Antibacterial and cytotoxic activities of wild mushroom Fomes fomentarius (L.) Fr., Polyporaceae

Full text

Industrial
Crops
and
Products
79
(2016)
110–115
Contents lists available at ScienceDirect
Industrial
Crops
and
Products
journal homepage: www.elsevier.com/locate/indcrop
Antibacterial
and
cytotoxic
activities
of
wild
mushroom
Fomes
fomentarius
(L.)
Fr.,
Polyporaceae
Marina
Kolundˇ
zi´
ca,
Nad¯a
Ð.
Grozdani´
cb,
Margarita
Dodevskac,
Marina
Milenkovi´
cd,
Francesca
Sistoe,
Alessandro
Mianie,
Giampietro
Farronatoe,
Tatjana
Kundakovi´
ca,∗
aDepartment
of
Pharmacognosy,
University
of
Belgrade,
Faculty
of
Pharmacy,
Belgrade,
Serbia
bInstitute
for
Oncology
and
Radiology
of
Serbia,
Pasterova
11,
11000
Belgrade,
Serbia
cCenter
for
Food
Analysis,
Belgrade,
Serbia
dDepartment
of
Microbiology
and
Immunology,
University
of
Belgrade,
Faculty
of
Pharmacy,
Belgrade,
Serbia
eDepartment
of
Biomedical,
Surgical
and
Dental
Sciences,
University
of
Milan,
Via
Festa
del
Perdono
7,
20122
Milan,
Italy
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
12
June
2015
Received
in
revised
form
14
August
2015
Accepted
12
October
2015
Keywords:
Antibacterial
activity
Cytotoxicity
a
b
s
t
r
a
c
t
The
antibacterial
activity
of
cyclohexane,
dichlormethane,
methanol
and
aqueous
extracts
of
tinder
fun-
gus
Fomes
fomentarius
(L.)
Fr
(Polyporaceae)
was
tested
against
9
bacterial
strains
(Staphylococcus
aureus,
Staphylococcus
epidermidis,
Micrococcus
luteus,
Bacillus
subtilis,
Enterococcus
feacalis,
Escherichia
coli,
Kleb-
siella
pneumoniae,
Pseudomonas
aeruginosa,
Salmonella
abony),
as
well
as
against
10
different
clinical
isolates
and
one
reference
strain
of
Helicobacter
pylori.
Minimal
inhibitory
concentrations
(MICs)
of
all
extracts
against
9
bacterial
strains
were
in
the
range
of
125–250
␮g/ml.
Methanol
and
aqueous
extracts
showed
significant
activity
against
H.
pylori
with
MIC
values
between
4–32
␮g/ml.
Also,
cytotoxicity
of
tested
extracts
was
significant.
Aqueous
extract
was
the
most
active
one
against
HeLa
cells
with
an
IC50
8.31
±
1.18
␮g/ml
and
N87
cells
with
IC50 64.46
±
3.13
␮g/ml
without
any
activity
against
normal
MRC5
cell
line
(>
200
␮g/ml).
©
2015
Elsevier
B.V.
All
rights
reserved.
1.
Introduction
Fomes
fomentarius
(L.)
Fr.,
Polyporaceae,
tinder
fungus
is
a
woody,
perennial
fungus,
large
in
size
which
develops
as
a
parasite
or
saprophyte
on
the
beech
(Fagus
sylvatica
L.)
and
other
decidu-
ous
species.
It
is
a
white
root
fungus
causing
heart
rot
of
the
wood
(Vˇ
etrovsk ´
y
et
al.,
2011).
In
Germany,
people
were
using
fruiting
bodies
in
ritual
smok-
ing
ceremonies,
but
F.
fomentarius
was
also
used
as
a
medicine.
Tinder
fungus
was
widely
used
as
a
styptic
by
surgeons
and
den-
tists
(Grienke
et
al.,
2014).
In
traditional
Chinese
medicine,
it
was
used
for
the
treatment
of
oral
ulcer,
gastroenteric
disorder,
inflam-
mations
and
various
cancers
(Chen
et
al.,
2008).
Some
studies
have
shown
that
F.
fomentarius
has
hypoglycemic,
anti-nociceptive,
anti-inflammatory,
anti-infective
and
anti-tumor
activities
(Seniuk
et
al.,
2011;
Yang
et
al.,
2008;
Park
et
al.,
2004).
Several
classes
of
metabolites
were
identified:
primary
metabolites
i.e.
proteins,
polysaccharides
(␤-glucans),
polysaccharide–protein
complexes,
and
secondary
metabolites
such
as
triterpene
glycosides
(1444001-
∗Corresponding
author.
Fax:
+381
11
3972840.
E-mail
address:
ktatjana@pharmacy.bg.ac.rs
(T.
Kundakovi´
c).
94-8,
tuberoside),
esters
and
lactones
(fungisterollinoleate,
betulin
28-O-acetate),
alcohols
(7-ergostenol,
␤-sitosterol),
aldehyde
and
ketones
(protocatechualdehyde;
(22E)-ergosta-7,22-dien-3-one),
organic
acids,
benzofurans
(paulownin),
coumarins
(daphnetin),
volatile
components
(Grienke
et
al.,
2014).
Mushrooms
are
an
important
source
of
nutritive
and
bioactive
compounds
with
different
biological
activities.
Modern
science
is
trying
to
establish
medicinal
properties
of
traditionally
used
mush-
rooms
and
plants
using
modern
scientific
methods
and
techniques.
The
most
important
compounds
with
clinically
beneficial
activity
are
␤-glucans.
In
vitro
studies
have
suggested
that
large
molecu-
lar
weight
or
particular
␤-glucans
can
directly
activate
leukocytes,
stimulating
their
phagocytic,
cytotoxic
and
antimicrobial
activities
including
the
production
of
reactive
oxygen
and
nitrogen
interme-
diates
(Akramiene
et
al.,
2007).
They
can
be
used
in
conjunction
with
chemotherapy
(Wasser,
2002).
Their
cytotoxic,
antimicrobial
and
imunostimulating
activities
make
them
promising
in
cancer
treatment,
including
H.
pylori
infection
associated
cancer
(Zhu
et
al.,
2015).
Extra-
and
intracellular
polysaccharides
from
F.
fomentarius
showed
in
vitro
anti-proliferative
effect
on
SGC
7091
human
gastric
carcinoma
cells
and
extracellular
polysaccharides
showed
power
to
promote
secretion
of
TNF-␣
IFN-␥
and
IL-2
and
to
enhance
mouse
humoral
immune
response
(Chen
et
al.,
2011;
Gao
et
al.,
2009).
http://dx.doi.org/10.1016/j.indcrop.2015.10.030
0926-6690/©
2015
Elsevier
B.V.
All
rights
reserved.

M.
Kolundˇ
zi´
c
et
al.
/
Industrial
Crops
and
Products
79
(2016)
110–115
111
The
second
class
of
biologically
important
compounds
from
mushrooms
are
polyphenols,
especially
phenolic
acids.
Polyphe-
nolic
compounds
possess
different
biological
activities,
including
antioxidant,
antimicrobial,
cytotoxic
and
some
other
activities
(Heleno
et
al.,
2015).
The
polypore
mushroom
F.
fomentarius
was
not
the
subject
of
extensive
research
concerning
its
chemical
and
biological
activities,
but
bearing
in
mind
previous
studies
and
its
traditional
use,
it
could
be
an
important
source
of
antioxidant,
antimicrobial
and
cyto-
toxic
compounds.
That
was
the
reason
why
we
tested
antimicrobial
activity
of
F.
fomentarius
extracts
of
different
polarity,
especially
the
antibacterial
activity
against
gram
negative
H.
pylori
which
has
not
yet
been
extensively
tested
and
connected
with
cytotoxic
activity
against
human
gastric
carcinoma
cell
lines
and
other
cell
lines.
H.
pylori
was
the
first
bacterial
species
proven
to
cause
cancer
and
was
classified
as
a
group
I
carcinogen
by
the
International
Agency
for
Research
on
Cancer
(Testerman
and
Morris,
2014).
Additionally,
we
tested
nutritive
value,
beta-glucane
content
and
total
fibres
in
F.
fomentarius,
as
well
as
total
phenolics
content
and
antiradical
activity.
2.
Materials
and
methods
2.1.
Reagents
and
chemicals
All
organic
solvents
were
HPLC
grade
and
were
purchased
from
J.T.
Baker
(Deventer,
The
Netherlands).
Coomassie
Brilliant
Blue
G-250
was
Bio-Rad
(Richmond,
CA,
USA).
Assay
kit
K-YBGL
(Megazyme)
was
used
for
determination
of
beta
glucan
content.
Reagents
for
antimicrobial
and
cytotoxic
activities
testing
were:
Mueller–Hinton
broth,
antibiotic
supplement,
Columbia
Blood
Agar
Base
(Difco,
USA),
Wilkins
Chalgren
broth
with
10%
horse
serum
(Seromed,
Biochrom
KG,
Berlin),
glycerol
(Merck,
USA),
yeast
extract
(Difco,
USA),
RPMI-1640
medium
(Sigma,
St.
Louis,
MO,
USA),
10%
heat
inactivated
fetal
bovine
serum
FBS
(GIBCO,
Thermo
Fisher
Scientific,
Life
Technologies).
All
other
reagents
were
pur-
chased
from
Sigma
(St.
Louis,
MO).
2.2.
Mushroom
material
Fruiting
bodies
of
mushroom
F.
fomentarius
were
collected
on
mountain
Avala
(Serbia)
in
May
2013.
Before
extraction,
the
mush-
room
had
been
dried
at
room
temperature
and
pulverized
in
a
laboratory
mill.
The
voucher
specimen
was
deposited
at
the
Faculty
of
Pharmacy,
Department
of
Pharmacognosy
(No.
23).
2.3.
Preparation
of
extracts
Extraction
was
carried
out
with
solvents
of
different
polarity
to
obtain
cyclohexane,
dichloromethane,
methanol
and
aque-
ous
extracts.
20
g
of
powdered
drug
was
submerged
into
200
ml
of
cyclohexane
and
extraction
went
on
for
two
days
at
room
temperature,
including
occasional
shaking.
The
same
procedure
was
repeated
with
dichloromethane
and
methanol
(successively).
Aqueous
extract
was
prepared
with
1
g
of
dried,
powdered
mush-
room
and
120
ml
of
distilled
water.
Extraction
was
carried
out
under
heating
in
a
water
bath,
for
30
min
at
100 ◦C
with
occa-
sional
shaking.
The
solvents
were
evaporated
under
low
pressure
and
dried
to
obtain
C6H12 (0.34
g),CH2Cl2(0.11
g),
CH3OH
(0.14
g),
H2O
(0.45
g)
extracts.
2.4.
Determination
of
nutritional
value
The
sample
material
was
analysed
for
the
percentage
of
mois-
ture,
proteins,
fats,
dietary
fiber
and
ash.
All
values
were
calculated
on
a
dry
weight
(dw)
of
mushroom.
Nutritional
value
was
deter-
mined
by
using
the
procedures
described
by
the
Association
of
Official
Analitycal
Chemists
(AOAC)
(1990).
The
moisture
and
ash
contents
were
estimated
using
gravimetric
methods
after
drying
on
105 ◦C,
and
incineration
at
550 ◦C,
respectively.
The
crude
protein
content
was
determined
by
the
Kjeldahl
method
and
the
nitrogen
factor
used
for
protein
calculation
was
6.25,
while
the
crude
fats
were
determined
using
the
extraction
procedure
on
Soxhlet
appa-
ratus,
with
petroleum
ether
as
a
solvent,
after
the
treatment
with
HCl.
Dietary
fibre
was
determined
by
gravimetric
method
(Prosky
et
al.,
1992).
Total
carbohydrates
were
calculated
as
the
residual
difference
after
subtracting
protein,
ash,
moisture,
total
fibre
and
crude
fat
content.
Total
energy
was
determined
by
the
calculation
of
energy
values
of
carbohydrate,
fat,
protein
and
fibre.
Energy,
kcal
=
9(crude
fat
content,
g)
+
4(protein
content,
g
+
carbohydrate
content,
g)
+
2(fibre
content,
g).
2.5.
Determination
of
ˇ-glucans
␤-glucans
were
determined
by
a
spectrophotometric
method
using
the
enzymatic
assay
kit
K-YBGL
(Megazyme
International
,
Ireland).
The
assay
was
performed
according
to
the
instruction
manual
of
the
kit
producer.
Content
of
␤-glucans
was
deter-
mined
from
dried,
powdered
mushroom
(100
mg)
and
it
was
calculated
indirectly
from
the
difference
between
the
content
of
total
glucans
and
␣-glucans.
Total
glucans
((1
→
3),
(1
→
6)-␤-d-
glucan,
(1
→
3)-␤-d-glucans
and
␣-glucans)
were
solubilised
in
concentrated
(37%;
10
N)
hydrochloric
acid
and
then
extensively
hydrolysed
by
1.3
N
HCl
at
100 ◦C
for
2
h.
Hydrolysis
to
d-glucose
was
completed
by
incubation
with
a
mixture
of
highly
puri-
fied
exo-(1
→
3)-␤-glucanase
and
␤-glucosidase.
Measurement
of
␣-glucans
was
performed
after
the
hydrolysis
with
mixture
of
amy-
loglucosidase
and
invertase.
The
content
of
total
and
␣-glucans
was
determined
spectrophotometrically,
measuring
absorbance
at
510
nm.
2.6.
Determination
of
total
dietary
fibre
Total
fibre
was
determined
by
enzymatic–gravimetric
method,
as
described
by
(Prosky
et
al.,
1992).
The
method
requires
phos-
phate
buffer,
pH
6.0
and
the
following
enzymes:
heat
stable
␣-amylase,
protease
and
amyloglucosidase.
Heat
stable
␣-amylase
depolymerises
starch,
protease
depolymerises
and
dissolves
pro-
teins,
while
amyloglucosidase
converts
starch
into
glucose.
The
residue
is
filtered,
washed
with
78–95%
ethanol,
and
acetone,
dried
and
weighed.
One
duplicate
is
analysed
for
protein
and
the
other
is
incubated
at
525 ◦C
to
determine
ash.
The
total
dietary
fibre
is
the
weight
of
the
filtered
and
dried
residue
less
the
weight
of
the
protein
and
ash.
2.7.
Total
phenolics
content
The
content
of
total
phenolics
in
methanol
(FFM)
and
aqueous
(FFA)
extracts
of
F.
fomentarius
was
determined
spectrophotomet-
rically,
as
described
by
Singleton
and
Rossi,
(1956).
The
100
␮l
of
sample
was
mixed
with
750
␮l
of
the
diluted
Follin–Ciocalteu
reagent
(FC-reagent)
(1
ml
FC
reagent
and
10
ml
distilled
water).
After
5
min,
750
␮l
of
60
g/l
Na2CO3solution
was
added
to
each
sample.
Firstly,
the
mixtures
were
shaken
and
then
incubated
for
90
min
in
the
dark
at
room
temperature.
Absorbance
was
measured
at
725
nm.
As
a
blank,
100
␮l
of
solvent
was
used.
Series
of
gallic
acid
dilutions
(1–10
mg/ml)
were
made,
the
absorbance
was
mea-
sured
and
the
calibration
curve
was
constructed.
The
content
of

112
M.
Kolundˇ
zi´
c
et
al.
/
Industrial
Crops
and
Products
79
(2016)
110–115
polyphenols
was
expressed
as
%
of
the
standard
substance
(gallic
acid
GA).
2.8.
DPPH
radical
scavenging
The
ability
of
methanol
extract
to
scavenge
free
radicals
was
measured
using
DPPH
test
(Kundakovi´
c
et
al.,
2008).
In
short,
test
samples
in
dilution
series
(10–110
␮l)
were
dissolved
in
methanol,
so
the
total
volumen
became
2
ml.
Into
these
solutions,
0.5
ml
DPPH
solution
(0.5
mM
DPPH
in
methanol)
was
added.
The
mixtures
were
incubated
in
a
dark
place
at
room
temperature.
After
30
min,
the
absorbance
of
the
solution
was
measured
at
517
nm
and
the
percent
of
inhibition
of
DPPH
radical
was
calculated.
Each
measurement
was
done
in
duplicate.
0.5
mM
DPPH
solution
in
methanol
was
used
as
control
and
the
sample
dissolved
in
methanol
was
used
as
blank.
The
results
are
expressed
as
a
percentage
of
inhibition-EC50 value
(concentration
of
the
test
sample
that
reduced
50%
of
the
DPPH
radicals).
2.9.
Antimicrobial
activity
For
antimicrobial
testing,
the
cyclohexane
(FFC),
dichloromethane
(FFD),
methanol
(FFM)
and
aqueous
(FFA)
extracts
were
used.
The
screening
of
antimicrobial
activity
of
the
tested
extracts
samples
was
evaluated
using
nine
different
laboratory
strains
of
bacteria—gram
positive:
Staphylococcus
aureus
(ATCC
25923),
Staphylococcus
epidermidis
(ATCC
12228),
Micrococcus
luteus
(ATCC
9341),
Bacillus
subtilis
(ATCC
6633),
Ente-
rococcus
feacalis
(ATCC
29212)
and
gram
negative:
Escherichia
coli
(ATCC
25922),
Klebsiella
pneumoniae
(NCIMB
9111),
Pseudomonas
aeruginosa
(ATCC
27853),
Salmonella
abony
(ATCC
13076).
The
broth
microdilution
method
was
used
to
determine
minimal
inhibitory
concentrations
(MICs)
of
tested
extracts
according
to
Clinical
and
Laboratory
Standards
Institute
(CLSI,
2005).
These
tests
were
performed
in
Müller–Hinton
broth.
Test
strains
were
suspended
in
medium
to
give
a
final
density
of
2.0
×
106cfu/ml
for
bacteria.
Samples
of
extracts
were
dissolved
in
dimethyl
sulfoxide
(DMSO)
in
concentrations
of
1.0
mg/ml.
Serial
dilutions
of
the
stock
solutions
in
broth
medium
were
prepared
in
a
microtitre
plate
(96
wells).
The
MIC
is
defined
as
the
lowest
concentration
of
the
extract
at
which
the
microorganism
does
not
demonstrate
visible
growth.
All
microbial
tests
were
performed
in
duplicate
and
two
positive
growth
controls
were
included.
The
MICs
of
ampicillin–amikacin
were
determined
in
parallel
experiments.
2.10.
Anti-H.
pylori
activity
Ten
clinical
strains
of
H.
pylori
isolated
from
patients
with
duodenal
ulcer
or
gastritis
were
tested.
All
of
these
strains
had
known
resistance
patterns.
Three
were
resis-
tant
to
clarithromycin—CLR
(MIC>1
␮g/ml),
one
resistant
to
metronidazole—MNZ
(MIC>16
␮g/ml),
two
resistant
to
both
MNZ
and
CLR,
and
four
susceptible
to
CLR
and
MNZ.
H.
pylori
ATCC
43.504
was
used
as
control
in
each
daily
assay
session.
H.
pylori
bacte-
ria
were
grown
on
Columbia
agar
base
supplemented
with
10%
horse
serum
and
0.25%
bacto
yeast
extract
under
microaerophilic
conditions
(5%
O2,
10%
CO2,
85%
N2)
in
85%
humidity
in
an
incu-
bator
at
37 ◦C.
Fresh
plates
were
started
from
glycerol
stocks
and
subcultured
every
48
h.
2.10.1.
Broth
microdilution
assay
MICs
were
determined
by
broth
microdilution
test
performed
by
using
Mega-CellTM
RPMI-1640
medium
containing
3%
fetal
calf
serum
(FCS),
as
previously
described
(Sisto
et
al.,
2009).
Two
fold
dilutions
of
each
antimicrobial
extracts
were
prepared
in
96-well
microtiter
plates.
An
inoculum
equivalent
to
1
McFarland
standard
was
prepared
in
Wilkins
Chalgren
broth
and
diluted
in
MegaCell
RPMI-1640
medium.
Each
well
was
inoculated
with
H.
pylori
at
a
final
concentration
of
approximately
5
×
105CFU/well.
The
plates
were
incubated
at
37 ◦C
under
microaerophilic
conditions
(5%
O2,
10%
CO2,
85%
N2in
a
gas
incubator).
The
plates
were
examined
visually
after
72
h
of
incubation.
2.11.
Cytotoxicity
assay
Cytotoxicity
of
four
extracts
of
F.
fomentarius
was
tested
against
human
epithelial
cervical
cancer
cells
HeLa,
NCI-N87
human
gastric
carcinoma
cell
lines
and
healthy
MRC-5
human
embryonic
lung
fibroblast
cell
lines.
2.11.1.
Cell
lines
Human
epithelial
cervical
cancer
cells
HeLa,
NCI-N87
human
gastric
carcinoma
cells,
and
normal
MRC-5
human
embryonic
lung
fibroblast
cell
lines
were
obtained
from
the
American
Type
Culture
Collection
(Manassas,
VA,
USA).
All
cancer
cell
lines
were
main-
tained
in
the
recommended
RPMI-1640
medium
supplemented
with
10%
heat-inactivated
(56 ◦C)
fetal
bovine
serum,
l-glutamine
(3
mM),
streptomycin
(100
mg/ml),
penicillin
(100
IU/ml)
and
25
mM
HEPES
and
adjusted
to
pH
7.2
by
bicarbonate
solution.
Cells
were
grown
in
a
humidified
atmosphere
of
95%
air
and
5%
CO2at
37 ◦C.
2.11.2.
Treatment
of
cell
lines
Stock
solutions
(30
mg/ml)
of
the
extracts,
were
made
in
DMSO,
and
dissolved
in
corresponding
medium
to
the
required
working
concentrations.
Target
neoplastic
HeLa
(2000
cells
per
well),
NCI-
N87
(10,000
cells
per
well)
and
MRC-5
(5000
cells
per
well)
were
seeded
into
96-well
microtiter
plates.
After
the
cell
adherence,
24
h
later,
five
different
double
diluted
concentrations
of
investi-
gated
compounds
were
added
to
the
wells.
For
the
cells
control
only
nutrient
medium
was
added.
Final
concentrations
achieved
in
treated
wells
were
200,
100,
50,
25
and
12.5
␮g/ml
for
investi-
gated
mushroom
extracts.
The
final
concentration
of
DMSO
solvent
never
exceeded
0.5%,
which
is
non-toxic
to
cells.
Nutrient
medium
was
RPMI
1640
medium,
supplemented
with
l-glutamine
(3
mM),
streptomycin
(100
mg/ml),
and
penicillin
(100
IU/ml),
10%
heat
inactivated
(56 ◦C)
FBS
and
25
mM
HEPES
and
was
adjusted
to
pH
7.2
by
bicarbonate
solution.
The
cultures
were
then
incubated
for
72
h.
2.11.3.
Determination
of
cell
survival
The
effects
of
tested
extracts
on
cancer
cells
survival
were
determined
by
MTT
test,
according
to
Mosmann
(1983)
with
modi-
fication
by
Ohno
and
Abe
(1991),
72
h
upon
addition
of
the
extracts,
as
it
was
described
previously
(Mosmann,
1983;
Ohno
and
Abe,
1991).
Briefly,
20
␮l
of
MTT
solution
(5
mg/ml
PBS)
were
added
to
each
well.
Samples
were
incubated
for
further
4
h
at
37 ◦C
in
5%
CO2and
humidified
air
atmosphere.
Then,
100
␮l
of
10%
SDS
were
added
to
dissolve
the
insoluble
product
formazan,
resulting
from
the
conversion
of
the
MTT
dye
by
viable
cells.
The
number
of
viable
cells
in
each
well
was
proportional
to
the
intensity
of
the
absorbance
of
light
(A),
which
was
then
read
in
an
ELISA
plate
reader
at
570
nm.
To
obtain
the
percentage
of
the
cell
survival
(%),
A
of
a
sample
with
cells
grown
in
the
presence
of
various
concentrations
of
extracts
was
divided
with
control
optical
density
(the
A
of
con-
trol
cells
grown
only
in
nutrient
medium)
and
multiplied
by
100.
It
was
implied
that
A
of
the
blank
was
always
subtracted
from
A
of
the
corresponding
sample
with
target
cells.
IC50 concentration
is
defined
as
the
concentration
of
an
agent
inhibiting
cell
survival
by
50%,
compared
with
a
vehicle-treated
control.
All
experiments
were
done
in
triplicate.
The
cis-DDP
was
used
as
positive
control.

M.
Kolundˇ
zi´
c
et
al.
/
Industrial
Crops
and
Products
79
(2016)
110–115
113
Table
1
Nutritional
value
of
dried
F.
fomentarius.
Parameter
Amount
Moisture
(g/100
g)
12.28
±
0.13
Ash
(g/100
g) 2.33
±
0.06
Fat
(g/100
g)
1.45
±
0.19
Proteins
(g/100
g)
12.96
±
0.17
Carbohydrates
(g/100
g)
2.06
±
0.33
Total
dietary
fibre
(g/100
g)
68.92
±
0.81
␤-glucan
(g/100
g)
20.32
±
0.29
Energy
(kcal/100
g)
210.99
±
1.75
Energy
(kJ/100
g) 881.95
±
7.31
Table
2
Content
of
total
phenolics
in
methanol
and
aqueous
extracts
of
Fomes
fomentarius
and
anti-DPPH
activity.
Extract
Total
phenolicsbDPPHc
FFMa26.41
5.86
FFAa13.18
n.t.d
aFF—F.
fomentarius
extracts
(M—methanol,
A—aqueous).
bTotal
phenolics
were
expressed
as
the
percentage
(%)
of
gallic
acid
(GA).
cAnti-DPPH
activity
was
expressed
as
EC50 (␮g/ml).
dn.t.—not
tested.
3.
Results
and
discussion
3.1.
Nutritional
value
of
dried
F.
fomentarius
The
health
benefits
of
mushrooms
as
food
have
been
recognized
for
hundreds
of
years,
because
they
contain
low
levels
of
calo-
ries
and
fat,
but
are
rich
in
dietary
fibres
and
proteins
(Cheung,
2010).
F.
fomentarius
is
a
lignified,
non-edible
mushroom
signifi-
cant
because
of
its
biological
activities.
The
nutritional
parameters
confirmed
the
following:
the
total
fiber
was
68.92
g/100
g
and
␤-glucan
20.32
g/100
g,
total
carbohydrates
were
2.06
g/100
g,
pro-
teins
12.96
g/100
g,
and
fats
only
1.45
g/100
g.
The
results
of
nutritional
parameters
are
presented
in
Table
1.
3.2.
ˇ-glucans
content
Interesting
compounds
extracted
from
mushrooms
are
␤-
glucans,
glucose
polymers,
classified
according
to
their
interchain
linkage
as
␣-
and
␤-glucans
(Barsanti
et
al.,
2011).
Previous
studies
have
shown
that
content
of
␤-glucans
in
some
mushrooms
is
in
the
range
of
3.2–53
g/100
g
of
dry
matter
(Manzi
and
Pizzoferrato,
2000;
Lee
et
al.,
2011).
Nowadays,
one
of
the
most
important
source
of
␤-glucans
is
Shiitake
mushroom
(Lentinus
edodes)
which
contains
22
g/100
g
of
dry
matter
(Manzi
and
Pizzoferrato,
2000;
Rop
et
al.,
2009).
The
content
of
␤-glucans
in
the
dried
mushroom
F.
fomen-
tarius
was
20.32
±
0.3
g/100
g
and
it
is
potentially
a
good
source
of
these
polysaccharides.
3.3.
Total
phenolic
content
and
antioxidant
activity
Polyphenols
have
the
ability
to
become
donors
of
hydrogen
atoms
and
to
form
phenoxy
radicals
that
are
more
stable
due
to
delocalization
of
electrons
and
existence
of
multiple
resonant
forms.
That
makes
them
good
antioxidants.
Up
to
now,
the
only
polyphenol
identified
in
F.
fomentarius
is
coumarin
daphnetin.
In
this
test,
we
determined
the
content
of
total
phenolics
in
methanol
and
aqueous
extracts
of
F.
fomentarius.
The
results
for
the
content
of
total
phenolics
are
presented
in
Table
2.
The
methanol
extract
exhibited
DPPH
scavenging
activity
with
an
EC50 =
5.86
␮g/ml.
The
result
was
significant,
when
compared
with
the
activity
of
well-known
antioxidants
such
as
vitamin
C
and
tocopherol
(EC50
4.22
␮g/ml
and
62.43
␮g/ml,
respectively)
(Kosani´
c
et
al.,
2012).
Likewise,
our
results
were
in
accordance
with
the
results
of
another
group
of
authors
(Karaman
et
al.,
2014).
3.4.
Antimicrobial
activity
The
results
of
antimicrobial
activity
of
the
tested
extracts
are
shown
in
Table
3.
Based
on
the
results,
it
is
apparent
that
the
MICs
were
in
the
range
from
125–250
␮g/ml.
Also,
methanol
and
aque-
ous
extracts
showed
higher
antimicrobial
activity
probably
due
to
the
polar
components
that
they
contain
(Petrovi´
c
et
al.,
2014).
The
tested
extracts
had
lower
antimicrobial
activity
when
com-
pared
to
semi-synthetic
antibiotics
such
as
ampicillin
and
amikacin.
It
is
interesting
to
note
that
all
extracts
showed
a
considerable
antimicrobial
activity
against
gram
negative
bacteria
E.
coli
(MIC
125
␮g/ml).
The
previous
systematic
researches
of
antimicrobial
activities
of
F.
fometarius
are
negligible,
so
this
is
the
first
report
on
the
significant
antimicrobial
activity
of
tinder
fungus
extracts
against
9
bacterial
strains.
The
results
of
(Zhao
et
al.,
2013)
have
shown
weak
antimicrobial
activity
of
isolated
phenyl–ethanediols
from
fruiting
bodies
of
F.
fomentarius.
The
presence
of
polysaccharides
in
the
polar
extracts
was
considered
very
important
because
pre-
viuos
study
of
(Seniuk
et
al.,
2011)
has
shown
that
water-soluble
melanin–glucan
complex
(contained
80%
of
melanins
and
20%
of
␤-
glucans)
completely
inhibited
the
growth
of
C.
albicans.
Methanol
and
aqueous
extracts
were
rich
in
total
polyphenols
content.
Previ-
ous
studies
have
shown
high
content
of
polyphenols
in
mushrooms
with
strong
antimicrobial
activity
(Alves
et
al.,
2013).
Hence,
the
high
polyphenols
and
␤-glucan
(Zhu
et
al.,
2015)
content
in
tinder
fungus
could
be
connected
with
significant
antimicrobial
activity.
3.5.
Anti
H.
pylori
activity
The
results
regarding
anti-Helicobacter
activity
of
mushrooms
were
very
scarce,
besides
the
fact
that
the
mushrooms
and
fungi
could
be
very
promising
sources
of
antimicrobial
compounds.
Quinolone
and
phthalide
compounds
from
the
actinomycete
Pseudonocardia
sp.
CL38489
and
from
the
basidiomycete
Phane-
rochaete
velutina
CL6387
were
very
active
and
specific
against
H.
pylori
with
low
MIC
values
up
to
0.1–5
ng/ml
(Dekker
et
al.,
1997,
1998)
Ethylacetate
fraction
of
Hericium
erinaceus
extract
was
active
against
9
clinical
isolates
of
H.
pylori,
with
MIC
values
ranging
between
62.5–250
␮g/ml,
and
a
MBC
(minimal
bactericide
concen-
tration)
value
of
200
␮g/ml
for
the
strain
ATCC
43.504
(Shang
et
al.,
2013).
Also,
in
the
same
experiment
fourteen
basidiomycetes
were
tested
by
agar
diffusion
method
and
for
twelve
ethanol
extracts
MIC
values
were
lower
than
3
mg/ml.
Triterpenoid
methylantcinate
B
from
the
mushroom
Antrodia
camphorata
showed
anti-H.
pylori
activity
and
inhibited
H.
pylori-associated
inflammation
in
human
gastric
epithelial
AGS
cells
(Geethangili
et
al.,
2010).
Only
one
clinical
study
was
performed
and
showed
negative
results
when
2
g/day
of
submerged
cultivated
Tremella
mesenterica
mycelium
was
administered
to
patients
with
H.
pylori
infection
for
10
days
(Lachter
et
al.,
2012).
Polysaccharides
from
algae
and
plants
were
studied
against
H.
pylori
infection
because
of
their
antiadhesive
properties
and
since
they
prevent
H.
pylori
from
binding
to
porcine
gastric
mucin,
but
without
direct
bactericidal
effect
(Ayala
et
al.,
2014).
Therefore,
the
polysaccharide
fraction
from
the
mushrooms
could
have
valuable
role
to
prevent
or
to
treat
H.
pylori
infection
as
a
source
of
polysac-
charides
because
mushrooms
are
frequently
included
in
everyday
diet.
Anti-H.
pylori
activity
ranged
from
4
␮g/ml
for
methanol
extract
of
F.
fomentarius
to
256
␮g/ml
for
cyclohexane
extract.
Polar
extracts
(aqueous
and
methanol)
were
more
active
than
non
polar
cyclohexane
extract
with
very
low
MIC
values
ranging
from
4
to

114
M.
Kolundˇ
zi´
c
et
al.
/
Industrial
Crops
and
Products
79
(2016)
110–115
Table
3
Antimicrobial
activity
of
Fomes
fomentarius
extracts
(MIC,
␮g/ml).
Bacterial
strains
MIC
(␮g/ml)
FFCaFFDaFFMaFFAaAmpicillin
Amikacin
Staphylococcus
aureus
(ATCC
25923)
250
125
125
125
0.5
n.t.b
Staphylococcus
epidermidis
(ATCC
12228)
250
250
125
125
1.5
n.t.
Micrococcus
luteus
(ATCC
9341)
250
250
125
125
2.0
n.t.
Bacillus
subtilis
(ATCC
6633) 250
250
125
125
1.8
n.t.
Enterococcus
feacalis
(ATCC
29212)
250
250
125
125
n.t.
n.t.
Escherichia
coli
(ATCC
25922)
125
125
125
125
2.0
1.5
Klebsiella
pneumoniae
(NCIMB
9111)
250
125
250
125
2.8
2.0
Pseudomonas
aeruginosa
(ATCC
27853)
250
125
125
125
n.t.
2.5
Salmonella
abony
(ATCC
13076)
250
250
125
250
n.t.
n.t.
aFF—F.
fomentarius
extracts
(C—cyclohexane,
D—dichloromethane,
M—methanol,
A—aqueous).
bn.t.—not
tested.
Table
4
Anti-Helicobacter
pylori
activity
of
cyclohexane,
dichlormethane,
methanol,
and
aqueous
extract
of
Fomes
fomentarius
presented
in
␮g/ml.
Helicobacter
pylori
strains MIC
(␮g/ml)
FFCaFFDaFFMaFFAaMNZbCLRc
1.
128
128
8
32
S
R
ATCC
43504
256
128
16
32
R
S
2.
256
128
8
32
S
S
3.
128
64
8
32
S
S
4.
128
64
4
16
S
R
5.
256
128
8
32
R
S
6.
256
256
4
16
S
S
7.
128
64
8
16
S
R
8.
128
64
8
16
R
R
9.
128
64
8
32
R
R
10.
256
128
16
32
S
S
aFF—F.
fomentarius
extracts
(C—cyclohexane,
D—dichloromethane,
M—methanol,
A—aqueous).
bMNZ—metronidazole.
cCLR—clarithromycin;
S—sensitive;
R—resistant.
8
␮g/ml
for
methanol
extract
(FFM).
MNZ
resistant
strains
of
H.
pylori
(ATCC
43.504,
5,
8,
9)
were
very
sensitive
to
the
methanol
extract
(MIC
8
and
16
␮g/ml),
while
both
methanol
and
aqueous
extracts
were
very
active
against
CLR-
resistant
strains
(1,
4,
7,
8,
9)
with
MIC
values
ranging
from
4
␮g/ml
(methanol
extract;
strain
4)
to
32
␮g/ml
(aqueous
extract;
strains
1
and
9).
Both
MNZ-
and
CLR-
resistant
strains
(8–9)
were
very
sensitive
to
the
methanol
extract
with
MIC
value
of
8
␮g/ml.
The
results
are
presented
in
Table
4.
Very
active
methanol
extract
contains
significant
amount
of
polyphenolic
compounds
which
could
be
active
against
H.
pylori.
Recent
studies
have
shown
beneficial
effects
of
several
classes
of
polyphenols
against
H.
pylori
infection
and
peptic
ulcer
disease
(Farzaei
et
al.,
2015).
Only
one
study
considered
anti-Helicobacter
activity
of
water-soluble
melanin–glucan
complex
and
showed
antimicrobial
effect
against
H.
pylori
identical
to
erithromycine
and
stronger
than
other
tested
antibiotics
(Seniuk
et
al.,
2011).
For
the
moment,
it
is
not
possible
to
make
definite
conclusion
which
compounds
in
methanol
and
aqueous
extracts
are
responsible
for
strong
anti-H.
pylori
activity.
Further
studies
regarding
chemistry
and
bioactivity
of
isolated
compounds
from
complex
extracts
will
give
more
information
about
active
constituents
of
F.
fomentarius.
3.6.
Cytotoxic
activity
The
cyclohexane,
dichlormethane,
methanol
and
aqueous
extracts
were
tested
against
two
malignant
and
one
healthy
cell
line.
The
results
are
presented
in
Table
5.
Overall
F.
fomentarius
extracts
showed
good
cytotoxic
activ-
ity.
Aqueous
extract
showed
the
best
selectivity
and
had
no
effect
on
normal
MRC5
cells
while
exhibiting
strong
cytotoxic
effect
on
Table
5
Concentrations
of
Fomes
fomentarius
tested
extracts
that
induced
a
50%
decrease
in
HeLa,
MRC5
and
N87
cell
survival
(expressed
as
IC50 (␮g/ml)).
Compounds
IC50 (␮g/ml)
HeLa
N87
MRC5
FFCa25.10
±
7.16
>
200
>
200
FFD
51.87
±
5.33
>
200
111.59
±
17.06
FFM
20.10
±
1.08
184.56
±
1.44
>
200
FFA
8.31
±
1.18
64.46
±
3.13
>
200
cis-DDPb3.18
±
0.29
12.31
±
0.74
13.45
±
0.62
aFF—F.
fomentarius
extracts
(C—cyclohexane,
D—dichlormethane,
M—methanol,
A—aqueous).
bUsed
as
a
positive
control.
malignant
cells,
both
HeLa
and
N87.
Methanol
extract
followed
with
strong
effect
on
HeLa
cells,
moderate
effect
on
N87
and
no
effect
on
MRC5.
Dichlormethane
and
cyclohexane
extracts
had
sig-
nificant
effect
on
HeLa,
but
no
effect
on
N87.
Only
dichlormethane
extract
affected
normal
MRC5
cells,
while
cyclohexane
extract
had
no
effect.
These
results
suggest
that
the
extracts
exhibit
a
specific
mechanism
of
action
that
affects
certain
types
of
cancer
cells
and
are
not
toxic
to
non-malignantly
transformed
cells.
The
high
selec-
tivity
of
these
extracts
toward
malignant
cells,
put
them
in
order
of
candidates
for
further
investigations.
Chen
et
al.,
(2011)
have
shown
that
polysaccharide
fraction
and
ethanol
extract
of
F.
fomentarius
in
doses
of
0.05–1.6
mg/L
inhib-
ited
the
growth
of
gastric
carcinoma
cells
SGC-7901
and
MKN-45
in
dose
dependent
manner.
The
ethanol
extract
was
more
active
against
tested
carcinoma
cells.
Promising
result
from
these
authors
was
that
normal
gastric
cells
GES-1
were
less
sensitive
than
the
carcinoma
cells,
but
were
also
affected
by
tested
agents.
Kim
et
al.,
(2014)
further
analysed
the
mechanism
that
under-
lies
F.
fomentarius
ethanol
extract
cytotoxicity
which
included
an
increase
of
inducible
NO
synthase
levels,
production
of
tumor
necrosis
factor-␣,
interleukin
(IL)-1␤,
and
IL-6
in
RAW
264.7
cells.
4.
Conclusion
According
to
the
obtained
results,
all
four
tested
extracts
were
active
against
10
tested
bacteria,
including
10
strains
of
H.
pylori.
Also,
DPPH
radical
scavenging
activity
was
significant
and
cor-
related
with
the
content
of
total
polyphenolics
in
the
methanol
extract.
All
four
extracts
were
active
against
tested
HeLa
cell
line
with
significant
activity
and
methanol
and
water
extracts
were
also
active
against
N87
gastric
carcinoma
cell
line.
Extracts
were
not
cytotoxic
to
MRC-5
healthy
cells
(except
dichlormethane
extract)
which
could
be
promising
for
further
research
of
preventive
role
of
F.
fomentarius
extracts
in
vivo
and
research
into
the
determination
of
the
active
compounds’
chemical
structure.

M.
Kolundˇ
zi´
c
et
al.
/
Industrial
Crops
and
Products
79
(2016)
110–115
115
Conflict
of
interest
The
authors
declare
that
this
article
content
has
no
conflict
of
interest.
Acknowledgment
The
authors
are
grateful
to
the
Ministry
of
Education,
Science
and
Technological
Development
of
Republic
of
Serbia
for
financial
support
(Grant
No.
173021
and
TR
34012).
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