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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.
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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
Correspondence should be addressed to Magdalena Jaszek;
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
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
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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Volume 2014, Article ID 743812, 10 pages
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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-
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
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
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
described by Hope and Burns [] with some modications.
was obtained by subtraction of reducing sugars from the total
2.4.3. Determination of Proteins and Phenolic Compounds.
e protein concentration was estimated by the Coomassie
brilliant blue (G-) dye-binding method []usingBio-
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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
.solution (. mg/mL in
ethanol). Trolox standards well known for their strong antiox-
idant activity were used as a positive control. Absorbance at
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
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
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 %
(1) MTT Assay. e MTT assay is a colorimetric cytotoxicity
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
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
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
solution (nal concentration  mg/mL) was added to each
well and incubated for hours. Next, 𝜇LoftheSDS
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
4cells/mL) and HSF (3×10
4cells/mL) were seeded
in a -well microtiter plate and cultivated under standard
conditions (% CO2at C)forhours.Aerwards,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 (
105cells/mL) were dierentiated using  ng/mL of PMA for
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-
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
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
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-
3. Results and Discussion
Fungal species belonging to genus Ganoderma are known
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.PCRAmplicationoftheITSRegion. 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
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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
Reducing sugar
Total phenolic
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
−1 was character-
istic of the presence of 𝛽-glucans [,]. Additionally, a
4000 3500 3000 2500 2000 1500 1000 500
Wavenumbers (cm−1)
F : FT-IR spectra of the exopolysaccharides from G. applana-
tum (GpEPS).
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 .
BioMed Research International
0 0.02285 0.2285 2.285 22.85 228.5
Ca Ski
Concentration (𝜇g/mL)
(% of control)
00.02285 0.2285 2.285 22.85 228.5
Ca Ski
Concentration (𝜇g/mL)
(% of control)
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).
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
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
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 0.02285 0.2285 2.285 22.85 228.5
Ca Ski
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)
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
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 [].
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
Concentration (pg/mL)
24 h
24 h
Concentration (pg/mL)
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).
Chol Tg Gluc
24 h
Substances bound to GpEPS (%)
24 h
Substances bound to GpEPS (%)
Mg Fe
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
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
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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... Related to this, G. applanatum contain exobiopolymer (EXP), which is composed of mannose and glucose, while the protein mainly consisted of amino acids as serine, glycine and aspartic acid . G. applanatum also possesses β-glucans (Osińska-Jaroszuk et al. 2014;Deveci et al. 2019) and α/β-glucans, composed of glucose, fucose, mannose and galactose as monosaccharide units (Kozarski et al. 2012;Deveci et al. 2019). ...
... Ganoderma applanatum: Similar to the other species of the Ganoderma family, G. applanatum contains about 400 diverse mycochemicals. Based on literature data, G. applanatum produces various bioactive compounds which exhibit antiallergic, anticancer, antifibrotic, antihyperglycemic, antimicrobial, antioxidant, antitumor, hepatoprotective, hypoglycemic, immunomodulatory, liver protective properties as well as inhibition of aldose reductase enzyme, Epstein-Barr and influenza virus (Chairul and Hayashi 1994;Smânia et al. 1999;Acharya et al. 2005;Lee et al. 2006;Karaman et al. 2010Karaman et al. , 2022Osińska-Jaroszuk et al. 2014;Luo et al. 2015a;Teplyakova and Kosogova 2015;Hapuarachchi et al. 2017;Rašeta et al. 2016Rašeta et al. , 2020aErmoshin et al. 2022;Sułkowska-Ziaja et al. 2022). Currently, G. applanatum and other species of the genus Ganoderma are used in China and Japan for the treatment and prevention of hepatitis, hypertension, chronic bronchitis, bronchial asthma, hyperglycemia, rheumatism, connective tissue and oesophageal cancer (a dangerous tumor with epithelial cells), arthritis, tuberculosis and many other diseases (Paul et al. 2002;Yong-Tae et al. 2008;Kumaran et al. 2017). ...
... The protective effect of G. applanatum polysaccharides against gastric ulcer by strengthen gastric mucosa barrier by improving the level of PGE2, GMBF and the secretion of gastric mucus (Yang et al. 2005). The polysaccharides also showed antibacterial activity against S. aureus and Vibrio fischeri (Osińska-Jaroszuk et al. 2014). A positive correlation between radical scavenging activity of exopolysaccharides against hydroxyl and superoxide radicals and exopolysaccharide concentration was reported by Liu et al. (2015). ...
Ganoderma adspersum (Schulzer) Donk; Ganoderma applanatum (Pers.) Pat.; Ganoderma lucidum (Curtis) P. Karst.; Ganoderma resinaceum Boud. - GANODERMATACEAE
... Also, the antimicrobial activity of anionic polysaccharides, such as sulfated polysaccharides, occurs through different mechanisms, including their chelation activities and the deprivation of metal, trace elements, or essential nutrients, which restrict the growth of microorganisms and limit microbial development [56]. Ganoderma applanatum exopolysaccharides were discovered to have antimicrobial properties against Staphylococcus aureus and to be poisonous to Vibrio fischeri [57]. Li et al. [58] showed that EPSs produced from the Hirsutella strain has antibacterial efficiency against Bacillus subtilis and Micrococcus tetragenus. ...
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Background Exopolysaccharides (EPSs) produced by microbes are recognized as biomacromolecules of great significance. EPSs from fungi are widely used in a variety of biotechnological fields, including medicine, bioremediation, and agriculture. Results In this study, ten fungal isolates were isolated from Kafir El-Dair, Qalubia Governorate, Egypt. Isolate 5 produced more exopolysaccharides than the other examined fungi. According to microscopic morphological traits and genetic confirmation by the 18S rRNA gene, isolate 5 was identified as Fusarium nygamai strain AJTYC1. The present study showed that Czapek’s broth media, which contains 6 g/100 ml of sucrose, 10 g/100 ml of peptone, pH 6, and 1.8 × 10⁵ CFU/ml of inoculum size and is incubated at 30 °C for 9 days, was suitable for the production of EPSs from Fusarium nygamai strain AJTYC1 by using static conditions. Fourier transform infrared (FT-IR) was employed in the characterization of EPSs, which exhibited the presence of carboxyl groups, hydroxyl groups, carbonyl groups, and glycosidic bonds. High-performance liquid chromatography (HPLC) detected that EPSs consist of sucrose and glucose. The scavenging activity indicates that EPSs have good antioxidant activity. The partially purified exopolysaccharides produced from F. nygamai strain AJTYC1 exhibited excellent antioxidant and antimicrobial activity against gram positive, gram negative and fungal strains. The EPSs at a dose of 1000 µg/ml exhibited anticancer activity against colorectal colon cancer (HCT116), breast cancer (MCF7), and hepatocellular cancer cell lines. Moreover, EPSs is an effective emulsifier of a variety of vegetable oils, and the emulsion it produces is generally stable for up to 168 h. Conclusions The production of EPSs from F. nygamai strain AJTYC1 can be used as antioxidants, antimicrobials, anticancer, and emulsifiers.
... G. applanatum has been used in ethnomedicine because of its high antioxidant capacity and antimicrobial properties, including antibacterial and antifungal activity against pathogenic bacteria [3][4][5]. Osińska-Jaroszuk et al. proved that substances from G. applanatum showed efficient antibacterial activities against Staphylococcus aureus and a toxic effect against Vibrio fischeri cells (82.8% cell damage) [6]. ...
Traditional Asian remedies have mainly employed the macrofungus Ganoderma applanatum, which belongs to the family Ganodermataceae, as a medicinal mushroom due to its high antibacterial and antioxidant activity. Extracts of the fungus can be synthesized into nanoparticles, which are subsequently produced as plaster gels. Synthesized silver nanoparticle-mediated G. applanatum was discovered to have the greatest ability to inhibit bacterial growth in S. epidermidis. When applied to the skin, the prepared plaster gel converted from a gel to a film; thus, both gel and film generation are characteristic of its formulation. The plaster gel that was made was found to be consistent and attractive, and the yellow color had darkened. Its viscosity and pH were appropriate for the application and allowed it to remain on the skin without dripping or reacting with the skin until it dried. A shorter duration for film formation is possible. The film's tensile was slightly reduced, and it exhibited excellent thermal stability. Decomposition of the generated film occurred at a slower rate, which constrained the polymer chain's ability to move. The semi-crystalline structure was characteristic of the film. It was found that particles were distributed in the film. Rapid release from plaster gel within 4 h was seen, and this was followed by a period of a slowly declining release rate over 12 h. The accurate first-order kinetic used to estimate the release rate of the formulation. The plaster gel demonstrated greater antibacterial activity than the MIC value indicated. The in vivo evaluation was positive and showed no skin irritation. The formulation showed good stability. Therefore, this indicated that the prepared plaster gel is appropriate for topical pharmaceutical delivery and safe for skin application.
... The diameter of the inhibition zone was measured in mm [51] as shown in Fig. 7 and Table 3. Table 4). The MTT assay is a proliferation detection and colorimetric cytotoxicity test, this approach is depending on the metabolic effects of normal cells [52]. The tetrazolium salts are usually decreased in variable cells to a blue-coloured formazan, which is proportional to the number of active cells. ...
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Pleurotus ostreatus is a common cultivated edible mushroom worldwide. The fruiting bodies of P. ostreatus is a rich source of a $\beta$-glucans polysaccharide. The current study aimed to investigate the effectiveness of $\beta$-glucans as a natural polysaccharide produced by P. ostreatus as an antioxidant, antimicrobial, and anticancer. The molecular identification of P. ostreatus isolate was confirmed by Internal Transcribed Spacer (ITS) sequence. The sequence alignment and phylogenetic evolutionary relationship of studied ITS sequence were performed against some deposited sequences in GenBank. The analysis of high-performance liquid chromatography (HPLC) as well as the result of fourier transform infrared spectroscopy (FTIR) has confirmed the presence of $\beta$-glucans polysaccharide in the tested samples. The percentage of antioxidant activity of $\beta$-glucans showed a gradual increase from 8.59% to 12.36, 18.56, 23.69, 44.66 and 80.36% at the concentrations of 31.2, 64.4, 125, 250, 500, and 800 $\mu$g/ml, respectively. In addition, all concentrations of $\beta$-glucans showed higher antioxidant activities when compared with standard antioxidant (Vitamin C). The highest antimicrobial activity of $\beta$-glucans polysaccharide was against P. aeruginosa with a zone of inhibition (45 mm), while the lowest activity was against S. aureus (13 mm) both at 100 mg/mL. The percentage of growth-inhibiting of MCF-7 a humanbreast cancer cell line and normal WRL-68 cell line affected by $\beta$-glucans were determined by 3-(4,5)-dimethylthiazol (-z-y1)-3,5-di-phenytetrazoliumromide (MTT assay).
... There has been extensive research conducted on the chemical composition of the Ganoderma applanatum fruiting bodies, which revealed the presence of diverse groups of chemical compounds. These compounds include polysaccharides, sterols, proteins, and fatty acids [3][4][5]. Additionally, tannins, saponins, phenolic compounds, and flavonoids have also been identified [6][7][8]. Over 380 terpenoids (ganoderic/lucidenic acids, meroterpenoids) were isolated from the fruiting bodies [9]. ...
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Abiotic elicitation, a well-known strategy in mushroom biotechnology, promotes increased accumulation of secondary metabolites in mycelial cultures. The study aimed the effects of methyl jasmonate (MeJA) on the production of triterpenes in submerged cultures of Ganoderma applanatum. Further, the study evaluated the cytotoxic activity of the extract corresponding to the optimal elicitation variant in selected human cancer cell lines as well as the selectivity against normal cells. MeJA was added on days 1, 4, 6, and 8 in the 10-day growth cycle at concentrations of 10, 50, 100, 150, and 200 µM MeJA. The HPLC-DAD was used to analyze the triterpenes. The cytotoxic activity was tested using the MTTFc assay in grouped panels of skin, prostate, and gastrointestinal cancer cells. The results of the quantitative analyses confirmed the stimulating effect of MeJA on the production of ganoderic acid A and ganoderic acid C. The greatest increase in total triterpenes was found on day 6 of the culture cycle compared to the control group—with the concentration of MeJA—150 µM. Compared to the control samples, mycelial culture extract after the most productive elicitation variant showed significant cytotoxic activity against prostate cancer cells and moderate effects on melanoma cells. Ganoderma applanatum mycelial cultures can be proposed as a model to study the dynamics of the accumulation of compounds with therapeutic values through abiotic elicitation.
Exopolysaccharides are high-molecular-weight polysaccharides with a variety of forms secreted by microbes (EPSs). EPSs play a variety of roles that help microorganisms grow in diverse environments. Due to their biocompatibility, biodegradability, nontoxic nature, and unique physicochemical features, several EPSs are industrial relevant polymers. Exopolymeric materials offer distinctive properties. Adhesion, aggregation, binding activity, energy and nutrient source, water retention, and sorption are a few of its functional characteristics. Fungal exopolymeric compounds are one type of exopolymer. Pullulan, scleroglucan, fungus β-glucans, botryosphaeran, and other exopolysaccharides are examples of fungi. The diverse applications of fungal exopolymeric materials, including the bioabsorption of heavy metals, wastewater treatment, and agriculture, make them important goods.
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Since prehistoric times, ethnic cultures have used wild mushrooms as food and medicine. Despite the extensive use of these important resources in ethnomycology, little is known about them, and the documentation that does exist is insufficient. The goal of the current study was to record the traditional knowledge of the Subanen populations regarding the use of different types of wild mushrooms. Field walks to collect the indicated mushroom species were undertaken after an actual interview utilizing a semi-structured questionnaire. To support diverse therapeutic claims, the local name, specific usage, method of preparation, and range of uses for wild mushrooms are described. In the ten barangays of Matugnaw, Uwayan, Sicot, Paiton, Taguite, Katagan, Kimat, Baluc, Salimpuno, and Caniangan, the study discovered various naturally occurring mushrooms. Leaf litter, soil, and rotting logs were used to identify the ten different mushroom species. Termitomyces cartilaginous, Auricularia auricularia-judae, Volvariella volvacea, Schizophyllum commune, Auricularia polytricha, Ganoderma applanatum, Trametes polyzona, Pycnoporus sanguineus, Trametes elegans and Lenzites betulinus were the species of wild mushrooms that the Subanen tribe used for food and medicine, respectively. These precious mushrooms were used by some ethnic tribes in the Philippines and other nations as well, and their broad use may support their therapeutic claims.
Biological characteristics of natural polymers make microbial polysaccharides an excellent choice for biopharmaceuticals. Due to its easy purifying procedure and high production efficiency, it is capable of resolving the existing application issues associated with some plant and animal polysaccharides. Furthermore, microbial polysaccharides are recognized as prospective substitutes for these polysaccharides based on the search for eco-friendly chemicals. In this review, the microstructure and properties of microbial polysaccharides are utilized to highlight their characteristics and potential medical applications. From the standpoint of pathogenic processes, in-depth explanations are provided on the effects of microbial polysaccharides as active ingredients in the treatment of human diseases, anti-aging, and drug delivery. In addition, the scholarly developments and commercial applications of microbial polysaccharides as medical raw materials are also discussed. The conclusion is that understanding the use of microbial polysaccharides in biopharmaceuticals is essential for the future development of pharmacology and therapeutic medicine.
The Ganoderma genus is known for its diverse use as a functional food and therapeutic agent. This fungus has over 428 species, with Ganoderma lucidum being the most studied. The Ganoderma species produce several secondary metabolites and bioactive compounds like polysaccharides, phenols, and triterpenes, which are largely responsible for their therapeutic properties. Throughout this review, several extracts obtained from Ganoderma species have been studied to delve into their therapeutic characteristics and mechanisms. Such properties like immunomodulation, antiaging, antimicrobial, and anticancer activities have been demonstrated by several Ganoderma species and are supported by a large body of evidence. Although its phytochemicals play a vital role in its therapeutic properties, identifying the therapeutic potentials of fungal-secreted metabolites for human health-promoting benefits is a challenging task. Identification of novel compounds with distinct chemical scaffolds and their mechanism of action could help suppress the spread of rising pathogens. Thus, this review provides an updated and comprehensive overview of the bioactive components in different Ganoderma species and the underlying physiological mechanisms.
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The book is a photographic field guide on wild-edible, inedible, medicinal and poisonous mushrooms of Mizoram compiled from a collection of more than 300 specimens accumulated over a span of 5 years. It contains 81 species of mushrooms (including 13 genus-level identified species) belonging to 50 genera of 30 families. It provides visual and written directories on a vast variety of fungi across the state to aid farmers, consumers, research scholars, mycologists, agri-horti personnels and laymen in verifying important characteristics of wild mushrooms. The book also emphasizes the ways and means of mushroom collection and preservation along with significant identification patterns. It includes local names in Mizo language for easier classification along with ethnomycological detail and uses for promoting the wide scope and significance of wild mushroom foraging among the locals. The book is anticipated to be an essential step towards eradicating mycophobia and unfortunate misidentifications.
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A Slovenian Ganoderma lucidum strain MZKI G97 was isolated and cultivated in a 10 L stirred tank reactor, on potato dextrose substrate. Biomass up to 15.2 g L-1 and fungal polysaccharides were produced. The extracellular polysaccharide fraction was obtained by the precipitation method with ethanol. Four intracellular polysaccharide fractions were obtained by the hot-water extraction and precipitation with ethanol, by ammonium oxalate extraction, and by extraction with sodium hydroxide, followed by precipitation with acetic acid and precipitation with ethanol. Immunostimulatory effects of isolates were tested on induction of cytokine (TNF-α, IFN-γ) synthesis in primary cultures of human mononuclear cells (PBMC) isolated from a buffy coat. Results have shown the potential of isolates to induce moderate amounts of TNF-α (max. 630 pg mL-1 of a culture supernatant), and IFN-γ in trace amounts (max. 11.5 pg mL-1), respectively. The TNF-α inducing activity is comparable to romurtide, which has been used as a supporting therapy in cancer patients treated with radiotherapy and/or chemotherapy.
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Three bioactive fractions, extracellular laccase (ex-LAC), crude endopolysaccharides (c-EPL), and a low molecular subfraction of secondary metabolites (ex-LMS), were isolated from the idiophasic cultures of the white rot fungus Cerrena unicolor. For the first time, we determined the antioxidant properties of these samples by chemiluminometric measurement (a) and assessment of the scavenging effect on ABTS (b) and the DPPH reduction rate (c). The highest reducing capability was found for the ex-LMS fraction: 39-90% for (a), 20-90% for (b), and 10-59% for (c) at the concentration of 6.25-800 µg/mL. The scavenging abilities of the C. unicolor c-EPL were between 36 and 70% for (a), 2 and 60% for (b), and 28 and 32% for (c) at the concentration of 6.25-800 µg/mL. A very high prooxidative potential was observed for the ex-LAC probes. The preliminary toxicity tests were done using the Microtox system and revealed the following percentage of the toxic effect against Vibrio fischeri: 85.37% for c-EPL, 50.67% for ex-LAC, and 99.8% for ex-LMS, respectively. The ex-LAC sample showed the antibacterial activity against Escherichia coli, c-EPL against Staphylococcus aureus, and ex-LMS against both bacterial strains, respectively, but the stronger inhibitory effect was exerted on S. aureus.
To study the antioxidant capacity of the exopolysaccharides peptides (EPSP) from Ganoderma lucidum, four methods including 1,1-diphenyl-2- picrylhydrazyl (DPPH) radical scavenging assay, reducing power assay, self-oxidation of 1,2,3-phentriol assay and hydroxyl radical assays were employed in vitro. The sample demonstrated scavenging capacity in a dose-dependent manner in every assay. Among them, the scavenging ability on hydroxyl radical of the sample was potent, which was more than two times that of ascorbic acid at each concentration (range of 1-5 mg ml -1). Especially, at the low level (1 mg ml -1), the scavenging effect of the sample (25.95±1.48%) was about 4.17 fold that of ascorbic acid (6.22±0.38%). The findings obtained in this work showed that EPSP from G. lucidum broth might be good sources for antioxidant-related and healthy functional food additive.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
Lamium album, commonly known as "white dead nettle," is a perennial herb widely used in folk medicine. The present paper presents the toxic, anti-proliferative, and free radical (DPPH) scavenging activities of methanol and ethyl acetate extracts of that plant. In order to determine the biologically active compounds, the plant extracts were separated by high performance thin layer chromatography (HPTLC) on silica gel Si 60 F254 and high performance liquid chromatography (HPLC) combined with densitometry. Methanol extract was rich mainly with flavonoids and phenolic acids. Ethyl acetate extract contained mainly triterpenes. Both extracts showed no toxic effects against normal human skin fibroblasts (HSF) in the range of applied concentrations (25-225 μg/ml). Anti-proliferative activity revealed that methanol extract expressed lower inhibitory properties than ethyl acetate one. The MTT test was, however, less sensitive than Neutral Red (NR) assay. Ethyl acetate extract did not exhibit DPPH radical scavenging activity. Methanol extract reduced the radical of about 29% at the highest applied concentration (225 μg/ml). Both extracts slightly influenced cellular cytoskeleton organization and amount, and size of agyrophilic nucleolar organizer regions (AgNOR) protein deposits. These findings suggest that extracts of Lamium album exhibit potential usefulness in preparation of new natural formulations.
Most methods of DNA preparation from fungi are time-consuming due to the need to first make protoplasts, expensive for chemicals such as cesium chloride, or suitable only for small scale preparations. We have developed a simple method for total DNA preparation, yielding a product of quality suitable for restriction digestion and library construction. To extract DNA, 0.5 to 2.0 g of fresh or frozen mycelium is ground in liquid nitrogen. The mycelial powder is divided between two Sorvall tubes each containing 15 ml of cold spermidine-SDS buffer (4 mM spermidine, 10 mM EDTA, 0.1 M NaCl, 0.5% SDS, 10 mM ß-mercaptoethanol, 40 mM Tris-HCl pH 8.0), and thoroughly shaken. The mixture is then immediately extracted with 1 vol double distilled phenol. After a second phenol extraction, the aqueous phase is extracted with 1 vol chloroform. To the resulting aqueous phase is added 10% of that volume of 3M sodium acetate pH 5.5. DNA is then precipitated by addition of 2 vols of cold absolute ethanol, and recovered either by spooling it out or by centrifugation at 10,000 x g for 10 minutes. After washing with 70% cold ethanol, the DNA is air-dried at room temperature, redissolved in TE buffer (20 mM Tris-HCl pH 7.5, 0.1 mM EDTA) to a DNA concentration circa 0.5 mg/ml and stored at -20°C. Purity and average fragment size are checked by electrophoresis. The method is highly reproducible, and results over 70% recovery of good quality DNA (A260/A280 circa 1.85). With optimal grinding and nuclease inhibition, the DNA is of high molecular weight (>23 kb), with minimal smear on electrophoresis. The method has been used for Neurospora crassa, Aspergillus nidulans, Dactylium dendroides and Humicola grisea. For all four species, the DNA prepared has been successfully restricted and religated. DNA from this method has been used in the construction of genomic libraries from Humicola grisea and Dactylium dendroides.
Mechanochemical‐assisted extraction (MCAE) method was developed to an effective method for polysaccharides extraction from Ganoderma lucidum spores. The MCAE parameters and the antioxidant activity of polysaccharides were investigated. Through response surface methodology design experiments, the processing conditions were optimised as follows: material/solid reagent (Na2CO3) 5 g·g−1, milling time 20 min, solution/material ratio twenty (mL·g−1) and extraction time 130 min. Under these conditions, the yield of polysaccharides was (5.92 ± 0.13)%, which was in close agreement with the predicted value. Compared with the heat‐reflux extraction method, the MCAE method had higher extraction yield, shorter extraction time and lower extraction temperature. In addition, the polysaccharides obtained from MCAE exhibit significant antioxidant activity.