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Hot water extracts of 2 groups of medicinal mushrooms have been tested from the genera Agaricus, Antrodia, Auricularia, Coprinus, Cordyceps, Hericium, Grifola, Ganoderma, Lentinus, Phellinus, and Trametes for ROS-generating activity in human cells and for DPPH-TEAC antioxidant activity. Group 1 comprised 39 commercial extracts (7 species), and group 2 comprised 12 fruiting body extracts made from 11 different species of culinary-medicinal mushrooms. For both groups, the ROS-generating activity and the antioxidant activity were strongly correlated, as were their respective polysaccharide and polyphenol contents. The extracts differ in their amounts of the latter components but not in the ratio of the two. The slopes of the correlation curves were different for both groups, which is related to the higher polyphenol content of the commercial extracts. It is suggested that possible excess cell defense-related intracellular ROS generated by mushroom extracts may be downregulated by the antioxidant components present in the same extracts.
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315
International Journal of Medicinal Mushrooms, 10(4):315–324 (2008)
1521-9437/08/$35.00
© 2008 by Begell House, Inc.
Pro- and Antioxidative Properties of Medicinal
Mushroom Extracts
Song Wei & 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, Wagen-
ingen University and Research Centre, POB 16, 6700 AA Wageningen, The Netherlands; leo.vangriensven@wur.nl
ABSTRACT: Hot water extracts of 2 groups of medicinal mushrooms have been tested from the gen-
era Agaricus, Antrodia, Auricularia, Coprinus, Cordyceps, Hericium, Grifola, Ganoderma, Lentinus,
Phellinus, and Trametes for ROS-generating activity in human cells and for DPPH-TEAC antioxidant
activity. Group 1 comprised 39 commercial extracts (7 species), and group 2 comprised 12 fruiting
body extracts made from 11 different species of culinary-medicinal mushrooms. For both groups, the
ROS-generating activity and the antioxidant activity were strongly correlated, as were their respective
polysaccharide and polyphenol contents. The extracts differ in their amounts of the latter components
but not in the ratio of the two. The slopes of the correlation curves were different for both groups, which
is related to the higher polyphenol content of the commercial extracts. It is suggested that possible
excess cell defense–related intracellular ROS generated by mushroom extracts may be downregulated
by the antioxidant components present in the same extracts.
KEY WORDS: β-glucan, polyphenol, ROS, antioxidant, pro-oxidant, medicinal mushrooms, Agari-
cus bisporus, Agaricus brasiliensis, Antrodia camphorata, Auricularia polytricha, Coprinus comatus,
Cordyceps sp., Trametes versicolor, Ganoderma lucidum, Grifola frondosa, Hericium erinaceus,
Lentinus edodes, Phellinus linteus
ABBREVIATIONS
Afu: arbitrary uorescence unit; Apaf: apoptosis-activation factor; BSA: bovine serum albumin; CR3: complement
receptor type 3; DCF-DA: dichloro uorescein diacetate; DEAE: diethyl diamino ethyl; DPPH-TEAC: diphenyl
picryl hydrazyl-based trolox equivalent antioxidant activity; FCS: fetal calf serum; GAE: gallic acid equivalent
activity; HMW: high molecular weight; PBMC: peripheral blood monocytic cell; PBS: phosphate-buffered saline;
R: correlation coef cient; ROS: reactive oxygen species; RPMI 1640: Roswell Park Memorial Institute cell culture
medium 1640; RT: room temperature; TLR: Toll-like receptor.
I. INTRODUCTION
Medicinal mushrooms, such as Agaricus brasilien sis
S. Wasser et al. (= A. blazei Murrill s. Heinem.),
Coprinus comatus (O. F. Müll.) Pers., Trametes
(= Coriolus) versicolor (L.: Fr.) Lloyd, Gano-
derma lucidum (W. Curt.: Fr.) P. Karst., Lentinus
edodes (Berk.) Singer, Phellinus linteus (Berk et
W. Curt.) Teng, and many others, have traditionally
been used as a health food or supplement for the
prevention and cure of a range of diseases, includ-
ing atherosclerosis, cancer, chronic hepatitis, and
diabetes. The preventive and therapeutic effects of
these mushrooms and their components have been
well documented in mouse and rat model systems
and in cancer cell lines.1,2 Many studies have led
to important knowledge regarding the effects of
mushroom extracts and their modes of action. This
316 International Journal of Medicinal Mushrooms
W. SONG & L. J. L. D. VAN GRIENSVEN
includes immunomodulatory effects in various
forms, for example, proin ammatory activity or
adjuvant effects, as well as apoptosis, mostly with
the involvement of chemokines as mediators that
are produced by the cells of the immune system.
Mushroom extracts contain several biologically
or medicinally active components. The in uence
of mushroom extracts on oxygen metabolism, both
in vitro and in vivo, may be a common denomina-
tor. Redox reactions strongly in uence a majority
of the physiological processes. Several higher
Basidiomycetes contain antioxidant phenolic com-
pounds.3 Agaricus bisporus (J. Lge) Imbach, which
contains high amounts of antioxidants,4 has been
reported to additionally harbor the pro-oxidative
4-(hydroxymethyl)-phenyl radical.5
Although phenolic compounds may gener-
ally have antioxidative properties, some are able
to activate the intracellular formation of reactive
oxygen species (ROS).6,7 ROS play an important
role in the prevention of infection by destroying
potential intracellular pathogens.8
The most fundamental role of ROS might,
however, be signal transduction and regulation
of diverse processes, including neurotransmis-
sion, phagocyte activation, cell proliferation, and
apoptosis.9 Cellular generation of ROS is central
to redox signaling.10 Molecular damage caused 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, to either
apoptosis11,12 or autophagous cell death.13 Tumor
cell lines appear to be particularly sensitive to ROS-
induced apoptosis,14 and many antitumor agents,
in fact, exhibit their activity via ROS-dependent
activation of apoptotic cell death.15 In cancer cells,
apoptosis is thought to be generally suppressed16
because of K+ channel inhibition resulting from
mitochondrial damage, which, in turn, leads to
attenuated mitochondrial function and enhanced
glycolysis dependency.17,18 If ROS and, especially,
H2O2 are generated in a cancer cell, cytochrome c
will be released from the intermembrane space into
the cytosol where it binds to the apoptosis activation
factor (Apaf-1) and activates the caspase cascade,
leading to death.17,19
On the other hand, the presence of intracellular
ROS may also have adverse consequences. There is
accumulating evidence that ROS play major roles in
the initiation and progression of cardiovascular dys-
function associated with diseases such as diabetes,
hypertension, ischemic heart disease, and chronic
heart failure.20 Pancreatic β-cells are particularly
vulnerable to oxidative stress, which may induce
apoptosis and a decrease in β-cell mass, leading to
dysfunction of insulin secretion and the onset of
type 1 and 2 diabetes.21 In mast cells, high levels
of ROS contribute to degranulation and to harm-
ful effects in patients suffering from in ammatory
disease.22,23 These harmful effects associated with
prolonged exposure to ROS present an additional
motive to study the properties of the different frac-
tions of fungal cell wall extracts commonly sold
as medicinal mushroom glucans.
In the present study, we describe the in uence
of medicinal mushroom extracts on ROS synthe-
sis and on diphenyl picryl hydrazyl-based trolox
equivalent antioxidant activity (DPPH-TEAC)
antioxidation in the human leukemia cell line
K562. Earlier, we demonstrated that polysaccharide
extracts of various mushrooms are highly active in
ROS generation.24 When hot water–extracted poly-
saccharides of Agaricus bisporus were subjected
to DEAE cellulose–adsorption chromatography,
a nonadsorbed colorless high molecular weight
(HMW) 1,4 glucan was obtained that was unable
to generate ROS, suggesting the inability of some
glucans to generate ROS. The DEAE-adsorbed
fractions were eluted by salt gradient and were
found to consist of a hazel-colored polysaccharide–
polyphenol complex that was very active in ROS
generation. This led us to the tentative conclusion
that A. bisporus glucan needs to be associated
with polyphenolic compounds to become an active
ROS generator.24
In the present article, we describe the ROS-
generating capacity, the concentration of polysac-
charides and polyphenols, and the antioxidant
effects of extracts of a collection of commercially
available medicinal mushroom preparations of
several different species that were obtained from
the P. R. China, Korea, Thailand, Europe, and
the United States. Further, we report the ROS-
generating activity, the polyphenol content, and the
antioxidant activity of extracts of various cultivated
medicinal mushrooms.
Volume 10, Issue 4, 2008 317
PRO- AND ANTIOXIDATIVE PROPERTIES OF MEDICINAL MUSHROOM EXTRACTS
II. MATERIALS AND METHODS
A. Materials
RPMI-1640 medium with L-glutamine and sodium
bicarbonate was obtained from Sigma Chemical
Corp (St. Louis, MO, USA). Serum supreme and
fetal bovine serum (of South American origin)
were from BioWhittaker (Walkersville, MD, USA).
27-dichloro uorescin-diacetate and DPPH were
obtained from Sigma. Trolox was obtained from
Hoffmann-Laroche (Switzerland).
B. Cells
K562 cells25 were cultivated as described before24
and cultured in a humidi ed incubator with 5%
CO2 at 37°C in RPMI 1640 containing 10% fetal
calf serum (FCS) and 100 U.mL–1 penicillin + 100
µg.mL–1 streptomycin.
C. Mushrooms
Fresh stipe tissues of cultivated Agaricus bisporus
and fruitbodies of A. brasiliensis were obtained from
Innerlife B.V. (Venlo, The Netherlands). Coprinus
comatus strain S 435 and Lentinus edodes dried
fruitbodies were obtained from the (former) Mush-
room Experimental Station (Horst, The Netherlands).
Dried hot water extract of wild-type Phellinus linteus
was kindly provided by Amazing Grace (Thailand).
Gano derma lucidum spore extract Grade A was ob-
tained from Fujian Xianzhilou Biological Science and
Technology Co. Ltd. (P. R. China). Dried fruitbod-
ies of Auricularia polytricha, Trametes versicolor,
Grifola frondosa, and Hericium erinaceus were from
commercial sources, and Cordyceps militaris (L.)
Link was a kind gift from Dr. J. M. Sung of Kangwon
National University (Chuncheon, Korea).
D. Polysaccharide Extracts
The mushroom polysaccharide extract was obtained
by hot water extraction and ethanol precipitation,
as described previously.24
A collection of 39 commercial medicinal mush-
room preparations (Table 1) was obtained from
dispensaries and health shops in the United King-
dom, P. R. China, Germany, Taiwan, Thailand, the
Netherlands, and the United States. They consisted
of 18 samples derived from Ganoderma lucidum, 5
from Cordyceps sp., 4 from Trametes versicolor, 4
from Phellinus sp., 2 from Grifola frondosa, 2 from
Agaricus brasiliensis, 1 from Antrodia camphorata
(M. Zhang & C.H. Su) Sheng H. Wu, Ryvarden et
T.T. Chan, and 3 were mixtures of several mush-
rooms. Two of the Phellinus samples were water
extracts; all the others were dry powders.
The dry samples were subjected to hot water
extraction analogously to fruiting-body extraction
and used without further alcohol precipitation.
E. Detection of Intracellular ROS
To detect intracellular ROS, 2,7-dichloro uorescin
diacetate (DCF-DA) was used. This non uorescent-
uorescin analogue is oxidized to highly uorescent
2,7-dichloro uorescein by intracellular oxidants.26
Incubations were done 4-fold in Greiner black
96-well plates and repeated several times. The
uorescence was measured in a Tecan uores-
cence spectro photometer at excitation/emission =
485/535 nm. The results are shown as arbitrary
uorescence units (afu) at 535 nm (or as a percent-
age of change from baseline values). Values were
corrected for auto uorescence, that is, reaction
mixtures incubated with uorescein 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. Determination of Antioxidant Activity
Using the DPPH Method
TEAC of extracts was measured as described by
Sun et al.27 Watery extracts were diluted 5-fold
with methanol. The mixture was incubated for 15
minutes at 60°C in a 1.5-mL Eppendorf tube and
centrifuged at 10,000 g for 5 minutes. A DPPH
318 International Journal of Medicinal Mushrooms
W. SONG & L. J. L. D. VAN GRIENSVEN
TABLE 1
Contents of Polysaccharide, Protein, and Phenolic Compounds of Hot Water Extracts of
Different Commercially Available Medicinal Mushroom Preparations and Their ROS-Activating
Capacity and DPPH-TEAC Antioxidant Activity in K562 Cells
Polysaccharide Protein Total Phenolics Antioxidant
Units, species, (mg glucose (g BSA (g gallic ROS (10 g
and origin eq.mL–1) eq.mL–1) acid eq.mL–1) (AFU.L–2) TEAC.mL–1)
Ganoderma lucidum
1. China 5.9 ± 1.4 58.2 ± 1.3 236.2 ± 0.4 1350 ± 73 6.3 ± 0.3
2. Korea 23.3 ± 0.5 224.3 ± 1.8 1191.3 ± 4.2 3006 ± 35 17.8 ± 0.6
3. China 24.2 ± 0.0 292.4 ± 10.7 1904.6 ± 35 9362 ± 51 22.8 ± 0.1
4. China 32.1 ± 6.5 187.2 ± 3.5 1470.1 ± 19 8542 ± 105 17.8 ± 0.6
5. China, Hong Kong 28.4 ± 1.4 168.6 ± 1.2 1333.6 ± 4.8 4037 ± 24 10.4 ± 0.5
6. China, Hong Kong 20.0 ± 0.4 49.9 ± 0.9 475.2 ± 1.9 1697 ± 17 5.8 ± 0.2
7. China, Hong Kong 20.9 ± 0.5 286.5 ± 4.7 1490.4 ± 18.8 4889 ± 74 17.2 ± 0.4
8. Thailand 19.2 ± 3.8 85.4 ± 0.4 177.9 ± 2.9 2319 ± 70 2.3 ± 0.3
9. Thailand 9.7 ± 1.0 33.8 ± 2.1 404.7 ± 0.6 892 ± 9 6.5 ± 0.5
10. Thailand 1.6 ± 0.1 0 ± 0 128.2 ± 0.8 291 ± 1 5.1 ± 0.2
11. Thailand 46.6 ± 0.0 0 ± 0 2641.9 ± 2.1 8170 ± 43 40.2 ± 0.2
12. Singapore 10.6 ± 3.2 73.9 ± 14.4 213.8 ± 1 851 ± 8 4.5 ± 0.1
13. Thailand 23.6 ± 0.4 328.1 ± 2.1 1828.4 ± 12.3 10539 ± 186 20.7 ± 0.8
14. Singapore 49.8 ± 2.8 372.8 ± 2.7 2787.5 ± 36.2 11319 ± 61 34.2 ± 0.8
15. China 19.2 ± 0.1 134.8 ± 4.1 2656.9 ± 34.4 1528 ± 8 45.8 ± 0.4
16. Taiwan 19.1 ± 0.1 421.9 ± 3.4 1813.5 ± 38.5 3727 ± 155 17.8 ± 0.8
17. Japan 24.4 ± 1.9 264.3 ± 4.7 1769.7 ± 10 7722 ± 424 20.3 ± 0.4
18. Thailand 6.1 ± 0.7 80 ± 5 234.4 ± 2.5 594 ± 23 4.6 ± 0.5
Cordyceps sp.
19. United Kingdom 3.9 ± 0.2 75.3 ± 6.4 92.9 ± 3.8 426 ± 23 3.9 ± 0.3
20. China 6.7 ± 0.2 187.3 ± 3.6 814.2 ± 14.8 809 ± 36 7.1 ± 0.5
21. Netherlands 1.7 ± 0.4 52.3 ± 2.5 63 ± 3.5 245 ± 10 1.6 ± 0.2
22. China 6.1 ± 0.0 275.7 ± 2.8 956.4 ± 10.6 1068 ± 14 4.8 ± 0.6
23. China, Hong Kong 2.1 ± 0.3 284.8 ± 2.5 316.7 ± 6.9 353 ± 2 5.1 ± 0.8
Trametes versicolor
24. United Kingdom 2.9 ± 0.5 73.2 ± 0.7 45.3 ± 0.8 425 ± 21 2.6 ± 0.2
25. China 40.8 ± 0.3 353.5 ± 1.4 2389.4 ± 30.8 8716 ± 76 22.1 ± 0.7
26. China, Hong Kong 58.0 ± 3.8 415 ± 1.1 2900.4 ± 3.8 16760 ± 23 42.4 ± 1.0
27. Germany 10.3 ± 0.7 42.6 ± 1.5 2562.1 ± 20.4 1121 ± 42 46.4 ± 0.1
Phellinus sp.
28. Thailand 53.0 ± 2.8 2.3 ± 4.2 2657.3 ± 5.8 7730 ± 217 41.5 ± 0.4
29. Thailand 9.5 ± 0.1 52 ± 0.4 1452 ± 15 747 ± 35 27.8 ± 0.9
30. Thailand 43.3 ± 3.2 12.8 ± 2.3 2604 ± 41.5 7077 ± 132 41.2 ± 0.4
31. Thailand 14.2 ± 0.2 60 ± 21.4 2002.7 ± 10.4 1705 ± 0 28.7 ± 0.1
Grifola frondosa
32. United Kingdom 3.2 ± 0.4 74.1 ± 5 69.8 ± 0 387 ± 23 2.4 ± 0.2
33. China 20.3 ± 2.5 362.9 ± 0.3 1784.6 ± 17.7 5866 ± 129 12.9 ± 0.4
Agaricus brasiliensis
34. China 5.5 ± 0.8 165.8 ± 2.3 512.5 ± 14.6 825 ± 51 5.7 ± 0.2
35. Netherlands 3.9 ± 0.8 45.9 ± 3.7 85.3 ± 0.4 584 ± 17 2.6 ± 0.1
Antrodia camphorata
36. China 1.9 ± 0.2 10.8 ± 3.0 191.6 ± 5.0 546 ± 5.0 1.9 ± 0.2
Mixtures
37. China, Hong Kong 15.0 ± 0.7 77.6 ± 6.7 343.3 ± 2.7 2088 ± 93 5.2 ± 0.4
38. United States 1.2 ± 0.1 156.8 ± 4.1 213.8 ± 0.6 540 ± 1 1.7 ± 0.1
39. China, Hong Kong 6.2 ± 0.2 150.7 ± 3.6 647.8 ± 12.5 2088 ± 2 6.6 ± 0.3
Volume 10, Issue 4, 2008 319
PRO- AND ANTIOXIDATIVE PROPERTIES OF MEDICINAL MUSHROOM EXTRACTS
(Sigma D 9132) solution of 25 mg.L–1 was prepared
in 500 mL of methanol of which 1 mL of DPPH
was mixed with 10-, 20-, and 40-µL sample mix-
tures. The solution was incubated for 30 minutes
at room temperature (RT). Its absorbance was
measured in a Tecan multifunctional uorescence
spectrophotometer at 485 nm. As a standard refer-
ence, Trolox (0.5 mM) was used. The inhibition
percentage was calculated and compared with the
corresponding volume, so that a regression line
with a slope could be created. The ratio between
the slope of Trolox and the sample was calculated
and expressed as Trolox equivalent per milligram
dry weight of mushroom extract.
G. Protein Determination
Protein concentrations were determined by Brad-
ford’s28 method using bovine serum albumin (BSA)
(Sigma A7030) as the standard. Total protein content
is expressed as milligrams of BSA equivalents per
gram of dry weight.
H. Total Phenolics Determination
Total phenolic content was determined using
Folin-Ciocalteu reagent, as described in detail by
Ainsworth and Gillespie29 using gallic acid as the
standard. Total phenolics are expressed as milligrams
of gallic acid equivalents per gram of dry weight.
I. Polysaccharide Determination
Polysaccharide concentrations were determined by
the phenol-sulphuric acid method, using D-glucose
as the standard.30 Total polysaccharide is expressed
as milligrams of glucose equivalents per gram of
dry weight.
J. Statistical Analysis
Instat+™ for Windows, an interactive statistical
package from Reading University (UK), was used
for all statistical analyses. Quantitative measure-
ments of samples were done 4-fold, and all experi-
ments were repeated from two to six times.
III. RESULTS AND DISCUSSION
In the present study, we subjected 43 commercially
available medicinal mushroom preparations to hot
water extraction. We made 10% (w/v) solutions in
water and measured the polysaccharide content, the
protein and total phenolics content. ROS-generating
activity was measured in the leukemia cell line
K562 and the DPPH–TEAC antioxidant activity
was determined in vitro. The results are given in
Table 1. Statistical analysis of the correlation be-
tween the different collections of numbers, which
was done using Instat+™, shows interesting results
(Table 2).
The correlation coef cient of protein content
and the other component activities, respectively,
is rather small, indicating a limited or no role for
protein in the generation of ROS (R = 0.32) and in
scavenging activity (R = 0.02), respectively. This
is in contrast to the correlation of the polyphenol
content and the other component activities, respec-
tively. Table 2 shows high correlation coef cients
for the relationship of polyphenol content and ROS
or TEAC, respectively. Figure 1 shows the rela-
tionship of polysaccharide content and the phenol
content of the hot water extracts. The relationship
appears linear and has a correlation coef cient of
R = 0.82. The slope of the curve indicates that 1 mg
of polysaccharide is associated with approximately
50 µg of polyphenols, measured as gallic acid
equivalent (GAE) weight.
For comparison, we prepared semipuri ed
fruiting-body extracts of a number of different me-
dicinal mushrooms (Table 3). The polysaccharides
in these fruiting-body extracts were precipitated
with 70% ethanol and, after centrifugation, dis-
solved in water. The same parameters as for the
commercial extracts were measured, and the same
statistical relations were calculated. Semipuri ed
extracts have a much lower polyphenol content,
that is, 10 mg/g for the polysaccharide versus 50
mg/g for the commercial extracts.
We determined the correlation between the
concentration of phenolic compounds and the
320 International Journal of Medicinal Mushrooms
W. SONG & L. J. L. D. VAN GRIENSVEN
ROS-generating activity for the commercial extracts
and found R = 0.75 (Table 2). As there is a known
linear relation between total phenolics content
and antioxidant activity of mushroom extracts,3,31
we also determined this relation for the whole
collection of commercial extracts, as well as for
our own semipuri ed extracts. We found a linear
relationship between the concentration of phenolic
compounds and DPPH-TEAC antioxidant activity,
with a correlation coef cient of R = 0.93 and R =
0.90, respectively (Table 4).
The results further indicate that the ratio of
polysaccharide and phenolics is approximately
the same for all extracts of the particular group of
medicinal mushrooms we measured. The correla-
tion coef cient of ROS generation and concentra-
tion of phenolics seems lower than the correlation
coef cient of ROS generation and polysaccharide
concentration. ROS generation seems to be de-
termined by the concentration of the mixture of
polysaccharide and phenolics independent of the
species of mushroom or the brand of preparation.
Given the use of gallic acid (3,4,5 trihydroxy
benzoic acid; FW = 170.2) as a standard in the
Folin-Ciocalteu assay for phenolic compounds, the
GAE we measured of the extracts vary with the
composition of the phenolic compounds involved.
The GAE weight is different for the different phe-
nolics because the method applied (Folin-Ciocalteu)
measures phenolic OH groups as a determinant of
quantity. Moreover, the composition and contents
of the medicinal mushroom samples we tested are
disputable; all samples are of commercial origin,
without independent proof of quality. This may
TABLE 2
Correlation of the Contents of Different
Components of Medicinal Mushroom
Extracts and Their Respective
Activities
Correlation
Relation coef cient
Protein and polysaccharide content R = 0.26
Protein content and ROS R = 0.32
Protein content and TEAC R = 0.02
Polyphenol and polysaccharide content R = 0.82
Polyphenol content and ROS R = 0.75
Polyphenol content and TEAC R = 0.93
Polysaccharide content and ROS R = 0.88
Polysaccharide content and TEAC R = 0.73
FIGURE 1. The relationship between the concentration of polysaccharide and phenolic compounds of extracts of
a collection of medicinal mushrooms. Y axis: polyphenol content (mg.mL–1) weight equivalents of gallic acid. X axis:
polysaccharide content (mg.mL–1).
0
0.5
1
1.5
2
2.5
3
3.5
0 10203040506070
Polysaccharide mg.mL
-1
Polyphenol mg.mL
-1
Volume 10, Issue 4, 2008 321
PRO- AND ANTIOXIDATIVE PROPERTIES OF MEDICINAL MUSHROOM EXTRACTS
TABLE 3
Polysaccharide Content, Total Phenol Content, and ROS-Generating and
Antioxidant Activity (DPPH-TEAC) of Typical Hot Water Extracts of Different
Medicinal Mushrooms
Polysaccharide Total phenol ROS DPPH-TEAC
Species (mg.mL–1) (10 g.mL–1) (AFU.nL–1) (10 g. mL–1)
Agaricus bisporus 1.6 ± 0.0 2.0 ± 0.0 0.2 ± 0.0 2.3 ± 0.2
A. brasiliensis 36.4 ± 0.5 29.1 ± 0.1 4.1 ± 0.2 23.0 ± 0.2
Auricularia polytricha 1.3 ± 0.0 1.2 ± 0.1 0.4 ± 0.0 2.7 ± 0.1
Coprinus comatus 6.0 ± 0.3 14.1 ± 0.6 0.4 ± 0.0 9.0 ± 0.2
Cordyceps militaris 2.2 ± 0.5 5.0 ± 0.8 0.2 ± 0.0 5.5 ± 0.6
Trametes versicolor 1 34.6 ± 0.8 45.6 ± 0.8 2.6 ± 0.1 20.6 ± 0.3
T. versicolor 2 10.9 ± 0.1 15.6 ± 0.3 1.7 ± 0.0 6.1 ± 0.5
Grifola frondosa 10.8 ± 1.0 18.0 ± 0.1 1.8 ± 0.0 6.4 ± 0.9
Ganoderma lucidum 7.5 ± 0.1 11.4 ± 0.1 1.2 ± 0.0 8.3 ± 0.6
Hericium erinaceus 3.0 ± 0.5 1.7 ± 0.1 0.6 ± 0.0 3.5 ± 0.6
Lentinus edodes 7.5 ± 0.5 7.8 ± 0.3 0.4 ± 0.0 2.9 ± 0.6
Phellinus linteus 4.1 ± 0.0 6.4 ± 0.1 0.2 ± 0.0 4.2 ± 0.5
TABLE 4
The Statistical Relationships between the Concentrations of Polysaccharide (mg.mL–1)
and Phenolic (mg.mL–1) Compounds in Extracts of Medicinal Mushrooms and Their
ROS-Generating (AFU.mL–1) and Antioxidant Activities (mg TEAC.mL–1)
Units Commercial extracts Prepared extracts
Phenolics (Y) and polysaccharide (X) Y = 0.05X + 0.23 Y = 0.01X + 0.026
R = 0.82 R = 0.93
S.E.
slope: 0.006 S.E. slope: 0.001
95% C.I.slope: 0.039–0.063 95% C.I. slope: 0.072–0.130
ROS (Y) and phenolics (X) Y = 3.03X + 0.08 Y = 7.38X + 0.17
R = 0.75 R = 0.81
S.E.slope: 0.428 S.E. slope: 1.709
95% C.I.slope: 2.164–3.897 95% C.I. slope: 3.57–11.19
ROS (Y) and polysaccharide (X) Y = 0.22X – 0.41 Y = 0.09X + 0.17
R = 0.88 R = 0.93
S.E.
slope: 0.019 S.E. slope: 0.012
95% C.I.slope: 0.186–0.263 95% C.I. slope: 0.067–0.119
TEAC (Y) and phenolics (X) Y = 0.14X – 0.004 Y = 0.46X + 0.02
R = 0.93 R = 0.90
S.E.
slope: 0.0084 S.E. slope: 0.0723
95% C.I.
slope: 0.120–0.154 95% C.I.slope:0.302–0.625
TEAC (Y) and ROS (X) Y = 0.02X + 0.022 Y = 0.05X – 0.02
R = 0.60 R = 0.89
S.E.
slope: 0.005 S.E. slope: 0.01
95% C.I.
slope: 0.013–0.031 95% C.I. slope: 0.032–0.069
TEAC and polysaccharide Y = 0.007X + 0.034 Y = 0.005X + 0.02
R = 0.72 R = 0.96
S.E.
slope: 0.001 S.E. slope: 0.00
95% C.I.
slope: 0.005–0.009 95% C.I.slope:0.004–0.007
Note: R = correlation coef cient; S.E. = standard error; C.I. = con dence interval.
322 International Journal of Medicinal Mushrooms
W. SONG & L. J. L. D. VAN GRIENSVEN
also be deduced from the correlation coef cients
that are for all but one relationship lower for the
commercial extracts than for our own fruiting-body
extracts. Gallic acid itself could not generate ROS
in K562 cells (data not shown).
Ethanol-precipitated polysaccharides of Agari-
cus bisporus, A. brasiliensis, Trametes versicolor,
Ganoderma lucidum, Grifola frondosa, Lentinus
edodes, and Phellinus linteus were hazel colored.
Hericium erinaceus and Coprinus comatus yielded
white polysaccharides. All ethanol-precipitated and
water-dissolved polysaccharide extracts of higher
basidiomycetous mushrooms induced ROS genera-
tion in K562 cells, although with lower activity
than the nonpuri ed commercial extracts.
The results (Table 4) of a regression analysis
show a linear relation between the concentration
of phenolic compounds and the ROS activation,
with a high correlation coef cient (R = 0.81) but
lower than that found for the correlation between
polysaccharide content and ROS (R = 0.93) and
between polysaccharides and phenolic compounds
(R = 0.93), respectively. The relation between ROS
and antioxidant activity also appeared linear, with
a correlation coef cient R = 0.89.
The commercial extracts have not been further
puri ed by ethanol precipitation and are, therefore,
rich in ethanol-soluble polyphenols. It is interest-
ing to observe that the ROS-generating activity
per milli gram of phenolic compounds is two
times lower for the commercial extracts than for
fruiting-body extracts of our own laboratory, and
that the ROS-generating activity per milligram of
polysaccharides is two times lower. This is exactly
as expected for a complex of polysaccharide and
polyphenols that exerts the biological effects men-
tioned. When TEAC is considered for an analogous
comparison of correlation coef cients of the two
groups, it is shown that, again, the biological effect
is much higher for the prepared extracts than for
the commercial extracts per milligram polyphenols,
but almost identical per milligram of the polysac-
charide. This suggests that different polyphenols in
the extracts have different functions, that is, either
ROS generating or radical scavenging.
The apparent linear relationship between anti -
oxidant activity and concentration of phenolics,
having a correlation coef cient R = 0.90, is not
surprising. As demonstrated in earlier studies,30,32
phenols are the major antioxidant components of
mushrooms; Agaricus bisporus is an exception,
with ergothioneine.4
In an earlier study,24 we have shown for an
A. bisporus extract that puri ed glucan devoid of
measurable phenolic compounds cannot activate
ROS in peripheral blood monocytic cells (PBMCs)
and in K562 and Jurkat cells. The present data show
that commercial medicinal mushroom preparations
are able to generate ROS in the K562 cell line and
that the ROS-generating capacity is correlated with
the concentration of polysaccharides and phenolic
compounds in the extract. Not surprisingly,32 we
observed a strong correlation between the concen-
tration of phenolic compounds in the extracts and
the DPPH-TEAC antioxidant activity. The mush-
room extracts we tested contain both oxidative and
antioxidative compounds. Again, the commercial
extracts demonstrate lower antioxidant activity per
milligram of phenolic compounds than the fruiting-
body extracts from our own laboratory. However,
when calculated per milligram of polysaccharide,
the value is almost the same.
Although phenolic compounds are widely be-
lieved to function as antioxidants, it is clear that
they can also generate reactive oxygens. The latter
was published previously for polyphenols when ac-
companied by Cu (II)33 and Fe (II), respectively.34
Recently Maeta et al.35 also demonstrated that
antioxidative green tea polyphenols function as pro-
oxidants and activate oxidative stress–responsive
transcription factors in yeast. The question remains,
however, if the same polyphenol molecule encom-
passes both functions or if this re ects the presence
of a mixture of polyphenols.
ROS generation is one of the causes of the
apoptosis-increasing ability of extracts of Phellinus
linteus, which consists mostly of polysaccharides,
in the cases of lung36 and prostate37 tumor cells.
Hypothetically, phenolic compounds may determine
ROS activation, depending on their primary struc-
ture and conformation. From our studies, it seems
likely that both polyphenols and polysaccharides
are involved in ROS generation and possibly also
in DPPH-TEAC antioxidant activity.
Phenolic compounds present in mushrooms may
in fact be complexed to soluble β-D-glucan by weak
Volume 10, Issue 4, 2008 323
PRO- AND ANTIOXIDATIVE PROPERTIES OF MEDICINAL MUSHROOM EXTRACTS
chemical linkages. It has already been found that
polysaccharides are able to bind to colored pheno-
lic compounds by interaction with their carbonyl
groups38 or by hydrophobic interaction.39 Such
conformational changes have been demonstrated for
zearalenone complexation with β-(1,3)-D-glucans
branched by β-(1,6)-D-glucans, which depends
on the interaction between the glucan hydroxyl
groups and the ketone and hydroxyl groups of the
zearalenone.40 Given the latter’s structural analogy
to the oxidized monophenol derivatives that occur
as intermediary products in the tyrosinase-driven
browning of mushroom extracts,41 it seems likely
that the brown-colored mushroom extract consists
of such a complex.
As can be seen from Table 4, the DPPH-TEAC
antioxidant activity of the mushroom polyphenols
in the extracts is quantitatively comparable to that
of trolox itself. The amount of polysaccharide
that is isolated from the different mushrooms and
that complexes with small amounts of polyphenol
components seems to be the major determinant of
the ROS-generating and DPPH-TEAC antioxidative
activity of medicinal mushroom extracts.
It is tantalizing to speculate on the possible role
of mushroom extracts that combine pro- and anti-
oxidative properties. A mushroom glucan–phenol
complex may bind to one or several of the recep-
tors TLR2, TLR4, CR3,42 and Dectin-1,43,44 which
leads to the rapid generation of intracellular ROS
and to the activation of cytokine gene expression.
ROS may induce a protective oxidative state against
intracellular bacteria and viruses but, if not reduced,
is transported outside the cell where the receptor-
bound glucan–phenol complex is able to exercise its
antioxidative action. Several aquaporins45 have been
suggested to function in the transport of hydrogen
peroxide through the cell membrane.46 Cancer cells,
with their proven mitochondrial defects in oxidative
phosphorylation, may thus be driven into apoptosis
and cell death.18
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To clarify the pathogenicity of (1-3)-β-D-glucan (β-glucan), the immunomodulatory activity of three types of particulate (water-insoluble) β- glucan and three water-soluble β-glucans, were compared in respect to the production of reactive oxygen species (ROS) by human polymorphonuclear leukocytes (PMN) in vitro. The production of ROS was measured by a chemiluminescence (CL) assay in which luminol was added to the samples and the PMN were subsequently stimulated with a particulate β-glucan (curdlan, zymosan or zymocel) and a water-soluble β-glucan (CM-curdlan, laminarin or sonifilan). This assay was also used to test the ability of each of the three water-soluble β-glucans and lipopolysacchalide (LPS) to augment the priming effects on the production of ROS from PMN. Each particulate β-glucan induced the apparent CL of PMN in a dose-dependent manner, while water-soluble β- glucans and LPS could not at any concentrations tested. PMN were incubated for 60 min at 37°C with LPS and three types of water-soluble β-glucan, and the integrated CL response induced by phorbol myristate acetate (PMA) was measured for 20 min, which permitted comparisons of the priming effects of LPS in combination with each water-soluble β-glucans. Preincubation with LPS resulted in an increase in the CL response of PMN at concentrations of more than 100 ng/ml. Similar results were obtained in the PMN sample, which included a small amount of serum after only 10 min incubation with LPS. However, no significant priming effect was observed when PMN were incubated for 10 and 60 min with various concentrations (1 ng-10 μg/ml) of CM-curdlan, laminarin or sonifilan. PMN-CL induced by particulate β-glucans such as curdlan or zymocel and Candida albicans was significantly prevented when PMN was preincubated with more than 10 μg/ml of the water-soluble β-glucans for 60 min at 37°C and then exposed to particulate β-glucan and C. albicans. These inhibitory effects were dose-dependent. Also, the effect of three antifungal agents, amphotericin-B (AMPH-B), fluconazole (FLCZ) and miconazole (MCZ) on the CL response of PMN were studied in vitro. After 60 min incubation with more than 1 μg/ml of AMPH-B, CL values of PMN during phagocytosis of curdlan were significantly enhanced, whereas these values were suppressed with FLCZ at the concentration of more than 1 μg/ml. There was no significant effect on the CL response of PMN in treatment with MCZ. These findings indicate that these β-glucans and antifungal agents modulate the oxygen metabolism of human PMN, however, further studies are required to elucidate the mechanisms responsible for this immunomodulatory effect and to establish its clinical relevance.
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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.
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The beta-D-glucans from the cell wall of Saccharomyces cerevisiae have shown in vitro affinity for zearalenone. For this reason, their utilization as dietary adsorbent, to reduce the bioavailability of zearalenone, is of practical interest. Our study used powerful devices to elucidate the spatial conformation and molecular sites of interaction between ZEN and beta-D-glucans. In this respect, 1H NMR spectroscopy implicated the hydroxyl groups of the phenol moiety of zearalenone in the complexation by laminarin, a pure beta-(1,3)-D-glucan. X-ray diffraction determined that laminarin displays the conformation of a single-helix with six beta-D-glucopyranose residues per turn. At this stage, molecular modeling was useful to locate the interaction sites and to propose highly probable complexes of zearalenone with laminarin fragment. Interestingly, the beta-(1,3)-D-glucan chain favors a very stable intra-helical association with zearalenone, nicely stabilized by beta-(1,6)-D-glucans side chains. Both hydrogen bonds and van der Waals interactions were precisely identified in the complex and could thus be proposed as driving interactions to monitor the association between the two molecules.
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Electron paramagnetic resonance (EPR) spectroscopy is used to measure directly the generation of free radicals during a simulation of the mastication process. This involves the gentle grinding of the food product in the presence of a spin trap, a molecule which reacts selectively with unstable free radicals to generate (more) stable radical adducts, which can then be characterised. With mushrooms of the Agaricus family, adducts consistent with a carbon-centred radical are seen with a wide range of spin traps and this radical has been confirmed as 4-(hydroxymethyl)phenyl. In plant tissues that are rich in ascorbic acid, this molecule competes successfully with spin traps for the free radicals and the (monodehydro)ascorbate radical, formed by the 1-electron oxidation of ascorbic acid, is seen in the EPR spectra. However, with >50% of the plant tissue samples studied in the present experiment, free radicals resulting from oxidation of the spin traps were observed. The formation of such molecules, for which oxygen was found to be necessary, requires the existence of powerful oxidation processes as the plant tissue is broken down. Such pro-oxidant behaviour is contrary to the popular assumption that the beneficial effects of uncooked plant tissues are the result of their high levels of anti-oxidant molecules.
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Edible mushrooms are a good source of antioxidants. Methanol extracts of mushrooms such as Pleurotus sp., Agaricus bisporus, Morchella esculenta, Boletus edulis (approx. 2 mg mL−1) showed a high 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity close to 90%. Water extracts showed even higher antioxidant activity. In this case, B. edulis, Lentinus edodes and Amanita cesarea showed the highest 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) scavenging activity at approx. 0.14 mg mL−1. Other mushrooms such as Lactarius deliciosus and Cantharellus cibarius showed lower antioxidant activity in both extracts. Oxidative enzymes (peroxidases and polyphenol oxidases) present in the water fractions reduced their antioxidant activity by different extents since the phenols responsible for the antioxidant activity were not only those substrates of the oxidative enzymes. Other phenolic compounds and low-molecular-weight compounds were also involved in the antioxidant activity and differed depending on mushroom species. Copyright © 2007 Society of Chemical Industry
Article
Agaricus bisporus, Lentinula edodes, Pleurotus ostreatus, Pleurotus eryngii and Grifola frondosa were analyzed for antioxidant capacity, as measured by oxygen radical absorbance capacity (lipophilic and hydrophilic) (ORACtotal), hydroxyl radical averting capacity (HORAC), peroxynitrite radical averting capacity (NORAC), superoxide radical averting capacity (SORAC) assays, Folin-Ciocalteu reagent and ergothioneine (ERG) content. ORACtotalvalues ranged from 39 to 138 μmol of Trolox equivalents (TE)/g dry weight (dw). HORAC values ranged from 3.0 to 13.6 μmol of caffeic acid equivalents/g dw. NORAC values ranged from 2.0 to 9.0 μmol TE/g dw. SORAC values ranged from 0.37 to 2.6 kunit superoxide dismutate equivalents/g dw. Polyphenols ranged from 4.2 to 10.6 mg gallic acid equivalents/g dw. A. bisporus mushrooms, especially portabellas, had higher antioxidant capacity relative to the specialty mushrooms tested. ERG ranged from 0.21–2.6 mg/g dw with L. edodes, P. ostreatus, G. frondosa containing a statistically significant greater amount compared to A. bisporus. A good correlation was found between ORACtotal and polyphenols (R2 = 0.86).