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African Journal of Pharmacy and Pharmacology Vol. 5(2). pp. 281-288, February 2011
Available online http://www.academicjournals.org/ajpp
ISSN 1996-0816 ©2011 Academic Journals
Full Length Research Paper
Antibacterial and cytotoxic activity from basidiocarp
extracts of the edible mushroom Lactarius indigo
(Schw.) Fr. (Russulaceae)
Alejandra Ochoa-Zarzosa1, Ma. Soledad Vázquez-Garcidueñas2, Virginia A. Robinson-
Fuentes2 and Gerardo Vázquez-Marrufo1*
1Centro Multidisciplinario de Estudios en Biotecnología, Facultad de Medicina Veterinaria y Zootecnia.
2División de Estudios de Posgrado, Facultad de Ciencias Médicas y Biológicas “Dr. Ignacio Chávez”, Universidad
Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, México.
Accepted 18 February, 2011
Aqueous and organic basidiocarp extracts of the edible mushroom Lactarius indigo were evaluated for
their antibacterial and cytotoxic effects. 10, 20 and 30 mg of organic extracts were tested against
diarrheagenic Escherichia coli strains (EIEC, EPEC, ETEC-LT and ETEC-ST), Pseudomonas aeruginosa,
Enterobacter cloacae, Staphylococcus aureus and Salmonella enterica. 10 mg of hexane extract
showed activity against ETEC-LT (18.8 mm zone of inhibition) and P. aeruginosa (10.5 mm). All levels of
the ethyl acetate extract inhibited all the strains, with stronger activity against EIEC (19.0 mm) and P.
aeruginosa (21.0 mm) at 30 mg. Methanol extract inhibited all bacterial growth, but E. cloacae. 100 µg/ml
of aqueous extract showed antiproliferative activity against MCF7 cells, but not on HeLa, A549 and
normal bovine mammary epithelium cells. Methanol and ethyl acetate extracts inhibited proliferation of
HeLa cells (50 to 1000 ng/ml) but increased proliferation of A549 (100 ng/ml), as did methanol extract
(500 ng/ml). Methanol extract did not inhibit normal rabbit serum fibroblast cells, while hexane and ethyl
acetate extracts showed an inhibitory effect with 50 and 100 ng/ml, respectively, less than in
proliferation of HeLa cells. These results show that L. indigo basidiocarps contain substances with
antibacterial and cytotoxic activities.
Key words: Lactarius indigo extracts, antibacterial activity, cytotoxic activity.
INTRODUCTION
Fungi from the division Basidiomycota have been widely
studied as an alternative source of metabolites with
pharmacological properties, including anticancerigenous,
antitumor, immunomodulating, antibacterial and cytotoxic
activities (Wasser, 2002; Daba and Ezeronye, 2003; Fan
et al., 2006; Borchers et al., 2008). Antibiotic resistance
of human pathogenic bacteria has become a major
worldwide public health concern (Finch, 2002; Harbarth
and Samore, 2005), this is why the search for new
substances with antimicrobial activity is a priority
(Livermore, 2005). Antimicrobial activity has already been
documented in extracts from the mycelium (Suay et al.,
2000) and fruiting bodies (Zjawioney, 2004) of differ-
*Corresponding author. E-mail: gvazque-zmarrufo@yahoo.com.
mx. Tel/Fax: + 52 443 295 80 29.
rent wild species from Basidiomycota.
Another worldwide public health problem is cancer,
given that it is estimated that approximately 25 million
people suffer one of its different manifestations and 10
million new cases are annually reported (WHO, 2002); for
that reason, there is an increasing demand for more
effective anticancerigenous substances and therapies
(Lord and Ashworth, 2010). In that regard, several
studies have reported cytotoxic activity against cancer
cells of organic extracts of spores (Fukuzawa et al.,
2008), vegetative mycelium (Hu et al., 2002; Choi et al.,
2004) and basidiocarps (Takaku et al., 2001; Hu et al.,
2002) from several species of Basidiomycota. The genus
Lactarius (Russulaceae) includes species reported as
edible in different parts of the world (Boa, 2004) and
reports exist of the antimicrobial activity of methanol
extracts of Lactarius deterrimus, Lactarius sanguifluus,
Lactarius semisanguifluus, Lactarius piperatus, Lactarius
282 Afr. J. Pharm. Pharmacol.
deliciosus and Lactarius salmonicolor (Dulger et al.,
2002; Barros et al., 2007a, b). In addition, the organic
extracts of some Lactarius species have shown immuno-
modulating, cytotoxic, antiviral and antigenotoxic
activities (Krawczyk et al., 2003, 2005, 2006; Mlinaric et
al., 2004).
Lactarius indigo (Schw.) Fr. is an edible mushroom that
distributed from East Asia (China and Japan) (Wu and
Mueller, 1997) to Northeastern and Central America
(Hesler and Smith, 1979; Hutchinson, 1991; Mueller and
Halling, 1995; Montoya and Bandala, 1996; Wu and
Mueller, 1997). In México, L. indigo is associated to
diverse plant communities (Montoya and Bandala, 1996)
and is highly valued as food (Boa, 2004; Pérez et al.,
2006), being sold in local markets (Montoya et al., 2001;
Martínez-Carrera et al., 2005). It is known by the Spanish
common names indigo, hongo azul (blue mushroom) and
the combined Spanish-Nahuatl names tecax azul (blue
tecax) and tecosan morado (purple tecosan) (Montoya et
al., 2001, 2003).
A single report is known about the nutritional value of L.
indigo (León-Guzmán et al., 1997); however, there is not
a single report regarding the pharmacological properties
of this species. In the present work, the antibacterial and
cytotoxic activities of aqueous and organic extracts of L.
indigo are evaluated for the first time and the results are
compared with those from similar reports for other
species of Basidiomycota, mainly for the genus Lactarius.
MATERIALS AND METHODS
Basidiocarp collection
Basidiocarps of L. indigo were collected in the Parque Nacional
Insurgente José María Morelos in the municipality of Charo, state of
Michoacán, México (19° 39.918’ N, 101° 00.450’ W) on September
2006, and were authenticated by M.Sc. Marlene Gómez Peralta,
curator of the herbarium of the Facultad de Biología, Universidad
Michoacana de San Nicolás de Hidalgo, where a voucher specimen
(KM03) was deposited. The basidiocarps were frozen at -80°C the
same day of collection and freeze-dried after 24 h. The freeze-dried
material was preserved in the dark at 4°C in a container with silica
gel until processed.
Test microorganisms
Strains of Escherichia coli EPEC, ETEC, and EIEC pathotypes
used for the antibacterial activity assays were purchased from the
Institute of Diagnostic and Epidemic Reference of México (InDRE,
Table 1). The Children’s Hospital Eva Sámano de López Mateos of
Morelia, Michoacán, Mexico, donated the tested strains of Pseudo-
monas aeruginosa, Enterobacter cloacae, and Salmonella enterica
subsp. enterica. The tested strain of Staphylococcus aureus was
ATCC27543.
Cell lines
Several cell lines corresponding to human cancers were used for
the cytotoxicity assays: HeLa (cervicouterine cancer), MCF7 (breast
cancer) and A549 (lung cancer). As controls for evaluating the
effect on normal cells, bovine mammary epithelial cells (BME) were
used for assays with aqueous extracts, and rabbit skin fibroblasts
(RSF) for assays with organic extracts. The latter two cell lines were
generated in our laboratory. All cell lines were kept in Dulbecco’s
modified Eagle’s medium (DMEM)-F12K (1:1, Sigma, USA)
supplemented with 30 mM NaHCO3, 15% fetal bovine serum
(Gibco, USA), 50 U/ml of penicillin and 50 U/ml of streptomycin
(Gibco, USA). Cells were cultured at 37°C in an atmosphere with
5% CO2 and 95% humidity.
Extract preparation
For obtaining the aqueous extract 100 g (dry weight) of basidiocarp
mass was boiled in 700 ml of deionized water for 60 min,
continuously replacing the evaporated water during the extraction
time. After extraction was completed the residual biomass was
eliminated by filtration through sterile gauze. The recovered filtrate
was lyophilized and resuspended in deionized water at a
concentration of 500 mg/ml. The resulting solution of aqueous
extract was sterilized by filtering through a 0.25 µm pore size
membrane (Millipore, USA) and stored at 4°C.
Organic extracts were prepared by crushing 50 g of freeze-dried
basidiocarp until a fine powder was obtained; the resulting material
was successively extracted with 100 ml of each hexane, ethyl
acetate, and methanol during 72 h. The extracts were later
centrifuged at 1500 x g at room temperature and filtered. The
solvents were eliminated by rotoevaporation until dryness and the
dry extracts were stored at 4°C. Solutions for the bioassays were
prepared by resuspending the dry extracts in the same solvent used
for the extraction at a concentration of 500 mg/ml.
Bioassays
Antimicrobial activity by disk diffusion method
The antimicrobial assays were performed in vitro using the agar-
disk diffusion method (National Committee for Clinical Laboratory
Standards, 1993). Petri dishes with Mueller-Hinton Agar (Oxoid,
USA) were inoculated massively with a 200 µl suspension of each
of the bacterial cultures and grown to their mid-log phase, after
which the plate’s surfaces were air-dried. Filter paper sensidisks (6
mm in diameter) were impregnated with the necessary volume of
each of the extracts in order to reach the final levels of 10, 20, and
30 mg/disk. The impregnated sensidisks were air-dried before being
placed on the Petri dishes with the test microorganisms. The plates
were incubated for 24 h at 37°C and the inhibition areas were
measured in mm using a digital caliper (precision of 0.01 mm,
Model CD-6” C, Mitutoyo Corp., Japan). Controls consisted of
sensidisks impregnated with the corresponding pure solvent and
air-dried. The inhibition diameter of the control disk was subtracted
from the inhibition diameter resulting from the application of the
corresponding extracts of L. indigo. In some tests, control
sensidisks with ampicillin (AM) and carbenicillin (CB) (BioRad,
USA) were used (Table 1). All assays were performed in triplicate
and the results were reported as mean ± standard deviation (SD).
Cytotoxicity assays
For cytotoxicity assays four different concentrations were used of
the organic extracts and the same volume of the corresponding
solvent as a control. All tested cell lines were cultivated to
confluence as described above and were detached from the culture
plate using PBS saline solution at pH 7.4 supplemented with
antibiotics and 0.25% trypsin-EDTA (w/v, Sigma, USA) and agitated
for 10 min. Afterwards, 1 x 106 cells were seeded in OPTIMEM
(Gibco, USA) growth medium without serum or antibiotics in 96-well
microtitration plates. To these cultures the corresponding extract
was added incubating the treated plates during 24 to 48 h at 37°C.
After incubation, 10 µl of a 5 mg/ml solution of tetrazolium bromide
3-(4,5-dimetil-2-tiazol)-2,5-diphenil-2H-tetrazolium salt (MTT,
Sigma, USA) were added and further incubated for 4 h at 37°C.
Finally, 100 µl of 10% SDS were added in order to dissolve the
formazan crystals and the viability of cells was determined by
measuring the reduction of MTT at 595 nm in a microplate reader
(Bio-Rad, USA). Each assay was carried out at least in sextuplicate.
Statistical analysis
In order to evaluate the effect of the extract concentration on the
different studied strains and cell lines, the results were analyzed by
one-way analysis of variance (ANOVA) and Tukey tests (p<0.05),
using StatistiXL ver. 1.8.
RESULTS
Antibacterial activity
The controls showed that hexane alone did not cause
any inhibition to the tested strains whereas the ethyl
acetate and methanol caused a minimal inhibition that
was subtracted as described in Materials and Methods.
The hexane extract caused inhibition of P. aeruginosa
and ETEC-LT (Table 2). For the ETEC-LT strain, 10 and
20 mg of hexane extract did not show significant
differences in the inhibition zone diameter but 30 mg
caused a significantly larger inhibition zone diameter than
that for lower amounts. For P. aeruginosa, the maximum
inhibition was observed at 20 mg of hexane extract, since
no significant differences could be observed with 20 and
30 mg of the extract (Table 2). The compounds present in
the hexane extract had a greater effect on the ETEC-LT
strain than on P. aeruginosa. The inhibition caused by the
hexane extract is comparable with that caused by the
standard antibiotics AM and CB (Table 1).
The ethyl acetate extract inhibited all tested strains
(Table 2). In all strains, except for ETEC-LT and S.
enterica, an apparent dose-response effect could be
observed, since the inhibition increased in relation to the
applied level of ethyl acetate extract. No significant
differences could be observed in E. cloacae and S.
aureus when applying the two lower amounts of the
extract. At 10 mg of ethyl acetate extract, no differences
could be observed in the inhibition of any of the tested
microorganisms. However, at 20 and 30 mg of ethyl
acetate extract, significant differences were observed in
the inhibition of P. aeruginosa, EIEC, ETC-ST, and EPEC
(Table 2). At the highest level, all strains showed different
susceptibilities to the ethyl acetate extract, except for E.
cloacae and S. aureus. The inhibition of the different E.
coli pathotypes caused by 30 mg of the ethyl acetate
extract was comparable to that caused by AM and CB,
except for strain ETEC-LT (Table 1).
Zarzosa et al. 283
The methanol extract caused inhibition in all bacteria
tested. Only E. cloacae did not show susceptibility at 10
mg (Table 2). At this level, S. enterica and P. aeruginosa
were much more susceptible than the other tested
bacteria. With 20 mg of the methanol extract, the
inhibitory effect was greater in the ETEC-LT and EIEC
strains, whereas EPEC was less susceptible. Even
though the inhibition effect caused by the methanol
extract differed little from that caused by the other tested
extracts, the differences were statistically significant.
Using 30 mg of methanol extract, EIEC and E. cloacae
exhibited the greatest inhibition, followed by ETEC-ST
and S. aureus (Table 2). The increase in level of the
methanol extract had different effects among strains. The
strains EIEC, EPEC, E. cloacae, and S. aureus displayed
an apparent dose-response effect, with an increase in the
inhibition zone diameter corresponding to the increment
in amount of extract. ETEC and P. aeruginosa increased
their inhibition zone diameter as the level of methanol
extract augmented from 10 to 20 mg, but their inhibition
zone did not increase when the level was raised to 30 mg
of the extract. Finally, S. enterica showed no variation in
its inhibition zone diameter as the extract level increased.
The different E. coli pathotypes showed inhibition zone
diameters comparable to those caused by AM and CB,
except from ETEC-LT (Table 1).
Cytotoxic activity
The aqueous extract of L. indigo basidiocarp had different
effects on the various cell lines tested. No antiproliferative
effect of the aqueous extract was observed against the
A549 and MCF7 cell lines, but it significantly inhibited the
proliferation of HeLa cells at a concentration of 100 µg/ml
(Figure 1). In the case of primary BME cells, a significant
stimulation of prolife-ration was observed at a concen-
tration of 10 µg/ml and no effect was observed at any
other tested concen-tration.
The organic extracts of L. indigo also showed
contrasting effects between studied cell lines. Thus, in
normal RSF cells only the hexane extract showed an
inhibitory effect of proliferation relative to the control at all
concentrations tested (Figure 2), although the maximum
inhibition value was only of 25.63% compared to the
control at the maximum concentration of 1000 ng/ml. The
ethyl acetate extract showed significant inhibitory values
at the concentrations of 100, 500 and 1000 ng/ml.
Nevertheless, the maximum inhibition percentage was of
only 20.80% relative to the control. The methanolic
extract did not alter the capacity for proliferation of
fibroblasts at any concentration used (Figure 2). In the
case of HeLa cells, the three organic extracts had a
significant inhibitory effect on proliferation at all
concentrations evaluated. Using ethyl acetate extract, the
maximum inhibition was observed at a concentration of
100 ng/ml with a value of 95.74%, while for the metha-
284 Afr. J. Pharm. Pharmacol.
Table 1. Reference strains and clinical isolates used in the present study.
Strain Pathotype/comment Code AM
a
CB
a
E. coli Enterotoxigenic with heat-labile toxin (ETEC-LT) H10407 18.0 17.0
Enterotoxaemia with heat-stable toxin (ETEC-ST) 25611 16.0 18.0
Enteroinvasive (EIEC) E11 20.0 12.0
Enteropathogenic (EPEC) O111 15.0 12.0
S. aureus isolated from bovine mastitis ATCC27543 nt nt
P. aeruginosa clinical isolate VGPM01 nt nt
E. cloacae clinical isolate VGEM17 nt nt
S. enterica subsp. enterica clinical isolate VGSM33 nt nt
aInhibition zone diameter (mm) caused by 10 µg of ampicillin (AM) and 100 µg of carbenicillin (CB), nt, not tested.
Table 2. Antibacterial activity of the organic extracts of L. indigo*.
Bacteria
mg/disk of each extract
Hexane Ethyl acetate Methanol
10 20 30 10 20 30 10 20 30
E. coli
EIEC - - - 9.6 (0.58)1,a 14.0 (0.73)1,b 19.0 (0.58)1,c 11.3 (0.53)1,a 16.0 (0.31)1, b 20.6 (0.31)1,c
EPEC - - - 9.3 (0.52)1,a 12.0 (0.50)2,3,b 15.3 (0.15)2,c 11.0 (0.78)1,a 12.3 (0.22)2,b 14.3 (0.58)2,5,c
ETEC-LT 18.8 (0.73)1,a 18.6 (0.30)1,a 21.0 (0.80)1,b 10.5 (0.73)1,a 11.3 (0.53)3,5,a 12.0 (0.78)3,a 11.6 (0.15)1,a 17.0 (0.73)1,b 17.6 (0.15)3,b
ETEC-ST - - - 10.0 (0.19)1,a 13.0 (0.75)1,2,b 17.1 (0.53)4,c 10.7 (0.58)1,a 15.0 (0.31)3,b 16.0 (0.73)3,4,b
P. aeruginosa 10.5 (0.51)2,a 11.2 (0.75)2,ab 12.1 (0.52)2,b 9.6 (0.33)1,a 17.2 (0.25)4,b 21.0 (0.65)5,c 13.3 (0.53)2,a 15.1 (0.53)3,b 15.3 (0.15)4,b
E. cloacae - - - 9.7 (0.36)1,a 10.1 (0.75)5,a 13.5 (0.17)6,b - 13.6 (0.15)4,a 19.0 (0.73)1,b
S. aureus - - - 10.1 (0.29)1,a 11.3 (0.65)3,5,a 13.0 (0.73)3, 6,b 9.6 (0.18)3, a 14.0 (0.78)3,4,b 16.3 (0.29)3,c
S. enterica - - - 9.7 (0.75)1,a 11.0 (0.58)3,5,a 11.0 (0.59)7,a 13.2 (0.38)2,a 13.7 (0.17)4,a 13.6 (0.15)5,a
*The mean radius of the inhibition zone of three independent experiments is shown in mm (±SD). Values in the same row with the same superscript letter have non-significant differences
between them. Values in the same column with the same superscript number have non-significant differences between them. For both rows and columns, the significance test was
evaluated at p < 0.05. This significance analysis was made only for results within the same kind of extract for all levels tested.
-, no inhibition zone.
nolic extract the percentage inhibition value at the
same concentration was of 67.61%. The hexane
extract caused maximum inhibitory activity
(76.51%) at 500 ng/ml. MCF7 cells showed a
significant stimulation of proliferation at different
concentrations of hexane and methanol extracts
(Figure 2), the response was higher for the
hexane extract at a concentration of 100 ng/ml
with a value of 98.08%.
DISCUSSION
The present work shows that organic extracts
from L. indigo are active against E. coli
diarrheagenic strains and other bacteria that are
pathogenic to humans. This is the first study in
which basidiocarps of the genus Lactarius are
tested against different E. coli diarrheagenic
strains. Some of the tested strains showed
significant differences in their responses to the
extracts. The ETEC-LT pathotype was susceptible
Zarzosa et al. 285
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
0. 00 1.00 5.00 10.00 50.00 100.00
Optical density
Extract concentration (
µ
µµ
µ
g/ml)
BME HeLa A549 MCF7
Figure 1. Effect of aqueous extract of L. indigo basidiocarp on proliferation of the four cell types evaluated. Bars represent the
mean of sextuplicates with its relative standard error. Asterisks indicate extract doses having significant differences relative to
the control (deionized water).
to the hexane extract whereas the EIEC pathotype was
more susceptible to the ethyl acetate and methanol
extracts. Our results agree with those reported by Dulger
et al. (2002), who documented the activity against E. coli
of methanol extracts of different species of Lactarius,
although they used a non-pathogenic strain. There are
some reports about the effect of methanol extracts of
Lactarius deliciosus on E. coli, with very contrasting
results. On one side, extracts of L. deliciosus collected in
Portugal had no activity against non-diarrheagenic E. coli
(Barros et al., 2007a, b); on the other extreme,
basidiocarp extracts of the same species collected in
Turkey gave positive inhibitory results against a different
strain of non-diarrheagenic E. coli (Dulger et al., 2002).
Methanolic extracts of Lactarius piperatus inhibited
growth of E. coli, but its activity was dependent on the
maturity of the basidiocarp used to make the extracts
(Barros et al., 2007b). These results may be due
to differences in the susceptibilities of the different strains
tested, although differences in the chemical composition
of the basidiocarps and in the extraction methods cannot
be discarded. In relation to the other microorganisms
tested, the three extracts showed strong activity against
P. aeruginosa and only the methanol extract exhibited
activity against E. cloacae and S. aureus. These results
agree with other reports stating that P. aeruginosa and S.
aureus can be inhibited by methanol extracts of
basidiocarps of species in the genus Lactarius (Dulger et
al., 2002; Barros et al., 2007a, b). However, the inhibition
activity of the methanol extract of L. indigo on S. aureus
that was observed in the present work seems to be
greater than the activity previously reported for methanol
extracts of other species in the same genus (Dulger et
al., 2002). No previous reports were found about the
inhibition of E. cloacae using extracts of species of the
genus Lactarius.
286 Afr. J. Pharm. Pharmacol.
0.00
0.04
0.08
0.12
0.16
0.20
0 50 100 500 1000
Optical density
Extract concentration (ng/ml)
HX EA MeOH
*
*
*
*
*
*
*
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0 50 100 500 1000
Optical density
Extract concentration (ng/ml)
*
*
*
*
*
*
*
*
*
*
*
*
0.00
0.05
0.10
0.15
0.20
0.25
0 50 100 500 1000
Optical density
Extract concentration (ng/ml)
*
*
Figure 2. Effect of hexane (HX), ethyl acetate (EA) and methanolic
(MeOH) extracts of L. indigo basidiocarp on the proliferation of
normal RSF (upper panel), HeLa (middle panel) and MCF7 (bottom
panel) cell lines. Bars represent the mean of sextuplicates with its
relative standard error. Asterisks indicate extract doses with
significant differences relative to the control (pure solvent).
The inhibition caused by the ethyl acetate and
methanol extracts was observed in practically all tested
strains. Therefore, it can be hypothesized that the
basidiocarps from L. indigo contain a wide spectrum of
antibacterial compounds. It has been previously establi-
shed that compounds with antibacterial activity extracted
from species in the genus Lactarius are mainly sesqui-
terpenes (Anke et al., 1989; Vidari et al., 1995). Further
studies are now needed to elucidate the chemical
structure of the antibacterial compounds extracted from
basidiocarps of L. indigo.
In the present work the aqueous extract of L. indigo
significantly inhibited the proliferation of HeLa cells and
showed a significant stimulatory effect on the proliferation
of primary BME cells. These results contrast with reports
for other species of Basidiomycota, although at
significantly higher concentrations of aqueous extract
than those used in the present study. For example, the
aqueous extracts of Coprinellus sp., Flammulina velu-
tipes and Coprinus comatus were capable of inhibi-ting
the proliferation in vitro of MCF7 cells, with IC50 values of
120, 150 and 450 µg/ml, respectively (Gu and Leonard,
2006). Consequently, we cannot discard the possibility
that the aqueous extract of L. indigo may present
antiproliferative activity against the other cell lines
evaluated in the present work at higher concentrations
than those used by us, a possibility that needs to be
evaluated in future works. On the other side, the fact that
low concentrations of the aqueous extract had a
significant stimulatory effect on the proliferation of normal
BME cells is indicative of the possibility that the chemical
compounds in the aqueous extract of L. indigo, which
inhibit the proliferation of HeLa cells may be specific
against certain types of cancerous cells. This fact is
relevant given that not all aqueous extracts of Basidio-
mycota present a specific activity against cancer cells; for
example, the vegetative mycelia of Funalia trogii and of
Trametes (Coriolus) vers-icolor contain water-soluble
metabolites that inhibit the proliferation of both HeLa and
normal fibroblasts (Ünyayar et al., 2006). One possibility
is that the aqueous extract of L. indigo contains active
polysaccharides that are specific against cancer cell
lines. One polysaccharide has been isolated from the
aqueous extract of Pleuotus ostreatus that has an
antiproliferative effect against HeLa cells at
concentrations of 100, 200 and 400 µM, but that has no
significant inhibitory effect on normal human embryonic
kidney cells 293T (Tong et al., 2009). Recently, a polysa-
ccharide enriched with selenium was obtained from an
aqueous extract of vegetative mycelium of Ganoderma
lucidum that displays a high antiproliferative activity in
vitro against HeLa and MCF7 cells with IC50 values of
0.17 and 0.14 µM, respectively (Shang et al., 2009). The
determination and characterization of anticarcinogenic
polysaccharides in aqueous extracts of L. indigo is
certainly an essential task to be carried out.
The organic extracts of the basidiocarp of L. indigo did
not show cytotoxic activity against the MCF7 cell line but
were capable for inhibiting the proliferation of HeLa cells.
While ethyl acetate and methanol extracts showed a
significant inhibitory effect of proliferation on normal RSF,
this was relatively low. Regarding MCF7 cells, the results
of our study contrast with those of several previous
reports about the effect of organic extracts of
Basidiomycota. Thus, methanolic extracts of Pleurotus
ostreatus significantly inhibit the proliferation in vitro of
MCF7 cells at concentrations above 120 µg/ml (Jedinak
and Sliva, 2008). Such extracts induce the arrest of the
cell cycle at the G0/G1 transition by means of induction of
the expression of p53 and p21 proteins, which inhibit the
cyclinkinase complexes (CDK). Analyses have been
made of the antiproliferative effects of the organic
extracts of the Basidiomycota fungus Naematoloma
fasciculare as well as those of the unsaturated aliphatic
acids, ergosterol and ergosterol peroxide, isolated from
such extracts (Yan et al., 2009). The pure chemical
compounds showed an antiproliferative effect against MCF7
cells that is from 10 to 100 times larger than that of the
extracts; while for the former the IC50 values oscillated
between 0.16 and 1.54 µg/ml, in the latter case those values
varied from 13.80 to over 30.0 µg/ml (Yan et al., 2009).
Triterpenoids isolated by means of extraction with ethyl
acetate of basidiocarp of Leucopaxillus gentianeus showed
a high antiproliferative activity against MCF7 cells, some
of these compounds having IC50 values lower than 300
µM (Clericuzio et al., 2004).
In contrast with the case of the MCF7 cells, our results
with HeLa cells agree with a number of literature reports.
Thus, the inhibition of proliferation of HeLa cells by
ethanolic extracts of spores of G. lucidum has been
reported (Fukuzawa et al., 2008), an effect that was
attributed to long chain fatty acids; however, there is a
recent report of triterpenoids of the same species having
high cytotoxic potential against HeLa cells (Cheng et al.,
2010).
An interesting observation made during the present
work is the stimulation of the proliferation of the MCF7
cell line, an activity that has been previously reported for
aqueous extracts of Agaricus blazei in the presence of
the estrogenic compound nonylophenol (Talorete et al.,
2002). Such proliferative activity has been associated
with induction of the expression of the c-Jun protein by
the extract, which is synthesized by one of the genes
from the proto-oncogene family c-jun, characterized by
being induced by mitogenic agents (Talorete et al., 2002).
Because the MCF7 cell line is characterized by
presenting estrogen receptors, it is possible that the
organic extracts of L. indigo may contain a substance that
stimuli-tes those receptors, something to be analyzed in
the future.
Conclusion
Both aqueous and organic extracts of L. indigo basidio-
carp posses pharmacological activity; in some cases, due
Zarzosa et al. 287
to inhibition of the proliferation capacity of pathogenic
bacteria, and in others, that of carcinogenic cells, without
significantly affecting normal cells. Our results show that
the basidiocarp of the edible L. indigo is a source of
pharmacological substances having diverse therapeutic
applications, which makes it necessary to perform further
studies in that regard by isolating and characterizing the
molecules responsible for the observed activities.
ACKNOWLEDGEMENTS
The authors acknowledge Silvia Martínez Chávez and
Monia Hernández Ayala for their technical assistance in
the cytotoxicity assays, and Verónica García Quiroz for
her help in the antimicrobial experiments.
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