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Phenolic Compounds Present in Medicinal Mushroom Extracts Generate

Reactive Oxygen Species in Human Cells In Vitro

Article  in  International Journal of Medicinal Mushrooms · January 2008

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 International Journal of Medicinal Mushrooms, 10(1):1–13 (2008)

Phenolic Compounds Present in Medicinal Mushroom

Extracts Generate Reactive Oxygen Species in
Human Cells In Vitro
Song Wei, Johannes P. F. G. Helsper, & Leo J. L. D. Van Griensven*
Plant Research International B.V., Wageningen UR, Wageningen, The Netherlands

* Address all correspondence to Leo J. L. D. Van Griensven, Department of Bioscience, Plant Research International, Wageningen
University and Research Centre, POB 16, 6700 AA Wageningen, The Netherlands; leo.vangriensven@wur.nl

ABSTRACT: Hot-water extracts of the higher Basidiomycetes Agaricus bisporus (J. Lge) Imbach,
A. brasiliensis S. Wasser et al., Coprinus comatus (O.F. Mull.) Pers., Ganoderma lucidum (W. Curt.:
Fr.) P. Karst., and Phellinus linteus (Berk. et Curt.) Teng were made, and the resulting polysaccharide
mixtures were purified by DEAE–cellulose chromatography and ethanol precipitation. The extracts
were noncytopathic. A. bisporus, A. brasiliensis, and G. lucidum strongly generated reactive oxygen
species (ROS) in human PBMCs and K 562 cells. C. comatus and Ph. linteus extracts had low ROS-
generating capacity. In A. bisporus extract, two different classes of polysaccharides were found. The
colorless polysaccharide of MW > 250 kDa caused no intracellular ROS generation; acid hydrolysis
followed by HPLC analysis showed it to consist of only glucose residues, thus being a pure glucan.
The light brown polyphenol/polysaccharide complex of MW 75–200 kDa was found to strongly
generate ROS. ROS generation by medicinal mushrooms could therefore be due to the presence of
polyphenols rather than of glucan alone. A. bisporus polysaccharide extract showed a saturation level
of ROS generation at 2 mg mL–1. Generation appeared to continue in the presence of polysaccharide
for more than 14 hours. Purified colorless polysaccharides of C. comatus and Ph. linteus showed no
ROS generation in K562 cells; G. lucidum and A. brasiliensis polysaccharides could not completely
be cleaned of phenolic compounds and remained active. Competitive inhibition of ROS generation by
laminarin was not observed for any of the polysaccharide extracts, suggesting that polyphenol/glucan
complexes isolated from higher Basidiomycetes mushrooms are able to generate ROS without binding
to a dectin receptor.

KEY WORDS: β-glucan, polyphenols, ROS, dectin-1, innate immunity, immunomodulatory effects,
polysaccharides, medicinal mushrooms, Agaricus bisporus, Agaricus brasiliensis, Coprinus comatus,
Ganoderma lucidum, Phellinus linteus

ABBREVIATIONS

Afu: arbitrary fluorescence unit; AU: arbitrary unit; °Brix: degrees breaking index; BSA: bovine serum albumin;
CR3: complement receptor type 3; DCF-DA: dichlorofluorescein diacetate; DMSO: dimethyl sulfoxide; ELISA:
enzyme linked immunosorbent assay; FcR: Fc chain (of immunoglobulin) receptor; FCS: fetal calf serum;
FPLC: fast protein liquid chromatography; HPLC: high performance liquid chromatography; HMW: high
molecular weight; IL-10: interleukin 10; kDa: kilodalton; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; PAMP: pathogen-associated molecular patterns; PBMC: peripheral blood mono-
nuclear cell; PRR: pattern recognition receptor; ROS: reactive oxygen species; Rt: retention time; TFA:
trifluoroacetic acid; TLR: Toll-like receptor.

1521-9437/08/$35.00
© 2008 by Begell House, Inc. 1

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 SONG WEI ET AL.
I. INTRODUCTION
Medicinal mushrooms such as Agaricus brasiliensis
S. Wasser et al. (= A. blazei Murrill s. Heinem.),
Coprinus comatus (O. F. Müll) Pers., Trametes (=
Coriolus) versicolor (L.: Fr.) Lloyd, Ganoderma
lucidum (W. Curt.: Fr.) P. Karst., Lentinus edodes
(Berk.) Singer, Phellinus linteus (Berk.: Curt.) Teng,
and many others traditionally have been used as a
health food or supplement for the prevention and
cure of a range of diseases, including atherosclero-
sis, cancer, chronic hepatitis, and diabetes. The
preventive and therapeutic effects of those mush-
rooms and their components have been well docu-
mented in mouse and rat model systems and in
cancer cell lines.1,2 This has led to important knowl-
edge of the effects of mushroom extracts and of
their modes of action. In many cases these involve
immunomodulatory effects in their various forms:
proinflammatory or adjuvant activity, apoptosis,
mostly with cytokines and responses of cells of the
immune system as mediators.
It is generally accepted that mushroom extracts
contain multiple components, each of which may have
its own biological or medicinal effects. Mushroom-
derived β-glucans have immunomodulatory effects;
they affect both innate and acquired immunity. In
addition, mushroom triterpenoids and small proteins
have been found to influence immune response.
In all these effects, the influence on oxygen
metabolism both in vitro and in vivo may be a
common denominator. Redox reactions strongly in-
fluence the majority of physiological processes. Sev-
eral higher Basidiomycetes contain antioxidant phenolic
compounds.3 Agaricus bisporus (J. Lge) Imbach has
been reported to also harbor the prooxidative 4-
(hydroxymethyl)phenyl radical.4 Phenolic compounds
are able to activate the intracellular formation of
reactive oxygen species (ROS),5,6 which play an
important role in the prevention of infection. ROS are
involved in killing potential intracellular pathogens.7
Molecular damage elicited by ROS in normal cells
challenges the cells to repair the damage. Abnormal
or affected cells are activated toward the cell death
program—that is, either apoptosis or direct death.8
As anticipated, receptors play a crucial role in
activation of ROS after stimulation with fungal or
2 International Journal of Medicinal Mushrooms
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other elicitors. Human phagocytes recognize poten-
tial pathogens as bacteria and fungi by a wide variety
of pattern recognition receptors (PRRs) such as Toll-
like receptors (TLR), complement receptor type 3
(CR3), dectin-1, and scavenger receptors, and sig-
naling through the PRRs leads to activation of the
innate immune system. Recognition triggers actin-
dependent phagocytosis, induces fungal killing, pro-
duces cytokines and chemokines, and initiates the
development of adaptive immunity. Some of these
phenomena, such as the respiratory burst that leads
to the production of ROS, are only induced by
specific receptors, such as the β-glucan receptor
dectin-1, which was shown to synergize with TLR-2
to augment proinflammatory cytokine responses.9,10
However, others11 showed that the role of TLR-2/
dectin-1 and TLR-4 in host responses to Cryptococ-
cus neoformans was only very limited, suggesting
important functions for other pathogen-associated
molecular pattern (PAMP ) receptors such as man-
nose and complement receptors.
β-glucans may therefore induce proinflammatory
processes12 and also cause direct death of affected
cells through the induction of ROS and the down-
stream activation of caspases, leading to elevations of
mitochondrial membrane potential and apoptosis.13
Tumor cell lines appear particularly sensitive to ROS-
induced apoptosis.14
Next to its role as a general enhancer of innate
immunity, intracellular ROS generation may also
have adverse proinflammatory consequences. In mast
cells high levels of ROS contribute to degranulation
and to potential harmful effects in patients suffering
from inflammatory disease.15,16 This forms an addi-
tional motive to study the properties of the different
fractions of fungal cell wall extracts commonly sold
as medicinal mushroom glucans.
Because ROS generation appears to play a key
role in the innate immune defence system and its
sequential effects, we decided to study the influence
of medicinal mushroom extracts on ROS generation
in human PBMCs and in the leukemia cell line K562.
We also tried to separately characterize different
components—that is, glucans and phenolic com-
pounds—of A. bisporus, A. brasiliensis, C. comatus,
G. lucidum, and Ph. linteus extracts in terms of their
ROS-generating activity.
 PHENOLIC COMPOUNDS IN MEDICINAL MUSHROOM EXTRACTS GENERATE REACTIVE OXYGEN SPECIES
II. MATERIALS AND METHODS
A. Materials
RPMI-1640 medium with L-glutamine and sodium
bicarbonate was from Sigma Chemical Corp (St.
Louis, MO, USA). Serum supreme and fetal bovine
serum (South American origin) were obtained from
BioWhittaker (Walkersville, MD, USA). Zymosan
(Z4250) and 2′7′-dichlorofluorescin-diacetate
(D6883) were obtained from Sigma. Trammune®
brand of PSK, a proteoglycan concentrate from
Trametes versicolor, was obtained from Vertos
Healthcare Pty Ltd. (NSW, Australia).
B. Cells
Human PBMCs were prepared from buffy coats of
human blood supplied by the Netherlands Blood
Transfusion Service (Sanquin, Nijmegen, The Nether-
lands). Briefly, buffy coats were diluted 1× with
Dulbecco’s PBS and centrifuged for 15 minutes at
2500 × g over a layer of Histopaque 1077 (Sigma).
PBMCs were carefully collected at the interphase
and washed with PBS. Cells were counted and used
directly or were slowly frozen to –70°C at 5–10 × 106
mL–1 in RPMI-1640 containing 10% fetal calf serum
(FCS) and 20% DMSO. Cells were then stored in
liquid nitrogen for later use. Thawing was done
quickly at 37°C, after which the cells were washed
with PBS, counted, and cultured in a humidified
incubator with 5% CO2 at 37°C in RPMI 1640
containing 10% FCS and 100 U mL–1 penicillin +
100 µg mL–1 streptomycin.
The myelogenous erythroid leukemia cell line
K56217 was cultured in the same medium as the
PBMCs.
C. Mushrooms
Fresh stems of cultivated A. bisporus and whole
fruitbodies of A. brasiliensis were obtained from
Innerlife B.V. (Venlo, The Netherlands). C. comatus
S 435 dried fruitbodies were obtained from the
(former) Mushroom Experimental Station (Horst,
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The Netherlands). Dried hot water extract of wild
type Ph. linteus was kindly provided by Amazing
Grace (Bangkok, Thailand). G. lucidum spore ex-
tract Grade A was obtained from Fujian Xianzhilou
Biological Science and Technology Co. Ltd. (P.R.
China). Saccharomyces cerevisiae (BakersYeast,
Bruggeman, The Netherlands) was grown in malt
extract medium with 2% glucose and collected by
centrifugation.
D. Extraction of Polysaccharides
Mushroom crude extract was made from fresh or
dried tissue. The tissue was homogenized in 2 vol-
umes of deionized water in a Waring blender for 2
minutes at high speed and then incubated at 90–
100°C for 4–5 days. Solids were removed by cen-
trifugation at 10,000 g for 15 minutes. The resulting
fluid was concentrated by evaporation at 100°C to
35° Brix and stored at 4°C.
Polysaccharides were precipitated from crude
extract by the addition of 2 volumes of cold 96%
ethanol and collected by centrifugation at 10,000 g
for 15 minutes. The precipitate was then dissolved in
deionized water, reprecipitated by the addition of
ethanol to 70%, and stored at 4°C until further use.
Alternatively, the precipitate was dried in vacuo,
mortarized, and stored in a dry, cool place.
E. Detection of Intracellular ROS
To detect intracellular ROS, 2′,7′-dichlorofluorescin
diacetate (DCF-DA) was used. DCF-DA diffuses
into the cell and is hydrolyzed by intracellular esterases
to 2′,7′-dichlorofluorescein. This nonfluorescent
fluorescin analogue can be oxidized to highly fluores-
cent 2′,7′-dichlorofluorescein by intracellular oxi-
dants.18 Incubations were done in 4-fold in Greiner
black 96-well plates and repeated several times. The
fluorescence was measured in a Tecan fluorescence
spectrophotometer at excitation/emission = 485/535
nm. The results are shown as arbitrary fluorescence
units (afu) at 535 nm (or as a percentage change from
baseline values). Values were corrected for
autofluorescence—that is, reaction mixtures were
3
 SONG WEI ET AL.
incubated with fluorescein but without cells—and
also for the endogenous activity of the cells used.
Routinely 4 × 105 cells mL–1 were incubated in PBS
in the presence of 100 µg mL–1 polysaccharide and
25 µg mL–1 DCF-DA, unless stated otherwise.
F. Cytotoxicity Assay
The MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide] assay,19 which reflects mito-
chondrial dehydrogenase activity and accumulates
dark blue formazan crystals in healthy cells, was used
to test cytotoxicity. The assay was carried out in
Greiner 96-well plates. For this assay 20 µL of a
0.4% MTT solution was added to each well with
100 µL cell suspensions. After 2–4 hours, cells were
centrifuged and dissolved in DMSO; the violet color
of the formazan product, which is proportional to the
number of living cells, was then quantified in a Biorad
ELISA plate reader at 595 nm.
G. Column Chromatography
Size exclusion chromatography was done on a 90 ×
1.5 cm column of Sepharose 200, using MilliQ water
as an eluant. Fractions of 5–10 mL were collected
using a LKB-FPLC apparatus. Superose 6 was used
in a column of 30 × 1.0 cm; 0.5–1.0 mL fractions
were collected. For calibration in the estimation of
molecular weights of polysaccharides, blue dextran
(MW = 2 × 106 Da) and a range of dextrans of
Leuconostoc mesenteroides were used. For proteins
thyroglobulin (669 kDa), catalase (232 kDa), aldo-
lase (158 kDa), and RNase A (13.7 kDa) were used
as calibrants. Anion exchange chromatography was
performed on DEAE-cellulose (Sigma D0909) with
a linear gradient of 0.0–1.0 M NaCl in 0.05 M tris-
HCl (pH 7.4) as the mobile phase.
H. Protein Determination
Protein concentrations were determined by
Bradford’s20 method using bovine serum albumin
(BSA) (Sigma A7030) as the standard. Total protein
4 International Journal of Medicinal Mushrooms
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content is expressed as mg of BSA equivalents per
gram of dry weight. If necessary, protein was mea-
sured by Lowry assay.21
I. Total Phenolics Determination
Total phenolic content was determined using Folin-
Ciocalteu reagent, as described in detail by Ainsworth
and Gillespie,22 using gallic acid as the standard.
Total phenolics are expressed as milligrams of gallic
acid equivalents per gram of dry weight.
J. Carbohydrate Determination
Carbohydrate concentrations were determined by
the phenol–sulphuric acid method, using D-glucose as
the standard.23 Total carbohydrate is expressed as
mg of glucose equivalents per gram of dry weight.
K. Analysis of Monosaccharide
Composition
For analysis of monosaccharide composition, a 5 µL
aliquot of the glucan solution or suspension was
diluted in 0.3 mL 2M trifluoroacetic acid (TFA) and
hydrolyzed overnight at 100°C. The solution was
evaporated to dryness at 40°C under a N2-stream.
Residual TFA was removed by two evaporation
cycles in 0.5 mL methanol, as above. The final
residue was dissolved in 0.5 mL water and analyzed
for monosaccharides on a Dionex HPLC equipped
with pulsed amperometric electrochemical gold de-
tector. A PA1-column (250 × 4 mm), fitted with a
PA-1 guard column (10 × 32 mm), was used as the
stationary phase and 0.020 M NaOH as the mobile
phase at 1.0 mL min–1 (isocratic elution). Quantifi-
cation of monosaccharides was performed on the
basis of external standards.
III. RESULTS
Polysaccharide extracts were prepared from mush-
room fruit bodies through prolonged hot water ex-
 PHENOLIC COMPOUNDS IN MEDICINAL MUSHROOM EXTRACTS GENERATE REACTIVE OXYGEN SPECIES
traction of homogenized tissue followed by repeated
ethanol precipitation. This procedure led to brown
colored extracts and precipitates. The color intensity
increased with the duration of the extraction process
but had no influence on the amount of polysaccha-
ride isolated. When extracts were made in the pres-
ence of EDTA or at high pH, far less coloring
occurred, underlining the influence of fungal
phenoloxidases, as earlier observed by Flurkey24 and
many others.
Table 1 shows that carbohydrate is the predomi-
nant component, whereas protein and phenol are
minor constituents in typical polysaccharides derived
from medicinal mushrooms.
When PBMCs were compared to K562 cells
for the activation of ROS by polysaccharides of
A. bisporus, A. brasiliensis, and zymosan, we found
that the rate of activation was highly comparable
(Table 2).
Polysaccharide extracts of A. bisporus, A. brasili-
ensis, C. comatus, G. lucidum, P. linteus, commer-
cial PSK, and zymosan all induced ROS activation
in K562 cells, although of different intensity (Fig. 1).
C. comatus extract and zymosan were colorless; the
others were brown.
Laminarin, a colorless 13-mer β-(1,3)-(1,6)-β-
glucan of the sea weed Laminaria digitata caused
no activation of ROS in those cells.
Figure 2 shows the dose response curve of ROS
activation versus concentration of A. bisporus and
G. lucidum polysaccharides, measured after 4 hours
of incubation at room temperature. The polynomial
TABLE 1
Carbohydrate, Protein, and Total Phenolic Composition (Percentage
of Total Weight) of Zymosan and of Typical Polysaccharide Extracts
of Fruitbodies of Different Basidiomycetes
Species Carbohydrate
Agaricus bisporus 95.2 ± 6.4
A. brasiliensis 97.0 ± 8.4
Coprinus comatus 91.5 ± 10.5
Ganoderma lucidum 98.0 ± 0.3
Phellinus linteus 96.5 ± 3.6
Zymosan 66.0 ± 0.0
* Protein measured by Lowry assay.21
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trendlines, calculated using Microsoft Excel, show
that saturation is reached at approximately 2 mg/mL.
At this concentration, ROS activation was found to
increase almost linearly for over 4 hours for
A. bisporus polysaccharide (Fig. 3). At lower con-
centrations, ROS activation continued for at least 20
hours without sign of decline.
After precipitation by ethanol, A. bisporus
polysaccharides were brown colored, although the
ethanol supernatant had taken up 30%–50% of the
stain of the original extract. When this polysaccharide
was subjected to size exclusion chromatography on
Superose 200, two major peaks could be recovered,
one eluting with the void volume of the column and
having a MW > 200 kDa and a low molecular weight
peak. Both fractions were brown and contained
mostly carbohydrate. Materials of both peaks acti-
vated ROS identically in PBMCs and in K562 cells
(Fig. 4).
When the ethanol-precipitated polysaccharide
was extracted for 20 minutes in hot water in the
presence of activated charcoal, and the extract was
chromatographed on DEAE-cellulose and eluted by
a continuous gradient of 0.0–1.0 M NaCl, up to 70%
of the polysaccharide did not bind to the column. The
remaining 30% could be separated by elution with a
continuous salt gradient into two major fractions
eluting at 0.25 M and 0.55 M NaCl, respectively. The
unbound fraction was strongly opalescent and color-
less and could not generate ROS. Both other frac-
tions were brown and generated ROS in K562 cells
and PBMCs.
Protein Total phenols
0.8 ± 0.0 3.4 ± 0.0
0.9 ± 0.1 2.1 ± 0.3
3.1 ± 0.0 6.1 ± 0.3
0.2 ± 0.0 1.5 ± 0.0
1.7 ± 0.5 1.8 ± 0.0
34.8 ± 1.2* 0.3 ± 0.0
5
 SONG WEI ET AL.

TABLE 2
ROS Activation in Human PBMCs and in K562 Cells
Treated by Polysaccharides of Agaricus bisporus and
A. brasiliensis by Zymosan*

ROS activity
(arbitrary fluorescence units × 10–3)
Sample K562 PBMC

Agaricus bisporus 36.7 ± 0.1 48.4 ± 2.3

A. brasiliensis 22.5 ± 0.5 29.3 ± 1.4
Zymosan 15.3 ± 0.4 20.7 ± 1.3
Blank 2.1 ± 0.0 4.2 ± 0.1

* 4 × 105 cells mL–1 were incubated for 4 hours at room tempera-

ture with 2 mg mL–1 polysaccharide.

We then subjected the unbound fraction and the
0.25 M NaCl eluate to size exclusion chromatogra-
phy on a Superose 6 column. The unbound fraction
showed a single sharp, opalescent peak of an esti-
mated MW > 250 kDa (Fig. 5).
The 0.25 M NaCl eluate showed two compo-
nents: one apparently identical to the unbound ma-
terial and devoid of ROS-generating activity, the
second light brown and generating ROS in K562 cells
(Fig. 5b). Its estimated MW was about 100 kDa.
When the unbound material was hydrolyzed with
2M TFA to determine its monosaccharide composi-
tion, it was found to be composed of glucose as the
major component, with some minor proportions of
xylose and galactose (Fig. 6). This led to the conclu-
sion that the colorless polysaccharide, which was not
bound by DEAE cellulose, is (virtually) pure glucan.
Colorless glucan of high purity could also be
extracted from A. bisporus, C. comatus, Ph. linteus,
and PSK (from T. versicolor) by repeated DEAE

20
1. A. b isporus
2. A. b rasiliensis
15 3. C. comatus
4. G. lucidum
ROS (afu×10-3)

5. Ph. linteus
10 6. PSK
7. Zymosan
8. A. sativa
5

0
1 2 3 4 5 6 7 8

samples

FIGURE 1. Generation of reactive oxygen species in K562 cells by polysaccharide extracts of different mushrooms. ROS
generation was measured after 4 hours of incubation at room temperature. afu = arbitrary fluorescence units.

6 International Journal of Medicinal Mushrooms

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 PHENOLIC COMPOUNDS IN MEDICINAL MUSHROOM EXTRACTS GENERATE REACTIVE OXYGEN SPECIES

40

35
y = -10.243x 2 + 36.458x + 4.4374
30
R2 = 0.994

ROS (afu×10-3)
25

20

15

10 y = -4.7372x 2 + 18.09x + 7.4755 A. bisp.

5 R2 = 0.9555
G. luc.
0
0 0.5 1 1.5 2 2.5
-1
glucose concentration (mg mL )

FIGURE 2. Dose response relation for ROS generation in K562 cells incubated with polysaccharides from Agaricus
bisporus and Ganoderma lucidum. Generation was measured after 4 hours’ incubation at room temperature.

cellulose treatment. This appeared not possible for were slightly brown colored, contained phenolic
polysaccharides from A. brasiliensis and G. lucidum. compounds, and induced considerable ROS genera-
Table 3 shows the effect of such purification on the tion in K562 cells.
ROS-activating capacity of these glucans and on their We then carried out a competition experiment for
phenol content. The glucans of A. bisporus, C. comatus, ROS activation between the sea weed L. digitata-
Ph. linteus, and T. versicolor could not activate ROS derived (1,3)-(1,6)-β-glucan laminarin and A. brasili-
in K562 cells and were found to have very low phenol ensis and G. lucidum glucans. No competition was
content. The glucans of A. brasiliensis and G. lucidum observed (Fig. 7).

40

35
y = 1.5369x2 + 2.9426x + 0.7161
30 R 2 = 0.9994
-3
ROS (afu×10 )

25

20

15

10

0
0 1 2 3 4
time (hours)

FIGURE 3. Kinetics of Agaricus bisporus polysaccharide generated ROS in K562 cells. 4 × 105 cells mL–1 were incubated
at room temperature in ROS testing mixture containing 2 mg mL–1 polysaccharide.

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 SONG WEI ET AL.

1400 0.6

glucose concentration (mg mL -1)

1200
0.5

1000
0.4
ROS (afu)

800
0.3
600

0.2
400

0.1
200

0 0
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29

fraction number

FIGURE 4. Sepharose 200 size exclusion chromatography of Agaricus bisporus polysaccharide. 䉬: carbohydrate
concentration in mg mL–1; 䊏: arbitrary fluorescence units at 535 nm. Arrow: blue dextran marker (MW = 2 × 106 d).

IV. DISCUSSION personal communication). The ROS values mea-

Brown, ethanol-insoluble polysaccharide fractions,
obtained from hot water extracts of homogenized
fruitbodies of A. bisporus, A. brasiliensis, Ph. linteus,
and G. lucidum, were found to contain variable
proportions of protein and phenolic compounds (Table
1). The brown color could be removed to a certain
extent by repeated washing with ethanol, suggesting
that it is caused by noncovalently bound polyphe-
nols. It has been found that polysaccharides are able
to bind to colored polyphenols by interaction with
their carbonyl groups25 or by hydrophobic inter-
action.26 As became apparent from this study, these
polyphenols may play a much more important role in
ROS generation than was previously anticipated.
When tested for activation of ROS in human
cells, it was found that all mushroom polysaccharide
extracts and the yeast-derived crude glucan zymosan
were able to activate the intracellular generation of
ROS (Fig. 1). The low value for Ph. linteus extract
was most likely caused by its preceding extraction
with ethanol as a first step in the commercial extrac-
tion process (Amazing Grace, Bangkok, Thailand,
sured in PBMCs and in the myeloid leukemia cell
lines K562 (Table 2) and Jurkat (data not shown)
were highly comparable. The dose response curve
(Fig. 2) for A. bisporus-derived polysaccharide
reached saturation at approximately 2 mg mL–1 and
4 × 105 cells mL–1. The reaction kinetics at these
conditions showed an almost linear increase in intra-
cellular ROS concentration for more than 4 hours.
When lower amounts of polysaccharide were ap-
plied, the reaction was found to continue for more
than 14 hours without obvious decrease of intracel-
lular ROS activation (Fig. 3). MTT assays did not
show cytopathic effects when K562 cells were incu-
bated for 18 hours with up to 1 mg mL–1 of polysac-
charide extract.
Size exclusion chromatography of A. bisporus
polysaccharide on Sepharose 200 showed the pres-
ence of two peaks (Fig. 4), one eluting together with
the void volume of the column, indicating high mo-
lecular weight; and the second containing molecules of
much lower size. Both peaks were found to contain
predominantly polysaccharide with minor amounts of
protein (<0.1% of polysaccharide content) and poly-

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 PHENOLIC COMPOUNDS IN MEDICINAL MUSHROOM EXTRACTS GENERATE REACTIVE OXYGEN SPECIES

phenols. Because the polyphenol content is expressed Figure 4, which is representative for the extracts
as equivalent weight of the standard gallic acid (3,4,5- of all four mushrooms, shows that most of the isolated
trihydroxybenzoic acid, MW = 170.12), it may be polysaccharide is of high molecular weight (>250
argued that the actual content of polyphenol in the kDa). All these extracts could generate ROS in human
extract is different because the group of natural polyphe- cells—that is, in PBMCs and K562 leukemic cell lines.
nols consists of thousands of compounds of com- When the polysaccharide from fruitbody extracts was
pletely different size and structure. further purified by DEAE–cellulose chromatography,

0.18
0.16

0.14
absorption 280 nm

0.12
A 0.1

0.08
0.06
0.04

0.02
0
1 4 7 10 13 16 19 22 25 28 31 34 37 40
fraction number

70 0.8
glucose concentration (mg mL )
-1

60 0.7

0.6
ROS (afu×10 )
-3

50
0.5
B 40
0.4
30
0.3
20
0.2
10 0.1

0 0
1 4 7 10 13 16 19 22 25 28 31 34 37 40

fraction number

FIGURE 5. Superose 6 size exclusion chromatography of Agaricus bisporus polysaccharide fractions after DEAE
cellulose chromatography. Figure 5A (top) shows the unbound polysaccharide; the arrow indicates the peak fraction of the
blue dextran molecular weight marker used. Figure 5B (bottom) shows the elution pattern of the polysaccharide complex
eluted from the DEAE cellulose column at 0.25 M NaCl. Arrows indicate from left to right the position of (protein) molecular
weight markers of 669, 232, 158, and 13.7 kDa, respectively.

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 SONG WEI ET AL.

140
nC
AU
Inhbit = On

MinAr = 0.044

Inhbit = Off
Glucose
100 96.3 % A. bisporus

50
Galactose
2.9 % Xylose
1 - glucose - 4.750 0.80 %
-20 min

5 10 15 20 Rt (min)
FIGURE 6. HPLC pattern of the acid (TFA) hydrolysate of glucans from Agaricus bisporus. AU = arbitrary units; Rt =
retention time.

it was observed that ROS-inducing activity remained
in the brown-colored fractions. The colorless high
molecular weight material isolated from the extracts
was in all cases devoid of ROS-inducing activity.
Polysaccharide of A. bisporus so purified—that is, the
first peak eluted by Superose 6 size exclusion chroma-
tography—had an estimated MW of 250 kDa and was
found to consist of predominantly glucose with minor
quantities of galactose and xylose, as shown in Figure
6, and thus can be considered pure glucan. It is not
certain whether the minor quantities of galactose and
xylose are a result of contamination or an intrinsic part
of the glucan. Further chemical characterization of the
glucans of A. bisporus, A. brasiliensis, G. lucidum,
and Ph. linteus will be published at a later date.
When tested for the presence of α-glucan, we
found that only extracts of A. bisporus stems con-
tained α-glucan. No such material was found in
A. bisporus cap extracts or in the other fruitbody
extracts used in this study. Incubation of A. bisporus
stem extract with α-amylase (1,4-α-D-glucan-
glucanohydrolase, Sigma A3403) had no effect on its
ROS-generating activity (data not shown). The pres-
ence of α-glucan in mushroom stipes may be ex-
plained by the completely different developmental
stage and function of the stipe mycelium compared
to that of the mature fruiting body.27
Its homopolymeric nature makes A. bisporus
glucan comparable to the glucans isolated of A. brasil-
iensis,28 Astraeus hygrometricus (Pers.) Morgan,29

TABLE 3
Phenol/Glucan Ratio of Non-DEAE-Cellulose Bound HMW
Polysaccharide Fractions of Different Medicinal Mushrooms
and Their ROS Generation in K562 Cells*

ROS generation Phenols/glucan ratio

Species (afu × 10–3 ) µg/mg)
(µ

Agaricus bisporus –0.6 ± 0.3 1.6 ± 0.1

A. brasiliensis 34.9 ± 1.7 44.2 ± 1.8
Coprinus comatus 0.5 ± 0.2 1.6 ± 0.1
Ganoderma lucidum 27.9 ± 1.2 34.5 ± 0.4
Phellinus linteus 0.3 ± 0.3 4.4 ± 0.4
PSK (Trametes versicolor) 0.1 ± 0.6 ND

* 4 × 105 cells/mL were incubated with 1.0 mg glucan for 12 hours.

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 PHENOLIC COMPOUNDS IN MEDICINAL MUSHROOM EXTRACTS GENERATE REACTIVE OXYGEN SPECIES

35

30
A.brasiliensis
25
ROS (afu×10-3)

G.lucidum
20 Laminarin

15

10

0
0 100 250 500 1000
Laminarin added (µg mL-1)

FIGURE 7. Competition between laminarin and non DEAE-cellulose bound HMW polysaccharide of Agaricus brasiliensis
and Ganoderma lucidum. 4 × 105 K562 cells/mL were incubated in the ROS reaction mixture containing 1 mg polysac-
charide, to which increasing amounts of laminarin were added. Fluorescence was read after 12 hours’ incubation. Values
are corrected for autofluorescence of K562 cells and of glucans.

Boletus erythropus (Fr.) Krombh.,30 Pleurotus florida
(= P. ostreatus ‘florida’),31 and Termitomyces
eurhizus (Berk.) R. Heim.32
The second peak eluting from the size exclusion
chromatography column (Fig. 5) is estimated to
consist of molecules of about 100 kDa. The nature
of polyphenols, also present in this fraction, is as yet
unknown but is presently being characterized.
Polyphenols may in fact be complexed to soluble
β-D-glucan by weak chemical linkages and change
the conformation of the glucan and its apparent size.
Such conformational changes have been demon-
strated for zearalenone complexation with β-(1,3)-
D-glucans stabilized by β-(1,6)-D-glucans, which
depend on the interaction between the glucan hy-
droxyl groups and the ketone and hydroxyl groups of
the zearalenone.33 Given the latter’s structural anal-
ogy to the oxidized monophenol derivatives that
occur as intermediary products in the tyrosinase-
driven browning of mushroom extracts,34 it seems
likely that the brown-colored second peak eluted
from the Superose column consists of such a com-
plex. We have observed that ROS activation is
induced solely by this material and not by the pure
glucan preceding the brown colored material.
The highly purified HMW glucans of A. bisporus,
C. comatus, Ph. linteus, and PSK from T. (= Coriolus)
versicolor did not activate ROS in human cells in
vitro. It appeared impossible to isolate comparable
colorless glucan from A. brasiliensis and G. lucidum
using repeated DEAE–cellulose treatments (Table
3). Instead, these two glucans, as well as the brown-
colored polysaccharide/phenol complex of A. bis-
porus, were very active ROS generators, suggesting
the strong influence either of the conformation of
polysaccharides or of the oxidized phenol derivatives
present in the complex.
Purified glucans of A. brasiliensis and G. luci-
dum, which strongly activated ROS, were not com-
peted for by laminarin, which is known to block the
dectin-1 receptor.9 This suggests that ROS activation
by crude mushroom polysaccharides can be effected
through a receptor not interacting with laminarin,
such as TLR-4. TLR-4 has recently been found to
interact directly with NADPH oxidase, resulting in
the production of superoxide free radicals—that is,

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 SONG WEI ET AL.
ROS—and activation of NF-κB.35 Also, polyphenols
such as tannic acid, gallic acid, and catechin com-
pounds are known to interact with steroid recep-
tors.36 This may lead to a change of the mitochondrial
transmembrane potential37 and ultimately to a de-
crease or increase in ROS activation, depending on
cell systems used.
Although polyphenols are widely believed to
function as antioxidants, it appears that they can also
generate reactive oxygens in the presence of Cu(II)38
and Fe(II),39 which may be a major mechanism for
their apoptosis-inducing ability. Recently, Maeta et
al.40 demonstrated that antioxidative green tea polyphe-
nols function as prooxidants and activate oxidative
stress responsive transcription factors in yeast.
Proinflammatory effects of medicinal mushrooms
as caused by the generation of reactive oxygen
species could therefore be due to the presence of
polyphenols rather than to β-glucans alone.
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