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R E S E A R C H A R T I C L E

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(naphthodi-

hydropyran-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 com-

pound 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.

Keywords Chaetomium microcephalumIsochaetomium 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,Asper-

gillus, andFusarium(Sekita et al.1980; Koyama et al.1987;

Tsuchiya et al.1987; Koyama and Natori1988; Singh et al.

2003; Ugaki et al.2012). These compounds possess variousbioactivities such as HIV-1 integrase inhibition, LB and MB

cells proliferation inhibition, triacylglycerol synthesis inhi-

bition, and cytotoxicity. In searching for new bioactive com-

pounds from the fungusChaetomium microcephalum, a new

congener of chaetochromin, isochaetochromin A2 (1), toge-

ther with chaetochromins A (2) and B (3), was isolated. Here

the isolation, structural elucidation, and biological activities of

compounds13were described.

Materials and methods

General experimental procedures

High-resolution electrospray ionization mass spectra

(HRESIMS) were carried out on a BioTOF-Q mass spec-

trometer. Opticalrotations were measured on a Perkin-Elmer

341polarimeter. UV spectraand IR spectra were recorded on

a Perkin-Elmer S2 Lambda 35 UV/VIS spectrometer and a

Perkin-Elmer Spectrum One FT-IR spectrometer, respec-

tively. NMR spectra were performed on the Bruker Avance

600 spectrometer. CD experiments were executed on circu-

lar 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,

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. BaoKey Laboratory of Bio-resources and Eco-environment, Ministry

of Education, School of Life Sciences, Sichuan University,

Chengdu 610064, Peoples Republic of China

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Arch. Pharm. Res. (2014) 37:575579

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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 solidmedium 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 fur-

ther separated over Sephadex LH-20 column eluted with 1:1

CHCl3MeOH to obtain compound2 (300.0 mg) and a mix-

ture (1.6 g).The separation of themixture by HPLC (methanol/

water, 65/35) afforded compounds1 (6.0 mg)and3 (10.1 mg).

Isochaetochromin A2 (1)

Yellow amorphous powder; a 20D ?365(c0.1, CHCl3); UV

(CHCl3)kmaxnm (loge) 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; a 20D ?630(c0.1, CHCl3); UV(CHCl3)kmaxnm (loge) 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; a 20D ?518(c0.1, CHCl3); UV

(CHCl3)kmaxnm (loge) 259 (4.60), 282 (4.69), 312 (4.11),

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 acqui-

sition and ModFit analysis software (BectonDickinson).

Results and discussion

Compound 1 was obtained as yellow amorphous powder.

Its molecular formula of C30H26O10was established by the

quasi-molecular ion atm/z545.1453 [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 [dH1.16 (3H, d, 7.3 Hz), dH 1.30 (3H, d, 6.4 Hz)], two aro-

matic protons [(dH 5.93 (1H, s), dH 6.49 (1H, s)], two

methine protons [dH2.61 (1H, dq, 2.3, 7.8 Hz,dH4.50 (1H,

dq, 2.3, 6.2 Hz)], and three hydroxyls (dH15.20, 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 (dC165.4, 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

576 G.-B. Xu et al.

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Isochaetomium A2 from fungus C. microcephalum 579

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