Arch. Pharm. Res.

(2014) 37:575579
DOI 10.1007/s12272-013-0206-3

RESEARCH ARTICLE

Isochaetomium A2, a new bis(naphthodihydropyran-4-one)

with antimicrobial and immunological activities from fungus
Chaetomium microcephalum
Guo-Bo Xu Tao Yang Jin-Ku Bao
Dong-Mei Fang Guo-You Li

Received: 7 March 2013 / Accepted: 3 July 2013 / Published online: 2 August 2013
The Pharmaceutical Society of Korea 2013

Abstract Isochaetomium A2 (1), a new bis(naphthodihydropyran-4-one), along with chaetochromins A (2) and B
(3), was isolated from the solid-state fermented rice culture
of Chaetomium microcephalum. The structure of compound 1 was elucidated on the basis of 1D and 2D NMR
spectral data, and the relative configuration was confirmed
by CD spectrum. Compounds 13 possessed significant
antimicrobial activity against Escherichia coli 1.044,
Staphylococcus aureus 1.252, and Bacillus subtilis 1.079.
Moreover, compounds 13 showed obvious inhibitory
effects on mouse spleen cell proliferation with successive
IC50 values of 0.52, 0.19, and 0.24 lM.

Tsuchiya et al. 1987; Koyama and Natori 1988; Singh et al.

2003; Ugaki et al. 2012). These compounds possess various
bioactivities such as HIV-1 integrase inhibition, LB and MB
cells proliferation inhibition, triacylglycerol synthesis inhibition, and cytotoxicity. In searching for new bioactive compounds from the fungus Chaetomium microcephalum, a new
congener of chaetochromin, isochaetochromin A2 (1), together with chaetochromins A (2) and B (3), was isolated. Here
the isolation, structural elucidation, and biological activities of
compounds 13 were described.

Materials and methods

Keywords Chaetomium microcephalum 
Isochaetomium A2  Antimicrobial  Immunological
activity

Introduction
Chaetochromins, a group of bis(naphthyl-pyrone), were
isolated from fungi of the genera Chaetomium, Claviceps,
Penicillium, Cephalosporium, Verticillium, Nectoria, Aspergillus, and Fusarium (Sekita et al. 1980; Koyama et al. 1987;
G.-B. Xu  T. Yang  D.-M. Fang (&)  G.-Y. Li (&)
Chengdu Institute of Biology, Chinese Academy of Sciences,
Chengdu 610041, Peoples Republic of China
e-mail: fangdm@cib.ac.cn
G.-Y. Li
e-mail: ligy@cib.ac.cn
G.-B. Xu  J.-K. Bao
Key Laboratory of Bio-resources and Eco-environment, Ministry
of Education, School of Life Sciences, Sichuan University,
Chengdu 610064, Peoples Republic of China

General experimental procedures

High-resolution electrospray ionization mass spectra
(HRESIMS) were carried out on a BioTOF-Q mass spectrometer. Optical rotations were measured on a Perkin-Elmer
341 polarimeter. UV spectra and IR spectra were recorded on
a Perkin-Elmer S2 Lambda 35 UV/VIS spectrometer and a
Perkin-Elmer Spectrum One FT-IR spectrometer, respectively. NMR spectra were performed on the Bruker Avance
600 spectrometer. CD experiments were executed on circular dichroism spectrometer (Chirascan). The proliferation of
CFSE-labelled cells was analyzed by flow cytometry on a
FACSCalibur (BectonDickinson, San Jose, CA, USA).
Fungus material
Chaetomium microcephalum (cib-112) was isolated from a
soil sample collected in Yaan of Sichuan Province, China,
and identified by associate Professor Tao Yang in Chengdu
Institute of Biology, Chinese Academy of Sciences (CAS),
P. R. China. The test strains Escherichia coli 1.044,

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576

Staphylococcus aureus 1.252, and Bacillus subtilis 1.079

were obtained from Institute of Microbiology, Chinese
Academy of Sciences (CAS), P. R. China, which were
maintained on potato dextrose agar slant (PDA) at 4 C and
stocked in Chengdu Institute of Biology (CAS).
Fermentation and isolation
Seed culture medium was comprised of dextrose (20 g/L),
yeast extract (1 g/L), KH2PO4 (3.0 g/L), MgSO47H2O
(1.5 g/L), and potato extract (20 %, m/V). The pH of medium
was adjusted to 6.5 with 1 mol/L NaOH (aq.). Solid culture
medium consisted of rice and 0.3 % peptone. The sterilization
was carried out at 121 C under 15 psi for 30 min. The fresh
mycelium grown on PDA slant at 29 C for 4 days was
inoculated into 500 mL flasks containing 120 mL sterilized
seed medium. Flasks with inoculated medium were placed in
rotary shaker at 29 C and incubated at 150 rpm for 3 days.
The seed culture was inoculated into sterilized rice solid
medium for further fermentation at 29 C for 18 days.
The fermented solid rice medium (4 kg) was soaked with
ethyl acetate (7 L 9 3, 1 day for each time) at 50 C. The
solution was evaporated under reduced pressure to afford a
residue (11.0 g). The residue was divided into four fractions
(Fr.1, 2, 3 and 4) over silica gel column (200 g, 300400 mesh,
U 75 mm 9 400 mm), eluted with petroleum ether-acetone
(6:1, 3:1, 1.5:1, 0:1, successively). Fraction 2 (3.1 g) was further separated over Sephadex LH-20 column eluted with 1:1
CHCl3MeOH to obtain compound 2 (300.0 mg) and a mixture (1.6 g). The separation of the mixture by HPLC (methanol/
water, 65/35) afforded compounds 1 (6.0 mg) and 3 (10.1 mg).

G.-B. Xu et al.

402 (3.89); HRESIMS m/z 545.1414 [MH]- (545.1453

calcd. for C30H25O10).
Biological assays
The anti-bacterial activity was evaluated according to the
reported procedure with little modification (Carlson et al.
1946; Schlorke and Zeeck 2006; Shaaban et al. 2012).
Activity was determined using the Oxford cup method with
medium (dextrose 20.0 g/L, beef infusion 10 g/L, NaCl
5 g/L, agar 17 g/L) respectively inoculated with strains of
E. coli, S. aureus, and B. subtilis. To each cup was added
200 lL sample (compounds 13) dissolved in DMSO.
Equivalent amounts of DMSO were used as controls in
each test. The plates were incubated at 37 C for 24 h. The
antimicrobial activity was evaluated by measuring the
diameter zone of growth inhibition against the test
microorganism.
The immunological activity was determined complying
with the literature (Liu et al. 2013). The mouse (Bal b/c)
spleen cell proliferation was determined by flow cytometry
analysis with CFSE labelling. The spleen cells (106 cells/
mL) were stained with CFSE (2.5 mM) for 10 min at
37 C, and stopped by RPMI 1640 complete medium.
Subsequently, CFSE-labelled mouse spleen cells were
stimulated by plate-bound anti-CD3 (2.0 mg/mL) plus
soluble anti-CD28 (1.0 mg/mL) monoclonal antibodies
(mAbs). Then these activated cells were all incubated at
37 C for 96 h with compounds 13 dissolved in DMSO.
The proliferation of CFSE-labelled cells was analyzed by
flow cytometry on a FACSCalibur using CellQuest acquisition and ModFit analysis software (BectonDickinson).

Isochaetochromin A2 (1)
Yellow amorphous powder; a20
D ?365 (c 0.1, CHCl3); UV
(CHCl3) kmax nm (log e) 259 (4.62), 281 (4.71), 312 (4.16),
402 (3.96); HRESIMS m/z 545.1453 [MH]- (545.1453
calcd. for C30H25O10). CD (dioxane) kmax (De) ?295 nm
(?31.7), -265 nm (-30.3).
Chaetochromin A (2)
Yellow amorphous powder; a20
D ?630 (c 0.1, CHCl3); UV
(CHCl3) kmax nm (log e) 258 (4.63), 281 (4.72), 312 (4.14),
403 (3.94); HRESIMS m/z 545.1420 [MH]- (545.1453
calcd. for C30H25O10). CD (dioxane) kmax (De) ?305 nm
(?35.8), -255 nm (-34.7).
Chaetochromin B (3)
Yellow amorphous powder; a20
D ?518 (c 0.1, CHCl3); UV
(CHCl3) kmax nm (log e) 259 (4.60), 282 (4.69), 312 (4.11),

123

Results and discussion

Compound 1 was obtained as yellow amorphous powder.
Its molecular formula of C30H26O10 was established by the
quasi-molecular ion at m/z 545.1453 [MH]- in HRESIMS
spectrum. The IR spectrum of 1 revealed the presence of
hydroxyl (3,430 cm-1) and carbonyl (1,632 cm-1). From
the 1H NMR spectrum (in CDCl3) and HSQC experiment,
the following groups were recognized: two methyls [dH
1.16 (3H, d, 7.3 Hz), dH 1.30 (3H, d, 6.4 Hz)], two aromatic protons [(dH 5.93 (1H, s), dH 6.49 (1H, s)], two
methine protons [dH 2.61 (1H, dq, 2.3, 7.8 Hz, dH 4.50 (1H,
dq, 2.3, 6.2 Hz)], and three hydroxyls (dH 15.20, 9.69, and
5.71, each 1H, s). The fifteen 13C NMR signals could be
attributed to one carbonyl carbon (dC 202.9), ten aromatic
carbons (dC 165.4, 160.2, 161.2, 101.6, 105.9, 100.0, 102.1,
142.2, 99.5, 156.2), two methine carbons (dC 75.8, 44.7),
and two methyl carbons (dC 16.8, 9.7). In view of the
molecular formula C30H26O10, compound 1 should be a

Isochaetomium A2 from fungus C. microcephalum

577

symmetric dimer with 18 unsaturated degrees. The NMR

and UV spectra were similar with those of compound 2
(Table 1) and ustilaginoidin D (Koyama and Natori 1988).
The most difference among compounds 1, 2, and ustilaginoidin D in the 1H NMR spectra was that J23/J20 30 values
(3.2 Hz) in 1 were smaller than those in 2 (J23/
J20 30 = 11.0) and ustilaginoidin D (J23/J20 30 = 10.7).
Thus, compound 1 was possibly a stereoisomer of compound 2 and ustilaginoidin D.
From the HMBC correlations of OH-6/C-6 C-5a, and
C-7, H-7/C-5a, C-8, and C-9, and H-10/C-9, C-5a, C-4a,
and C-10a (Fig. 2), a naphthyl motif in compound 1 was
concluded. The HMBC correlations of 2-CH3 to C-2 and
C-3, 3-CH3 to C-3, C-2 and C-4, H-3 to C-4 and C-4a
suggested the presence of c-pyrone fused with naphthyl
motif. The planar structure of compound 1 was finally
elucidated as shown in Fig. 1.
The relative stereochemistry of C-2, C-3, C-20 , and C-30
in compound 1 was investigated by J values. The cis oriented vicinal methyl groups (CH3-2 and CH3-3, CH3-20 and
CH3-30 ) were determined by the J values (J23/
J20 30 = 2.3 Hz) in compound 1. The CD curve of compound 1 showed positive Cotton effect at 295 nm and
negative Cotton effect at 265 nm, which were similar with
those of chaetochromin A (positive Cotton effect: 305 nm;
negative Cotton effect: 255 nm) (Koyama et al. 1987).
Hence, the absolute axis stereochemistry of compound 1
was elucidated to be S (C-9, 90 ).

Compounds 2 and 3 were identified as chaetochromins

A (2) and B (3) (Fig. 1) by comparing their IR, UV, NMR
spectra, and optical rotation values with those reported
(Koyama and Natori 1987).
The fungi of Chaetomium genera are widely distributed
in soil and air, and some of them are pathogenic to plant,
animal, and human beings. Its secondary metabolites play
important roles as chemical signals to communicate with
other organisms for survival (Berdy 2005), and serve as a
source for new drugs. Therefore, the antibacterial and
immunological inhibitory activities of compounds 13
were selectively examined.
As a result, compounds 13 (Table 2) exhibited stronger
antimicrobial activity against Gram-negative bacterial
(E. coli 1.044) than Gram-positive bacterial (S. aureus
1.252 and B. subtilis 1.079). Although isomers of chaetochromin A showed structureactivity relationship among
isochaetochromin A1, B1 and B2 on inhibiting triacylglycerol (TG) synthesis (Ugaki et al. 2012), this similar
relationship of compounds 13 was unconspicuous on
antimicrobial activity.
The in vitro immunological inhibitory activity of compounds 13 was evaluated by using mouse spleen cell
(SPC) proliferation method (Halloran 2004). The IC50
values of compounds 13 were 0.52, 0.19, and 0.24 lM,
respectively.
Isochaetochromin A2 is the first symmetric bis
(naphthodihydropyran-4-one) characteristic of cis oriented

Table 1 NMR data of isochaetochromin A2 (1) and chaetochromin A (2) in CDCl3 (1H: 600 MHz;
No.

C: 150 MHz)

Chaetochromin A

Isochaetochromin A2
dH (J in Hz)

13

dC

dH (J in Hz)

dC

2, 20

4.50 (1H, dq, 2.3, 6.2)

75.8

4.15 (1H, dq, 11.0, 6.1)

78.6

2.61 (1H, dq, 2.3, 7.8)

44.7
202.9

2.62 (1H, dq, 10.9, 6.9)

46.4
201.0

3, 3
4, 40
4a, 4a0

101.6

100.0

5, 50

165.4

164.6

5a, 5a0

105.9

105.7

6, 6

160.2

7, 70

6.49 (1H, s)

100.0

160.1
6.45 (1H, s)

99.6

8, 80

161.2

160.1

9, 90

102.1

102.2

9a, 9a0

142.2

142.2

10, 10

5.93 (1H, s)

10a, 10a0

99.5

5.91 (1H, s)

156.2

99.6
156.5

2-CH3, 20 -CH3

1.30 (3H, d, 6.4)

16.8

1.42 (3H, d, 6.2)

19.8

3-CH3, 30 -CH3

1.16 (3H, d, 7.3)

9.7

1.24 (3H, d, 7.0)

10.1

5-OH, 50 -OH
6-OH, 60 -OH
8-OH, 80 -OH

15.20 (s)
9.69 (s)
5.71 (brs)

15.23 (s)
9.60 (s)
5.78 (brs)

All data was assignment on the basis of HSQC and HMBC experiments

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G.-B. Xu et al.

7
8

6
9
9'

8'
7'

5a

9a
9'a

10a
10
10' 10'a

2'
3'
4'a

5'a

3
2

5'
6'

4a

4'

Isochaetochromin A2

Chaetochromin A

Chaetochromin B

Ustilaginoidin D

Fig. 1 Structures of isochaetochromin A2 (1), chaetochromins A (2), B (3) and ustilaginoidin D

6
7

5a

9a

5
4a

10

bioactivities of 13 may be attributed to the phenolic

hydroxyl groups of bis(naphthodihydropyran-4-one) (Singh
et al. 2003). Moreover, the biological activity of naphthopyrone was also related to other groups including the C-3
methyl group, 5/50 -methyl ester or 5,50 -dimethyl esters
(Barrow and McCulloch 2009). Thus the structureactivity
relationships of antimicrobial and immunological activities
for naphthopyrone are not clear and deserve to be further
studied in the future.

4
3

10a

Acknowledgments This study was financially supported by the

National Natural Science Foundation of China (Nos. 20902089 and
21272228) and the Knowledge Innovation Program of the Chinese
Academy of Sciences (KSCX2-EW-G-13-04).

References
Fig. 2 Key HMBC correlations of isochaetochromin A2 (1)
Table 2 Antimicrobial activities of compounds 13
1a

2a

3a

Meticillinb

DMSO

S. aureus 1.252

3.3 0.6

3.2 1.0

3.3 0.3

4.0 0

B. subtillis 1.079

4.5 0.9

4.2 1.2

4.0 0.7

4.2 0.3

E. coli 1.044

7.2 1.3

6.0 0.9

6.5 1.5

8.3 0.6

The activity expressed as value of diameter halo (mean SD, mm, n = 3,

three replicates; result was expressed by deducted the value of Oxford cup
diameter)
DMSO dimethyl sulfoxide
a

All the concentrations of compounds 13 were 0.19 lg/200 lL

0.50 lg/200 lL

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