Phytomedicine 15 (2008) 512–519
In vitro cytostatic and immunomodulatory properties of the medicinal
mushroom Lentinula edodes
C. Israilidesa,?, D. Kletsasb, D. Arapogloua, A. Philippoussisc, H. Pratsinisb,
A. Ebringerova ´d, V. Hrˇı´balova ´e, S.E. Hardingf
aBiotechnology Laboratory, National Agricultural Research Foundation (NAGREF), 1, Sof. Venizelou St.,
14123 Lycovrissi, Athens, Greece
bLaboratory of Cell Proliferation & Ageing, National Center of Scientific Research (N.C.S.R.) ‘‘Demokritos’’,
Institute of Biology, 15310 Athens, Greece
cNAGREF, Institute of Agricultural Engineering, Laboratory of Edible Fungi, 61 Democratias St.,
13561 Ag. Anargyri, Athens, Greece
dInstitute of Chemistry, Slovak Academy of Sciences, Du ´bravska ´ cesta 9, 845 38 Bratislava, Slovakia
eNational Institute of Public Health, Sˇroba ´rova 48, 100 42 Prague, Czech Republic
fUniversity of Nottingham, MCMH, School of Biosciences, Sutton Bonington, Leicestershire LE 12 5RD, UK
Lentinula edodes, known as ‘‘shiitake’’ is one of the widely used medicinal mushrooms in the Orient. Antitumour
activity of extracts of this mushroom has been widely demonstrated in animals and humans. However, this activity was
shown to be host mediated and not by direct cytotoxic activity to cancer cells. This study demonstrates cytotoxic and
cell growth inhibitory (cytostatic) effect of aqueous extracts of the mushroom on MCF-7 human breast
adenocarcinoma cell line using an MTT cytotoxicity assay. Such effect was demonstrated with fruit body and
mycelial extracts, the difference being that there was no significant suppression on normal cells with the latter.
Furthermore mycelial extracts did not induce any cytostatic effect in both cancer and normal cell lines based on a
DNA synthesis assay. The significant suppression of the proliferation of cancer cells was reflected by the comparatively
low IC50values and the simultaneous higher respective values on normal fibroblast cells. The immunostimulatory
activity of both fruit body and mycelial extracts was tested by the lymphocyte transformation test (LTT), which is
based on the capacity of active immunomodulators to augment the proliferative response of rat thymocytes to T
mitogens in vitro. Both fruit body and mycelial preparations were able to enhance the proliferation of rat thymocytes
directly and act as co-stimulators in the presence of the T-mitogen PHA. Interestingly both extracts, similarly to
zymosan showed SIcomit/SImitratios of about 2, indicating adjuvant properties. Overall L. edodes aqueous extracts
have demonstrated direct inhibition of the proliferation of human breast cancer cells in vitro and immunostimulatory
properties in terms of mitogenic and co-mitogenic activity in vitro.
r 2007 Elsevier GmbH. All rights reserved.
Keywords: Lentinula edodes; Cancer; Cytotoxic; Cytostatic; Antitumour; Mitogenic and comitogenic activity
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0944-7113/$-see front matter r 2007 Elsevier GmbH. All rights reserved.
?Corresponding author. Tel.: +302102842676; fax: +302117508893.
E-mail address: firstname.lastname@example.org (C. Israilides).
Medicinal mushroom extracts have been considered
as important remedies for the prevention and treatment
of many diseases for thousands of years especially in the
Orient (Israilides and Philippoussis, 2003; Kidd, 2000;
Wasser and Weis, 1999). A plethora of medicinal effects
has been demonstrated for many traditionally used
mushrooms including antibacterial, antiviral, antifun-
gal, antitumour and immuno-potentiating activities
(Hobbs, 2003; Ooio and Liu, 1999). Among the various
bioactive components which have been demonstrated to
be most effective as antitumor and immunomodulatory
agents are polysaccharides and polysaccharopeptides.
Lentinula edodes is the source of many therapeutic
polysaccharide macromolecules among which the ones
with proven pharmacological effects are lentinan, LEM
and KS-2. Lentinan is a high molecular weight (about
one million) homopolysaccharide in a triple helix
structure, with linear chains consisting of (1-3)-b-D-
glucopyranosyl (Glcp) residues with two b-(1-6)-
linked Glcp branchings for every five b-(1-3)-Glcp
residues (Aoki, 1984). LEM is a mycelial extract
preparation of L. edodes harvested before the cap and
stem grow. It is a heteroglycan–protein conjugate
containing 24.6% protein and 44% sugars, comprising
mostly pentoses as well as glucose and smaller amounts
of galactose, mannose and fructose (Iizuka, 1986;
Sugano et al., 1982). It also contains nucleic acid
derivatives, B complex vitamins, ergosterol, eritadenine
(an anticholesteremic amino acid), and water-soluble
lignins (Sugano et al., 1985). KS-2 is a peptide–poly-
saccharide complex. The comparison of fruit body and
mycelial extracts was carried out for the following
1. The production of fruit bodies and mycelium in
L. edodes as well as in many other medicinal mush-
rooms, comprise the two main production methods
(Wasser and Weis, 1999). The production of fruit
bodies does not always guarantee a consistent product
while the mycelial growth in fermenters under
vigorously controlled conditions gives improved pro-
duct purity and reproducibility.
2. The main antitumor polysaccharide in L. edodes fruit
bodies is a single compound, lentinan. On the other
hand there are many different active compounds in
mycelia which have been demonstrated to have
‘‘antitumor’’ properties. This provides the opportu-
nity for enhanced activity from crude extracts of fruit
bodies or mycelium. The mechanism of antitumor
activity of either lentinan, which is the main
biologically active compound in L. edodes fruit
bodies, or the mycelial extract has not been fully
elucidated, but it has been reported as host mediated
by activating the host’s immune responses and not
attacking cancer cells directly (Aoki, 1984; Meiqin
et al., 1998). Therefore there is a need for comparison
of the two kinds of extracts in an effort to investigate
and differentiate tumor selective cytotoxicity.
Since many of the compounds, which are found in
L. edodes, have been shown to act synergistically
(Yamasaki et al., 1989), it is worth testing the cytotoxic
and/or cell growth inhibitory effects of the whole
mushroom and mycelium extract rather than its indi-
vidual components. This principle (synergy) is compa-
tible with similar natural biological products like the
essential oils, which allow the achievement of strong
effects when used as whole products, while quenching
or nullifying potential unwanted side-effects by the
presence of individual components.
The objectives of this project were to investigate the
cytotoxic and cell growth inhibitory effect on normal
and cancer cell lines of active Lentinula edodes extracts
produced from both the mushroom and mycelia as well
as their immunostimulatory activity with the ‘in vitro’
comitogenic rat thymocytes test (lymphocyte transfor-
mation test, LTT).
Materials and methods
The strain of L. edodes (Berk.) Pegler used in this
study, was originated from China and registered in the
fungal culture collection of the Edible Fungi Laboratory
of NAGREF with the code number AMRL 118. It was
selected for its phenotypic characteristics concerning
productivity and quality. The culture substrate prepara-
tion and growing procedure for sporophore production
has been previously described (Philippoussis et al.,
2007). The culture was maintained on a 2% potato
dextrose agar (PDA, Merk) for routine culture and
Mycelia were grown in a submerged liquid fermenta-
tion in a Bioengineering L1523, 7liter bench fermentor.
The initial pH was 5.50, temperature 281C, and the
aeration was 10liter/min. The substrate composition
was (w/v): malt extract 0.3%, yeast extract 0.3%,
peptone 0.3% and glucose 1.0%. The inoculum,
500ml, was grown in the same medium and the duration
of fermentation was 3 days. The fruiting bodies and
mycelia were dried by lyophilization and powdered. All
extracts were stored at ?401C.
Methanol and distilled water extracts from mush-
rooms and mycelia of L. edodes were prepared to an
initial concentration of 100mg/ml. The extracts were
incubated for 2h at room temperature under continuous
shaking. They were centrifuged for 30min at 1500g and
the supernatant was passed through a 0.2mm filter.
Samples were further diluted with plain culture medium
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C. Israilides et al. / Phytomedicine 15 (2008) 512–519513
(Dulbecco’s minimal essential medium (DMEM)) to the
defined concentrations as indicated.
Human breast adenocarcinoma cell line MCF-7 and
human normal fibroblasts from a 30 year-old healthy
volunteer were cultured in DMEM supplemented with
antibiotics and 10% fetal bovine serum (FBS) and they
were subcultured using trypsin-citrate (0.25–0.3%,
respectively) solution. In the incubation chamber the
gas mixture consisted of 5% CO2 and 95% air.
Furthermore, the humidity was adjusted to 85%, so as
to diminish evaporation of the culture medium and the
consequent changes to its osmolarity that could have
been detrimental to the cultured cells. Cells were tested
periodically and found to be mycoplasma-free. All cell
culture media were from Gibco–BRL.
For the assessment of the cytotoxic and cytostatic
activities of L. edodes extracts cells were seeded in 96-
well flat-bottomed microplates at a density of approxi-
mately 5000cells/well, in DMEM 10% FBS. After 18h
to ensure cell attachment, serial dilutions of the extracts
in culture medium were added and incubated for 24 or
48h. Then, cytotoxicity and DNA synthesis rate were
determined using the MTT assay and tritiated thymidine
In testing the cytotoxic or cytostatic effects of
different substances on cancer cells the ideal control is
always an issue. Such control ideally could be normal
epithelial cells originating from a neighboring area with
healthy tissue of the same patient. However, this is not
always feasible, especially regarding commercially avail-
able cancer cell lines. On the other hand, tumors in vivo
are surrounded by stroma, thus understanding the effect
of the studied substances on normal fibroblasts is
equally important. In this study we have chosen to use
a commercially available human cancer cell line, MCF-
7, which is one of the most popular cell lines in the
literature, because this would facilitate replication as
well as comparisons with similar work. As a control we
have used normal human stroma fibroblasts. Further-
more, MCF-7 cells and fibroblasts grow in the same
medium, while normal epithelial cells require special
serum-free, chemically-defined media for their culture,
which would introduce further unequal parameters in
The assay detects the reduction of MTT [3-(4,5-
Sigma] by mitochondrial dehydrogenase to blue for-
mazan product, which reflects the cell viability, as
well as the actual cell number of the culture. Following a
48-h-incubation of the cells with the extracts, the culture
medium was replaced with MTT dissolved at a final
concentration of 1mg/ml in serum-free, phenol-red-free
DMEM (Biochrom KG), for a further 4-h-incubation.
Then, the MTT-formazan was solubilized in isopropa-
nol and the optical density was measured at a
wavelength of 550nm and a reference wavelength of
690nm. The results were assessed based on IC50, the
concentration that reduced by 50% the optical density
of treated cells with respect to untreated controls.
DNA synthesis assay
In this assay, the rate of novel DNA synthesis in the
cell nuclei is monitored, based on the incorporation of
radiolabelled thymidine. Following a 24-h-incubation
of the cells with the extracts, fresh culture medium
was added along with [3H]-thymidine (0.15mCi/ml, 25
Ci/mmol) (Amersham, Buckinghamshire, UK). After
incubation for further 14h, the radioactivity incorpo-
rated in DNA was counted, by fixing the cells with
trichloroacetic acid (10% w/v), washing copiously under
running tap water and air-drying. Then DNA was
solubilised by addition of 0.3 N NaOH–1% SDS and
the lysates were subjected to scintillation counting.
Lymphocyte transformation test (LTT)
For the immunostimulatory activity testing (LTT)
test, samples were dissolved in physiological solution
(8.5g NaCl/1000ml d H2O) to a 2% (w/v) concentration
and mixed by a magnetic stirrer, until the suspension
was homogeneous. Then it was centrifuged at 3000rpm
and the supernatant was sterilized (at 1201C for 20min)
and was used for the test.
LTT was performed according to a slightly modified
method elaborated for muramyl glycopeptides (Iribe
and Koba, 1984), which do not stimulate thymocyte
proliferation markedly. On the other hand, several
polysaccharides were reported to be directly mitogenic
for rat thymocytes (Ebringerova ´ et al., 2002; Hroma ´ d-
kova ´ et al., 2003). Rat thymocytes (strain Wistar, males
weighing about 200g) in RPMI-1640 medium, HEPES
modification (Sigma) supplemented with 5% fetal calf
serum (Sigma) were cultivated at 1.5?106cells in 0.2ml
per well either without or with 25mg/ml phytohaemag-
glutinin (PHA). Test compounds were added at final
concentrations ranging from 3–2000mg/ml. After 72h
cultivation, thymocyte proliferation was measured by
incorporation of [3H]-thymidine expressed in counts per
minute (cpm). In each of at least two experiments,
means of the counts per minute (cpm) for each set of 4
replica experiments were used to calculate the stimula-
tion indices (SI). The direct mitogenic effect of the
compounds tested was expressed as: SImit¼ mean cpm
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C. Israilides et al. / Phytomedicine 15 (2008) 512–519514
for test compound/mean cpm for control. The comito-
genic effect was expressed as: SIcomit¼ (mean cpm for
PHA+test compound)/mean cpm for PHA. The even-
tual contamination with endotoxin was checked by
cultivation of the thymocytes in presence of polymyxin
B, which inhibits stoichiometrically its biological effect
including the mitogenic activity. As positive control the
commercial immunomodulator zymosan – a particulate
b-glucan from baker’s yeasts (Likospol Ltd., Bratislava,
Slovakia) was used. Zymosan gave a fine suspension of
non-sedimenting particles. Polymyxin B sulfate was
from Wellcome (UK) and PHA from Murex Biotech
Chemical and FTIR analyses
These were performed on lyophilised samples pre-
pared from the fruit body and mycelium of L. edodes as
described in a previous paper (Israilides and Philip-
Multiple extracts from both fruiting bodies and
mycelia were prepared and analysed on multiple
occasions. The results for the cytotoxicity assay are
presented as the mean of three independent experiments
performed in quadruplicate wells. Differences from
control cultures were considered significant when
pp0.01 (Student’s t-test). In Figs. 2 and 3 asterisks
above data points indicate significant differences from
In the LTT test, the means of SI in repeated testing of
the extracts were evaluated by analysis of variance
(ANOVA), and calculations were done by the EP16
Results and discussion
Chemical and FTIR analyses
The analytical characteristics of the extracts isolated
from the fruit body and mycelium of L. edodes are listed
in Table 1. The glucose content, indicating the presence
of glucan-type polysaccharides, is higher in the case of
the mycelium, whereas, the mannoglycan content was
twice as high in the fruit body. Mannose, galactose,
glucose as well as the minor sugars are components of
mannoglycans and mannoproteins of fungal cell walls
(Kim et al., 2003; Peng et al., 2003).
The FT-IR spectra (Fig. 1) of lentinan samples
showed the presence of considerable amount of proteins
indicated by the absorption bands c at 1660cm?1
(nC ¼ O, amide I) and d at 1550cm?1(dNH, amide II).
The bands a at 2850–3000cm?1(nCH2and nCH3) and b
at 1745cm?1and 1702cm?1(nCO), related to vibrations
of alkyl chain, ester and free carboxyl groups, respec-
tively, are indicative of lipids, and are particularly
intense in the fruit body. The absorption bands in the
mid-infrared region 1200–800cm?1are useful for the
identification of polysaccharides with different structure
and composition (Kac ˇ ura ´ kova ´ et al., 2000). In contrast
to the fruit body, the spectral pattern of the mycelium
in this region showed some similarity to that of the
b-glucan from S. cerevisiae (Hroma ´ dkova ´ et al., 2003).
L. edodes fruit body water extracts at 10 to 800mg/ml
exhibited significant dose-dependent inhibitory effects
on the proliferation of MCF-7 cells (Fig. 2) with more
than 90% suppression, and an average IC50 of
73714mg/ml. In normal cells under the same range of
extract concentration, there was a similar inhibitory
trend. However the number of normal cells always
remained higher at the same extract concentration
compared to cancer cells at all concentrations tested,
with an average IC50of 140730mg/ml.
Similar inhibitory effects were found with L. edodes
after incubation with mycelial extract (Fig. 3). The
difference was that MCF -7 proliferation was much less
suppressed in the case of mycelia, showing a much
higher average IC50value (11,23674856mg/ml). On the
other hand, the mycelia extract seem to induce a
suppressive effect on the proliferation of normal
fibroblast cells only at high doses (over 10mg/ml) with
an IC50of 15,49072310mg/ml). The data in Figs. 2 and
3 appear to suggest a biphasic effect. However these
differences were not statistically significant.
The same effect was also verified with a DNA
synthesis assay, which proved that the cytostatic effect
of this fruit body extract was much more potent on
cancer cells, compared to normal cells (IC50119723 vs
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Analytical data of L. edodes mushroom and
Klason lignin (wt%)
Neutral sugar composition (rel. wt%)
C. Israilides et al. / Phytomedicine 15 (2008) 512–519515
251774, respectively). However, this was not shown
with mycelial extracts where there was an absence of any
significant cytostatic effect, reflected by the high IC50
values (41000mg/ml) in both cancer and normal cell
lines (Table 2). Zhang et al., (2005) who used lentinan in
an MTT test to study cell proliferation in 5–180 sarcoma
tumor cells showed less than 50% inhibition even with
concentrations up to 500mg/ml. Although our test
results are expressed in a different way if appears that
the crude extracts of the fungus have higher activity.
The IC50in either mushroom or mycelia extracts in
absolute methanol was 42500 in all cases, indicating
that methanolic extracts contrary to aqueous ones did
not have any inhibitory (cytotoxic) effect on MCF-7
cancer cell line.
Mitogenic response of T-cells
The mitogenic and comitogenic activities of fruit body
and mycelium determined by the ‘in vitro’ rat thymocyte
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Absorbance (% of untreated)
0.01 0.10 1.0010.00 100.00
Fig. 2. Effects of L. edodes fruit body water extracts on the
proliferation of MCF-7 and normal cell lines (one representa-
tive experiment). *Statistically significant.
Absorbance (% of untreated)
* Statistically significant
Fig. 3. Effects of L. edodes mycelia extracts on the prolifera-
tion of MCF-7 and normal cell lines (one representative
experiment). *Statistically significant.
DNA synthesis (IC50)aof Lentinus edodes aqueous extracts on
MCF-7 and fibroblast cell lines
Concentration producing 50% cytostatic effect of
L. edodes extractIC50(mg/ml)
aIC50values were expressed as the mean7SD, determined from the
results of DNA synthesis assay in triplicate experiments.
600 80010001200 14001600 18002000 25003000 3500
Fig. 1. FT-IR spectra (in KBr) of lentinan samples and the immunologically active b-glucan from Saccharomyces cerevisiae. The
arrows indicate absorption bands typical of lipids (a and b), and proteins (c and d).
C. Israilides et al. / Phytomedicine 15 (2008) 512–519 516
test are shown in Table 3. Both samples, containing
glucan as the main polysaccharide component, showed
dose-dependent stimulatory activities, similar to the
immunomodulator zymosan. The mean values of cpm
for control cultures without any stimulant was 1084
(1024–1143) and for the PHA-stimulated cultures 1320
Both the fruit body and mycelium showed dose-
dependent stimulatory activities. The lowest concentra-
tion, at which the mitogenic and comitogenic responses
showed a significant increase in comparison to the
controls, appeared with fruit body and mycelium at
200 and 600mg/m, respectively. Maximum stimulation
indices were achieved at 1500mg/ml. At higher doses, the
decrease of the responses indicated an inhibitory effect.
This has been observed also for zymosan (Kardosˇova ´
et al., 2003) and other polysaccharides (Ebringerova ´
et al., 2002; Ebringerova ´ et al., 2003) The biological
responses might also be affected by contaminants
present in the tested preparations. The presence of
polymyxin B in the cultivation medium excluded the
influence of potential endotoxin contamination. The
results suggest that both samples were able to enhance
the proliferation of rat thymocytes directly and act as
co-stimulators in the presence of the T-mitogen, PHA.
The SIcomit/SImit ratio was ?2. In accord with the
interpretation of the comitogenic test the ratio indicated
adjuvant properties of the tested extracts similar to
zymosan (Iribe and Koba, 1984; Rovensky ` et al., 1990).
There are not significant differences in the immuno-
enhancing effect of both extracts. Interestingly, the poly-
saccharides isolated from the mycelium of Ganoderma
tsugae have been reported to possess the same antitumor
activity as those from the L. edodes fruiting bodies (Zhang
et al., 1994; Wang et al., 1993). More pronounced
immunostimulatory effect in the comitogenic rat thymo-
cyte test was reported with the particulate b-glucan from
S. cerevisiae (Hroma ´ dkova ´
structurally related to the b-glucan lentinan (Aoki, 1984;
Surenjav et al., 2006; Zheng et al., 2005) but is only
et al., 2003), which is
sparsely branched. As the b-1,3-glucan (lentinan) has been
suggested to be the dominating antitumor and immunos-
timulatory component in L. edodes, it could be suggested
that the low immunostimulatory activity of the fruit body
and mycelium is due to the low glucose content, which
comprises only about 60 and 70% (on dry weight basis),
respectively, of the sugar components. However, the
mannoglycans and protein-containing mannans have been
reported to posses biological activities as well (Kriz ˇ kova ´ et
al., 2001; Tizard et al., 1989), but their biological response
in the comitogenic rat thymocyte test have not been
In using L. edodes extracts in comparison to lentinan
one has to consider the fact that L. edodes extracts are
given as dietary supplements and lentinan as a drug.
Although lentinan is considered fairly safe in doses used
by i.v. administration (0.001–30mg/kg) for 5–6 weeks,
the long term clinical use of lentinan is not recom-
mended and there have been reports of its toxicity
(Aoki, 1984; Mori, et al., 1977). Also lentinan has very
little oral activity and may cause gastrointestinal
disturbances and allergic reactions if taken orally.
On the other hand mycelial extracts of L. edodes like
LEM have the advantage to be used per os for long term
as dietary supplements, without reported side effects
even in massive doses (over 50mg/day for a week). In
the future the L. edodes extracts have the potential of
being used more widely than lentinan.
Aqueous extracts of Lentinula edodes can significantly
suppress the proliferation of cancer cell line MCF-7 in
vitro. This is reflected by the comparative low IC50
values and the simultaneous higher IC50 values on
L. edodes mushroom water extracts are more cyto-
toxic than mycelial aqueous extracts. Methanolic
extracts of either mushroom or mycelia of L. edodes
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Table 3.Mitogenic and comitogenic activities of the lentinan samples
Sample20 60 200600 15002000
a,bThe first lowest concentration which gives significant increase of SI mit (p-values 0.802, 0.075) in comparison to the control: SINE 1084
c,dThe first lowest concentration which gives significant increase of SI comit (p-values 0.011, 0.017) in comparison to the control: PHA 1320
C. Israilides et al. / Phytomedicine 15 (2008) 512–519517
do not exhibit any inhibitory (cytostatic) effect on
MCF-7 cancer cell line.
Both fruit body and mycelial extracts are able to
enhance the proliferation of rat thymocytes directly and
act as co-stimulators in the presence of the T-mitogen,
PHA. The SIcomit/SImitratio about 2, indicated adjuvant
properties of the tested extracts.
This paper supports the direct cytostatic/cytotoxic
action of the L. edodes extracts on cancer cells, which is
in parallel action with its host-mediated antitumour
Furthermore it was demonstrated that L. edodes
can act as an immunomodulator to augment the
proliferative response of rat thymocytes to T mitogens
in vitro, indicating another mechanism for immunosti-
mulatory activity. Overall there seems to be a ther-
apeutic advantage in using L. edodes extracts orally
administered instead of a single substance like lentinan
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