Exopolysaccharide from Ganoderma applanatum as a Promising Bioactive Compound with Cytostatic and Antibacterial Properties

Abstract

A new exopolysaccharide preparation isolated from stationary cultures of the white rot fungus Ganoderma applanatum (GpEPS) was tested in terms of its bioactive properties including its cytotoxic and immunostimulatory effect. The results indicate that the tested GpEPS (at concentrations above 22.85 µg/mL and 228.5 µg/mL) may exhibit selective activity against tumor cells (cell lines SiHa) and stimulate production of TNF-α THP-1-derived macrophages at the level of 752.17 pg/mL. The GpEPS showed antibacterial properties against Staphyloccoccus aureus and a toxic effect against Vibrio fischeri cells (82.8% cell damage). High cholesterol-binding capacity and triglycerides-binding capacity (57.9% and 41.6% after 24 h of incubation with the tested substances, resp.) were also detected for the investigated samples of GpEPS.

Full text

Research Article
Exopolysaccharide from Ganoderma applanatum as
a Promising Bioactive Compound with Cytostatic and
Antibacterial Properties
Monika OsiNska-Jaroszuk,1Magdalena Jaszek,1Magdalena Mizerska-Dudka,2
Adriana BBachowicz,2Tomasz Piotr Rejczak,2Grzegorz Janusz,1Jerzy Wydrych,3
Jolanta Polak,1Anna Jarosz-WilkoBazka,1and Martyna Kandefer-SzerszeN2
1Department of Biochemistry, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland
2Department of Virology and Immunology, Maria Curie-Sklodowska University, Akademicka 19, 20-033 Lublin, Poland
3DepartmentofComparativeAnatomyandAnthropology,MariaCurie-SklodowskaUniversity,Akademicka19,20-033Lublin,Poland
Correspondence should be addressed to Magdalena Jaszek; magdalena.jaszek@poczta.umcs.lublin.pl
Received  February ; Accepted  June ; Published  July 
Academic Editor: Oluwatoyin A. Odeku
Copyright ©  Monika Osi´
nska-Jaroszuk et al. is is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
A new exopolysaccharide preparation isolated from stationary cultures of the white rot fungus Ganoderma applanatum (GpEPS)
was tested in terms of its bioactive properties including its cytotoxic and immunostimulatory eect. e results indicate that the
tested GpEPS (at concentrations above . 𝜇g/mL and . 𝜇g/mL) may exhibit selective activity against tumor cells (cell lines
SiHa) and stimulate production of TNF-𝛼THP--derived macrophages at the le vel of. pg /mL. e GpEPS showed antibacterial
properties against Staphyloccoccus aureus and a toxic eect against Vibrio scheri cells (.% cell damage). High cholesterol-binding
capacity and triglycerides-binding capacity (.% and .% aer  h of incubation with the tested substances, resp.) were also
detected for the investigated samples of GpEPS.
1. Introduction
Numerous fungal preparations are used in traditional Eastern
medicine for prevention and treatment of diseases, such as
migraine, hypertension, arthritis, bronchitis, asthma, dia-
betes, hypercholesterolemia, and hepatitis. Among many
species, the genus Ganoderma seemstobethemostinterest-
ing mainly due to its wide therapeutic eect []. According to
the available research, biologically active substances obtained
from G. applanatum canbeusedincancertreatment;more-
over, they show a therapeutic eect against HIV [].
Recent explosion of interest in isolation and charac-
terization of bioactive compounds with unique properties
from family Ganodermaceae may be observed. Among them,
polysaccharides, especially glucans, deserve special attention
[]. Polysaccharides include a large and diverse group of
substances that play an important role in the structure and
function of fungal cell walls, which is the main polysaccharide
source. However, it should be mentioned that, depending on
thecultureconditions,somefungalspeciesalsoeectively
produce fractions extracellular polysaccharides.
One of the most frequently studied biological properties
of fungal polysaccharides is their antitumor activity. e anti-
tumor eect depends on their immunomodulatory activities
aected by many physical and chemical properties such as
thechemicalcompositionofthemolecule,thedegreeof
branching, the type of glycosidic bonds, conformation, or
molecular weight []. Among the number of fungal polysac-
charides described, 𝛽-glucans containing mainly 𝛽(1→3)-
glycosidic bonds and having side chains linked by 𝛽(1→
6)-glycosidic bonds have been presented as the most active
[]. It is supposed that inhibition of tumor cell growth is
the result of 𝛽-glucan-dependent stimulation of macrophages
and dendritic cells followed by secretion of various cytokines
including TNF-𝛼,IFN-𝛾,andIL-𝛽, and stimulation of NK T
and B cells [,]. Another possible mechanism of the impact
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BioMed Research International
Volume 2014, Article ID 743812, 10 pages
http://dx.doi.org/10.1155/2014/743812

BioMed Research International
of 𝛽-glucans on immune cells is the interaction of these
polysaccharides with the CR receptors [,]. Besides their
action on immune cells, 𝛽-glucans also inhibit angiogenesis
by cutting o the supply of nutrients to tumor cells and, in
consequence, inhibiting their development [].
Redox processes in living organisms are the basis for
obtaining energy necessary for the proper conduct of meta-
bolic changes. However, uncontrolled production of highly
reactive forms of free radical compounds can be a cause
of damage to genetic material, initiation of carcinogenesis,
and cell degradation associated with aging processes. To pre-
vent radical-mediated disorders, many natural compounds
exhibiting antioxidant properties can be used and polysac-
charides are the main group of them. Some reports have
indicated that the antioxidant properties of the intracellular
polymers produced by G. lucidum and G. applanatum may
be correlated with the content of polyphenolic compounds
in the samples. Polyphenols have been described as powerful
antioxidants due to their redox potential, which allows them
to act as reducing agents and hydrogen donors as well as
singlet oxygen scavengers [–].
ere are many available reports describing antibacterial
properties of fungal polysaccharides in relation to both gram-
positive and gram-negative bacteria. For example, it has
been discovered that the lentinan obtained from the fungus
Lentinus edodes exhibits antibacterial properties. Hirasawa
et al. [] proved that substances from dried Shiitake mush-
rooms (L. edodes) showed ecient antibacterial activities
against Streptococcus spp., Lactobacillus spp., Actinomyces
spp., Porphyromonas spp., and Prevotella spp. of oral origin.
e above ndings suggest that exploration of the world
of fungal extracellular polysaccharides seems to be a very
interesting issue for medicinal application, given the ease of
isolation and production thereof, compared with intracellular
polysaccharide preparations. e aim of the present work
was isolation of the extracellular polysaccharide (GpEPS)
produced by stationary cultivated G. applanatum and char-
acterization of its chemical composition, structure, and
biological (antimicrobial, antitumor, immunostimulatory,
and antioxidative) activities. Additionally, the cholesterol-
binding capacity, triglyceride-binding capacity, and glucose-
bindingcapacityoftheGpEPSpreparationwerealsoana-
lyzed.
2. Materials and Methods
2.1. Microorganism and Culture Conditions. e G. applana-
tum strain was obtained from the Fungal Collection (FCL)
of the Biochemistry Department, Maria Curie-Sklodovska
University,Lublin,Poland.eculturesweremaintainedon
potato-dextrose-agar (PDA) plates, which were inoculated
and incubated at ∘C for  days and stored at ∘C. e
experimental inocula were prepared in  mL Elenmeyer
askscontainingmLofthePDAmediumat
∘Cfor
days. Aer inoculation with % (v/v) of homogenate, rotary
shaking cultures were incubated in  mL Erlenmeyer asks
containing  mL medium. e media consisted of the fol-
lowing components:  g/L glucose,  g/L (NH4)2SO4, . g/L
KH2PO4, . g/L MgSO4×H2O, . g/L FeSO4zH2O, and
 g/L yeast extract. e experiments were performed at ∘C
in a rotary shaker ( rpm) for  days. Aer this time,
the culture liquid was separated from the mycelium by
centrifugation for  min in ∘C at . rpm.
2.2. Genomic DNA Isolation and Amplication of ITS Seque-
nces. AcultureofGanoderma applanatum was grown sta-
tionary in Lindeberg and Holm medium []atroomtem-
perature (∘C) for  days. Mycelia were harvested through
Miracloth (Merck, Whitehouse Station, NJ, USA), washed
twice with TE buer, and frozen in liquid nitrogen. DNA
was isolated according to Borges et al. []. e purity
and quantity of the DNA samples were evaluated using an
ND- spectrophotometer (ermo Scientic, West Palm
Beach, FL, USA).
PCRs were performed using Sigma RedTaq in a Tpersonal
thermal cycler (Biometra, Goettingen, Germany). To conrm
the identity of the fungus, the ITS region in the nuclear
ribosomal repeat unit was determined by direct sequencing
of the PCR products amplied with ITS-ITS primers as
described previously [].
2.3. Extraction of Exopolysaccharides. Crude exopolysaccha-
rides in the culture liquid were precipitated with cold %
ethanol in the ratio  :  (v/v) and kept overnight at ∘C. e
resulting preparation was centrifuged (  rpm,  min.),
washed three times with ethanol, dissolved in distilled water,
and lyophilized.
2.4. General Properties of Crude Exopolysaccharides
2.4.1. FT-IR Spectroscopy Analysis. Complete acid hydroly-
sis of the exopolysaccharides was carried out with .N
triuoroacetic acid (TFA) at ∘C in a heating block for
 h, and next the mixture was cooled, evaporated, and then
analyzed using infrared spectroscopy. e FT-IR spectra of
the exopolysaccharides were recorded on a ermo-Nicolet
Model  A spectrophotometer with a FT Ramana Nicolet
NXR module (ermo Scientic, USA). e spectra were
recorded in a wavelength range of – cm−1 using the
KBr disc technique.
2.4.2. Determination of Total Carbohydrate and Reducing
Sugar. e total carbohydrate content of the exopolysac-
charides was determined according to Dubois et al. []
using the phenol-sulfuric acid assay with D-glucose as a
standard. e concentration of reducing sugars was measured
bytheSomogyi-Nelsonmethodbasedontheprocedure
described by Hope and Burns [] with some modications.
eamountoftotalreducingsugarswascalculatedwithD-
glucoseasastandard.enaltotalvalueofpolysaccharides
was obtained by subtraction of reducing sugars from the total
carbohydrates.
2.4.3. Determination of Proteins and Phenolic Compounds.
e protein concentration was estimated by the Coomassie
brilliant blue (G-) dye-binding method []usingBio-
Raddyestocksolutionwithbovineserumalbumin(Sigma)as

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a standard. e total phenolic compounds content of GpEPS
was determined with diazosulfanilamide by the DASA test
[]. e changes in absorbance were measured at  nm
and compared with the standard curve of vanillic acid.
2.4.4. Microscopic Imaging of Exopolysaccharide Using Con-
focal Laser Scanning Equipment. e visualization of GpEPS
morphology was conducted according to the method
described in the earlier report []. Fluorescence Brightener
 was used for proper detection of 𝛽-linked polysaccharides.
As presented in the earlier report, the lyophilized samples of
extracted exopolysaccharides ( mg) were washed with MQ
water and aer water removal they were stained for  min
with  𝜇Lof𝜇g/mL Fluorescence Brightener . en
the sample was washed twice with water to remove the dye,
placed on a glass slide and estimated under a microscope.
An inverted microscope Axiovert M equipped with an
LSM  Pascal head (with magnication x) was used for
visualization of the GpEPS structure.
2.5. Biological Properties of Crude Exopolysaccharides
2.5.1. DPPH Radical Scavenging Activity. Free radical scav-
enging activity of the crude exopolysaccharides was esti-
mated by the ,-diphenyl--picrylhydrazyl (DPPH.)assay,
described by Paduch et al. []. e tested compound
(. mL) at concentrations ranging from . to  𝜇g/mL
wasaddedto.mLofDPPH
.solution (. mg/mL in
ethanol). Trolox standards well known for their strong antiox-
idant activity were used as a positive control. Absorbance at
nmwasestimatedaer,,,,,andmin.of
incubation at room temperature. e capability of scavenging
DPPH.radicals was calculated by the following formula:
DPPH.scavenging eect (%)=[𝑋0−𝑋1
𝑋0]×100, ()
where 𝑋0istheabsorbanceofthecontroland𝑋1is the
absorbance of the tested compound/standard. e inhibition
curves were prepared and EC50 values were obtained as
described previously [].
2.5.2. Estimation of the Toxicity Eect Using the Microtox
Protocol. e toxic eect of the tested EPS from G. applana-
tum cultures towards marine bacterium Vibrio scheri was
estimatedusingtheMicrotoxModelAnalyzerdetection
system according to the procedure described in the earlier
report []. e toxicity test used in the present report is
based on the study of luminescence intensity of genetically
modied bacteria. Any changes of cell respiration are closely
correlated with the cellular activity and they cause a reduction
ofluminescence.eintensityoflightofV.scheri cells was
measured at , , and  min aer the treatment with the
GpEPS fraction. e research method applied was conducted
according to the Screening Test Protocol of the Microtox
assay.
2.5.3. Cytotoxic Activity of GpEPS. Cervical carcinoma cell
lines SiHa (ATCC, HTB-) and Ca Ski (ATCC, CRL )
were used to determine the antitumor activity of this prepa-
ration. e SiHa cell line was established from squamous cell
carcinoma (primary tumor), containing HPV serotype .
is cell line was maintained in MEM supplemented with %
fetal bovine serum (FCS). e Ca Ski cell line was established
from epidermoid carcinoma derived from a metastatic site in
the small intestine. e cells contained HPV serotypes  and
. is cell line was maintained in RPMI  supplemented
with % fetal bovine serum (FCS). In this study, a human
skin broblast (HSF) cell line was used as a model of normal
cells. e HSF cell line was established from skin explant and
maintained in DMEM/MEM ( : ) supplemented with %
FCS.
(1) MTT Assay. e MTT assay is a colorimetric cytotoxicity
andproliferationdetectionassay,basedonthemetabolic
activity of viable cells. Tetrazolium salts (MTT) are reduced
only by metabolically active cells, namely, by a mitochondrial
enzyme, to a blue colored fromazan, whose amount is
proportional to the number of viable cells. Cytotoxicity assay.
e SiHa and Ca Ski cell lines (5×10
5cells/mL) and HSF
(1×10
5cells/mL) were seeded in a -well microtiter plate
and cultivated under standard conditions (% CO2at ∘C)
for  hours. In the case of the immunomodulatory activity
assay, the cytotoxicity of the tested fraction was determined
toward THP--derived macrophages. e culture medium
was discarded from the wells and the cells were incubated
forandhourswithvariousconcentrationsoffraction
samples as indicated in the gure. e fraction samples
were prepared using a medium with % FCS appropriate
for the cell line. Cells in the medium with % FCS alone
wereusedasapositivecontrol.Aerincubation,theMTT
solution (nal concentration  mg/mL) was added to each
well and incubated for  hours. Next,  𝜇LoftheSDS
solutionwasaddedtoeachwelltodissolveformazancrystals.
e plates were incubated for  hours at ∘C. e optical
density was measured on a microtitre plate reader (Bio-
Tek Instruments, Inc.) at  nm. e cytotoxicity of the
tested fraction was determined from absorbance values and
expressed as percentage relative to the control (% of
living cells). Proliferation assay. SiHa and Ca Ski cell lines
(5×10
4cells/mL) and HSF (3×10
4cells/mL) were seeded
in a -well microtiter plate and cultivated under standard
conditions (% CO2at ∘C)forhours.Aerwards,the
medium was discarded and the cells were incubated for 
hours with various concentrations of fraction samples as
indicated in the gure. e fraction samples were prepared
using a medium with % FCS appropriate for the cell line.
Cells in the medium with % FCS alone were used as a
positive control. e MTT assay was carried out as described
above (the cytotoxicity assay).
2.5.4. Immunomodulatory Activity. Monocytic cell lines of
varying degrees of dierentiation are frequently used as a
macrophage model. In this case, THP-, an acute mono-
cytic leukemia cell line (ATCC) was used to determine
the immunomodulatory activity of the G. applanatum
GpEPS fraction. Before the assay, the THP- cells were
treated with phorbol--myristate--acetate (PMA). PMA

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treatment, which activates protein kinase C (PKC), induces
dierentiation of THP- cells into macrophages.
(1) THP-1 Cell Dierentiation. e THP- cell line was main-
tained in RPMI  supplemented with % fetal bovine
serum (FCS) and  mM/L L-glutamine. e THP- cells (5×
105cells/mL) were dierentiated using  ng/mL of PMA for
daysin%CO
2at ∘C. Aerwards, the PMA-containing
medium was discarded and adherent cells were gently washed
three times with RPMI  (without FCS). Next, THP-
derived macrophages were cultivated in RPMI (% fetal
bovine serum (FCS) and  mM/L of L-glutamine) for three
days with daily changes of the medium. e macrophages cell
cultures obtained were used to determine the immunomod-
ulatory activity of the G. applanatum GpEPS fraction, which
was followed by determination of the cytotoxicity (MTT
assay) of the tested fraction (described above).
(2) Immunomodulatory Activity Assay.eimmunomodula-
tory activity of the G. applanatum exocellular polysaccharide
fraction was determined using THP--derived macrophages
that were able to synthesize and secrete IL- and TNF-𝛼.e
level of cytokines was measured using the ELISA method
(BD OptEIA, BD Biosciences) in cell culture supernatants
of macrophages treated with a noncytotoxic concentration
of the tested fraction. e THP--derived macrophages cul-
tivatedinRPMIwith%FCSwerethenegativecontrol,
whereas cells treated with LPS of E. coli, serotype  : B
( 𝜇g/mL), constituted the positive control. e cell cultures
were incubated for  and  hours in % CO2at ∘C.
Aer incubation, cell culture supernatants were collected and
centrifuged for  min, ( rpm) at ∘C. e samples were
stored at −∘CuntiltheELISAassay.eELISAassaywas
carried out according to manufacturer’s instruction. e IL-
level was determined aer  hours, whereas TNF-𝛼aer 
and  hours of incubation.
2.5.5. Analysis of Antibacterial Activity. Antibacterial activity
of the GpEPS fractions was tested using reference bacterial
strains Escherichia coli (ATCC ) and Staphylococcus
aureus (ATCC ). E. coli and S. aureus inocula (∘
in McFarland scale) were kept under sterile conditions on
Mueller-Hinton Agar II (Lab M, IDG plc, UK) (on Petri
dishes). e isolated exopolysaccharide fractions ( mg/mL)
were applied to these agar plates in an amount of  𝜇Lper
well. e plates were incubated for  h at room temperature
andthenforhat
∘C. Subsequently, the E. coli and
S. aureus inhibition zones were measured. e minimum
inhibitory concentration (MIC) of the GpEPS fractions
obtained was measured according to the recommendations of
the National Committee for Clinical Laboratory Standards.
2.5.6. Testing the Ability of Exopolysaccharides to Bind
Cholesterol, Triglycerides, Glucose, and Magnesium and Iron
Ions. Standard human serum containing appropriate test
substances (cholesterol, triglycerides, glucose, and magne-
sium and iron ions) were mixed with exopolysaccharides
( mg/mL) in a proportion of . : . (v/v). e nal con-
centrations of the test substances in the human serum were
as follows: cholesterol  mg/dL, triglycerides  mg/dL,
glucose  mg/dL, magnesium ions . mg/dL, and iron
ions  𝜇g/dL. e samples were incubated for  and 
hours at room temperature. Aer this time, the samples were
centrifuged and assayed towards appropriate biochemical
parameters. e comparative control was a sample of human
serum containing distilled water instead of GpEPS. e con-
centration of plasma triglycerides, cholesterol, glucose, and
magnesium,andironionswasevaluatedbycommercially
available biochemical test kits (Alpha Diagnostics, Poland).
2.6. Statistical Analysis. All the results are expressed as mean
±SD from three experiments (𝑛=3). Data were analyzed
using one-way ANOVA followed by a post hoc Tukey’s test.
Values o f 𝑃 ≤ 0.05 were only reported as statistically signi-
cant.
3. Results and Discussion
Fungal species belonging to genus Ganoderma are known
fortheirabilitytoproduceanumberofsubstanceswith
promising biomedical properties. e fruiting bodies of
G. applanatum are very oen used in traditional Chinese
medicinal therapies. ey are known as very ecient anti-
cancer, immunostimulatory, and antiviral factors [,].
Hitherto, many papers have been published indicating that G.
applanatum mycelia comprise certain amounts of saponins,
avonoids, cordial glycosides, steroids, and polysaccharides
[,]. A particularly interesting and still poorly stud-
ied group of compounds is extracellular polysaccharides
extracted from G. applanatum.
3.1.PCRAmplicationoftheITSRegion. e strain of G.
applanatum used in this study was genetically identied by
determination of ITS sequences. One product of  bp was
obtained from PCR with ITS-ITS primers and followed by
direct sequencing. e complete sequences of this product
indicated over % identity to the G. applanatum ITS
sequences and was deposited in GenBank under accession
number JN.
3.2. General Properties of Crude GpEPS Preparation. In the
present work, -day-old rotary shaken cultivated cultures
of G. applanatum were used in order to obtain culture
uid for extraction of exopolysaccharides. Similarly, in their
study, Lee et al. [] showed that the highest production of
exopolysaccharides was obtained from -day-old culture of
G. applanatum. Currently, some fungal polysaccharides are
obtained from fruiting bodies by means of time-consuming,
multistep procedures for isolation and fractionation con-
sisting in sugar ethanol precipitation, repeated extraction
with boiling water and ammonium oxalate solutions of
NaOH. e extracted polysaccharides are then puried by
a variety of steps of chromatographic techniques. In this
work, the preparation of extracellular polysaccharides was
obtained by a simple ethanol precipitation procedure from
the culture liquid of G. applanatum. Our initial experiments
proved that the new extraction method with a yield of

BioMed Research International 
T : Amount of proteins, total polysaccharides, reducing sugar, and total phenolic compounds content of GpEPS. All results are expressed
as mean ±SD from three experiments (𝑛=3).
Sample Extraction
yieldaProtein contents
(mg/gb)
Total
carbohydrate
(mg/gb)
Total
polysaccharide
(mg/gb)
Reducing sugar
(mg/gb)
Total phenolic
compounds
(𝜇M/gb)
GpEPS . ±. . ±.  ±. . ±.±.  ±.
ag/ g dr y weight basis.
bg dry weight of crude exopolysaccharide.
about .% (Tab l e  ) might be the most ecient method
for isolation of exopolysaccharides from dierent strains of
family Ganodermaceae. In contrast, Zhao et al. []received
four times lower extraction eciency (.%) for the crude
polysaccharide obtained from Ganoderma lucidum.
e total carbohydrate content of the exopolysaccharide
extracts from G. applanatum was  mg/g dcw (dry weight
of crude polysaccharide) of the extract (.%). e amounts
obtained in the present work are higher than the quanti-
ties of crude hot water extracted polysaccharides yielded
by G. applanatum and G. lucidum,whichwere%and
%, respectively []. Telles et al. [] found .% total
carbohydrate in native extracellular polysaccharides from
Pleurotus sajor-caju.However,Cordyceps sinensis was shown
to comprise from % to % of total sugar, depending on
the culture day []. e total polysaccharide content of the
presented exopolysaccharides (. mg/g dcw) (Table )was
signicantly higher than those reported for crude hot water
extracted polysaccharides from G. lucidum (. mg/g), Agar-
icus bisporus (. mg/g), and Phellinus linteus (. mg/g)
[]. e exopolysaccharides extracted from the tested strain
showed high content of reducing sugar (.mg/g) (Table ).
e concentration of phenolic compounds in the crude
exopolysaccharides was  𝜇Mandthatwasalmostsix
times lower than the values obtained for crude extracts
of endopolysaccharides from Cerrena unicolor []. e
total protein contents of the crude exopolysaccharide of G.
applanatum was 22.6±0.7mg/g dwc (.%) (Table ). Polysac-
charide fractions from another strain of the Ganoderma
genus,G.lucidum, also contained proteins (about .%)
characterized as glycopeptides []. Crude exopolysaccha-
rides isolated from mycelium of Cordyceps sinensis described
by Leung et al. [] contained –% of sugar and about
% of proteins, suggesting their polysaccharide-protein
character. Cui and Chisti [] reported additionally that
polysaccharides-peptides complexes from Coriolus versicolor
contained peptides mainly consisting of aspartic and glu-
tamic acids.
e FT-IR spectrum of the ethanol-extracted exopolysac-
charides of G. applanatum showed a typical carbohydrate pat-
tern (Figure ). e absorption band at . cm−1 indicates
the presence of the hydroxyl group (–OH) characteristic for
molecular interactions of polysaccharide chains []. e two
bands towards . and . cm−1 are correlated with the
presence of the deprotonated carboxylic group (–COO−). e
bands at ., , and  cm−1 suggested the presence
ofC–Obands,andthebandatcm
−1 was character-
istic of the presence of 𝛽-glucans [,]. Additionally, a
797.421
840.8115
1026.909
1073.674
1132.01
1186.971
1437.672
1661.856
3292.375
720.7645
4000 3500 3000 2500 2000 1500 1000 500
Wavenumbers (cm−1)
T(%)
F : FT-IR spectra of the exopolysaccharides from G. applana-
tum (GpEPS).
A
200 𝜇m
F : Morphology of exopolysaccharides bers using confocal
laser scanning microscopy. e tested samples of GpEPS were
stained with Fluorescence Brightener  commonly used in order
to detection of 𝛽-linked polysaccharides. For visualization of the
GpEPS, the inverted microscope Axiovert  M equipped with
an LSM  Pascal head (with magnication x) was used. e
letter (A) indicates the luminous bers exhibiting visible 𝛽-linked
polysaccharide fragments.
characteristic peak at . cm−1 indicating 𝛼-linked glycosyl
residues was observed by Kozarski et al. []. In addition,
staining of the exopolysaccharides with Fluorescence Bright-
ener  conrmed the presence of 𝛽-linked bonds in the
polysaccharides studied (Figure ). Chemical characteriza-
tion of the exopolysaccharides properties is presented in
Table .

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0
30
60
90
120
150
180
210
0 0.02285 0.2285 2.285 22.85 228.5
SiHa
Ca Ski
HSF
Concentration (𝜇g/mL)
(% of control)
(a)
0
30
60
90
120
150
180
210
00.02285 0.2285 2.285 22.85 228.5
SiHa
Ca Ski
HSF
Concentration (𝜇g/mL)
(% of control)
(b)
F : e cytotoxic eect of exopolysaccharides from G. applanatum (GpEPS) against carcinoma cell lines (SiHa and Ca Ski) and human
skin broblast (HSF) aer h (a) and  h (b) incubation. Each value is expressed as mean ±SD (𝑛=3).
T : e antibacterial activities and the toxicity eect of GpEPS
( mg/mL) isolated from G. applanatum submerged cultures. All
results are expressed as mean ±SD from three experiments (𝑛=3).
Sample
Diameters of
inhibition zone (mm) Tox i c e  e c t ( % )
E. coli S. aureus V.  s c he r i
GpEPS a— . ±. . ±.
aNot detected.
3.3. Biological Properties of the Crude GpEPS Preparation
3.3.1. Toxic, Antimicrobial, and Antioxidant Properties. It is
known that a number of substances isolated from mushrooms
may exhibit antibacterial activity. Zhu et al. [] showed the
antibacterial activity of polysaccharides from spent mush-
room substrate against E. coli and S. lutea.Resultsfromour
preliminary toxicity tests obtained using the Microtox detec-
tion system showed that the exposure of genetically modied
marine bacterium Vibrio scheri to tested exopolysaccharides
caused .% cell damage. e antibacterial activity of GpEPS
was analyzed using E. coli and S. aureus strains. Exopolysac-
charide samples showed antibacterial properties against the
S. aureus strain with the inhibition zone of . mm and
MIC values  mg/mL (Tab l e  ). e results indicate an evident
antibacterial eect of the tested preparation.
Polymeric carbohydrates from mushrooms have been
reported as modulators of inammatory response systems.
Among others, antioxidant properties of polysaccharides
produced by fungi such as Agaricus bisporus,Agaricus
brasiliensis,Ganoderma lucidum,andPhellinus linteus have
been shown [,–].
e tested GpEPS exhibited relatively weak antioxidant
properties and an ability to scavenge free radicals. In the
present study, the ability of exopolysaccharide to reduce
DPPH was conrmed, but the degree of reduction did not
exceed % of the antioxidant properties, in comparison to
the control. Similar to Kozarski et al. [], we found that
the ability to scavenge free radicals by polymers is related to
the presence of large amounts of phenolic compounds. e
results obtained may prove the thesis proposed by Kozarski et
al. [] that the low levels of free radical scavenging exhibited
by the tested preparation are related to the results of phenolic
compounds.
3.3.2. Antitumor Activity. e crude exopolysaccharides
extracted from G. applanatum were subjected to in vitro
cytotoxicity assays against carcinoma cell lines (SiHa and Ca
Ski)andahumanskinbroblast(HSF)line.Aerhours
of incubation, slight changes in cell viability were observed
(HSF, SiHa, Ca Ski), but they were not statistically signicant,
compared to the control. In contrast, the results obtained aer
 hour incubation were more varied. Our results showed
that the isolated polysaccharides exhibited cytotoxic activity
against the SiHa carcinoma cell line. A .% and %
decrease in cell viability at . 𝜇g/mL and . 𝜇g/mL con-
centrations of the exopolysaccharides studied, respectively,
was noted (Figures (a) and (b)). On the other hand, a ca.
% and % increase in the metabolic activity of Ca Ski
cells for the exopolysaccharide concentrations of . and
. 𝜇g/mL, respectively, was observed. In turn, a ca. –%
increase in the activity was observed in the case of broblasts
(HSF). To our knowledge, there are currently no reports
on the cytotoxic properties of exopolysaccharides from G.
applanatum.Lietal.[] showed that the polysaccharides
from G. atrium inhibited tumor growth in S-bearing mice
via induction of apoptosis through mitochondrial pathways
and immunoenhancement eects. Recently, polysaccharide-
protein peptide conjugates with anticancer or immunomodu-
lation properties were isolated from G. lucidium. For example,
a GIPP fraction (polysaccharide-peptide conjugation) was
indicated to inhibit proliferation of HUVECs by inducing

BioMed Research International 
0
20
40
60
80
100
120
140
0 0.02285 0.2285 2.285 22.85 228.5
SiHa
Ca Ski
HSF
Concentration (𝜇g/mL)
(% of control)
F : Proliferative activity of carcinoma cell lines (SiHa and
Ca Ski) and human skin broblast (HSF) in the presence of
exopolysaccharides from G. applanatum (GpEPS).Each value is
expressed as mean ±SD (𝑛=3).
cell apoptosis and decrease the expression of secreted VEGF
in human lung cancer cells [,]. e results of our
study indicate that the tested polysaccharide fraction of G.
applanatum may exhibit selective cytotoxic activity against
SiHa cell lines at concentration above . 𝜇g/mL.
In addition to the cytotoxic activity, antiproliferation
activity of exopolysaccharides was also determined. In the
presence of the exopolysaccharide from G. applanatum,there
were no statistically signicant changes in cell proliferation
activity of the tested cell lines (HSF, SiHA, Ca Ski). e
dierences in proliferative activity in comparison with the
control cells were approximately –% for HSF, –% for
Ca Ski, and –% for SiHa cell lines (Figure ).
3.3.3. Immunomodulatory Activity. e isolated from G.
applanatum exopolysaccharides were tested for their ability to
regulate immune response mechanisms. e immunomodu-
latory properties of the exopolysaccharides were determined
by means of THP- cells dierentiated into macrophages,
capableofproductionofIL-andTNF-𝛼. A preliminary
study of the cytotoxic activity of exopolysaccharides against
THP--derived macrophages revealed that this fraction was
not toxic at all concentrations (Figure ). Exopolysaccharides
at the concentration of . 𝜇g/mL were used for further
study of the immunomodulatory activity. e study pre-
sented in this paper indicated that aer  hours of incu-
bation the extracellular polysaccharides from G. applanatum
stimulated secretion of IL- by macrophages at a level of
. pg/mL (Figure (a)). Similar results were observed for
polysaccharides isolated from Ganoderma lucidum which
increased the level of proinammatory cytokines (IL-𝛽,
TNF-𝛼, and IL-) secreted by macrophages isolated from
rat bone marrow []. For comparison, Wang et al. []
obtained IL- at a level of . pg/mL in the culture
supernatant, incubating peripheral blood mononuclear cells
00.02285 0.2285 2.285 22.85 228.5
Concentration (𝜇g/mL)
(% of control)
0
20
40
60
80
100
120
140
160
24 h
48 h
F : e cytotoxic activity of exopolysaccharides from G.
applanatum (GpEPS) against macrophages (THP-). Each value is
expressed as mean ±SD (𝑛=3).
at a density of 1×10
6cells/mL for  days with a fraction of
G. lucidum polysaccharide at a concentration of  mg/mL
[]. Taking into account the fact that Wang et al. []
used higher cell density and a longer time of incubation,
the results (lower level of IL-) obtained for the fractions
examinedinthisstudymaynotresultnecessarilyfrom
weaker immunomodulatory activity of the tested fractions
but rather from the experimental conditions. e -hour
incubation of extracellular polysaccharides isolated from G.
applanatum at a concentration of . 𝜇g/mL resulted in
production of TNF-𝛼by THP--derived macrophages at the
level of . pg/mL, representing an approximately -fold
increase compared to the negative control (Figure (b)). In
turn, a decrease in the TNF-𝛼level to . pg/mL was
observed aer -hour incubation, although the cytokine
level remained higher in comparison with the negative con-
trol. Habijaniˇ
cetal.[] studied the dierent polysaccharide
fractions from G. lucidum. Aer -hour incubation with
human peripheral blood mononuclear cells, this polysaccha-
ride (concentration of  𝜇g/mL) induced appearance of
TNF-𝛼at concentrations of approximately – pg/mL in
the culture supernatant. e fungal polysaccharide fractions
tested in this study stimulated macrophage production of
cytokines at a higher level. Mucopolysaccharides, particu-
larly 𝛽-glucans, operate as so-called PAMPs, which aer
nonspecic recognition by the immune system stimulate the
immune mechanisms [].
3.3.4.TestingtheAbilityofGpEPStoBindCholesterol,
Triglycerides, Glucose, and Magnesium and Iron Ions. e
ability of fungal polysaccharides to reduce levels of choles-
terol and the lipids in blood remains one of the important
pharmacological properties. Chen and Huang []conducted
experiments proving the ability of 𝛽-glucans to reduce
cholesterol levels in blood by partial inhibition of absorp-
tion thereof. We conrmed that exopolysaccharides from
G. applanatum were able to bind in vitro cholesterol and

BioMed Research International
0
200
400
600
800
1000
1200
CGpEPS LPS
a
b
c
IL-6
Concentration (pg/mL)
6h
24 h
(a)
0
500
1000
1500
2000
2500
3000
3500
4000
C GpEPS LPS
a
b
c
d
6h
24 h
TNF-𝛼
Concentration (pg/mL)
(b)
F : Immunostimulatory activity of exopolysaccharides from G. applanatum (GpEPS):(a) the level of IL- in the culture uid aer  h
of treatment with the GpEPS fractions (. 𝜇g/mL), (b) the changes of TNF-𝛼level aer  and  h of treatment with the GpEPS fractions
(. 𝜇g/mL) (C-negative control-cultures provided in the RPMI medium with the content of % of serum, LPS-positive control, and E. coli
lipopolysaccharide ( 𝜇g/mL)). All results are expressed as mean ±SD from three experiments (𝑛=3), values marked with the dierent
letters are signicantly dierent (𝑃 ≤ 0.05).
0
10
20
30
40
50
60
70
80
90
100
Chol Tg Gluc
a
b
c
a
bb
aa
0
2h
24 h
Substances bound to GpEPS (%)
(a)
0
10
20
30
40
50
60
70
80
90
100
0
2h
24 h
Substances bound to GpEPS (%)
Mg Fe
abb
ab
c
(b)
F : Testing the ability of exopolysaccharides from G. applanatum (GpEPS)to binding: cholesterol, triglycerides, glucose, (a) and
magnesium and iron ions (b) expressed as a percentage of the test substance bound to exopolysaccharides. All results are expressed as mean
±SD from three experiments (𝑛=3); values marked with the dierent letters are signicantly dierent (𝑃 ≤ 0.05).
triglycerides (Figure (a)). e amount of bound cholesterol
increased during the incubation time (.% aer  hours
and .% aer  hours of incubation). is correlation
was not observed for triglycerides, where the level of bound
substances was stable despite the time of incubation ( hours,
.% and  hours, .%). e conducted experiments also
provedthepossibilityofglucosebindingtothetestedGpEPS.
e incorporated glucose level was amounted to .% and
was independent of the incubation time. Magnesium and iron
ions attachment ability (Figure (b)) revealed weak capacity
of exopolysaccharides to absorption of these substances
(.% for Mg2+ and .% for Fe2+). In conclusion, all

BioMed Research International 
these data indicate that exopolysaccharides extracted from G.
applanatum possess a high capability of binding cholesterol
and triglycerides; however, further tests in vivo are required
to conrm their hypocholesterolemic properties.
4. Conclusions
e weight of evidence suggests that exopolysaccharides
isolated from the white rot fungus Ganoderma applanatum
are characterized by a lot of important biomedical proper-
ties.e conducted experiments have evidently shown an
anticancer, immunomodulating, and antibacterial eect. e
results of our tests proved that the crude GpEPS preparation
exhibited antitumor activity against carcinoma cells (lines
SiHa) and stimulated production of Il- and TNF-𝛼by
macrophage line THP-. At the same time, the antibacte-
rial tests also indicated good antiseptic properties of the
exopolysaccharides studied against S. aureus and V.  s c h e r i
strains. e biological properties described as well as the
hypocholesterolemic eect of the tested substances suggest
that the present studies should be continued in view of future
pharmacological applications. However, it is worth noting
that further studies comprising preventive and therapeutic
actions of the GpEPS fraction are needed. In the promotion of
the described preparation as a promising bioactive product,
the simple and economical way of production and isolation
thereofaswellasthepossibilityofstandardizationand
control of the production conditions should be emphasized.
Conflict of Interests
e authors declare that there is no conict of interests
regarding the publication of this paper.
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