800), the National Natural Science Foundation of China (No. 30973680 & 30670219),
and from PCSIRT (No. IRT0944). The cytotoxicity assay was performed by M.Y. Geng’s group at the
Shanghai Institute of Materia Medica, CAS.

Mar. Drugs 2011, 9
1377
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