JOURNAL OF

FOOD COMPOSITION
AND ANALYSIS
Journal of Food Composition and Analysis 20 (2007) 337345
Original Article
Determination of antioxidant activity and antioxidant compounds in
wild edible mushrooms
Mahfuz Elmastas
a,
, Omer Isildak
a
, Ibrahim Turkekul
b
, Nuri Temur
a
a
Gaziosmanpas -a University, Faculty of Science and Arts, Department of Chemistry, 60240 Tokat, Turkey
b
Gaziosmanpas -a University, Faculty of Science and Arts, Department of Biology, 60240 Tokat, Turkey
Received 13 July 2005; received in revised form 20 June 2006; accepted 3 July 2006
Abstract
The methanolic extracts of dried Agaricus bisporus, Polyporus squamosus, Pleurotus ostreatus, Lepista nuda, Russula delica, Boletus
badius, and Verpa conica were analyzed for antioxidant activity in different systems including reducing power, free radical scavenging,
superoxide anion radical scavenging, total antioxidant activity, and metal chelating activities. Those various antioxidant activities were
compared to standard antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and a-tocopherol. The
percentage inhibition methanolic extracts of dried Russula delica, Boletus badius, Agaricus bisporus, Polyporus squamosus, Pleurotus
ostreatus, Lepista nuda and Verpa conica at 100 mg/mL concentrations on peroxidation in linoleic acid system were 99.7%, 99.2%, 98.8%,
98.4%, 98.3%, 97.9% and 97.7%, respectively, and greater than those 400 mg/mL of a-tocopherol, BHA and BHT (77%, 85%, and
97%). Among methanolic extracts from seven wild edible mushrooms, the reducing power of Russula delica and Verpa conica were
excellent, and were 1.32 and 1.22 at 200 mg/mL, respectively. Methanolic extract from Verpa conica, Boletus badius and Russula delica
proved to be better at scavenging O
2
d
than other mushroom species. The scavenging effects of methanolic extracts from mushroom
species and standards on the DPPHd radical decreased in the order of BHA4a-tocopherol4Lepista nuda4Russula delica4Polyporus
squamosus4Pleurotus ostreatus4Agaricus bisporus4Verpa conica4Boletus badius and were, at the concentration of 180 mg/mL, 97.4,
95.4, 91.3, 86.1, 82.8, 81.3, 77.5, 75.7 and 68.7, respectively. The metal scavenging effect of extract of the mushroom species and
standards decreased in the order of Verpa conica4Lepista nuda4Russula delica4Boletus badius4Polyporus squamosus4BHT4Pleur-
otus ostreatus4Agaricus bisporus4BHA4a-tocopherol. On the other hand, total phenolic compounds, a-tocopherol, and b-carotene
were determined in the methanolic extracts of dried Agaricus bisporus, Polyporus squamosus, Pleurotus ostreatus, Lepista nuda, Russula
delica, Boletus badius, and Verpa conica.
r 2006 Elsevier Inc. All rights reserved.
Keywords: Antioxidant activity; Antioxidant compound; Mushroom; Edible mushroom; Food safety
1. Introduction
Free radicals are produced in normal and/or pathologi-
cal cell metabolism. Oxidation is essential to many living
organisms for the production of energy to fuel biological
processes. However, the uncontrolled production of oxy-
gen-derived free radicals is involved in the onset of many
diseases such as cancer, rheumatoid arthritis, cirrhosis and
arteriosclerosis as well as in degenerative processes
associated with ageing. Exogenous chemical and endogen-
ous metabolic processes in the human body or in the food
system might produce highly reactive free radicals,
especially oxygen derived radicals, which are capable of
oxidizing biomolecules, resulting in cell death and tissue
damage (Halliwell and Gutteridge, 2003). Almost all
organisms are well protected against free radical damage
by oxidative enzymes such as superoxide dismutase (SOD)
and catalase (CAT), or chemical compounds such as
a-tocopherol, ascorbic acid, carotenoids, polyphenol com-
pounds and glutathione (Niki et al., 1994). When the
mechanism of antioxidant protection becomes unbalanced
by factors such as ageing, deterioration of physiological
ARTICLE IN PRESS
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0889-1575/$ - see front matter r 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2006.07.003

Corresponding author. Fax: +90 3562521585.

E-mail addresses: elmastas@gop.edu.tr (M. Elmastas),
omeris@gop.edu.tr (O. Isildak).
functions may occur, resulting in diseases and accelerated
ageing. However, antioxidant supplements or antioxidant-
containing foods may be used to help the human body to
reduce oxidative damage (Halliwell and Gutteridge, 2003;
Mau et al., 2001; Gu lc-in et al., 2002a, b).
Many species of fruits, vegetables, herbs, cereals, sprouts
and seeds have been investigated for antioxidant activity
during the past decade (Elmastas et al., 2005, 2006a;
Kahkonen et al., 1999, Velioglu et al., 1998, Gu lc-in et al.,
2002a, b). Natural antioxidants are being extensively
studied for their capacity to protect organisms and cells
from damage brought on by oxidative stress, the latter
being considered a cause of ageing and degenerative
diseases (Cazzi et al., 1997). Herbs and spices are, in
general, harmless sources for obtaining natural antioxi-
dants. Plant tissue antioxidant capacity is clearly associated
with the activity of free radical scavenging enzymes
(superoxide dismutase, catalase, peroxidase, etc.) and with
the contents of antioxidant substances, mainly phenolic
compounds, carotenoids, tocopherol and ascorbic acid
(Bartosz, 1997). It is evident that there is an increasing
demand to evaluate the antioxidant properties of direct
plant extract (McClements and Decker, 2000).
Fruit bodies of mushrooms are appreciated, not only for
texture and avour but also for their chemical and
nutritional properties. Wild edible mushrooms are tradi-
tionally used in many Asian countries in both food and
medicine (Isildak et al., 2004; Tu rkekul et al., 2004; Manzi
et al., 1999; Sanmee et al., 2003). Mushrooms have also
been reported as therapeutic foods that are useful in
preventing diseases such as hypertension, hypercholester-
olemia and cancer. These functional characteristics are
mainly due to their chemical composition (Manzi et al.,
2001). In most countries, there is a well-established
consumer acceptance for cultivated mushrooms (Agaricus
bisporus, Pleurotus spp., Lentinus edodes, Volveriella
volvacea, Auricularia spp., etc.). Although the edible wild
mushrooms command higher prices than cultivated mush-
rooms, people prefer to consume them due to their avour
and texture. Nevertheless, wild edible mushrooms are
becoming more and more important in our diet for their
nutritional and pharmacological characteristics (Breene,
1990; Crisan and Sands, 1978; Bobek et al., 1991; Manzi
et al., 2001).
Although there are many studies on cultivated and
wild edible mushrooms in the northern hemisphere, there is
little information available about antioxidant properties of
wild edible mushrooms of Turkey, and this is the rst study
on the antioxidant statue of wild edible mushrooms
collected from northern Turkey. Our objective was to
evaluate the antioxidant properties of methyl alcohol
extract of seven wild edible mushroom species (Agaricus
bisporus, Polyporus squamosus, Pleurotus ostreatus, Lepista
nuda, Russula delica, Boletus badius, Verpa conica),
including total antioxidant activity, superoxide anion
scavenging activity, reduction power and free radical
scavenging activity, metal chelating activity and determi-
nation of a-tocoferol, b-carotene and total phenolic
compounds.
2. Materials and methods
2.1. Chemicals
1,1-Diphenyl-2-picryl-hydrazyl (DPPHd), ferrous chlor-
ide, polyoxyethylenesorbitan monolaurate (Tween-20), a-
tocopherol, 3-(2-pyridyl)-5,6-bis (4-phenyl-sulfonic acid)-
1,2,4-triazine (Ferrozine), butylated hydroxyanisole
(BHA), and trichloraceticacid (TCA) were purchased from
Sigma (Sigma-Aldrich GmbH, Sternheim, Germany).
Ammonium thiocyanate and butylated hydroxytoluene
(BHT) were purchased from E. Merck. All other chemicals
were analytical grade and obtained from either Sigma-
Aldrich or Merck.
2.2. Plant material and extraction
Fresh and mature mushrooms were harvested from
the Black Sea region of Turkey and were authenticated by
Dr. Ibrahim Tu rkekul, Department of Biology, Faculty of
Science and Arts, Gaziosmanpas-a University, Tokat,
Turkey. They were stored at the Gaziosmanpasa Uni-
versity Faculty of Art and Science Microbiology Herbar-
ium Laboratory (Herbarium numbers: Agaricus bisporus
Turk 1920, Polyporus squamosus Turk 1470, Pleurotus
ostreatus Turk 1950, Verpa conica Turk 2152, Lepista nuda
Turk 2104, Russula delica Turk 930, Boletus badius
Turk 1832).
Fresh mushrooms from each species were divided into
seven parts and then air-dried in an oven at 40 1C before
analysis. For methyl alcohol extraction, 10 g of dried
mushroom samples was weighed and ground into a ne
powder in a mill, then mixed with 100 mL methyl alcohol at
room temperature at 150 rpm for 24 h. The residue was
re-extracted under the same conditions until the extraction
solvents became colourless. The extract obtained was
ltered over Whatman no. 1 paper and the ltrate was
collected, then methyl alcohol was removed using a rotary
evaporator (Laborata 4001, Heidolph WB, Germany, and
Serial no. 069800367) at 40 1C to obtain dry extract. The
extract were placed in a plastic bottle and then stored at
4 1C to prevent oxidative damage until analysis for nearly a
week.
2.3. Total antioxidant activity determination
The total antioxidant activities were determined accord-
ing to the thiocyanate method (Mitsuda et al., 1996). Ten
milligrams of dried extract of each mushroom species
were dissolved in 10 mL methyl alcohol. Various concen-
trations of methanolic extract from mushroom species
(50400 mg/mL) in 2.5 mL of potassium phosphate buffer
(0.04 M, pH 7.0) were added to linoleic acid 2.5 mL of
emulsion in potassium phosphate buffer (0.04 M, pH 7.0).
ARTICLE IN PRESS
M. Elmastas et al. / Journal of Food Composition and Analysis 20 (2007) 337345 338
The control solution consisted a 5 mL solution consisting
of 2.5 mL linoleic acid emulsion and 2.5 mL potassium
phosphate buffer (0.04 M, pH 7.0). The mixed solution was
incubated at 37 1C in a glass ask and in the dark. During
incubation analysis performed every 5 h. After reaction
with FeCI
2
and thiocyanate, the peroxide values were
determined at 3-min intervals by reading absorbance at
500 nm in a spectrophotometer (Jasco V-530 UV/VIS
Spectrophotometer, Japan Servo Co. Ltd., Japan, Serial
no: B099560512), after reaction with FeCI
2
and thiocya-
nate at intervals during incubation. During the linoleic acid
oxidation, peroxides formed which oxidized Fe
+2
to Fe
+3
.
The Fe
+3
ions form a complex with SCN

, and this
complex has maximum absorbance at 500 nm. The solu-
tions without added extract were used as blank samples.
All data about total antioxidant activity were the average
of triplicate analyses. The inhibition of lipid peroxidation
(as %) was calculated by the following equation:
% Inhibition 100 A
1
=A
0
100,
where A
0
was the absorbance of the control reaction and
A
1
the absorbance in the presence of the sample of
mushroom species extract (Elmastas et al., 2006b; Gu lc-in
et al., 2003a, b).
2.4. Determination of reducing power
The reducing power of methanolic extract of mushroom
species was determined according to the method of Oyaizu
(1986). Various concentrations of mushroom species
extract (25, 50, 100, 200, 400, 600 mg/mL) in 1 mL of
methyl alcohol were mixed with phosphate buffer (2.5 mL,
0.2 M, pH 6.6) and 2.5 mL of 1% potassium ferricyanide
[K
3
Fe(CN)
6
] The mixture was incubated at 50 1C for
20 min, then 2.5 mL of trichloroacetic acid (10%) was
added to the mixture, which was then centrifugation for
10 min at 1000g (MSE Mistral 2000, UK, Serial no: S693/
02/444). The upper layer of solution (2.5 mL) was mixed
with distilled water (2.5 mL) and FeCl
3
(0.5 mL, 0.1%), and
the absorbance was measured at 700 nm. A higher
absorbance indicates a higher reductive capability.
2.5. Superoxide anion radical scavenging activity
Superoxide radicals of methanolic extract of mushroom
species were determined according to the method of
Zhishen et al. (1999). All solutions were 0.05 M in
phosphate buffer (pH 7.8). The photo-induced reactions
were performed in an aluminium foil-lined box with two
20 W uorescent lamps. The distance between reactant and
lamp was adjusted until the intensity of illumination
reached 4000 lx. The total volume of reactant was 5 mL
and the concentration of riboavin, methionine and nitro
blue tetrazolium (NBT) were 3 10
6
, 1 10
2
and
1 10
4
M, respectively. The reactant was illuminated at
25 1C for 25 min. The photochemically reduced riboavins
generated O
2
d
, which reduced NBT to form blue
formazan. The un-illuminated reaction mixture was used
as a blank. Absorbance (A) was measured at 560 nm.
Methanolic extract of mushrooms species and standard
were added to the reaction mixture, in which O
2
d
was
scavenged, thereby inhibiting the NBT reduction. Absor-
bance (A
1
) was measured and decrease in O
2
d
was
represented by AA
1
. The degree of scavenging was
calculated by the following equation:
% Scavenging A A
1
=A 100.
2.6. Free radical scavenging activity
The free radical scavenging activities of mushroom
species were measured by 1,1-diphenyl-2-picryl-hydrazil
(DPPHd) using the method of Blois (2002). Briey, 0.1 mM
solution of DPPH in methyl alcohol was prepared and
1 mL of this solution was added to 3 mL of methanolic
extract mushroom species at different concentrations
(20, 40, 60, 80, 120, 180 mg/mL). The mixture was shaken
vigorously and allowed to stand at room temperature for
30 min. Then the absorbance was measured at 517 nm in a
spectrophotometer. Lower absorbance of the reaction
mixture indicated higher free radical scavenging activity.
The capability to scavenge the DPPHd radical was
calculated using the following equation:
DPPHd scavenging effect% A
0
A
1
=A
0
100.
where A
0
was the absorbance of the control reaction and
A
1
the absorbance in the presence of the sample of
mushroom species extract.
2.7. Chelating effect on ferrous ions
The chelating of ferrous ions of mushroom species was
estimated by the method of Dinis et al. (1994). Briey,
different concentrations of methanolic extract of mushrooms
species (25, 50, 100, 200, 300 mg/mL) were added to a solution
of 2mM FeCl
2
(0.05mL). The reaction was initiated by
adding 5mM ferrozine (0.2mL) and the mixture was shaken
vigorously and left standing at room temperature for 10min.
After the mixture had reached equilibrium, the absorbance of
the solution was then measured spectrophotometrically at
562nm. All tests and analyses were run in triplicate and
averaged. The percentage of inhibition of ferrozineFe
2+
complex formation is given by this formula:
% Inhibition A
0
A
1
=A
0
100,
where A
0
was the absorbance of the control and A
1
the
absorbance in the presence of the sample of mushroom species
extract and standards. The control did not contain FeCl
2
or
ferrozine, complex formation molecules (Oktay et al., 2003).
2.8. Determination of antioxidant components
b-Carotene was extracted and analyzed as described by
Rundhaug et al. (1988). Methanolic extract of mushroom
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M. Elmastas et al. / Journal of Food Composition and Analysis 20 (2007) 337345 339
species (20 mg) was extracted with a solution of 1%
pyrogallol (Sigma) in 10 mL of methyl alcohol/dichlor-
omethane (1:1, v/v) for 45 min at room temperature,
ltered through Whatman no. 4 lter paper and the
volume adjusted to 10 mL using the same solution. The
ltrate was ltered using a 0.45-mm CA lter paper prior
to injection onto a high-performance liquid chromatograph
(HPLC). The HPLC system was a Perkin Elmer series 3
instrument which consisted of a dual channel pump and
Rheodyne injection valve with a 20 ml sample loop and a
Spectra system SN 4000 (Thermo separation pruducts,
USA) and Prodigy 5 25 cm, ODS reversed phase colum
(Phenomenex Inc., Torrance, CA, USA). The mobile phase
was methyl alcohol/acetonitrile, 2:8 (v/v), at a ow rate
of 0.8 mL/min and UV detection at 470 nm. Content of
b-carotene was calculated on the basis of the calibration
curve of authentic b-carotene (Sigma).
Tocopherols were extracted and analyzed according to
the method of Carpenter (1979). Methanolic extract from
mushrooms (50 mg) was suspended in 6 mL of pyrogallol
(6% in 95% ethanol) and 4 mL of 60% potassium
hydroxide aqueous solution, and the resulting mixture
was saponied at 70 1C for 20 min. Deionized water
(15 mL) was added and the mixture was extracted with
15 mL of n-hexane. The organic layer was washed with
deionized water to neutral, dried over anhydrous sodium
sulphate, and rotary evaporated to dryness. The residue
was redissolved in 5 mL of n-hexane and ltered prior to
HPLC injection in the same manner as in the b-carotene
assay.
The HPLC system was the same as for the b-carotene
assay. The mobile phase was methyl alcohol/acetonitrile,
2:8 (v/v), at a ow rate of 1.0 mLmin and UV detection at
295 nm. The content of each tocopherol was calculated on
the basis of the calibration curve of each authentic
tocopherol (Sigma).
Total phenolic compounds in the mushroom species of
methanolic extract were determined with FolinCiocalteau
reagent according to the method of Slinkard and Singleton
(1977) using gallic acid as a standard phenolic compound.
Briey, 1 mL of extract solution (containing 1 mg extracts)
was transferred into a volumetric ask diluted with distilled
water (46 mL); 1 mL of FolinCiocalteau reagent was
added and the content of the ask mixed thoroughly.
After 3 min, 3 mL of Na
2
CO
3
(2%) was added then the
mixture was allowed to stand for 2 h with intermittent
shaking. The absorbance was measured at 760 nm. The
concentration of total phenolic compounds in the diluted
extract determined as microgram of gallic acid equivalent
by using an equation that was obtained from standard
gallic acid graph (R
2
0:995).
Absorbance 0:001 Total phenols
gallic acid equivalent mg 0:0154.
2.9. Statistical analysis
All the experimental results were the mean (7standard
deviation) of three parallel measurements. Data were
evaluated by using one-way variance analysis (Statmost,
1995). P values o0.05 were regarded as signicant and P
values40.01 as very signicant.
3. Results and discussion
The families and habitat of the seven species of wild
mushrooms are given in Table 1 (Philips, 1981). Numerous
antioxidant methods and modications have been pro-
posed to evaluate antioxidant activity and to explain how
antioxidants function. Of these, total antioxidant activity,
reducing power, DPPH assay, metal chelating, active
oxygen species such as H
2
O
2
, O
2
d
, and OHd quenching
assay are most commonly used for the evaluation of
antioxidant activities of extracts (Duh et al., 1999; Chang
et al., 2002).
3.1. Total antioxidant activity determination in linoleic acid
emulsion
Using the thiocyanate method, methanolic extract of
seven wild edible mushrooms showed different patterns of
total antioxidant activities (Fig. 1). The antioxidant activity
of extract increased with increasing concentration. At the
100 mg/mL concentrations, mushroom species showed
higher antioxidant activities than those 400 mg/mL con-
centration of a-tocopherol, BHT and BHA. The percentage
inhibition of methanolic extract of Russula delica, Boletus
badius, Agaricus bisporus, Polyporus squamosus, Pleurotus
ostreatus, Lepista nuda and Verpa conica at 100 mg/mL
ARTICLE IN PRESS
Table 1
Families and habitat of seven edible wild mushroom species
Mushroom species Habitat
Agaricus bisporus (Lange) Pila t On manure heaps, garden waste and roadsides
Polyporus squamosus Huds. ex. Fr. In parasitic growth on deciduous trees
Pleurotus ostreatus (Jacp. ex. Fr.) Kummer Often in large clusters on stumps and fallen or standing trunks, usually of deciduous trees
Verpa conica Swartz ex Pers. In gardens and abandoned terrain
Lepista nuda (Bull. ex. Fr.) Cooke In woodland, hedgerows and gardens
Russula delica Fr. Under both broad-leaved and coniferous trees
Boletus badius Fr. In mixed woods
M. Elmastas et al. / Journal of Food Composition and Analysis 20 (2007) 337345 340
concentrations on peroxidation in linoleic acid system were
99.7%, 99.2%, 98.8%, 98.4%, 98.3%, 97.9% and 97.7%,
respectively, and higher than those of the 400 mg/mL
concentrations of a-tocopherol, BHA and BHT (77%,
85%, 97%). At the 100 mg/mL concentration, Russula
delica and Boletus badius showed excellent antioxidant
activities in this study. On the other hand, the methanolic
extract from the wild edible mushrooms Russula delica and
Boletus badius showed the highest antioxidant activities of
the group under analysis (Agaricus bisporus, Polyporus
squamosus, Pleurotus ostreatus, Lepista nuda and Verpa
conica).
In previous studies, the antioxidant activities of metha-
nolic extraction of several commercial and medicinal
mushrooms have been reported (Yang et al., 2002; Mau
et al., 2004). Those studies claimed that the methanolic
extractions of mushroom species showed high antioxidant
activity on the lipid peroxidation.
3.2. Determination of reducing power
The reducing capacity of a compound may serve as a
signicant indicator of its potential antioxidant activity
(Meir et al., 1995). For the measurements of the reductive
ability, we investigated the Fe
3+
Fe
2+
transformation in
the presence of the mushroom species extract samples using
the method of Oyaizu (1986). Fig. 2 indicates the reductive
capabilities of methanolic extract of mushroom species.
Among methanolic extracts from seven wild edible mush-
rooms, the reducing power of Russula delica and Verpa
conica were excellent and were 1.32 and 1.22 at 200 mg/mL,
respectively (Fig. 2). The antioxidant activity of putative
antioxidants have been attributed to various mechanisms,
among which are prevention of chain initiation, binding of
transition metal ion catalysts, decomposition of peroxides,
prevention of continued hydrogen abstraction, reductive
capacity and radical scavenging (Diplock, 1997; Gu lc-in
et al., 2003a, b). The reducing powers of methanolic
extracts from Boletus badius, Lepista nuda, Agaricus
bisporus, Pleurotus ostreatus, and Polyporus squamosus
increased with increasing concentration. All of the amounts
of methanolic extracts of mushroom species showed higher
activities than BHA, BHT and a-tocopherol, and these
differences were statistically very signicant (Po0.01).
Reducing power of methyl alcohol extract of mushroom
species and standard compounds followed the order:
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0
0.2
0.4
0.6
0.8
1
1.2
1.4
0 5 10 15 20 25 30 35 40
Incubation time (h)
A
b
s
o
r
b
a
n
c
e

(
5
0
0

n
m
)
Control
A. bisporus
P. squamasus
P. ostreatus
L. nuda
R. delica
B. badius
V. conica
Fig. 1. Determination of total antioxidant activity of methanolic extract of mushroom species at 100 mg/mL in the linoleic acid emulsion was determined
by the thiocyanate method.
0
0.5
1
1.5
2
2.5
0 100 200 300 400 500 600
Concentration (g/ml)
A
b
s
o
r
b
a
n
c
e

(
7
0
0

n
m
)
A. bisporus
P. squamasus
P. ostreatus
L. nuda
R. delica
B. badius
V. conica
BHA
Tocopherol
BHT
Fig. 2. Comparision of reducing power of different concentrations of methanolic extract of mushroom species, BHA, BHT, and a-tocopherol by
spectrophotometric detection of the Fe
+3
Fe
+2
transformation at 700 nm.
M. Elmastas et al. / Journal of Food Composition and Analysis 20 (2007) 337345 341
Russula delica4Verpa conica4Boletus badius4Lepista
nuda4Agaricus bisporus4Pleurotus ostreatus4Polyporus
squamosus4BHA4BHT4a-tocopherol.
3.3. Superoxide anion scavenging activity
The decrease of absorbance at 560 nm with antioxidants
indicates the consumption of superoxide anion in the
reaction mixture. Fig. 3 shows the % inhibition of super-
oxide radical generation by 10, 20, 30, 40, 50mg/mL
concentration of methanolic extract of wild edible mush-
room species. Metahnolic extract from Verpa conica, Boletus
badius and Russula delica proved to be better at scavengind
O
2
d
than Lepista nuda, Pleurotus ostreatus, Agaricus
bisporus and Polyporus squamosus. This may be explained
by the interaction of the different avonoids in these extracts
(Zhishen et al., 1999). The mushroom species have strong
superoxide radical scavenging activity and exhibited higher
superoxide radical scavenging activity. The percentage
inhibition of superoxide generation by 50mg/mL concentra-
tion of methanolic extract of wild edible mushroom species
was found as 99% for Verpa conica, 98% for Boletus badius,
97% for Russula delica, 94% for Lepista nuda, 87% for
Pleurotus ostreatus, 71% for Agaricus bisporus and 78% for
Polyporus squamosus, respectively.
3.4. Free radical scavenging activity
The model of scavenging the stable DPPHd radical is a
widely used method to evaluate antioxidant activities in a
relatively short time compared with other methods. The
effect of antioxidants on DPPHd radical scavenging was
thought to result from their hydrogen donating ability
(Baumann et al., 2002). DPPHd is a stable free radical and
accepts an electron or hydrogen radical to become a stable
diamagnetic molecule (Soares et al., 1997). The reduction
capability of DPPH radicals was determined by the
decrease in its absorbance at 517 nm induced by antiox-
idants. The absorption maximum of a stable DPPHd
radical in methyl alcohol was at 517 nm. The decrease in
absorbance of DPPHd radical caused by antioxidants,
because of the reaction between antioxidant molecules and
the radical, progresses, which results in the scavenging of
the radical by hydrogen donation. It is visually noticeable
as a discoloration from purple to yellow. Hence, DPPHd is
usually used as a substrate to evaluate antioxidative
activity of antioxidants (Duh et al., 1999; Chang et al.,
2002).
Scavenging effects of methanolic extracts from mush-
room species on DPPHd radicals increased with increased
concentrations (Fig. 4). As shown in Fig. 4, the decrease in
the concentration of DPPHd radical due to the scavenging
ability of methanolic extracts from mushroom species
and standards is very signicant (Po0.01). Methyl alcohol
extract of the mushroom species was showed strong
DPPHd scavenging activity. We used BHA and
a-tocopherol as standards. The scavenging effects of
methanolic extracts from mushroom species and standards
on the DPPHd radical decreased in the order of BHA4
a-tocopherol4Lepista nuda4Russula delica4Polyporus
squamosus4Pleurotus ostreatus4Agaricus bisporus4Ver-
pa conica4Boletus badius and were 97.4, 95.4, 91.3, 86.1,
82.8, 81.3, 77.5, 75.7 and 68.7 at the concentration of
180 mg/mL, respectively. These results indicated that
methanolic extracts of mushroom species have a noticeable
effect on scavenging free radical. However, the scavenging
effect of BHA and a-tocopherol are higher than all
methanolic extracts of mushrooms species (Fig. 4). At the
previous studies, the same situations were reported (Yang
et al., 2002; Mau et al., 2004; Yen and Wu, 1993).
These results revealed that methanolic extracts of
mushroom species were free radical scavengers, acting
possibly as primary antioxidants. Methanolic extracts from
wild edible mushroom species might react with free
radicals, which are the major initiator of the autoxidation
chain of fat, thereby terminating the chain reaction
(Frankel, 1991; Gordon, 1990).
3.5. Metal chelating activity
The binding of ferrous ions by methanolic extracts of
mushroom species was estimated by the method of Dinis
ARTICLE IN PRESS
0
20
40
60
80
100
0 10 20 30 40 50
S
c
a
v
e
n
g
i
n
g

(
%
)
A. bisporus
P. squamasus
P. ostreatus
L. nuda
R. delica
B. badius
V. conica
Concentration (g/ml)
Fig. 3. Comparison of superoxide anion radical scavenging activity of different concentrations of methanolic extract of mushroom species.
M. Elmastas et al. / Journal of Food Composition and Analysis 20 (2007) 337345 342
et al. (1994). Ferrozine can quantitatively form complexes
with Fe
2+
. In the presence of chelating agents, the complex
formation is disrupted with the result that the red colour of
the complex is decreased. Measurement of colour reduction
therefore allows estimation of the chelating activity of the
coexisting chelator (Yamaguchi et al., 2000). In this assay,
methanolic extracts of mushroom species and standard
antioxidant compounds interfered with the formation of
ferrous and ferrozine complex, suggesting that they have
chelating activity and capture ferrous ion before
ferrozine.
Iron can stimulate lipid peroxidation by the Fenton
reaction, and also accelerates peroxidation by decomposing
lipid hydroperoxides into peroxyl and alkoxyl radicals that
can themselves abstract hydrogen and perpetuate the chain
reaction of lipid peroxidation (Halliwell, 1991; Chang
et al., 2002).
As shown in Fig. 5, the formations of the Fe
2+
-ferrozine
complex were prevented by methanolic extracts of mush-
room species. The absorbance of Fe
2+
-ferrozine complex
was linearly decreased dose dependently (25, 50, 100, 200,
300 mg/mL). The percentage of metal chelating capacity of
100 mg/mL concentration of methanolic extract of Russula
delica, Boletus badius, Agaricus bisporus, Polyporus squa-
mosus, Pleurotus ostreatus, Lepista nuda and Verpa conica,
BHA, a-tocopherol, and BHT were found as 83.2%,
77.6%, 58.5%, 74.2%, 62.5%, 85.0%, 99.1%, 47.8%,
45.7% and 68.5%, respectively (Fig. 5). However, there
was statistically a very signicant difference between
100 mg/mL of methanolic extracts of some mushroom
species (Verpa conica, Lepista nuda, Russula delica and
Boletus badius) and 100 mg/mL of BHT, BHA and
a-tocopherol (Po0.01). The metal scavenging effect of
metanolic extracts of Pleurotus ostreatus and Agaricus
bisporus are lower than BHT, but their metal chelating
effects are higher than BHA and a-tocopherol. The metal
scavenging effect of extract of the mushroom species and
standards decreased in the order of Verpa conica4Lepista
nuda4Russula delica4Boletus badius4Polporus squamo-
sus4BHT4Pleurotus ostreatus4Agaricus bisporus4
BHA4a-tocopherol.
The data obtained from Fig. 5 reveal that the metanolic
extract of wild edible mushrooms in this study demon-
strated a marked capacity for iron binding, suggesting that
ARTICLE IN PRESS
0
20
40
60
80
100
0 20 40 60 80 100 120 140 160 180 200
Concentration (g/ml)
S
c
a
v
e
n
g
i
n
g

e
f
f
e
c
t

(
%
)
A. bisporus
P. squamasus
P. ostreatus
L. nuda
R. delica
B. badius
V. conica
BHA
Tocopherol
Fig. 4. Free radical scavenging activity of different concentrations of methanolic extract of mushroom species, BHA, BHT, and a-tocopherol by
1,1-diphenyl-2-picrylhydrazyl radicals.
0
20
40
60
80
100
0 50 100 150 200 250 300 350
Concentration (g/ml)
C
h
e
l
a
t
i
n
g

e
f
f
e
c
t
s

(
%
)
A. bisporus
P. squamasus
P. ostreatus
L. nuda
R. delica
B. badius
V. conica
tocopherol
BHA
BHT
Fig. 5. Metal chelating effects of different concentrations of methanolic extract of mushroom species, BHA, BHT, and a-tocopherol on ferrous ions.
M. Elmastas et al. / Journal of Food Composition and Analysis 20 (2007) 337345 343
their action as peroxidation protector may be related to its
iron binding capacity.
3.6. Determination of a-tocopherol, b-caroten and total
phenolic compounds
The highest quantities of a-tocopherol were found in
Agaricus bisporus , Boletus badius and Russula delica
species (9.2070.14, 8.8870.12 and 4.2070.08 mg/g, re-
spectively). The a-tocopherol content in other mushroom
species was lower than 1.40-mg/g; b-caroten was not
detected in Pleurotus ostreatus or Boletus badius. However,
b-caroten contents in other musroom species were found in
small amounts (from 6.75 10
3
to 3.58 10
2
mg/g)
(Table 2).
Total phenolic compound the major natural antioxidant
components found in the methanolic extracts from wild
edible mushrooms and their contents were in order of
Russula delica4Boletus badius4Verpa conica4Polyporus
squamosus4Agaricus bisporus4Pleurotus ostreatus4
Lepista nuda (Table 2). Phenols are important plant
constituents because of their scavenging ability due to
their hydroxyl groups (Hatano et al., 1989). The phenolic
compounds may contribute directly to the antioxidative
action (Duh et al., 1999). In addition, it was reported that
phenolic compounds were associated with antioxidant
activity and play an important role in stabilizing lipid
peroxidation (Yen et al., 1993). Polyphenols such as BHT
and gallate were known to be effective antioxidants
(Halliwell and Gutteridge, 2003). The highest content of
total polyphenolics in Russula delica, Boletus badius and
Verpa conica might be the key components accounting for
the better results found in total antioxidant activity,
reducing power, metal chelating activity and superoxide
anion scavenging activity abilities as compared to other
mushroom species. Numerous studies have conclusively
showed that consumption of foods high in phenolic content
can reduce the risk of heart disease by slowing the
progression of atherosclerosis, because they act as anti-
oxidants (Halliwell and Gutteridge, 2003; Kaur and
Kapoor, 2002; Kahkonen et al., 1999).
It revealed that in addition to these antioxidant
activities, other factors contribute in part to the anti-
oxidant properties of wild edible mushrooms. To study the
antioxidant mechanisms by some other potential anti-
oxidant components, the fractionation of the methanolic
extract and further identication are in progress.
4. Conclusion
According to the results of this study, it is clearly
indicated that the methanolic extract of mushroom species
has signicant antioxidant activity against various anti-
oxidant systems in vitro; moreover, the mushroom species
can be used as an easily accessible source of natural
antioxidants and as a possible food supplement or in
pharmaceutical industry. The various antioxidant mechan-
isms of the mushroom species extract may be attributed to
strong hydrogen-donating ability, a metal-chelating ability,
and their effectiveness as good scavengers of superoxide
and free radicals. Phenolic compounds seem to be the main
compenents responsible for the antioxidant activity of all
the mushroom species extracts.
Acknowledgement
This work was performed with nancial support from
the Scientic Research Commission of Gaziosmanpasa
University, Project no: 2003/32.
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Table 2
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