Microbiological Research 168 (2013) 204 210
Contents lists available at SciVerse ScienceDirect
Microbiological Research
journal homepage: www.elsevier.com/locate/micres
Paecilomide, a new acetylcholinesterase inhibitor from Paecilomyces lilacinus
Ana Paula C. Telesa,b, Jacqueline A. Takahashib,"
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 a b s t r a c t
Article history:
Fungi are some of the most important organisms in the production of bioactive secondary metabolites.
Received 2 August 2012
This success is related to the advances in biotechnology and also to the possibility of working with
Received in revised form 6 November 2012
techniques such as the  OSMAC (one strain-many compounds) to achieve different fungal secondary
Accepted 11 November 2012
metabolites profiles upon modifying the culturing conditions. Using this approach, the fungal species
Available online 6 December 2012
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.
Keywords:
S. typhimurium was added alive or after inactivation by autoclave or microwave irradiation in different
Paecilomyces lilacinus
stages of fungal growth. Extracts were prepared by liquid liquid extraction using ethyl acetate, a medium
OSMAC
polarity solvent in order to avoid extracting culturing media components. Production of fatty acids of rele-
Acetylcholinesterase inhibition
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 Ä… 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 Ä… 5.50% of acetylcholinesterase
inhibition.
© 2012 Elsevier GmbH. All rights reserved.
Introduction also been used in order to promote metabolic diversification in
fungi (Cueto et al. 2001; Wang et al. 2011; Huang et al. 2011).
Fungi are important organisms in the production of bioactive Many drugs currently in the market, possessing a variety
secondary metabolites. Around 38% of the active compounds iso- of activities such as antitumor, immunosuppressants, antibi-
lated until 2005 were of fungal origin (Bérdy 2005), and this context otics, hipocolesterolemic agents, antifungals, antiparasites, anti-
has not changed much in the late years. The success of fungal inflammatory and enzyme inhibitors, were obtained from fungal
metabolites can be attributed to many factors, like the advances metabolism (Bérdy 2005; Kingston 2011). Fungal metabolites have
in the industrial production of biotechnological metabolites and been shown their potential in the production of novel com-
the possibility of working with techniques such as the  OSMAC pounds (Zhang et al. 2011; Houghton et al. 2006) for treatment
(one strain-many compounds) (Bode et al. 2002). OSMAC is based of Alzheimer s disease, a progressive and irreversible neurodegen-
on the premise that a single fungal species, upon submission to erative disorder that leads to memory loss and cognitive disorders
different cultivation conditions, can produce a great diversity of (Lima et al. 2009).
new bioactive molecules. Among the parameters that can be varied The symptoms of Alzheimer s disease are connected to the
using OSMAC strategy, can be pointed the composition of cul- reduction of brain neurotransmitters, such as acetylcholine, nora-
ture medium, aeration, period of cultivation, pH, temperature and drenalin and serotonin (Bryne 1998). Therefore, the treatment
addition of agents to induce or inhibit the production of metabo- is based on the attempt to restore cholinergic function, using
lites (Saleem et al. 2009; Bugni and Ireland 2004). Some stressing inhibitors of acetylcholinesterase (AChE), an enzyme that acts on
factors such as high osmotic levels, addition of a competitive micro- acetylcholine degradation in the synaptic cleft (Lleo et al. 2006).
organism in the medium (co-culturing), and water restraint have 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
"
Corresponding author at: Departamento de Química, Universidade Federal de
quick and sensitive screening bioassays to be used in the evalu-
Minas Gerais, Av. Antonio Carlos, 6627, 31270-901 Belo Horizonte, MG, Brazil.
ation of acetylcholinesterase inhibitory effect caused by organic
Tel.: [phone]; fax: [phone].
compounds (Ellmann 1961; Rhee et al. 2001).
E-mail address: [e-mail] (J.A. Takahashi).
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
HO
H
OH O
N O
OH
N O
NH2
OH
12
Fig. 1. Structures of Militarinone A (1) and Huperzine A (2).
The fungal species used in work was Paecilomyces lilacinus. Pae- Fermentations conditions and extracts preparation
cilomyces genus is divided into two sections, section Paecilomyces
and section Isarioidea. The later contains mesophile members The experiments started with the inoculation of pre-inoculum
including P. lilacinus (Samson 1974). P. lilacinus 18S rRNA genes into 14 Erlenmeyer flasks containing liquid medium (PDB,
(rDNA) were sequenced and compared to other filamentous fungi 200 mL/flask), after which the contents were extensively homoge-
species showing to be far differentiated from Paecilomyces vari- nized. Each flask was prepared to install the fungal growth in an odd
otii, a representative species from Paecilomyces genus (Wu et al. condition on a medium containing Salmonella typhimurium in order
2003). Species from Paecilomyces genus are capable of producing a to furnish biotic stress. S. typhimurium was added to the flasks con-
variety of secondary metabolites of different chemical classes and taining P. lilacinus in two different concentrations (1 and 10 mL), in
with varied biological activities, such as cytotoxic, antibacterial, and three different forms (alive, after microwave irradiation and after
immunostimulating agents (Kyong et al. 2001; Isaka et al. 2007; Liu inactivation by autoclave) and in two different phases of P. lilaci-
et al. 2011; Xu et al. 2010). Neurotrophic pyridone alkaloids such nus development (1st and 8th day of fungal growth). P. lilacinus S.
ć%
as Militarenone A (1) have been reported from Paecilomyces mil- typhimurium were co-cultivated at room temperature (25 Ä… 3 C),
itaris (Schmidt et al. 2002). These compounds possess structural in static condition. Controls without addition of bacterial material
resemblance with Huperzine A (2), a potent acetylcholinesterase were run in parallel. After a period of 21 days, the growth of P.
inhibitor (Liu et al. 1986). Structures of compounds 1 and 2 can be lilacinus was interrupted by addition of EtOAc in the flasks. The
found in Fig. 1. culture media were individually filtered under vacuum through a
No reports on the AChE inhibitors biosynthesis have been filter paper, to separate the broths from the mycelia. The broths
described for P. lilacinus under natural fermentation conditions. were exhaustively extracted with ethyl acetate on a separator fun-
Therefore, OSMAC approach was exploited in order to create suit- nel. This procedure was repeated three times. The mycelia have
able stressing conditions with the aim of modulate the production also been extracted with ethyl acetate, and both extracts (broth and
of secondary metabolites with AChE inhibitory activity by P. lilaci- mycelium) obtained in each of the 14 co-culturing conditions were
nus. From the existing stressing factors, such as increase of osmotic combined, concentrated under vacuum and transferred to clean
and atmospheric pressure, and decrease of nutrients availability, in bottles.
this work, the stress was achieved by addition of bacterial genetic
material in the culturing medium used to grow P. lilacinus. This Gas chromatography (GC) analytical conditions
strategy has been successfully employed to induce the production
of bioactive secondary metabolites by other fungal species (Oh et al. GC analysis was carried out on an HP5890 Gas Chromatograph
2005; Du et al. 2011). Several extracts were prepared, assayed and equipped with Flame Ionization Detector (FID) to obtain the fatty
the most active extract for AChE inhibition was prepared in large acids profiles. A HP-INNOWax (HP) column (15 m × 0.25 mm) was
ć% ć%
sale. An active metabolite was isolated from this extract and identi- used at the following temperature gradient: 150 C, 1 min, 7 C/min
ć% ć%
fied. Fatty acids profiles were determined for all extracts obtained. until 240 C; injector (split of 1/50) at 250 C and detector at
ć%
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
Materials and methods comparison with SUPELCO37 fatty acid methyl esters (FAMEs) stan-
dard. The percentages of FAMEs were also compared with soybean
Source, maintenance and culturing conditions of the fungus P. oil (12.0 mg) hydrolyzed, methylated and analyzed under the same
lilacinus conditions.
P. lilacinus was isolated from soil and it is deposited in the Preparation of samples for GC analysis
micro-organisms collection of the Biotechnology and Bioassays
Laboratory (UFMG, MG, Brazil). P. lilacinus was maintained on All extracts and some fractions from extract 5 (10.0 mg) were
ć%
potato dextrose agar culture medium (PDA) on a refrigerator (8 C) dissolved, on a 2.0 mL cryogenic tube, in 100 L of a 1 mol/L potas-
(Schürmann et al. 2010). Prior to the experiments, the fungus was sium hydroxide solution (5%) in ethanol (95%). After vortex stirring
transferred to freshly prepared PDA and grown at room temper- for 10 s, the material was hydrolyzed on a microwave oven, during
ć%
ature (25 Ä… 3 C). For pre-inoculum preparation, P. lilacinus was 5 min. After cooling, 400 L of hydrochloric acid 20% (w/v), NaCl and
inoculated to Erlenmeyer flasks containing 200 mL of potato dex- 600 L of ethyl acetate were added, stirred by 10 s and let to stand
trose broth (PDB) and cultivated during seven days, under stirring for 5 min. An aliquot of 300 L of the organic layer was transferred
ć%
(150 rpm), at 25 Ä… 3 C. This procedure was performed to gener- to a tube and dried by evaporation, to obtain the free fatty acids
ate enough amount of biomass (pre-inoculum) to start the fungal (Segall et al. 2006). Free fatty acids were methylated with 100 L
ć%
cultivations. BF3/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 containing 0.1% of bovine serum albumin (BSA) fraction V; (III)
by gas chromatography. 50 mM Tris/HCl pH 8, containing 0.1 M of NaCl and 0.02 M of
MgCl2·6H2O; (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
Nuclear magnetic resonance (NMR) conditions
enzyme was dissolved in buffer solution I to make a 1000 U/mL
1 13 1
stock solution.
The spectra of H and C NMR, sub spectra DEPT 135 and H 1H
1 1 1
COSY, H 1H NOESY, H 13C HMBC, and H 13C HSQC experi-
ments were obtained on BRUKER AVANCE DPX200 and DRX400
Thin layer chromatography (TLC) assay
spectrometers at the High Resolution Magnetic Resonance Labo-
ratory (LAREMAR) from the Chemistry Department of the Federal
The qualitative evaluation of the inhibitory activity of the
University of Minas Gerais (MG, Brazil). Spectra were measured at
enzyme acetylcholinesterase was performed according to the
400 (100) or 200 (50) MHz at 300 K, and the spectrometers were
enzymatic assay (Ellmann 1961; Rhee et al. 2001), with minor mod-
1
equipped with inverse detection 5 mm multinuclear head H/13C.
ifications. The extracts to be assayed were prepared (10 mg/mL) by
The compound was dissolved in piridine-d5, and transferred to a
dissolution of the extracts on a suitable solvent and applied (5.0 L)
5 mm o.d. NMR tube. Chemical shifts (1
) were registered in ppm.
in ready made TLC plates (0.2 mm thickness, Merck). The TLC plates
Tetramethylsilane (TMS) was used as an internal reference stan- were developed and, after eluent evaporation, they were sprayed
dard to chemical shift 1
0. Bi-dimensional (2D) NMR spectra were
with solutions VI and VII. After 5 min, the TLC plates were sprayed
acquired under standard conditions. Data processing was carried
with acetylcholinesterase solution (3 U/mL). After 10 min, a yel-
out on SGI workstation using the Bruker (DRX 400) software.
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
Electrospray ionization mass spectroscopy (MS-ESI)
control.
High resolution mass spectra were obtained on a Bruker
Daltonics Mass Spectrometer, model MicroTOF, equipped with
Microplates assay
electrospray ionization source (Chemistry Institute, USP, SP, Brazil).
Solutions of the compound in methanol/water was prepared and
To quantify the extension of acetylcholinesterase inhibition, a
analyzed in the zone of m/z between 200 and 500 Da.
further bioassay was conducted in 96 well microplates, and inhibi-
tions were quantitatively determined on a Thermo Plate microplate
Evaluation of acetylcholinesterase inhibitory activity reader, using absorbance of 406 nm, based on the Ellmann s method
(1961). There were tested all extracts, controls and compound 1.
Bioassay for acetylcholinesterase inhibition was carried out In this assay, acetylcholinesterase hydrolyzes the substrate, gener-
with Sigma reagents: albumin bovine serum; 5,5 -dithiobis(2- ating thiocholine as the product. The later reacts with Ellmann s
nitro-benzoic acid) (DTNB); Tris/HCl 0,1 M pH 8; acetylthiocholine reagent, producing 2-nitrobenzoate-5-mercaptothiocolin and 5-
iodide (ATCI); acetylcholinesterase from electric eel, type V-S. The thio-2-nitrobenzoate, which can be detected and quantified at
following standard solutions I VII were prepared to be used in 406 nm. In each microplate wells, there were added 25 L of stan-
the bioassay: (I) 50 mM Tris/HCl pH 8; (II) 50 mM Tris/HCl pH 8, 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
Fig. 3. TLC profile of Paecilomide (1) sprayed with Dragendorff s reagent.
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
the FAME profiles. Analysis of the FAMEs chromatograms obtained
P18, eluted by ethyl acetate:methanol (95:5). Extract 5 and com-
revealed that these fractions were mainly constituted by a mix-
pound 1 showed positive reaction when sprayed with Dragendorff s
ture of palmitic (C16:0), stearic (C18:0), oleic (C18:1 9 cis), linoleic
reagent (Wagner and Bladt 2001).
(C18:2 9,12 cis) and linolenic acids (C18:3) (Table 1). Fig. 2 presents
the FAME chromatogram of fraction P03, as an example of the FAME
Results profile.
Fractions P12 (250.0 mg), P13 (13.0 mg) and P14 (93.0 mg) were
1
In this work, production of secondary metabolites active for characterized as waxes of yellow color and analyzed by H and
13 1
AChE inhibition by the fungus P. lilacinus was promoted by addi- C NMR and DEPT. In the spectrum of NMR of H there were
tion bacterial genetic material to the liquid medium used to grow observed signals between 1
H 1.27 and 1.31 ppm, assigned to hydro-
P. lilacinus. Modulation of secondary metabolites production by gen atoms of methylene groups and signals between 1
H 0.8 and
13
co-culturing of P. lilacinus and S. typhimurium was monitored in 0.9 ppm, assigned to hydrogen atoms of methyl groups. In the C
fourteen different fermentation conditions. The respective crude NMR spectra there were observed signals at 1
C 173.4 176.3 ppm,
extracts were obtained by using liquid liquid extraction in ethyl corresponding to carboxyl carbons, at 1
C 22.9 32.14 ppm (methy-
acetate. This medium polarity solvent is widely used for fungal lene carbons signals) and, at 1
C 14.3 ppm, which are classical signals
extracts preparation since it is able to extract most non-polar of terminal methyl groups. These fractions were methylated and
secondary metabolites, avoiding extracting polar culturing media analyzed by GC, revealing to have the same FAMEs profiles observed
components such as proteins. The extracts were qualitative and for the less polar fractions.
quantitatively evaluated for acetylcholinesterase inhibition. The Fraction P18 gave a pure compound (69.0 mg; 6.3% yield) that
qualitative evaluation of AChE inhibition was verified by forma- was obtained as a yellow wax; on TLC analysis, this compound gave
tion of white halos in the TLC plates assay. Of the fourteen extracts positive result after sprayed with Dragendorff s reagent (Fig. 3), a
tested, ten (71%) produced white halos in the plate, indicating AChE very useful reagent used to identify alkaloids (Wagner and Bladt
inhibition. In relation to the amount of fungal inoculum (1 or 10 mL) 2001). Therefore, it was inferred the presence of nitrogen in the
added in the experiments, it has been observed that addition of molecule. In the high resolution mass spectrum of this compound,
smaller quantity of the inoculum (1 mL) generated an active extract, it was observed a peak with m/z 261.1284, compatible with the
while addition of a large volume of inoculum (10 mL) led to an sodium adduct [238+Na+] of a compound with molecular formula
13
inactive extract. C12H15NO4. Inthe C NMR spectrum, there were observed 12 sig-
Since AChE inhibition test in TLC is only a qualitative evaluation, nals, four of them did not appear in DEPT sub-spectrum, showing
Ellmann s methodology was used to quantify the AChE inhibition that there were four non-hydrogenated carbons in the molecule:
presented by the extracts obtained in this study. By this methodol- a carbonyl from an amide group at 1
C 165.8 ppm (Silverstein et al.
ogy, it was found that the extract most able (91 Ä… 2.91% inhibition) 1991), a carboxylic group (1
C 169.8) and other two quaternary car-
to inhibit that enzyme was the one resulting addition of 1 mL of the bons that showed resonances at 1
H 127.7 ppm and 1
C 157.8 ppm.
bacterial inoculum (deactivated by autoclave) in PDB, 7 h after fun- From the eight hydrogenated carbon atoms, four showed signs
gal inoculation that was grown under static condition. This extract between 1
22.5 ppm and 1
45.3 ppm, being characterized as methy-
was prepared again, with the same conditions in large scale and lene carbons. Signs of four methine groups were observed at 1
C 57.0,
fractionated by chromatography column, obtaining a total of 32 59.4 ppm, 116.2 and 1
131.3 ppm. Structure elucidation of this com-
fractions (P01 P32). pound was accomplished by careful examination of data obtained
1
The less polar fractions P01 P06 (eluted with n-hexane) pre- in the bidimensional NMR contour maps HSQC, H 1H COSY and
sented an oily aspect, typical of fatty acids. Since FAME profiles HMBC (Table 2).
are important to categorize fungi, the fractions containing fatty Correlations between carbon and hydrogen atoms participat-
acids were hydrolyzed, methylated and analyzed by GC, to obtain 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 (C16:0) Stearic (C18:0) Oleic (C18:1 9.cis) Linoleic (C18:2 9,12 cis) Linolenic (C18:3)
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.
OH
H
1
COOH
3' H
3
H
H
2
2' H
H
4'
4
2
H
7
4
H
3
COOH
5' OH
5
1'
N O
H H
6 R
7
H
6
H H
A B
5
H
H
H
1 N O
H
H-1H COSY Correlations
H
R =
1
Fig. 4. Fragments proposed based in the H 1H COSY correlations.
Fig. 5. Spatial correlations of Paecilomide (1) based in NOESY correlations map.
connectivity between the carbons of the molecule and the neigh- both H-6 and H-5 as well as between H-7 and H-5 , according to
Fig. 6 (A and B, respectively).
bor hydrogen were determined by HMBC contour map. Afterwards,
1
analysis of H 1H COSY spectrum allowed correlating the hydro- Table 2 presents the complete NMR assignment of hydrogens
and carbons of Paecilomide (1), as well as a list of bi-dimensional
gens couplings. Two fragments A and B were established by this
NMR correlations found for atoms of this molecule. Paecilomide
analysis as shown in Fig. 4.
(1) was tested in the AChE inhibition bioassays, giving a positive
The fragment B corresponds to a 1
-lactam and correlates to the
fragment A as observed in the HMBC contour map. Further correla- spot (white halo) when assayed by the TLC methodology. In the
1
AChE microplates assay, P. lilacinus extract showed 91.0 Ä… 2.91%
tions observed in the H 1H COSY contour map, led to the proposal
of acetylcholinesterase inhibition while Paecilomide (1) presented
of a bicycle molecule, by bonding both fragments A and B linked
57.5 Ä… 5.50% of inhibition. The results of the inhibitory potential of
through carbons 7 and 4 .
The most favorable conformation of 1,2-disubstituted a cyclo- the original P. lilacinus extract compared to activity of 1 are shown
in Table 3.
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 Discussion
between H-4 and H-5 , H-3 and H-2 , H-6 and H-7 , as shown
in Fig. 5. The presence of bacterial material in the medium furnished a
Due to possibility of free rotation of the C7 C4 sigma bond, it stressing condition able to promote the biosynthesis of novel bioac-
was also observed correlations in the NOESY contour map between tive metabolites. The bacterium used was S. typhimurium, a suitable
Table 2
1 13 1
H and CNMR, H 1H COSY and HMBC correlations for Paecilomide (1).
Carbon Number Type 1
C (ppm) 1
H (ppm) COSY HMBC
1 COOH 169.8
2 CH 59.4 4.15 H2 × H3b H2 × C3; C1
1
4.20
3CH2 29.5 H3a  1.90 H3a × H3b H3a × C2, C1
H3b  2.20 1
2.20
4CH2 22.5 H4a  0.80 H4a × C5
H4b  1.60
H5a  3.40 H5a × H4b
5 CH2 45.3 H5b × C4
1
3.40
H5b  3.70 H5b × H4b
1
3.65
6CH2 36.2 H6a  3.30 H6a × C7, C3 , C5
H6b  3.60 H6b × C5
7 CH 57.0 4.60 H7 × H6a;
H7 × H6b
1
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
described to start in the stationary phase of fungal growth (Lucas
Inhibition (%) of acetylcholinesterase by P. lilacinus extract and Paecilomide (1) in
et al. 2007), activation of certain biosynthetic routes seems to occur
microplate by Ellmann s methodology.
early in the fermentation.
Sample % inhibition (mean Ä… sd)
P. lilacinus extract showed higher activity than isolated com-
pound Paecilomide (1). This can be due to the presence of other
P. lilacinus extract 91 Ä… 2.91
Paecilomide 57.5 Ä… 5.50
synergic active compounds in the extract since Paecilomide cor-
Positive control (Eserine) 98.0 Ä… 1.66
responds only to 6.3% of the secondary metabolites present in
Negative control 0 Ä… 0
the extract. According to Adsersen et al. (2006), percentages of
sd: standard deviation.
inhibition greater than 50% can be considered of high acetyl-
cholinesterase inhibitory potential.
(A) (B)
Conclusions
H H
OH
H H
H
H
H
H
H 6 Production of secondary metabolites by fungi depends on the
5' 3'
H
H
biosynthetic capacity from microorganism and of the fermenta-
H 2'
H 7
7 NH
4'
4'
tion conditions. Thus, the manipulation of the fermentation process
H
H
parameters can alter the expression of the secondary metabolites
H
H
H 5'
H 3' 1'
H 1'
H N O
O
produced (Bills et al. 2008). Using this strategy, a novel compound
H
HO 2' H
with acetylcholinesterase inhibition has been isolated from P. lilac-
inus, after selection of a favorable growth conditions among 14
1
conditions evaluated. The isolated compound showed capacity of
inhibiting AChE (57.5%) in the doses of 10 mg/mL. Further modu-
Fig. 6. Spatial correlations to Paecilomide (1) based in NOESY data. (A) and (B)
represent two possible rotational forms of Paecilomide (1).
lation experiments can lead to culture media able to induce this
species to produce compounds with improved biological activities.
species to be used for this purpose that does not produce secondary
metabolites able to interfere in the experiment. S. typhimurium Acknowledgements
has been previously reported as able to grown in the presence
of filamentous fungi (Brandl et al., 2011). The results showed that We thank Brazilian institutions Universidade Federal de Ouro
co-culturing of P. lilacinus and S. typhimurium led to extracts with Preto, FAPEMIG and CNPq for financial resources and Chemistry
acetylcholinesterase inhibitory activity except when the bacterium Department from Universidade Federal de Minas Gerais for pro-
was autoclaved prior its addition to the fermentative medium. viding facilities.
It was interesting to observe that inoculum amount altered the
biological activity, showing that the proportion of co-cultured
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