Microbiological Research 168 (2013) 204–210

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Microbiological Research

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Paecilomide, a new acetylcholinesterase inhibitor from Paecilomyces lilacinus

Ana Paula C. Teles

a

,

b

, Jacqueline A. Takahashi

b

,

∗

a

Escola de Farmácia, Universidade Federal de Ouro Preto, Ouro Preto, MG, Brazil

b

Departamento de Química, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil

a r t i c l e i n f o

Article history:
Received 2 August 2012
Received in revised form 6 November 2012
Accepted 11 November 2012
Available online 6 December 2012

Keywords:
Paecilomyces lilacinus
OSMAC
Acetylcholinesterase inhibition

a b s t r a c t

Fungi are some of the most important organisms in the production of bioactive secondary metabolites.
This success is related to the advances in biotechnology and also to the possibility of working with
techniques such as the “OSMAC” (one strain-many compounds) to achieve different fungal secondary
metabolites profiles upon modifying the culturing conditions. Using this approach, the fungal species
Paecilomyces lilacinus was cultivated in potato dextrose broth under 14 different fermentative conditions
by adding the bacterium Salmonella typhimurium to the growing medium in order to provide biotic stress.
S. typhimurium was added alive or after inactivation by autoclave or microwave irradiation in different
stages of fungal growth. Extracts were prepared by liquid–liquid extraction using ethyl acetate, a medium
polarity solvent in order to avoid extracting culturing media components. Production of fatty acids of rele-
vance for the pharmaceutical and food industries was enhanced by the modified fermentative conditions
and they were identified and quantified. The extracts were evaluated for acetylcholinesterase inhibition
and the more active extract (91

± 2.91% inhibition) was prepared in large scale. From this active P. lilaci-

nus extract, a novel pyridone alkaloid, named Paecilomide, was isolated and its structure was elucidated
by modern nuclear magnetic resonance techniques and mass spectrometric analyses. Paecilomide (1)
was also evaluated for acetylcholinesterase inhibition, presenting 57.5

± 5.50% of acetylcholinesterase

inhibition.

© 2012 Elsevier GmbH. All rights reserved.

Introduction

Fungi are important organisms in the production of bioactive

secondary metabolites. Around 38% of the active compounds iso-
lated until 2005 were of fungal origin (

Bérdy 2005

), and this context

has not changed much in the late years. The success of fungal
metabolites can be attributed to many factors, like the advances
in the industrial production of biotechnological metabolites and
the possibility of working with techniques such as the “OSMAC”
(one strain-many compounds) (

Bode et al. 2002

). OSMAC is based

on the premise that a single fungal species, upon submission to
different cultivation conditions, can produce a great diversity of
new bioactive molecules. Among the parameters that can be varied
using OSMAC strategy, can be pointed the composition of cul-
ture medium, aeration, period of cultivation, pH, temperature and
addition of agents to induce or inhibit the production of metabo-
lites (

Saleem et al. 2009; Bugni and Ireland 2004

). Some stressing

factors such as high osmotic levels, addition of a competitive micro-
organism in the medium (co-culturing), and water restraint have

∗ Corresponding author at: Departamento de Química, Universidade Federal de

Minas Gerais, Av. Antonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil.
Tel.: [phone]; fax: [phone].

E-mail address:

[e-mail]

(J.A. Takahashi).

also been used in order to promote metabolic diversification in
fungi (

Cueto et al. 2001; Wang et al. 2011; Huang et al. 2011

).

Many drugs currently in the market, possessing a variety

of activities such as antitumor, immunosuppressants, antibi-
otics, hipocolesterolemic agents, antifungals, antiparasites, anti-
inflammatory and enzyme inhibitors, were obtained from fungal
metabolism (

Bérdy 2005; Kingston 2011

). Fungal metabolites have

been shown their potential in the production of novel com-
pounds (

Zhang et al. 2011; Houghton et al. 2006

) for treatment

of Alzheimer’s disease, a progressive and irreversible neurodegen-
erative disorder that leads to memory loss and cognitive disorders
(

Lima et al. 2009

).

The symptoms of Alzheimer’s disease are connected to the

reduction of brain neurotransmitters, such as acetylcholine, nora-
drenalin and serotonin (

Bryne 1998

). Therefore, the treatment

is based on the attempt to restore cholinergic function, using
inhibitors of acetylcholinesterase (AChE), an enzyme that acts on
acetylcholine degradation in the synaptic cleft (

Lleo et al. 2006

).

Tacrine, Rivastigmine and Galantanine, AChE inhibitors available
in the market, have a high cost, making necessary the search for
new substances for treatment of Alzheimer’s disease. Currently,
this screening can be readily accomplished since there are some
quick and sensitive screening bioassays to be used in the evalu-
ation of acetylcholinesterase inhibitory effect caused by organic
compounds (

Ellmann 1961; Rhee et al. 2001

).

0944-5013/$ – see front matter © 2012 Elsevier GmbH. All rights reserved.

http://dx.doi.org/10.1016/j.micres.2012.11.007

A.P.C. Teles, J.A. Takahashi / Microbiological Research 168 (2013) 204–210

205

N

O

H

NH

2

N

HO

OH

O

O

OH

OH

1

2

Fig. 1. Structures of Militarinone A (1) and Huperzine A (2).

The fungal species used in work was Paecilomyces lilacinus. Pae-

cilomyces genus is divided into two sections, section Paecilomyces
and section Isarioidea. The later contains mesophile members
including P. lilacinus (

Samson 1974

). P. lilacinus 18S rRNA genes

(rDNA) were sequenced and compared to other filamentous fungi
species showing to be far differentiated from Paecilomyces vari-
otii, a representative species from Paecilomyces genus (

Wu et al.

2003

). Species from Paecilomyces genus are capable of producing a

variety of secondary metabolites of different chemical classes and
with varied biological activities, such as cytotoxic, antibacterial, and
immunostimulating agents (

Kyong et al. 2001; Isaka et al. 2007; Liu

et al. 2011; Xu et al. 2010

). Neurotrophic pyridone alkaloids such

as Militarenone A (1) have been reported from Paecilomyces mil-
itaris (

Schmidt et al. 2002

). These compounds possess structural

resemblance with Huperzine A (2), a potent acetylcholinesterase
inhibitor (

Liu et al. 1986

). Structures of compounds 1 and 2 can be

found in

Fig. 1

.

No reports on the AChE inhibitors biosynthesis have been

described for P. lilacinus under natural fermentation conditions.
Therefore, OSMAC approach was exploited in order to create suit-
able stressing conditions with the aim of modulate the production
of secondary metabolites with AChE inhibitory activity by P. lilaci-
nus. From the existing stressing factors, such as increase of osmotic
and atmospheric pressure, and decrease of nutrients availability, in
this work, the stress was achieved by addition of bacterial genetic
material in the culturing medium used to grow P. lilacinus. This
strategy has been successfully employed to induce the production
of bioactive secondary metabolites by other fungal species (

Oh et al.

2005; Du et al. 2011

). Several extracts were prepared, assayed and

the most active extract for AChE inhibition was prepared in large
sale. An active metabolite was isolated from this extract and identi-
fied. Fatty acids profiles were determined for all extracts obtained.

Materials and methods

Source, maintenance and culturing conditions of the fungus P.
lilacinus

P. lilacinus was isolated from soil and it is deposited in the

micro-organisms collection of the Biotechnology and Bioassays
Laboratory (UFMG, MG, Brazil). P. lilacinus was maintained on
potato dextrose agar culture medium (PDA) on a refrigerator (8

◦

C)

(

Schürmann et al. 2010

). Prior to the experiments, the fungus was

transferred to freshly prepared PDA and grown at room temper-
ature (25

± 3

◦

C). For pre-inoculum preparation, P. lilacinus was

inoculated to Erlenmeyer flasks containing 200 mL of potato dex-
trose broth (PDB) and cultivated during seven days, under stirring
(150 rpm), at 25

± 3

◦

C. This procedure was performed to gener-

ate enough amount of biomass (pre-inoculum) to start the fungal
cultivations.

Fermentations conditions and extracts preparation

The experiments started with the inoculation of pre-inoculum

into 14 Erlenmeyer flasks containing liquid medium (PDB,
200 mL/flask), after which the contents were extensively homoge-
nized. Each flask was prepared to install the fungal growth in an odd
condition on a medium containing Salmonella typhimurium in order
to furnish biotic stress. S. typhimurium was added to the flasks con-
taining P. lilacinus in two different concentrations (1 and 10 mL), in
three different forms (alive, after microwave irradiation and after
inactivation by autoclave) and in two different phases of P. lilaci-
nus development (1st and 8th day of fungal growth). P. lilacinus–S.
typhimurium were co-cultivated at room temperature (25

± 3

◦

C),

in static condition. Controls without addition of bacterial material
were run in parallel. After a period of 21 days, the growth of P.
lilacinus was interrupted by addition of EtOAc in the flasks. The
culture media were individually filtered under vacuum through a
filter paper, to separate the broths from the mycelia. The broths
were exhaustively extracted with ethyl acetate on a separator fun-
nel. This procedure was repeated three times. The mycelia have
also been extracted with ethyl acetate, and both extracts (broth and
mycelium) obtained in each of the 14 co-culturing conditions were
combined, concentrated under vacuum and transferred to clean
bottles.

Gas chromatography (GC) analytical conditions

GC analysis was carried out on an HP5890 Gas Chromatograph

equipped with Flame Ionization Detector (FID) to obtain the fatty
acids profiles. A HP-INNOWax (HP) column (15 m

× 0.25 mm) was

used at the following temperature gradient: 150

◦

C, 1 min, 7

◦

C/min

until 240

◦

C; injector (split of 1/50) at 250

◦

C and detector at

250

◦

C. Hydrogen was used as carrier gas (2.0 mL/min) and injec-

tion volume was 2.0

␮L. Identification of compounds was made by

comparison with SUPELCO37 fatty acid methyl esters (FAMEs) stan-
dard. The percentages of FAMEs were also compared with soybean
oil (12.0 mg) hydrolyzed, methylated and analyzed under the same
conditions.

Preparation of samples for GC analysis

All extracts and some fractions from extract 5 (10.0 mg) were

dissolved, on a 2.0 mL cryogenic tube, in 100

␮L of a 1 mol/L potas-

sium hydroxide solution (5%) in ethanol (95%). After vortex stirring
for 10 s, the material was hydrolyzed on a microwave oven, during
5 min. After cooling, 400

␮L of hydrochloric acid 20% (w/v), NaCl and

600

␮L of ethyl acetate were added, stirred by 10 s and let to stand

for 5 min. An aliquot of 300

␮L of the organic layer was transferred

to a tube and dried by evaporation, to obtain the free fatty acids
(

Segall et al. 2006

). Free fatty acids were methylated with 100

␮L

BF

3

/methanol (14%), heated for 10 min in water bath at 80

◦

C to

206

A.P.C. Teles, J.A. Takahashi / Microbiological Research 168 (2013) 204–210

Fig. 2. Chromatogram of fraction P03 obtained by GC in comparison with standard fatty acid esters profile.

produce the fatty acid methyl esters (FAMEs) that were analyzed
by gas chromatography.

Nuclear magnetic resonance (NMR) conditions

The spectra of

1

H and

13

C NMR, sub spectra DEPT 135 and

1

H–

1

H

COSY,

1

H–

1

H NOESY,

1

H–

13

C HMBC, and

1

H–

13

C HSQC experi-

ments were obtained on BRUKER AVANCE DPX200 and DRX400
spectrometers at the High Resolution Magnetic Resonance Labo-
ratory (LAREMAR) from the Chemistry Department of the Federal
University of Minas Gerais (MG, Brazil). Spectra were measured at
400 (100) or 200 (50) MHz at 300 K, and the spectrometers were
equipped with inverse detection 5 mm multinuclear head

1

H/

13

C.

The compound was dissolved in piridine-d

5

, and transferred to a

5 mm o.d. NMR tube. Chemical shifts (

ı) were registered in ppm.

Tetramethylsilane (TMS) was used as an internal reference stan-
dard to chemical shift

ı 0. Bi-dimensional (2D) NMR spectra were

acquired under standard conditions. Data processing was carried
out on SGI workstation using the Bruker (DRX 400) software.

Electrospray ionization mass spectroscopy (MS-ESI)

High resolution mass spectra were obtained on a Bruker

Daltonics Mass Spectrometer, model MicroTOF, equipped with
electrospray ionization source (Chemistry Institute, USP, SP, Brazil).
Solutions of the compound in methanol/water was prepared and
analyzed in the zone of m/z between 200 and 500 Da.

Evaluation of acetylcholinesterase inhibitory activity

Bioassay for acetylcholinesterase inhibition was carried out

with Sigma reagents: albumin bovine serum; 5,5



-dithiobis(2-

nitro-benzoic acid) (DTNB); Tris/HCl 0,1 M pH 8; acetylthiocholine
iodide (ATCI); acetylcholinesterase from electric eel, type V-S. The
following standard solutions I–VII were prepared to be used in
the bioassay: (I) 50 mM Tris/HCl pH 8; (II) 50 mM Tris/HCl pH 8,

containing 0.1% of bovine serum albumin (BSA) fraction V; (III)
50 mM Tris/HCl pH 8, containing 0.1 M of NaCl and 0.02 M of
MgCl

2

·6H

2

O; (IV) 3 mM of DTNB or Ellman Reagent, (V) 15 mM of

ATCI, (VI) 1 mM of DTNB and (VII) 1 mM of ATCI. Lyophilized AChE
enzyme was dissolved in buffer solution I to make a 1000 U/mL
stock solution.

Thin layer chromatography (TLC) assay

The qualitative evaluation of the inhibitory activity of the

enzyme acetylcholinesterase was performed according to the
enzymatic assay (

Ellmann 1961; Rhee et al. 2001

), with minor mod-

ifications. The extracts to be assayed were prepared (10 mg/mL) by
dissolution of the extracts on a suitable solvent and applied (5.0

␮L)

in ready made TLC plates (0.2 mm thickness, Merck). The TLC plates
were developed and, after eluent evaporation, they were sprayed
with solutions VI and VII. After 5 min, the TLC plates were sprayed
with acetylcholinesterase solution (3 U/mL). After 10 min, a yel-
low color appeared in the plates. White halos showed inhibition
of the enzyme by some compounds present in the extracts. In this
experiment, caffeine solution (10 mg/mL) was used as a positive
control.

Microplates assay

To quantify the extension of acetylcholinesterase inhibition, a

further bioassay was conducted in 96 well microplates, and inhibi-
tions were quantitatively determined on a Thermo Plate microplate
reader, using absorbance of 406 nm, based on the

Ellmann’s method

(1961)

. There were tested all extracts, controls and compound 1.

In this assay, acetylcholinesterase hydrolyzes the substrate, gener-
ating thiocholine as the product. The later reacts with Ellmann’s
reagent, producing 2-nitrobenzoate-5-mercaptothiocolin and 5-
thio-2-nitrobenzoate, which can be detected and quantified at
406 nm. In each microplate wells, there were added 25

␮L of stan-

dard solution V; 125

␮L of solution IV; 50 ␮L of solution II and

A.P.C. Teles, J.A. Takahashi / Microbiological Research 168 (2013) 204–210

207

25

␮L of the sample to be analyzed, dissolved in MeOH (10 mg/mL);

absorbance was measured every 60 s, 8 times. Then, the enzyme
solution (0.222 U/mL) was added; absorbance was measured again
each 60 s, 10 times. The percentages of inhibition were calculated
comparing the rates of reactions promoted by the samples with
the rate of reaction resulting from the control (solvent used to
dissolve the samples) using the formula: % inhibition = 100

− (rate

of sample reaction/rate of control reaction

× 100). Eserine (Sigma,

St. Louis, USA) solution (10 mg/mL) was used as control positive.
Solvent used to dissolve the samples was used as negative control.

Chromatography conditions and compound isolation

Column chromatography (CC) was carried out using silica gel

60 (70–230 mesh, Merck) and for thin layer chromatography (TLC)
there were employed precoated silica gel plates. After assaying for
acetylcholinesterase inhibition, the most active extract (extract 5)
was prepared in large scale. There were produced 20.0 L of cul-
ture medium, obtaining 1.098 g of crude extract. This extract was
fractionated on a silica gel column chromatography, using as elu-
ents n-hexane, ethyl acetate and methanol in increasing polarities.
There were collected 138 fractions of 100 mL which that were
combined in 32 groups (P01–P32) according to the similarities of
chromatographic profiles in TLC and revelation with iodine vapor.
Compound 1 (69.0 mm) was obtained as a pure compound in group
P18, eluted by ethyl acetate:methanol (95:5). Extract 5 and com-
pound 1 showed positive reaction when sprayed with Dragendorff’s
reagent (

Wagner and Bladt 2001

).

Results

In this work, production of secondary metabolites active for

AChE inhibition by the fungus P. lilacinus was promoted by addi-
tion bacterial genetic material to the liquid medium used to grow
P. lilacinus. Modulation of secondary metabolites production by
co-culturing of P. lilacinus and S. typhimurium was monitored in
fourteen different fermentation conditions. The respective crude
extracts were obtained by using liquid–liquid extraction in ethyl
acetate. This medium polarity solvent is widely used for fungal
extracts preparation since it is able to extract most non-polar
secondary metabolites, avoiding extracting polar culturing media
components such as proteins. The extracts were qualitative and
quantitatively evaluated for acetylcholinesterase inhibition. The
qualitative evaluation of AChE inhibition was verified by forma-
tion of white halos in the TLC plates assay. Of the fourteen extracts
tested, ten (71%) produced white halos in the plate, indicating AChE
inhibition. In relation to the amount of fungal inoculum (1 or 10 mL)
added in the experiments, it has been observed that addition of
smaller quantity of the inoculum (1 mL) generated an active extract,
while addition of a large volume of inoculum (10 mL) led to an
inactive extract.

Since AChE inhibition test in TLC is only a qualitative evaluation,

Ellmann’s methodology was used to quantify the AChE inhibition
presented by the extracts obtained in this study. By this methodol-
ogy, it was found that the extract most able (91

± 2.91% inhibition)

to inhibit that enzyme was the one resulting addition of 1 mL of the
bacterial inoculum (deactivated by autoclave) in PDB, 7 h after fun-
gal inoculation that was grown under static condition. This extract
was prepared again, with the same conditions in large scale and
fractionated by chromatography column, obtaining a total of 32
fractions (P01–P32).

The less polar fractions P01–P06 (eluted with n-hexane) pre-

sented an oily aspect, typical of fatty acids. Since FAME profiles
are important to categorize fungi, the fractions containing fatty
acids were hydrolyzed, methylated and analyzed by GC, to obtain

Fig. 3. TLC profile of Paecilomide (1) sprayed with Dragendorff’s reagent.

the FAME profiles. Analysis of the FAMEs chromatograms obtained
revealed that these fractions were mainly constituted by a mix-
ture of palmitic (C

16:0

), stearic (C

18:0

), oleic (C

18:1

9 cis

), linoleic

(C

18:2

9,12 cis

) and linolenic acids (C

18:3

) (

Table 1

).

Fig. 2

presents

the FAME chromatogram of fraction P03, as an example of the FAME
profile.

Fractions P12 (250.0 mg), P13 (13.0 mg) and P14 (93.0 mg) were

characterized as waxes of yellow color and analyzed by

1

H and

13

C NMR and DEPT. In the spectrum of NMR of

1

H there were

observed signals between

ı

H

1.27 and 1.31 ppm, assigned to hydro-

gen atoms of methylene groups and signals between

ı

H

0.8 and

0.9 ppm, assigned to hydrogen atoms of methyl groups. In the

13

C

NMR spectra there were observed signals at

ı

C

173.4–176.3 ppm,

corresponding to carboxyl carbons, at

ı

C

22.9–32.14 ppm (methy-

lene carbons signals) and, at

ı

C

14.3 ppm, which are classical signals

of terminal methyl groups. These fractions were methylated and
analyzed by GC, revealing to have the same FAMEs profiles observed
for the less polar fractions.

Fraction P18 gave a pure compound (69.0 mg; 6.3% yield) that

was obtained as a yellow wax; on TLC analysis, this compound gave
positive result after sprayed with Dragendorff’s reagent (

Fig. 3

), a

very useful reagent used to identify alkaloids (

Wagner and Bladt

2001

). Therefore, it was inferred the presence of nitrogen in the

molecule. In the high resolution mass spectrum of this compound,
it was observed a peak with m/z 261.1284, compatible with the
sodium adduct [238+Na

+

] of a compound with molecular formula

C

12

H

15

NO

4

. In the

13

C NMR spectrum, there were observed 12 sig-

nals, four of them did not appear in DEPT sub-spectrum, showing
that there were four non-hydrogenated carbons in the molecule:
a carbonyl from an amide group at

ı

C

165.8 ppm (

Silverstein et al.

1991

), a carboxylic group (

ı

C

169.8) and other two quaternary car-

bons that showed resonances at

ı

H

127.7 ppm and

ı

C

157.8 ppm.

From the eight hydrogenated carbon atoms, four showed signs
between

ı 22.5 ppm and ı 45.3 ppm, being characterized as methy-

lene carbons. Signs of four methine groups were observed at

ı

C

57.0,

59.4 ppm, 116.2 and

ı 131.3 ppm. Structure elucidation of this com-

pound was accomplished by careful examination of data obtained
in the bidimensional NMR contour maps HSQC,

1

H–

1

H COSY and

HMBC (

Table 2

).

Correlations between carbon and hydrogen atoms participat-

ing of the same chemical binding were established and then, the

208

A.P.C. Teles, J.A. Takahashi / Microbiological Research 168 (2013) 204–210

Table 1
Fatty acids composition of the fractions analyzed.

Fractions

% fatty acid in fraction

Palmitic (C

16:0

)

Stearic (C

18:0

)

Oleic (C

18:1

9.cis

)

Linoleic (C

18:2

9,12 cis

)

Linolenic (C

18:3

)

P01

18.99

14.08

N.D.

N.D.

N.D.

P02

31.29

8.63

17.54

18.45

0.71

P03

38.2

9.43

4.9

5.94

10.64

P04

40.97

5.17

4.83

7.37

0.91

P05

22.49

8.95

8.16

5.52

N.D.

P06

23.14

8.39

7.44

N.D.

1.71

N.D.: not detected in the fraction.

COOH

H

H

H

H

H

H

1

2

3

4

5

6

7

A

N
H

O

OH

1'

2'

3'

4'

5'

B

1

H-

1

H COSY Correlations

Fig. 4. Fragments proposed based in the

1

H–

1

H COSY correlations.

connectivity between the carbons of the molecule and the neigh-
bor hydrogen were determined by HMBC contour map. Afterwards,
analysis of

1

H–

1

H COSY spectrum allowed correlating the hydro-

gens couplings. Two fragments A and B were established by this
analysis as shown in

Fig. 4

.

The fragment B corresponds to a

ı-lactam and correlates to the

fragment A as observed in the HMBC contour map. Further correla-
tions observed in the

1

H–

1

H COSY contour map, led to the proposal

of a bicycle molecule, by bonding both fragments A and B linked
through carbons 7 and 4



.

The most favorable conformation of 1,2-disubstituted a cyclo-

hexane would bear the two substituents in equatorial positions.
This proposal is in accordance with the spatial correlations
observed in NOESY contour map, which showed correlations
between H-4

␣ and H-5␣, H-3␤ and H-2␤, H-6␣ and H-7␣, as shown

in

Fig. 5

.

Due to possibility of free rotation of the C7 C4



sigma bond, it

was also observed correlations in the NOESY contour map between

H

H

H

H

H

H

H

H

H

COOH

H

R

2

3

4

5

6

7

R =

N

H

O

OH

Fig. 5. Spatial correlations of Paecilomide (1) based in NOESY correlations map.

both H-6 and H-5



as well as between H-7 and H-5



, according to

Fig. 6

(A and B, respectively).

Table 2

presents the complete NMR assignment of hydrogens

and carbons of Paecilomide (1), as well as a list of bi-dimensional
NMR correlations found for atoms of this molecule. Paecilomide
(1) was tested in the AChE inhibition bioassays, giving a positive
spot (white halo) when assayed by the TLC methodology. In the
AChE microplates assay, P. lilacinus extract showed 91.0

± 2.91%

of acetylcholinesterase inhibition while Paecilomide (1) presented
57.5

± 5.50% of inhibition. The results of the inhibitory potential of

the original P. lilacinus extract compared to activity of 1 are shown
in

Table 3

.

Discussion

The presence of bacterial material in the medium furnished a

stressing condition able to promote the biosynthesis of novel bioac-
tive metabolites. The bacterium used was S. typhimurium, a suitable

Table 2

1

H and

13

C NMR,

1

H–

1

H COSY and HMBC correlations for Paecilomide (1).

Carbon Number

Type

ı

C

(ppm)

ı

H

(ppm)

COSY

HMBC

1

COOH

169.8

2

CH

59.4

4.15

H2

× H3b

ı 4.20

H2

× C3; C1

3

CH

2

29.5

H3a – 1.90
H3b – 2.20

H3a

× H3b

ı 2.20

H3a

× C2, C1

4

CH

2

22.5

H4a – 0.80
H4b – 1.60

H4a

× C5

5

CH

2

45.3

H5a – 3.40

H5a

× H4b

ı 3.40

H5b

× C4

H5b – 3.70

H5b

× H4b

ı 3.65

6

CH

2

36.2

H6a – 3.30
H6b – 3.60

H6a

× C7, C3



, C5



H6b

× C5



7

CH

57.0

4.60

H7

× H6a;

H7

× H6b

ı 4.60

1



C O

165.8

2



CH

116.2

7.15

H2



× C3



, C4



3



C

127.7

4



C

157.8

5



CH

131.3

A.P.C. Teles, J.A. Takahashi / Microbiological Research 168 (2013) 204–210

209

Table 3
Inhibition (%) of acetylcholinesterase by P. lilacinus extract and Paecilomide (1) in
microplate by Ellmann’s methodology.

Sample

% inhibition (mean

± sd)

P. lilacinus extract

91

± 2.91

Paecilomide

57.5

± 5.50

Positive control (Eserine)

98.0

± 1.66

Negative control

0

± 0

sd: standard deviation.

H

H

H

H

H

H

H

H

H

H

6

7

NH

H

O

HO

4'

5'

1'

2'

3'

H

H

H

H

H

H

H

H

H

H

7

N

H

OH

O

H

4'

5'

1'

2'

3'

(A)

1

(B)

Fig. 6. Spatial correlations to Paecilomide (1) based in NOESY data. (A) and (B)
represent two possible rotational forms of Paecilomide (1).

species to be used for this purpose that does not produce secondary
metabolites able to interfere in the experiment. S. typhimurium
has been previously reported as able to grown in the presence
of filamentous fungi (

Brandl et al., 2011

). The results showed that

co-culturing of P. lilacinus and S. typhimurium led to extracts with
acetylcholinesterase inhibitory activity except when the bacterium
was autoclaved prior its addition to the fermentative medium.
It was interesting to observe that inoculum amount altered the
biological activity, showing that the proportion of co-cultured
microorganisms influences the fungal secondary metabolism.

It has been noticed an increased production of fatty acids in

the extracts where P. lilacinus was grown on stressing conditions
in comparison to the control. Therefore, the FAME contents were
analyzed. The major unsaturated fatty acids detected in extracts of
P. lilacinus were the very important omegas 3 (

␣-linolenic acid),

6 (linoleic acid) and 9 (oleic acid) which produce reduction of
triglycerides and cholesterol LDL plasma levels. They also have
important roles in allergies and inflammatory processes, being
required for the formation of inflammatory prostaglandins, throm-
boxanes and leukotrienes (

Das 2006

).

Park et al. (2004)

also isolated

oleic, linoleic and linolenic acids from P. lilacinus cultures, grown in
PDB by seven days, corroborating the present work.

Upon addition of a 7 h old S. typhimurium culture deactivated by

autoclave, P. lilacinus yielded a novel compound that, by the best of
our knowledge, has not been obtained from another natural source
so far. This compound was named Paecilomide (1) and its structure
was fully determined by mono and bi dimensional NMR techniques
as composed by a pyridone ring attached to a 1,2-disubstituted
cyclohexane. NOESY experiments were crucial to determine spa-
tial correlations for this compound. Paecilomide can be classified
as a pyridone alkaloid and some compounds of this class have been
previously reported such as Militarinone A, a substituted polar rep-
resentative of this class (2,

Fig. 1

), isolated from P. militaris (

Schmidt

et al. 2002

). More recently, alkaloids from this class have also been

isolated from other fungal species (

Isaka et al. 2010

).

Paecilomide was only detected in the extract prepared in this

specific condition, showing that addition of bacterial genetic mate-
rial prior to the fungus inoculation does not furnish enough stress to
activate the biosynthesis of Paecilomide. The same occurred when
genetic bacterial material was added 8 days after fungal inocu-
lation. Therefore, although secondary metabolites biosynthesis is

described to start in the stationary phase of fungal growth (

Lucas

et al. 2007

), activation of certain biosynthetic routes seems to occur

early in the fermentation.

P. lilacinus extract showed higher activity than isolated com-

pound Paecilomide (1). This can be due to the presence of other
synergic active compounds in the extract since Paecilomide cor-
responds only to 6.3% of the secondary metabolites present in
the extract. According to

Adsersen et al. (2006)

, percentages of

inhibition greater than 50% can be considered of high acetyl-
cholinesterase inhibitory potential.

Conclusions

Production of secondary metabolites by fungi depends on the

biosynthetic capacity from microorganism and of the fermenta-
tion conditions. Thus, the manipulation of the fermentation process
parameters can alter the expression of the secondary metabolites
produced (

Bills et al. 2008

). Using this strategy, a novel compound

with acetylcholinesterase inhibition has been isolated from P. lilac-
inus, after selection of a favorable growth conditions among 14
conditions evaluated. The isolated compound showed capacity of
inhibiting AChE (57.5%) in the doses of 10 mg/mL. Further modu-
lation experiments can lead to culture media able to induce this
species to produce compounds with improved biological activities.

Acknowledgements

We thank Brazilian institutions Universidade Federal de Ouro

Preto, FAPEMIG and CNPq for financial resources and Chemistry
Department from Universidade Federal de Minas Gerais for pro-
viding facilities.

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