International Journal of Food Properties

ISSN: 1094-2912 (Print) 1532-2386 (Online) Journal homepage: www.tandfonline.com/journals/ljfp20

Antioxidant Properties of Cultured Mycelia from

Four Pleurotus Species Produced in Submerged
Medium

Abdurrahman Dundar, Veysi Okumus, Sadin Ozdemir & Abdunnasir Yildiz

To cite this article: Abdurrahman Dundar, Veysi Okumus, Sadin Ozdemir & Abdunnasir Yildiz
(2013) Antioxidant Properties of Cultured Mycelia from Four Pleurotus Species Produced
in Submerged Medium, International Journal of Food Properties, 16:5, 1105-1116, DOI:
10.1080/10942912.2011.576793

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International Journal of Food Properties, 16:1105–1116, 2013
Copyright © Taylor & Francis Group, LLC
ISSN: 1094-2912 print / 1532-2386 online
DOI: 10.1080/10942912.2011.576793

ANTIOXIDANT PROPERTIES OF CULTURED MYCELIA

FROM FOUR PLEUROTUS SPECIES PRODUCED IN
SUBMERGED MEDIUM

Abdurrahman Dundar1 , Veysi Okumus2 , Sadin Ozdemir2 ,

and Abdunnasir Yildiz3
1
Medical Promotion and Marketing Program, Vocational Higher School of Health
Services, Mardin Artuklu University, Mardin, Turkey
2
Department of Biology, Faculty of Science and Arts, Siirt University, Siirt, Turkey
3
Department of Biology, Faculty of Science, Dicle University, Diyarbakir, Turkey

The ethanolic extracts of dried cultured mycelia of Pleurotus ostreatus, Pleurotus eryngii,
Pleurotus florida, and Pleurotus sajor-caju were analyzed for antioxidant activity in
different systems. Tests used are as follows: reducing power, free radical scavenging,
superoxide anion radical scavenging, total antioxidant activity, metal chelating activitiy,
etc.; total phenolic content was determined. The percentage inhibition of P. ostreatus,
P. eryngii, P. florida, and P. sajor-caju at 20 mg/mL concentration on peroxidation in
a β-carotene–linoleic acid system was 57.19, 60.68, 62.12, and 58.81%, respectively. The
reducing power of P. eryngii was higher than the other samples, and its value was 0.86 at
10 mg/mL concentration. P. ostreatus and P. sajor-caju proved to be better at scavenging
superoxide anion radicals than the P. eryngii and P. florida. In the scavenging effect of
DPPH radical test, P. ostreatus showed the highest activity potential and P. sajor-caju
showed the strongest metal chelating capacity.

Keywords: Pleurotus species, Cultured mycelia, Antioxidant, Submerged medium,

Oxidation.

INTRODUCTION
Oxidation is essential to many living organisms for the production of energy to carry
out biological processes. Free radicals are produced in normal and/or pathological cell
metabolism.[1] However, the uncontrolled production of oxygen-derived free radicals is
involved in the onset of many diseases, such as cancer, rheumatoid arthritis, cirrhosis, and
atherosclerosis, as well as in degenerative processes associated with ageing.[2] Almost all
the organisms are well protected against free radical damage by oxidative enzymes, such
as superoxide dismutase (SOD), glutathione peroxidise (GSHPx), and catalase (CAT),[3]
or chemical compounds, such as tocopherol, ascorbic acid, carotenoids, and polyphenol
compounds.[2] Phytochemicals, especially phenolics in fruits and vegetables, are suggested
to be the major bioactive compounds for health benefits and are found to be associated

Received 5 January 2011; accepted 28 March 2011.

Address correspondence to Abdurrahman Dundar, Medical Promotion and Marketing Program, Vocational
Higher School of Health Services, Mardin Artuklu University, Marin 47000, Turkey. E-mail: anzdundar@
gmail.com

1105
1106 DUNDAR ET AL.

with the inhibition of atherosclerosis and cancer.[4] Many species of fruits, vegetables,
herbs, cereals, sprouts, and seeds have been investigated for antioxidant activity in the
past.[5]
Mushrooms have been part of the human diet for thousands of years, involving a large
number of edible species. In most countries, there is a well-established consumer accep-
tance for cultivated mushrooms, probably due to their unique flavor and texture. Edible
wild mushrooms are traditionally used in many Asian countries as food and medicine.[6]
Recently, mushrooms have become an attractive functional food mainly because of their
chemical composition,[1] and this can be explained by the antioxidant capacity of mush-
rooms to scavenge free radicals, which are responsible for oxidative damage of lipids,
proteins, and nucleic acids.
Pleurotus species, the third largest commercially produced mushroom in the world,[7]
are found growing naturally on rotten wood material. The growing increase in consumption
of oyster mushroom is largely due to its taste and medicinal and nutritional properties.[7]
Pleurotus species have been used by human cultures all over the world for their nutritional
value, medicinal properties, and other beneficial effects. Pleurotus species are a good source
of dietary fiber and other valuable nutrients. They also contain a number of biologically
active compounds with therapeutic activities. Oyster mushrooms modulate the immune
system, inhibit tumor growth and inflammation, have hypoglycemic and antithrombotic
activities, lower blood lipid concentrations, prevent high blood pressure and atherosclerosis,
and have antimicrobial and other activities.[6] Mushrooms accumulate a variety of sec-
ondary metabolites, including phenolic compounds, polyketides, terpenes, and steroids.[8]
The antioxidants present in mushrooms are of great interest as protective agents to help
the human body reduce oxidative damage without any interference. They are known as
functional foods and as a source of physiologically beneficial components.[9]
Although there are many studies on cultivated and wild edible mushrooms in the
northern hemisphere, there is little knowledge available about antioxidant properties of
wild edible mushrooms of Turkey. As far as our literature survey could ascertain, there
is no report in the literature on cultured mycelia of these mushroom species collected
from the southeast of Turkey. Therefore, data given for the antioxidant statue of cultured
mycelia of wild mushrooms here could be regarded as the first report on this topic. Our
objective was to evaluate the antioxidant properties of ethanol extract of cultured mycelia
from four wild oyster mushroom species (Pleurotus ostreatus, Pleurotus eryngii, Pleurotus
florida, and Pleurotus sajor-caju), with the antioxidant tests, such as total antioxidant activ-
ity, superoxide anion scavenging activity, reducing power, free radical scavenging activity,
metal chelating activity, and determination of total phenolic compounds.

MATERIALS AND METHODS

Chemicals
1,1-Diphenyl-2-picryl-hydrazyl (DPPH), ferrous chloride, polyoxyethylenesorbi-
tan monolaurate (Tween-20), atocopherol, 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
(Darmstadt, Germany). All other chemicals were analytical grade and obtained from either
Sigma-Aldrich or Merck.
 CULTURED MYCELIA FROM FOUR PLEUROTUS SPECIES 1107

Preparation of Cultured Mycelia of Pleurotus Species

Cultured mycelia of Pleurotus species were obtained via submerged culture by using
the samples originally isolated from fresh specimens collected in the southeast of Turkey.
During the experimental work, the isolates were kept on petri dishes on Malt Extract Agar
(MEA, Fluka, Buchs, Switzerland) at 25 ± 1◦ C, and they were re-inoculated every 3 weeks
to maintain their viability and activity as described by Dundar et al.[10] Isolates were grown
in a malt extract liquid medium. Mycelia was cultured in 500-mL flasks, each of which
contains 100 mL of medium inoculated with 1 cm2 cuts of a 7-day-old culture from MEA,
and each of which is kept in a growth chamber at 25 ± 1◦ C with no agitation. In this
growth condition, the mycelia of mushrooms developed a solid pellicle aggregate and this
aggregate was harvested after 20 days of growth. The mycelial pellets were removed asep-
tically through filtration and washed three times with RO (reverse osmosis) water and then
air-dried in an oven at 40◦ C before analysis.

Mushroom Mycelia Extraction

For ethanol extraction, 5 g of dried cultured mycelia of mushroom samples were
weighed and ground into a fine powder in a mill, then mixed with 100 mL of ethanol
at room temperature at 150 rpm for 24 h. The residue was re-extracted under the same
conditions until the extraction solvents became colorless. The extract obtained was filtered
over Whatman No. 1 paper and the filtrate was collected, then ethyl alcohol was removed
using a rotary evaporator (Laborata 4001, Serial no. 069800367, Heidolph WB, Schwabach,
Germany) at 40◦ C to obtain dry extract. The dried extract was used directly for analyses of
total phenols or dissolved in ethanol up to a concentration of 5 mg/mL and stored at 4◦ C
for further antioxidant analyses.

Total Antioxidant Activitiy by the β-Carotene–Linoleic Acid Method

In this assay, antioxidant capacity was determined by measuring the inhibition of the
volatile organic compounds and the conjugated diene hydroperoxides arising from linoleic
acid oxidation.[11] A stock solution of β-carotene–linoleic acid mixture was prepared as
follows: 0.5 mg β-carotene was dissolved in 1 mL of chloroform (HPLC grade); 25 μL
of linoleic acid and 200 mg of Tween 40 were added to this solution. Chloroform was
completely evaporated using a vacuum evaporator. Then 100 mL of oxygenated distilled
water was added and it was shaken vigorously. Finally, 4.6 mL of this reaction mixture was
dispersed to test tubes and 0.4 mL of various concentrations (0.5, 2.5, 5.0, and 10.0 mg/mL)
of the extracts in ethanol was added and the emulsion system was incubated for up to 2 h at
50◦ C. The same procedure was applied for BHT, a-tocopherol, quercetin, and blank. After
this incubation period, absorbance of the mixtures was measured at 490 nm. Measurement
of absorbance was continued until the color of β-carotene disappeared. The bleaching rate
(R) of β-carotene was calculated according to Eq. (1):

R = ln(a/b)/t, (1)

where ln = natural log, a = absorbance at time 0, b = absorbance at time t (30, 60, 90,
120 min) (Cheung et al., 2003). The antioxidant activity (AA) was calculated in terms of
percentage inhibition related to the control using Eq. (2):
1108 DUNDAR ET AL.

AA = [(Rcontrol -Rsample )/Rcontrol ] × 100. (2)

Antioxidative activities of the extracts were compared with those of BHT, a-tocopherol, and
quercetin at 0.5 mg/mL and also compared with blank consisting of only 0.4 mL ethanol.

Determination of Reducing Power

The reducing power of ethanolic extracts dried cultured mycelia was determined
according to the method of Oyaizu.[12] Various concentrations of ethanolic extract (1, 2,
5, and 10 mg/mL) in 1 mL of ethanol were mixed with phosphate buffer (2.5 mL,0.2 M,
pH 6.6) and 2.5 mL of 1% potassium ferricyanide [K3 Fe(CN)6 ] was added. This mixture
was incubated at 50◦ C for 20 min, and 2.5 mL of trichloroacetic acid (10%) was added
to the mixture, and finally centrifugated for 10 min at 1000 g (MSE Mistral 2000, Serial
no: S693/ 02/444, Sanyo Gallenkamp PLC, Leicestershine, UK). The upper layer of solu-
tion (2.5 mL) was mixed with distilled water (2.5 mL) and FeCl3 (0.5 mL, 0.1%), and the
absorbance was measured at 700 nm.

Superoxide Anion Radical Scavenging Activity

Superoxide radicals of ethanolic extract of cultured mycelia were determined accord-
ing to the method of Zhishen et al.[13] 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 fluorescent lamps. The distance between reactant and lamp was adjusted until the
intensity of illumination reached 4000 lx. The total volume of the reactant was 5 mL and
the concentrations of riboflavin, 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◦ C for
25 min. The photochemically reduced riboflavins generated superoxide anion radicals, thus
reducing NBT to form blue formazan. The un-illuminated reaction mixture was used as a
blank. Absorbance (A) was measured at 560 nm. Ethanolic extracts of mushroom species
and standards were added to the reaction mixture, in which superoxide anion was scav-
enged, thereby inhibiting the NBT reduction. Absorbance (A1 ) was measured and decrease
in superoxide radical was represented by A – A1 . The degree of scavenging was calculated
by the following equation:

% Scavenging = [(A-A1 )/A] × 100. (3)

Free Radical Scavenging Activity

The free radical scavenging activities of mushroom species were measured by 1,1-
diphenyl-2-picryl-hydrazil (DPPH) using the method of Blois.[14] Briefly, 0.1 mM solution
of DPPH in methyl alcohol was prepared and 1 mL of this solution was added to 3 mL
of ethanolic extract of cultured mycelia mushroom species at different concentrations (1.0,
2.0, 5.0, and 10 mg/mL). The mixture was shaken vigorously and left standing at room tem-
perature for 30 min. Then, the absorbance was measured at 517 nm in a spectrophotometer.
Lower absorbance of the reaction mixture would indicate higher free radical scavenging
activity. The capability to scavenge the DPPH radical was calculated using the following
equation:
 CULTURED MYCELIA FROM FOUR PLEUROTUS SPECIES 1109

DPPH scavenging effect = [(A0 -A1 )/A] × 100, (4)

where A0 was the absorbance of the control reaction and A1 was the absorbance in the
presence of the sample of cultured mycelia extract.

Chelating Effect on Ferrous Ions

The chelation of ferrous ions of mushroom species was studied through the method of
Dinis et al.[15] Briefly, different concentrations of ethanolic extract of mushrooms species
(2, 5, and 10 mg/mL) were added to a solution of 2 mM FeCl2 (0.05 mL). The reaction was
initiated by adding 5 mM ferrozine (0.2 mL) and the mixture was shaken vigorously and
left standing at room temperature for 10 min. After the mixture reached equilibrium, the
absorbance of the solution was measured spectrophotometrically at 562 nm. All the tests
and analyses were carried out in triplicate and then averaged. The percentage inhibition of
ferrozine–Fe2+ complex formation is given with this formula:

% Inhibition = [(A0 -A1 )/A] × 100, (5)

where A0 was the absorbance of the control and A1 was the absorbance in the presence of
the sample of mushroom species extract and standards. The control did not contain FeCl2
or ferrozine, complex formation molecules.[16]

Determination of Total Phenolics

Total phenolic compounds of ethanolic extract of cultured mycelia were determined
with Folin–Ciocalteau reagent according to the method of Slinkard and Singleton[17] using
gallic acid as a standard phenolic compound. Briefly, 1 mL of extract solution (containing
1 mg extracts) was transferred into a volumetric flask diluted with distilled water (46 mL);
1 mL of Folin–Ciocalteau reagent was added and the content of the flask was mixed. After
the process of mixing (3 min), 3 mL of Na2 CO3 (2%) was added to the mixture and it was
left to be shaken intermittently for 2 h. The absorbance was measured at 760 nm. The con-
centration of total phenolic compounds in the diluted extract was determined as microgram
of gallic acid equivalent by using an equation that was obtained from the standard gallic
acid graph (R2 = 0.995):

Absorbance = 0.001 × Total phenols[gallic acid equivalent (μg)] − 0.0154. (6)

RESULTS AND DISCUSSION

Total Antioxidant Activity Determination by the β-Carotene–Linoleic
Acid Method
Using the β-carotene–linoleic acid method, ethanolic extract of mycelia of P. ostrea-
tus, P. eryngii, P. florida, and P. sajor-caju showed different patterns of total antioxidant
activities (Table 1). As the concentration increased, the antioxidant activity of extracts
increased. At 10 mg/mL concentrations, P. florida showed the highest linoleic acid thus
preventing capacity against the oxidative stress available in the medium. Antioxidant activ-
ity of this mushroom was found to be 62.12% and this is closely followed by P. eryngii
1110 DUNDAR ET AL.

Table 1 Antioxidant activity (%) of the ethanolic extracts of cultured mycelia and standards in β-carotene–
linoleic acid test system.a

Sample concentration (mg/mL)

Cultured mycelia and standards 0.5 2.5 5.0 10.0

P. eryngii 45.23 ± 0.02 52.12 ± 0.01 54.25 ± 0.02 60.68 ± 0.78

P. ostreatus 38.05 ± 0.01 43.11 ± 0.00 51.23 ± 0.01 57.19 ± 0.05
P. florida 47.03 ± 0.00 52.09 ± 0.03 57.20 ± 0.02 62.12 ± 0.41
P. sajor-caju 34.04 ± 0.01 41.08 ± 0.01 50.21 ± 0.04 58.81 ± 0.90
Trolox 58.34 ± 0.01 63.12 ± 0.01 69.25 ± 0.02 74.73 ± 0.01
BHT 47.93 ± 0.02 52.11 ± 0.00 58.16 ± 0.02 62.45 ± 0.56
BHA 49.50 ± 0.01 53.29 ± 0.03 60.54 ± 0.02 65.62 ± 0.61
α-tocopherol 27.63 ± 0.00 33.78 ± 0.01 40.76 ± 0.02 47.44 ± 0.06
a Values expressed are means ± S.D. of three parallel measurements.

(60.68%), P. sajor-caju (58.81%), and P. ostreatus (57.19%). In this study, the percent-
age inhibitions of positive controls were 74.73, 65.62, 62.45, and 47.44% for trolox,
BHA, BHT, and α-tocopherol, respectively. Lo[18] found that the antioxidant activity of
the ethanolic extracts from the basidiocarp of the P. ostreatus and P. eryngii was 58.60 and
64.50%, respectively, at 5 mg/mL concentration. Jayakumar et al.[19] mentioned that P.
ostreatus showed 56.20% antioxidant activity. Total antioxidant activity of methanolic
extract of the basidiocarp of P. ostreatus was found to be 98.3% by Elmastas et al.[1]
From these findings, we conclude that our ethanolic extract of cultured mycelia species
showed moderate antioxidant activity on the lipid peroxidation and can be used as a natural
antioxidant agent instead of the synthetic antioxidants. The differences between the values
obtained from the study of the antioxidant activity may be due to the diversity of mushroom
species used in this study.[20]

Determination of Reducing Power

The reducing capacity of a compound may serve as a significant indicator of its poten-
tial antioxidant activity. The antioxidant activity of putative antioxidants has been attributed
to various mechanisms, among which prevention of chain initiation, binding of transition
metal ion catalysts, decomposition of peroxides, prevention of continued hydrogen abstrac-
tion, reductive capacity, and radical scavenging.[21] In the present study, assay of reducing
activity was based on the reduction of Fe3+ /ferricyanide complex to the ferrous form in
presence of reductants (antioxidants) in the tested samples. The Fe2+ was then monitored
by measuring the formation of Perl’s Prussian blue at 700 nm.[12] Table 2 shows the reduc-
ing power of cultured mycelia ethanolic extracts as a function of their concentration. As
concentration increased (1–10 mg/mL), the reducing power of the ethanolic extracts from
the cultured mycelia increased. At 10.0 mg/mL concentration, the absorbance values were
higher than 0.73 for all of the extracts. According to these results, the most active mushroom
was P. eryngii with an absorbance value of 0.86. At the concentration value of 10 mg/mL,
this mushroom was followed by P. ostreatus, which was 0.81, P. florida, 0.75, and P. sajor-
caju 0.74. Lo[18] mentioned that at 20 mg/mL, reducing powers of ethanolic extracts of
basidiocarps of P. eryngii and P. ostreatus were 0.75 and 0.61, respectively. Jayakumar
et al.[19] reported that the ethanolic extract from P. ostreatus showed a reducing power of
1.367 at 10 mg/mL. BHT, BHA, and α-tocopherol exhibited 0.51, 0.49, and 0.99 activities
 CULTURED MYCELIA FROM FOUR PLEUROTUS SPECIES 1111

Table 2 Reducing power of cultured mycelia and standards.a

Sample concentration (mg/mL)

Cultured mycelia and standards 1.0 2.0 5.0 10.0

P. eryngii 0.05 ± 0.02 0.12 ± 0.01 0.25 ± 0.02 0.86 ± 0.23

P. ostreatus 0.05 ± 0.01 0.11 ± 0.00 0.23 ± 0.01 0.81 ± 0.34
P. florida 0.03 ± 0.00 0.09 ± 0.03 0.20 ± 0.02 0.75 ± 0.23
P. sajor-caju 0.04 ± 0.01 0.08 ± 0.01 0.21 ± 0.04 0.74 ± 0.03
BHT 0.06 ± 0.03 0.15 ± 0.01 0.30 ± 0.02 0.51 ± 0.12
α-tocopherol 0.14 ± 0.01 0.22 ± 0.00 0.53 ± 0.00 0.99 ± 0.21
BHA 0.09 ± 0.00 0.14 ± 0.01 0.17 ± 0.07 0.49 ± 0.16
a Values expressed are means ± S.D. of three parallel measurements.

at 10 mg/mL, respectively. Except for α-tocopherol, reducing powers of all of the ethanolic
extracts of cultured mycelia were superior to the BHT and BHA. It was clearly stated by
the researchers[1,18] that the synthetic antioxidants, such as BHT, BHA, and TBHQ, have
mutagenic activity. Therefore, the antioxidant capacity of natural resources could be used
as an alternative to these compounds mentioned.

Scavenging Effect on Superoxide Anion Radicals

As it was presented in Table 3, the superoxide radical-scavenging activity of the
ethanolic extracts of the cultured mycelia of P. ostreatus, P. florida, P.sajor-caju, and P.
eryngii was measured by the riboflavin-light system in vitro. The superoxide radical is
known to be very harmful to cellular components since it is a precursor of more reactive
oxygen species.[22] Photochemical reduction of flavin generates superoxide anion radi-
cals, which could reduce NBT, resulting in the formation of blue formazan.[23] In the
present study, cultured mycelia extracts were found to be notable scavengers of superoxide
radicals generated in the riboflavin-NBT light system. As in other tests, superoxide radical-
scavenging activity of ethanolic extracts from cultured mycelia increased with increasing
concentrations (1–10 mg/mL). The extracts in a concentration of 10 mg/mL inhibited the
formation of blue formazan and the percentages of inhibitions were 76.14 for P. ostreatus,
74.77 for P. florida, 75.91 for P. sajor-caju, and 67.92 for P. eryngii. Etahnolic extract from
P. ostreatus, P. florida, and P. sajor-caju proved to be better at scavenging superoxide anion
radicals than P. eryngii at scavenging superoxide anion radicals This may be explained by

Table 3 Superoxide anion radical scavenging effect (%) of cultured mycelia and standards.a

Sample concentration (mg/mL)

Cultured mycelia and standards 1.0 2.0 5.0 10.0

P. eryngii 12.67 ± 0.09 30.14 ± 0.12 48.29 ± 0.32 67.92 ± 0.23

P. ostreatus 17.69 ± 0.08 27.17 ± 0.09 51.94 ± 0.076 76.14 ± 0.34
P. florida 16.21 ± 0.01 30.25 ± 0.11 44.18 ± 0.10 74.77 ± 0.76
P. sajor-caju 18.72 ± 0.02 25.34 ± 0.15 52.74 ± 0.87 75.91 ± 0.62
BHA 91.10 ± 0.06 91.89 ± 0.08 94.86 ± 0.76 95.21 ± 0.29
α-tocopherol 91.89 ± 0.05 92.69 ± 0.02 94.75 ± 0.23 96.35 ± 0.85
a Values expressed are means ± S.D. of three parallel measurements.
1112 DUNDAR ET AL.

the interaction of the different flavonoids in these extracts.[13] BHA and α-tocopherol were
used as positive controls for comparison and displayed excellent activity and they were
95.21 and 96.35%, respectively. All of the tested concentration of standards showed higher
scavenging activity than ethanolic extracts of cultured mycelia. Jayakumar et al.[19] found
that the superoxide anion radical scavenging activity of ethanolic extract of P. ostreatus
basidiocarp was 60.02% and Elmastas et al.[1] found it to be 87%.

Free Radical Scavenging Activity

The model of scavenging the stable DPPH radical is a method that is widely used to
evaluate antioxidant activities in a relatively short time compared with other methods. The
effect of antioxidants on DPPH radical scavenging was thought to result from their hydro-
gen donating ability.[24] DPPH is a stable free radical and accepts an electron or hydrogen
radical in order to become a stable diamagnetic molecule.[25] The reduction capability of
DPPH radicals was determined by the decrease in its absorbance at 517 nm induced by
antioxidants. The maximum absorption of a stable DPPH radical in ethyl alcohol was at
517 nm. The decrease in absorbance of DPPH radical was caused by antioxidants because
of the reaction between antioxidant molecules and the radical, thus resulting in the scaveng-
ing of the radical by hydrogen donation. A discoloration from purple to yellow is visually
noticeable. Hence, DPPH is usually used as a substrate to evaluate antioxidative activ-
ity of antioxidants.[26] Scavenging abilities of cultured mycelia ethanolic extracts sharply
increased from 16.0 to 61.97%, from 16.74 to 68.01%, from 16.74 to 62.82%, and from
19.92 to 71.29% at 1.0 to 10 mg/mL for P. florida, P. eryngii, P. sajor-caju, and P. ostreatus,
respectively (Table 4). BHA and α-tocopherol exhibited higher radical scavenging activity
than cultured mushroom mycelia extracts at all the concentrations and their DPPH radical
scavenging activities were found to be 94.70 and 95.76% at 10 mg/mL concentrations,
respectively. Elmastas et al.[1] found that the methanolic extract from P. ostreatus showed
a high scavenging ability of 81.3% at 200 μg/mL. Lo[18] mentioned that the ethanolic
extracts from P. ostreatus and P. eryngii scavenged DPPH radicals with 74.4 and 92.2%
at 5 mg/mL, respectively. The findings of researchers mentioned above are higher than
ours. These results revealed that ethanolic extracts of cultured mycelia were free radi-
cal scavengers, acting possibly as primary antioxidants. Obviously, the extracts contained
antioxidant components, which could react rapidly with DPPH radicals, and reduce more
DPPH radical molecules. Ethanolic extracts from cultured mycelia might react with free
radicals, which are the major initiators of the autoxidation chain of fat, thereby terminating
the chain reaction.[27]

Table 4 Scavenging effect (%) of cultured mycelia and standards on 1,1-diphenyl-2-picrylhydrazyl.a

Sample concentration (mg/mL)

Cultured mycelia and standards 1.0 2.0 5.0 10.0

P. eryngii 16.74 ± 0.24 31.04 ± 1.23 52.01 ± 1.52 68.01 ± 1.39

P. ostreatus 19.92 ± 0.65 32.42 ± 1.98 58.16 ± 2.30 71.29 ± 1.21
P. florida 16.00 ± 0.52 27.44 ± 2.05 46.82 ± 0.61 61.97 ± 1.87
P. sajor-caju 16.74 ± 0.62 28.18 ± 1.01 47.14 ± 1.01 62.82 ± 0.48
BHA 91.31 ± 0.11 92.16 ± 0.54 93.64 ± 1.76 94.70 ± 0.56
α-tocopherol 92.06 ± 0.48 93.11 ± 0.65 94.49 ± 2.87 95.76 ± 2.43
a Values expressed are means ± S.D. of three parallel measurements.
 CULTURED MYCELIA FROM FOUR PLEUROTUS SPECIES 1113

Metal Chelating Activity

Transition metals have been proposed as the catalysts for the initial formation of
radicals. Chelating agents may stabilize transition metals in living systems and inhibit a
generation of radicals, consequently reducing free radical-induced damage. To better esti-
mate the antioxidant potential of the cultured mycelia extracts, its chelating activity was
evaluated against Fe2+ . The chelating effects of the ethanol extract of cultured mycelia and
standard antioxidants on ferrous ions increased with increasing concentrations (Table 5).
In this assay, ethanolic extracts of cultured mycelia 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 perox-
idation by the Fenton reaction, and also can accelerate peroxidation by decomposing lipid
hydroperoxides into peroxyl and alkoxyl radicals that can themselves abstract hydrogen
and perpetuate the chain reaction of lipid peroxidation.[28] The chelating effects of the cul-
tured mycelia were compared with α-tocopherol and BHT as standards. As can be seen
from the Table 5, chelating capacity of the ethanol extracts was increased with the increas-
ing concentration. At all of the concentrations studied (2.0, 5.0, and 10 mg/mL), ethanolic
extracts of mycelia showed higher chelating activites than the α-tocopherol and BHT. The
chelating effects of ethanolic extracts from cultured mycelia species and standards on the
ferrous ions decreased in the order of P. sajor-caju > P. eryngii > P. ostreatus > P. florida
> α-tocopherol > BHT and values were 69.5, 68.2, 65.6, 57.3, 35.9, and 20.5% at the con-
centration of 10 mg/mL, respectively. The chelating activity of methanolic extract from P.
ostreatus was found as 62.5% at 100 μg/mL. Elmastas et al.[1] and Jayakumar et al.[19]
reported the activity as 60.68% at 10 mg/mL concentration for the ethanolic extracts of P.
ostreatus basidiocarp. The percentage chelating activity of ethanolic extracts from P. eryn-
gii and P. ostreatus basidiocarp at 5 mg/mL was reported as 41.4 and 64.0 by Lo.[18]

Determination of Total Phenolic Compounds

Total phenolic compounds the major natural antioxidant components, found in the
ethanolic extracts from cultured mycelia and their contents, were in order of P. florida > P.
eryngii > P. ostreatus > P. sajor-caju (Table 6). Phenols are important plant constituents
due to their hydroxyl groups thus having scavenging ability.[29] The phenolic compounds
may contribute directly to the antioxidative action.[26] In addition, it was reported that

Table 5 Chelating effect (%) of cultured mycelia and standards.a

Sample concentration (mg/mL)

Cultured mycelia and standards 2.0 5.0 10.0

P. eryngii 20.20 ± 1.25 39.90 ± 1.43 68.20 ± 2.98

P. ostreatus 10.60 ± 1.01 35.80 ± 0.92 65.60 ± 1.12
P. florida 6.70 ± 0.65 32.80 ± 1.10 57.30 ± 1.34
P. sajor-caju 20.30 ± 0.87 41.00 ± 0.54 69.50 ± 1.29
Trolox 0.90 ± 0.20 5.70 ± 1.23 12.20 ± 1.27
α-tocopherol 12.10 ± 1.15 23.20 ± 1.43 35.90 ± 1.62
BHT 7.70 ± 1.12 14.60 ± 1.65 20.50 ± 2.56
BHA 10.70 ± 1.20 13.90 ± 0.12 18.90 ± 1.07
a Values expressed are means ± S.D. of three parallel measurements.
1114 DUNDAR ET AL.

Table 6 Total phenol content of cultured mycelia.a

Cultured mycelia Total phenol mg/g (dry weight)

P. eryngii 4.45 ± 0.03

P. ostreatus 4.37 ± 0.10
P. florida 4.56 ± 0.15
P. sajor-caju 3.97 ± 0.29
a Values expressed are means ± S.D. of three parallel measurements.

phenolic compounds were associated with antioxidant activity and played an important role
in stabilizing lipid peroxidation.[26] Polyphenols, such as BHT and gallate, were known
to be effective antioxidants.[29] Because of the similarities of total phenolic content of
ethanolic extracts from cultured mycelia, they showed close activities in the antioxidant
tests taken under study. Numerous studies have conclusively showed that food consumption
with high phenolic content can reduce the risk of heart disease by slowing the progression
of atherosclerosis, since they act as antioxidants.[30,31] From the study, we conclude that the
cultured mycelia of P. florida, P. eryngii, P. ostreatus, and P. sajor-caju mushroom species
showed effective antioxidant activity as basidiocarps of these mushrooms.[1,17,18]

CONCLUSION
The various antioxidant mechanisms of the ethanolic extracts of cultured mycelia
may be attributed to strong hydrogen-donating ability, metal-chelating ability, and their
effectiveness as good scavengers of superoxide and free radicals. In the present study, the
antioxidant properties of ethanol extracts from cultured mycelia of P. florida, P. eryngii, P.
ostreatus, and P. sajor-caju were described by using a series of testing systems in vitro like
the ones mentioned above. According to the results of this study, it is clearly seen that the
ethanolic extract of cultured mycelia of P. florida, P. eryngii, P. ostreatus, and P. sajor-caju
mushroom species has significant antioxidant activity against various antioxidant 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 the pharmaceutical industry.
Furthermore, it is known that production of mycelia is easier than the production of fun-
gus basidiocarps under laboratory conditions. Therefore, these cultured mycelia species
can be produced in a desired level in huge fermentors instead of basidiocarp production
of these mushrooms. In order to investigate the antioxidant mechanism of some poten-
tial antioxidant molecules, the fractionation and the identification of the ethanolic extract
containing the low molecular weight compounds are in progress.

ACKNOWLEDGMENTS
This work was performed with financial support from the Scientific Research Commission
of Dicle University, Project No. 08-FF-05. The authors wish to thank Emrah Eris for his support in
English.

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