Antimicrobial aflatoxins from the marine-derived fungus Aspergillus flavus 092008

Article abstract of DOI:10.1007/s12272-012-0808-1

A new aflatoxin, aflatoxin B2b (1), together with six known compounds, were isolated from the marine-derived fungus Aspergillus flavus 092008 endogenous with the mangrove plant Hibiscus tiliaceus (Malvaceae). The structure of 1 was determined by the spectroscopic and chemical methods. Compound 1 exhibited a moderate antimicrobial activity against Escherichia coli, Bacillus subtilis and Enterobacter aerogenes, with MIC values of 22.5, 1.7 and 1.1 μM, respectively. Compound 1 also showed a weak cytotoxicity against A549, K562 and L-02 cell lines, with IC50 values of 8.1, 2.0 and 4.2 μM, respectively. The results showed that hydration and hydrogenation of Δ8-double bond significantly reduces the cytotoxicity of aflatoxins, while the esterification at C-8 increases the cytotoxicity.

Full text of DOI:10.1007/s12272-012-0808-1

Arch Pharm Res Vol 35, No 8, 1387-1392, 2012
DOI 10.1007/s12272-012-0808-1
Antimicrobial Aflatoxins from the Marine-Derived Fungus
Aspergillus flavus 092008
1,
1,
4
1
3
1
1
2
*
*
Hui Wang , Zhenyu Lu , Hai-Jun Qu , Peipei Liu , Chengdu Miao , Tonghan Zhu ,Jing Li , Kui Hong ,
and Weiming Zhu1
1Key laboratory of Marine Drugs, Ministry of Education of China, School of Medicine and Pharmacy, Ocean University
of China, Qingdao 266003, China, 2Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Educa-
3
tion of China, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China, Guangzhou Microbiology
Institute, Guangzhou 510250, China, and Affiliated Hospital of Medical College, Qingdao University, Qingdao 266003,
4
China
(Received December 29, 2011/Revised February 21, 2012/Accepted February 23, 2012)
A new aflatoxin, aflatoxin B2b
marine-derived fungus Aspergillus flavus 092008 endogenous with the mangrove plant Hibiscus
tiliaceus (Malvaceae). The structure of was determined by the spectroscopic and chemical meth-
ods. Compound exhibited a moderate antimicrobial activity against Escherichia coli Bacillus
subtilis and Enterobacter aerogenes, with MIC values of 22.5, 1.7 and 1.1 M, respectively.
Compound also showed a weak cytotoxicity against A549, K562 and L-02 cell lines, with IC50
values of 8.1, 2.0 and 4.2 M, respectively. The results showed that hydration and hydrogenation
of
8-double bond significantly reduces the cytotoxicity of aflatoxins, while the esterification at C-
8 increases the cytotoxicity.
(1), together with six known compounds, were isolated from the
1
1
,
µ
1
µ
∆
Key words: Aflatoxin, Antibacterial activity, Aspergillus flavus, Cytotoxicity, Mangrove plant
2006) were isolated from
fungus flavus was reported to produce tetrathioas-
Mangrove plants (Wu et al., 2008) and endophytes pirochlorine, cyclo-(D- -methyl-Leu-L-Trp) (Klausmeyer
H. tiliaceus. In addition, the
INTRODUCTION
A.
N
(Zhang et al., 2006) are two prolific sources of structur- et al., 2005), and 1'-(2-phenyl-ethylene)-ditryptophen-
ally new and bioactive compounds for drug discovery. aline (Barrow et al., 1994). Chemical study resulted in
Our previous study identified several interesting the isolation and identification of a new aflatoxin,
compounds from mangrove fungi, such as expansols A aflatoxin B2b
and B (Lu et al., 2010), and ustusoranes A-F (Lu et aflatoxins B1 (
(
2
1
), along with six known compounds,
) (Asao et al., 1965; Brechbuhler et al.,
al., 2009). In the course of our ongoing search for new 1967; Moss et al., 1983) and B2 (
and bioactive compounds, from the mangrove fungi, 8-acetyloxyaflatoxin B1 ( ) (Buchi et al., 1967; Coles et
Aspergillus flavus 092008, whose fermentation broth al., 1980), averantin ( ) (Lee et al., 2010), ( )-ditrypto-
showed antibacterial activities against methicillin- phenaline ( ) (Movassaghi et al., 2008), and saadamycin
resistant Staphylococcus aureus and Enterobacter ) (EI-Gendy et al., 2010) from the EtOAc extract of
aerogenes, was isolated from the surface-sterilized roots the fermentation broth of flavus 092008. Aflatoxin
of the mangrove plant Hibiscus tiliaceus (Malvaceae). B2b ( ) showed a moderate antimicrobial activity against
Some stigmasteroids (Rosa et al., 2006), triterpenoids Escherichia coli Bacillus subtilis and aerogenes, with
(Feng et al., 2008), coumarins and amides (Chen et al., MIC values of 22.5, 1.7 and 1.1 M, respectively. Further,
aflatoxin B2b ( ) also showed a weak cytotoxicity against
A549, K562 and L-02 cell lines, with IC50 values of 8.1,
2.0 and 4.2 M, respectively.
3) (Zhou et al., 2005),
4
5
−
6
(7
A.
1
,
E.
µ
1
*These authors contributed equally to this work.
Correspondence to: Weiming Zhu, School of Medicine and Phar-
macy, Ocean University of China, Qingdao 266003, China
Tel, Fax: 86-532-82031268
µ
E-mail: weimingzhu@ouc.edu.cn (W. Zhu),
kuihong31@gmail.com (K. Hong)
1387

1388
H. Wang et al.
Fig. 1. Structures of compounds 1-7.
mL/flask) composed of (g/L): glucose (10), maltose (20),
mannitol (20), monosodium glutamate (10), KH2PO4
(0.5), MgSO4·7H2O (0.3), yeast extract (3), corn steep
MATERIALS AND METHODS
General experimental procedures
Optical rotations were obtained on a JASCO P-1020 liquor (1), CaCO3 (2) and seawater after adjusting its
digital polarimeter. IR spectra were taken on a Nicolet pH to 7.0.
NEXUS 470 spectrophotometer in KBr disks. UV spec-
tra were recorded on Waters 2487 Absorbance Detector.
CD spectra were measured on a JASCO J-810 spectro-
Extraction
The fermented whole broth (30 L) was filtered through
polarimeter. 1D and 2D-NMR spectra were recorded a cheese cloth, which separates the broth into filtrate
on a JEOL JNM-ECP 600 spectrometer, using TMS and mycelia. The filtrate was concentrated in vacuo to
as an internal standard, and the chemical shifts were about a quarter of the original volume, and then ex-
recorded as
δ values. ESI-MS was measured on a Q- tracted three times with ethyl acetate (15 L each time)
TOF ULTIMA GLOBAL GAA076 LC mass spectrometer. to give an ethyl acetate solution, while the mycelia
Semi-HPLC was performed using an ODS column were extracted three times with acetone. The acetone
[YMC-pack ODS-A, 10 × 250 mm, 5 µm, 4.0 mL/min]. solution was evaporated under a reduced pressure to
TLC and column chromatography (CC) were performed afford an aqueous solution. The aqueous solution was
on plates that were precoated with silica gel GF254 (10- extracted three times with EtOAc (5 L each time) to
40 µm), and over silica gel (200-300 mesh, Qingdao Ma- give another EtOAc solution. Both the EtOAc solutions
rine Chemical Factory) and Sephadex LH-20 (Amersham were combined and concentrated in vacuo to give an
Biosciences), respectively. Vacuum liquid chromatog- EtOAc extract (14.0 g).
raphy (VLC) was carried out over silica gel H (Qingdao
Marine Chemical Factory).
Purification
The EtOAc extract (14.0 g) was separated into 9 frac-
tions after subjection to VLC on a silica gel column,
A. flavus 092008 was isolated using step gradient elution with CHCl3-petroleum
Strain
The working strain
from the root of the mangrove plant,
H. tiliaceus (Mal- ether (0-100%), and then MeOH-CHCl3 (0-50%). Both
vaceae) from Wenchang, Hainan province of China. It fraction 8 and fraction 5 were further subjected to
was identified according to its morphological charac- VLC, using a step gradient elution with MeOH-CHCl3
teristics and analyses of ITS gene sequence (GenBank (0-100%) to give 7 fractions (Fr.8-1~8-7 and Fr.5-1~5-7,
accession No EU664477). Working strain was prepared respectively). Fr.8-1, 8-3 and 8-6 were subjected to CC
on potato dextrose agar slants, and was stored at 4oC. over Sephadex LH-20 (MeOH-CHCl3, v/v 1:1) to provide
Fr.8-1-1~8-1-3, Fr.8-3-1~8-3-3, and Fr.8-6-1~8-6-3, re-
spectively. Fr.8-1-3 was purified by an extensive semi-
flavus 092008 was grown under static PHPLC (68% MeOH-H2O) to yield (29.9 mg, tR 7.93
conditions at 25oC for 30 days in one hundred 1000- min). Fr.8-3-3 was further purified on semi-PHPLC
mL conical flasks, containing a liquid medium (300 (50% MeOH-H2O) to give compound (12.5 mg, tR 10.48
Fermentation
The fungus
A
.
6
2

Antimicrobial Aflatoxin from A. flavus
1389
min). Fr.8-6-3 was further separated into 5 subfrac- Aflatoxin B1 (2)
25
tions (Fr.8-6-3-1~8-6-3-5) by CC over ODS, using a step Yellow powder; [α D
]
−
506.7 (
c
0.14, CHCl3); ESIMS
c 0.05) λmax (∆ε) 353
gradient elution with H2O-MeOH. Compounds
1
(8.4 m/z 313.1 [M + H]+; CD (MeOH,
mg, tR 7.61 min),
3 (1.7 mg, tR 10.01 min) and 4
(18.7 (-5.1), 239 (+2.0), 217 (-2.2) nm; 1H-NMR (CDCl3, 600
mg, tR 9.47 min) were purified from Fr.8-6-3-3, Fr.8-6- MHz) and 13C-NMR (CDCl3, 150 MHz) see Table I.
3-2 and Fr.8-6-3-1 by semi-PHPLC eluting with 50%
MeOH-H2O, 50% MeOH-H2O and 45% MeOH-H2O, re- Aflatoxin B2 (3)
25
spectively. Fr.5-5 was subjected to CC over Sephadex Yellow powder; [α D
]
−
194.5 (
c
0.09, CHCl3); ESIMS
0.04) λmax ∆ε) 361
(3.0 (-4.2), 240 (+1.8), 219 (-4.6) nm; H-NMR and 13C-
LH-20 (MeOH-CHCl3, v/v 1:1), and then purified by m/z 315.1 [M + H]+; CD (MeOH,
an extensive semi-PHPLC (50% MeOH) to yield
c
(
1
5
mg, tR 6.34 min). Fr.5-7 was separated by a silica gel NMR were identical with those reported in literature
column, eluting with CHCl3-MeOH (1-100%), to afford (Zhou et al., 2005).
the four subfractions (Fr.5-7-1~5-7-4). Compound
7 (31.7
mg, tR 4.78 min) was isolated from Fr.5-7-2 by semi- 8-Acetoxyaflatoxins B2 (4)
25
PHPLC eluting with 40% MeOH-H2O.
Yellow powder; [α D
m/z 373.2 [M + H]+; CD (MeOH,
11.5), 238 (+10.5), 215 (-4.0) nm; 1H-NMR (CDCl3, 600
0.11, CHCl3); HR-ESIMS MHz) and 13C-NMR (CDCl3, 150 MHz) see Table I.
]
−
744.3 (
c
0.12, CHCl3); ESIMS
c
0.05) λmax ∆ε) 333 (-
(
Aflatoxin B2b (1)
25
Yellow powder; [α D
]
−
262.9 (
c
m/z 445.1149 [M + H]+ (calcd for C22H21O10, 445.1135);
UV (MeOH) max (log ) 232 (3.53), 256 (3.48), 354 (3.76)
0.04) λmax ∆ε) 356 (-12.2), 238 (+5.7),
max cm−1 (KBr) 3442, 1755, 1728, 1630, mL) and the solution was added 0.8 mg of KOH. After
λ
ε
Chemical transformation
Compound (5.0 mg) was dissolved in MeOH (1.0
nm; CD (MeOH,
218 (-5.6) nm; IR
c
ν
(
4
1600, 1555, 1488, 1315, 1160, 975. 1H-NMR (CDCl3, 600 stirring for 1 h at 16oC, the solution was neutralized
MHz) and 13C-NMR (CDCl3, 150 MHz) see Table I.
with a saturated solution of NH4Cl, and then the mix-
ture was evaporated to dryness. The residue was dis-
Table I. 1H- and 13C-NMR (600 and 150 MHz) data of
1, 2 and 4 in CDCl3
1
2
4
Position
1H (
J
in Hz)
13C
1H (
J
in Hz)
13C
201.7 qC
1H (
J
in Hz)
13C
1
/
201.3 qC
/
/
201.3 qC
35.0 CH2
29.0 CH2
152.8 qC
103.7 qC
161.7 qC
90.2 CH
165.8 qC
114.0 CH
98.4 CH
2
3
3a
3b
4
2.64 (2H, t, 4.4)
3.42 (2H, t, 4.4)
/
/
35.0 CH2 2.64 (2H, t, 5.5)
28.5 CH2 3.40 (2H, t, 5.5)
35.3 CH2 2.66 (2H, t, 4.4)
29.3 CH2 3.44 (2H, t, 4.4)
153.2 qC
104.0 qC
161.8 qC
/
/
/
153.2 qC
104.3 qC
161.8 qC
/
/
/
/
5
6.39 (1H, s)
/
6.51 (1H, d, 6.6)
6.38 (1H, dd, 4.4, 4.4)
90.9 CH 6.43 (1H, s)
165.6 qC
112.8 CH 6.81 (1H, d, 7.7)
98.4 CH 6.47 (1H, dd, 3.3, 2.2) 145.5 CH 6.46 (1H, d, 4.4)
91.1 CH 6.38 (1H, s)
166.0 qC
113.8 CH 6.56 (1H, d, 5.5)
5a
6a
8
/
/
9
2.60 (1H, ddd, 14.3, 9.9, 4.4), 36.7 CH2 5.47 (1H, dd, 3.3, 2.2) 102.9 CH 2.54 (1H, ddd, 14.3, 8.8, 4.4), 36.7 CH2
2.46 (1H, ddd, 14.3, 5.5, 4.4)
2.51 (1H, ddd, 14.3, 5.5, 4.4)
9a
9b
9c
11
11a
4.27 (1H, ddd, 9.9, 5.5, 4.4)
42.2 CH 4.76 (1H, dd, 7.7, 2.2) 48.2 CH 4.23 (1H, ddd, 8.8, 5.5, 4.4) 42.2 CH
/
/
/
/
107.6 qC
154.9 qC
177.1 qC
117.3 qC
56.5 CH3 3.96 (3H, s)
172.4 qC
/
/
/
/
108.1 qC
155.5 qC
177.4 qC
117.6 qC
/
/
/
/
108.0 qC
155.1 qC
177.1 qC
117.2 qC
56.5 CH3
169.3 qC
20.9 CH3
4-OCH3 3.97 (3H, s)
56.8 CH3 3.98 (3H, s)
1'
2'
3'
4'
/
/
2.67 (2H, d, 6.6)
2.66 (2H, d, 6.6)
/
29.1 CH2
1.74 (3H, s)
29.0 CH2
171.1 qC
4'-OCH3 3.71 (3H, s)
51.9 CH3

1390
H. Wang et al.
solved in 200
µL of H2O, and then extracted 5 times gently replaced by DMSO and pipetted to dissolve any
each with 4 mL of CHCl3. The organic layers were com- formazan crystals formed. Absorbance was then deter-
bined and evaporated to the dryness to yield aflatoxin mined on a Spectra Max Plus plate reader at 540 nm.
B2a (1a) (2.0 mg, 45% yield) that was identified by 1H- In the SRB assay, 200
µ
L of the A549 and L-02 cell
NMR (Trost et al., 2003), ESIMS, and specific rotation. suspensions were plated in 96-well plates at a density
Using the same procedure, hydrolysis of compound L of the test
of 2 × 105 cell/mL, respectively. Then, 2
1
µ
(2.0 mg) also afforded 1a (0.8 mg, 54%), which was solutions (in MeOH) were added to each well, and the
identified by HPLC and TLC analysis with above afla- culture was further incubated for 24 h. The cells were
toxin B2a, and specific rotation.
fixed with 12% trichloroacetic acid and the cell layer
stained with 0.4% SRB. The absorbance of the SRB
solution was measured at 515 nm.
Aflatoxin B2a (1a)
25
Yellow powder; [
α
]
−158.8 (c 0.13, 10:1 CHCl3-meth-
D
anol); ESIMS m/z 331.1 [M + H]+; H-NMR (DMSO-
d6, 600 MHz): 6.51 (d, = 6.1 Hz, 1H, H-6a), 6.50 (s,
1H, H-5), 5.62 (d, = 5.0 Hz, 1H, H-8), 4.12 (dd,
9.3 Hz, 6.1 Hz, 1H, H-9a), 3.90 (s, 3H, CH3O-4), 3.27 mined to be C22H20O10, according to the HR-ESIMS peak
1
RESULTS AND DISCUSSION
δ
J
J
J
=
The molecular formula for compound 1 was deter-
(t,
2.29 (ddd,
(d, = 13.2 Hz, 1H, H-9
J
= 5.5 Hz, 2H, H-3), 2.51 (t,
= 13.2 Hz, 9.3 Hz, 4.9 Hz, 1H, H-9
).
J
= 5.5 Hz, 2H, H-2), at m/z 445.1149 [M + H]+. The IR spectrum showed the
J
α
), 2.06 presence of carbonyls (1755 and 1728 cm−1), a double
band (1630 cm−1) and a benzene nucleus (1600 and
1554 cm−1). Analysis of the 1H-, 13C-, and DEPT-NMR
J
β
data for
E. aerog- five methylenes, four methenyl groups, and eleven
S. aureus quaternary carbons, including four carbonyl carbons.
and Candida albicans were evaluated by an agar One aromatic proton, at
H 6.39, showed in the 1H-NMR
1 revealed the presence of two methoxy groups,
Antimicrobial assays
The antimicrobial activities against E. coli
enes B. subtilis Pseudomonas aeruginosa
,
,
,
,
δ
dilution method (Zaika, 1988). The tested strains were spectrum and molecular high degree of unsaturation
cultivated in LB agar plates for bacteria and in YPD indicated the presence of a penta-substituted benzene
agar plates for
and positive controls were dissolved in 5% DMSO-H2O tra with the known analogues revealed that
at different concentrations from 100 to 0.025 g/mL by the same structure nucleus to 8-acetyloxyaflatoxin B1
the continuous 2-fold dilution method. A 5 L quantity ) (Buchi et al., 1967; Coles et al., 1980), and the acetyl
of test solution was absorbed by a paper disk (5 mm signals in were replaced by the corresponding signals
C.
albicans at 37oC. Compounds
1-4
ring. Careful comparison of its 1H- and 13C-NMR spec-
1
contained
µ
µ
(4
4
diameter) and placed on the assay plates. After 24 h of a methoxy, two carbonyls and two methenes in 1.
1
incubation, zones of inhibition (mm in diameter) were This deduction was further supported by the H-1H
recorded. The minimum inhibitory concentrations COSY of H-6a/H-9a/H2-9/H-8 and H2-2/H2-3 and HMBC
(MICs) were defined as the lowest concentration, at correlations from H-6a (
which no microbial growth could be observed. Cipro- 8 ( C 98.4) and C-9b ( C 107.6), from H-5 (
floxacin lactate was used as the positive control for 3b (δC 104.0) and C-5a, and from H-3 (δH 3.42) to C-1
coli subtilis aerogenes and P. aeruginosa, with C 201.3) and C-11a ( C 117.3) (Fig. 2). Besides, the C-
M, respectively. 8 was shifted upfield. The 1H-1H COSY between H2-2'
and H2-3', in addition to the HMBC correlations from
the methoxy proton ( H 3.71) to C-4' ( C 171.1) and from
H 2.67) to C-1' ( C 172.4), which indicated that
δ
H 6.51) to C-5a (
δ
C 165.6), C-
δ
δ
δH 6.39) to C-
E
.
,
B
.
,
E
.
(δ
δ
MIC values of 0.3, 0.15, 0.08 and 0.15
µ
δ
δ
Cytotoxic assays
Cytotoxicity was assayed by the MTT (Mosmann, H2-2' (
δ
δ
1983) and SRB (Skehan et al., 1990) methods. In the these groups are connected into 4-methoxy-4-oxobu-
MTT assay, K562 cell line was grown in RPMI-1640
supplemented with 10% FBS under a humidified
atmosphere of 5% CO2 and 95% air, at 37oC. Cell sus-
pension, 200
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 were 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
µ
L, at a density of 5 × 104 cell/mL was
µ
µ
Fig. 2. 1H-1H COSY, HMBC and key NOESY correlations
for
1.
µ

Antimicrobial Aflatoxin from A. flavus
1391
pathogens, indicating that esterification at C-8 with
the longer fatty acid helps to increase the antibac-
terial activity.
The cytotoxic effects of compounds
02 cell line and the new compound
were assayed by the SRB method (Skehan et al.,
1990). The cytotoxicity of compound against K562 cell
line was also assayed by the MTT method (Mosmann,
1983). The results showed that compound exhibited
a weak cytotoxicity against the A549 and K562 cell
lines with IC50 values of 8.1 and 2.0 M, respectively.
In addition, compounds and exhibited cytotoxic
activity against the human normal liver cell line (L-
02) with IC50 values of 4.2, 3.7 and 6.4 M, respectively.
These data suggested that the hydration and hydro-
genation of
8-double bond, significantly, reduce the
1
-
4
and 1a on L-
1
on A549 cell line
1
1
µ
1,
2
4
µ
Fig. 3. CD curves of compounds 1-4.
∆
cytotoxicity of aflatoxins, while the esterification at C-
8 increases the cytotoxicity of aflatoxin B2a.
tanoyl moiety. These data supported that compound
is the derivative of 4-methoxy-4-oxobutanoyl substi-
tution for acetyl of . The NOESY correlations among
H-6a (δH 6.51), H-8 (δH 6.38) and H-9a (δH 4.27) sug-
gested their cis- configuration (Fig. 2). After subjection
1
4
ACKNOWLEDGEMENTS
This work was supported by grants from the National
to a basic hydrolysis, both
product, ( )aflatoxin B2a (1a) (Trost et al., 2003). Further, the National Natural Science Foundation of China (No.
the similar CD Cotton effects at 356 (-12.2), 238 (+5.7), 21172204, 30973680 & 30670219), from the Major Pro-
218 (-5.6) nm between and were observed (Fig. 3), gram for Technique Development Research of New
which indicates the same absolute configuration, i.e. Drugs in China (No. 2009ZX09103-046), and the Special
6a , 8 , 9a . Thus, structure of was determined as Fund for Marine Scientific Research in the Public In-
-(4-methoxy-4-oxobutanoyloxy)aflatoxin B2.
Compounds were assayed for antimicrobial ac-
tivities against aureus coli aerogenes
subtilis aeruginosa, and albicans, evaluated by
1 and 4 produced the same Basic Research Program of China (No. 2010CB833800),
−
1
4
S
S
S
1
8S
terest of China (No. 2010418022-3).
1-4
S.
,
E
C.
.
,
E.
, B.
REFERENCES
,
P
.
Asao, T., Buchi, G., Abdel-Kader, M. M., Chang, S. B., Wick,
E. L., and Wogan, G. N., The structures of aflatoxins B and
G1. J. Am. Chem. Soc., 87, 882-886 (1965).
Brechbuhler, S., Buchi, G., and Milne G., The absolute con-
figuration of the aflatoxins. J. Org. Chem., 32, 2641-2642
(1967).
Barrow, C. J. and Sedlock, D. M., 1'-(2-phenyl-ethylene)-di-
tryptophenaline, a new dimeric diketopiperazine from
Aspergillus flavus. J. Nat. Prod., 57, 1239-1244 (1994).
Buchi, G., Foulkes, D. M., Kurono, M., Mitchell, G. F., and
Schneider, R. S., The total synthesis of racemic aflatoxin
an agar dilution method (Zaika, 1988). Compound
exhibited a moderate antibacterial activity against
1
E
.
coli
22.5, 1.7 and 1.1
exhibited a moderate antibacterial activity against
aerogenes with MIC values of 3.2, 3.2 and 2.7 M,
respectively. Compound also showed a weak anti-
bacterial activity against aeruginosa with a MIC
value of 32.1 M. These data showed that aflatoxin
B2b ) is the most active compound against the test
,
B
.
subtilis and
E. aerogenes with MIC values of
M, respectively (Table II). Compounds
µ
2-4
E.
µ
2
P.
µ
(
1
Table II. MIC values of antibacterial activities against
albicans for compounds M)
S.
aureus
,
E.
coli
,
E.
aerogenes
,
B.
subtilis
,
P.
aeruginosa, and
C
.
1-4 (µ
No.
P.
aeruginosa
E.
coli
E.
aerogenes
B.
subtilis
C. albicans
1
2
3
4
>100
32.1
>100
>100
0.15
-
22.5
>100
>100
>100
0.3
1.1
3.2
3.2
2.7
0.08
-
1.7
>100
>100
>100
0.15
-
>100
>100
>100
>100
-
Ciprofloxacin
Ketoconazole
-
0.02

1392
H. Wang et al.
B1. J. Am. Chem. Soc., 89, 6745-6753 (1967).
assays. J. Immunol. Methods, 65, 55-63 (1983).
Chen, J. J., Huang, S. Y., Duh, C. Y., Chen, I. S., Wang, T. C.,
and Fang, H. Y., A new cytotoxic amide from the stem wood
Moss, E. J., Judah, D. J., Przybylski, M., and Neal, G. E., Some
mass-spectral and n.m.r. analytical studies of a glutathione
of Hibiscus tiliaceus
.
Planta Med., 72, 935-938 (2006).
conjugate of aflatoxin B1. Biochem. J., 210, 227-233 (1983).
Coles, B. F., Welch, A. M., Hertzog, P. J., Smith, J. R. L., and
Garner, R. C., Biological and chemical studies on 8,9-di-
hydroxy-8,9-dihydro-aflatoxin B1 and some of its esters.
Carcinogenesis 1, 79-90 (1980)
El-Gendy, M. M. A. and EL-Bondkly, A. M. A., Production
and genetic improvement of a novel antimycotic agent,
saadamycin, against dermatophytes and other clinical
fungi from endophytic Streptomyces sp. Hedaya48. J. Ind.
Microbiol. Biotechnol., 37, 831-841 (2010).
Movassaghi, M., Schmidt, M. A., and Ashenhurst, J. A., Concise
total synthesis of (+)-WIN 64821 and (-)-ditryptophenaline.
Angew. Chem. Int. Ed., 47, 1485-1487 (2008).
Rosa, R. M., Melecchi, M. I. S., Halmenschlager, R. D. C., Abad,
F. C., Simoni, C. R., Caramao, E. B., Henriques, J. A. P., Saffi,
J., and Ramos, A. L. L. D. P., Antioxidant and antimutagenic
properties of Hibiscus tiliaceus L. methanolic extract. J.
Agric. Food Chem., 54, 7324-7330 (2006).
Skehan, P., Storeng, R., Scudiero, D., Monks, A., McMahon,
J., Vistica, D., Warren, J. T., Bokesch, H., Kenney, S.,
Boyd, M. R., Tan, N. H., and Zhou, J., New colorimetric
Feng, C., Li, X. M., Ji, N. Y., and Wang, B. G., Triterpenoids
from the mangrove plant Hibiscus tiliaceus. Helv
. Chim.
Acta, 91, 850-855 (2008).
cytotoxicity assay for anticancer-drug screening. J. Natl.
Klausmeyer, P., McCloud, T. G., Tucker, K. D., Cardellina,
J. H., II, and Shoemaker, R. H., Aspirochlorine class com-
pounds from Aspergillus flavus inhibit azole-resistant
Cancer Inst., 82, 1107-1112 (1990).
Trost, B. M. and Toste, F. D., Palladium catalyzed kinetic
and dynamic kinetic asymmetric transformations of
ã-
Candida albicans
Lee, Y. M., Li, H. Y., Hong, J., Cho, H. Y., Bae, K. S., Kim, M.
A., Kim, D. K., and Jung, J. H., Bioactive metabolites from
.
J. Nat. Prod., 68
,
1300-1302 (2005).
acyloxybutenolides. Enantioselective total synthesis of
(+)-aflatoxin B1 and B2a. J. Am. Chem. Soc., 125, 3090-3100
(2003).
the sponge-derived fungus Aspergillus versicolor
Pharm. Res., 33, 231-235 (2010).
Lu, Z. Y., Zhu, H. J., Fu, P., Wang, Y., Zhang, Z. H., Lin, H.
P., Liu, P. P., Zhuang, Y. B., Hong, K., and Zhu, W. M.,
Cytotoxic polyphenols from the marine-derived fungus
.
Arch.
Wu, J., Xiao, Q., Xu, J., Li, M. Y., Pan, J. Y., and Yang, M. H.,
Natural products from true mangrove flora: source,
chemistry and bioactivities. Nat. Prod. Rep., 25, 955-981
(2008).
Zaika, L. L., Spices and herbs: their antimicrobial activity
Penicillium expansum
.
J.
Nat
.
Prod., 73, 911-914 (2010).
and its determination. J. Food Safety, 9, 97-118 (1988).
Lu, Z. Y., Wang, Y., Miao, C. D., Liu, P. P., Hong, K., and Zhu,
W. M., Sesquiterpenoids and benzofuranoids from the
Zhang, H. W., Song, Y. C., and Tan, R. X., Biology and chem-
istry of endophytes. Nat. Prod. Rep., 23, 753-771 (2006).
Zhou, G. and Corey, E. J., Short, enantioselective total syn-
thesis of aflatoxin B2 using an asymmetric [3+2]-cyclo-
addition step. J. Am. Chem. Soc., 127, 11958-11959 (2005).
marine-derived fungus Aspergillus ustus 094102.
Prod., 72, 1761-1767 (2009).
J. Nat.
Mosmann, T., Rapid colorimetric assay for cellular growth
and survival: Application to proliferation and cytotoxicity