Food Chemistry 157 (2014) 323–331

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Antioxidant/antiradical properties of microwave-assisted extracts

of three wild edible mushrooms
Mustafa Özyürek ⇑, Mustafa Bener, Kubilay Güçlü, Resßat Apak
Department of Chemistry, Faculty of Engineering, Istanbul University, Avcilar, Istanbul, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: A microwave-assisted extraction (MAE) process for polyphenols from three wild edible mushrooms was
Received 15 October 2013 studied. The optimal extraction conditions were found to be methanol concentration of 80%, extraction
Received in revised form 7 February 2014 temperature of 80 °C, and extraction time of 5 min. Different antioxidant assays (i.e., total antioxidant
Accepted 12 February 2014
capacity (TAC) and total phenolic content (TPC)) were utilized to evaluate the antioxidant capacity of
Available online 22 February 2014
the methanolic extracts of Terfezia boudieri Chatin, Boletus edulis, and Lactarius volemus. The reactive spe-
cies scavenging activities of these extracts were also investigated in vitro. High contents of phenolic and
Keywords:
flavonoid compounds may be the major contributors to the observed high antioxidant activities of these
Wild edible mushrooms
Microwave-assisted extraction
extracts. B. edulis showed the higher TAC and TPC; highest inhibitory effect on DPPH and on other studied
Total phenolic content reactive oxygen species (ROS). MAE showed obvious advantages of high extraction efficiency with lower
Total antioxidant capacity solvent consumption in terms of high antioxidant capacity/activity of extracts achieved within the short-
Antioxidant activity est time.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction scavenge free radicals (Hirano et al., 2001). Wild edible mush-
rooms are widely consumed in many countries, especially by local
‘Oxidative stress’ conditions emerge as a result of the genera- populations of lower income, and have been found to possess good
tion of an unbalanced excess of reactive oxygen species concomi- antioxidant properties well correlated with their phenolic content
tant with a change in cellular redox status, in which biological (Barros, Ferreira, Queiros, Ferreira, & Baptista, 2007).
macromolecules (i.e., proteins, lipids and nucleic acids) can suffer Anatolian Peninsula (i.e., the Asian part of Turkey) is rich in the
oxidative damage causing tissue injury leading to various diseases diversity of mushrooms as well as medicinal plants. Turkish people
(Halliwell & Gutteridge, 1999). Almost all organisms have estab- have a tradition of using a number of wild edible mushrooms
lished antioxidant defences to protect them against oxidative dam- essentially for food rather than for medicinal purposes, e.g., treat-
age. Exogenous dietary antioxidants, which can scavenge free ment of infectious diseases (Akyuz, Onganer, Erecevit, & Kirbag,
radicals and oxidants, contribute to the defence system as benefi- 2010). Anti-proliferative and anti-tumour effects, primarily in hu-
cial protecting agents for the human body (Mayakrishnan et al., man cell lines, have been reported for polysaccharides, i.e., acidic
2013). As a result, the consumption of dietary antioxidants have and neutral compounds with different types of glycosidic linkages,
been suggested for preventing serious diseases originating from as well as some that are bound to protein or peptide residues such
oxidative stress. as polysaccharide–protein complexes, extracted from various
The consumption of natural foods, such as fruits, vegetables and mushrooms; prevention of oncogenesis, enhancement of immune
juices provides protection against various important diseases responses, and induction of apoptosis of tumour cells are possible
(Ames, Shigenaga, & Hagen, 1993). The search for new products mechanisms governing the antitumour and anti-proliferative ef-
with antioxidative properties is a very attractive field of research. fects of polysaccharides in mushrooms and their extracts (Wasser,
Mushrooms have been used for many years as nutritional food 2002). Terfezia boudieri Chatin is a kind of desert truffles. Desert
and food flavouring materials in soups and sauces, due to their truffles are seasonal and socio-economically important fungi.
unique and subtle flavour. Mushrooms contain various polypheno- These truffles are edible and grow wild in the southeast Anatolian
lics recognized as excellent antioxidants due to their ability to part of Turkey. Desert truffles are a rich source of protein, amino
acids, fatty acids, minerals and carbohydrates (Bokhary & Parvez,
1993). Boletus edulis is a delicious mushroom growing in many dis-
⇑ Corresponding author. Tel.: +90 212 4737070; fax: +90 212 4737180. tricts of Europe, North America, and Asia. Fresh and dried species
E-mail address: [email protected] (M. Özyürek).

http://dx.doi.org/10.1016/j.foodchem.2014.02.053
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
324 M. Özyürek et al. / Food Chemistry 157 (2014) 323–331

are found in oriental restaurants, gourmet and health food stores. system is specially designed to operate at elevated temperature
The flavour of dried B. edulis including odour and taste is marvel- monitored by a fibre optic temperature probe. The effects on the
lous-nutty, earthy, and meaty all at once (Tsai, Tsai, & Mau, composition and quantity of the phenolics of interest depend on
2007). Lactarius volemus is widely distributed in the warm temper- many factors involved in MAE (Li et al., 2012). Due to the many
ate or northern districts of the Northern Hemisphere, and is known factors such as microwave temperature, extraction time and sol-
as an edible mushroom (Shimono, Hiroi, Iwase, & Takamatsu, vent concentration that influence MAE, optimization of the extrac-
2007). The seasonal character of wild mushroom collection cause tion protocol is required. In the present study, a rapid and effective
difficulties in the distribution and marketing as fresh products, microwave-assisted method for extracting antioxidant compounds
and consequently, there is very little information concerning the from wild edible mushroom species (T. boudieri Chatin, B. edulis, L.
antioxidant/antiradical activities of the species tested in this study, volemus) was optimized for the first time.
such as B. edulis (Fernandes et al., 2013), and L. volemus (Ozen, Dar-
can, Aktop, & Turkekul, 2011). To fill this literature gap, the antiox- 2. Materials and methods
idant capacity of wild edible mushrooms was determined in this
work by using the optical sensor-based CUPRAC method (Bener, 2.1. Standards and reagents
Özyürek, Güçlü, & Apak, 2010). The chromogenic oxidizing reagent
of the CUPRAC assay (Apak, Özyürek, Güçlü & Karademir, 2004), The following chemical substances of analytical reagent grade
bis(2,9-dimethyl-1,10-phenanthroline)copper(II), is simple, di- were supplied from the corresponding sources: Neocuproine (Nc)
versely applicable to both hydrophilic and lipophilic antioxidants, (2,9-dimethyl-1,10-phenanthroline), DPPH (2,2-diphenyl-1-pic-
stable and easily available at low cost. The CUPRAC method has rylhydrazyl), catalase from bovine liver (1340 U mg 1 solid), b-nic-
been successfully applied to food plants (apricot, herbal teas, wild otinamide adenine dinucleotide reduced dipotassium salt (NADH),
edible plants, herby cheese, etc.), and human serum for evaluating nitroblue tetrazolium chloride (NBT), methanol (MeOH), ethanol
antioxidant properties. The main CUPRAC method was modified for (EtOH) and the Folin–Ciocalteau reagent were purchased from
measuring the hydroxyl radical scavenging activities of polyphen- Sigma–Aldrich (St. Louis, MO, USA); NafionÒ 115 perfluorinated
olics (Özyürek, Bektasßoğlu, Güçlü, & Apak, 2008), xanthine oxidase membrane (thickness 0.005 in.) was purchased from Aldrich
(XO) inhibition activity (Özyürek, Bektasßoğlu, Güçlü, & Apak, (Steinheim, Germany); Potassium sodium tartarate tetrahydrate,
2009), hydrogen peroxide scavenging activity of polyphenolics in copper(II) sulphate, copper(II) chloride dihydrate, iron(II) chloride
the presence of Cu(II) catalyst (Özyürek, Bektasßoğlu, Güçlü, tetrahydrate, dimethyl sulfoxide (DMSO), hydrogen peroxide
Güngör, & Apak, 2010), and development of a CUPRAC-based anti- (30%, by wt.), Na2HPO42H2O, NaH2PO42H2O, ammonium acetate
oxidant sensor on a Nafion membrane (Bener et al., 2010). As an- (NH4Ac), sodium hydroxide, and sodium carbonate were pur-
other easy, rapid and low-cost assay, the DPPH free radical chased from E. Merck (Darmstadt, Germany); Disodium–EDTA,
scavenging method (Sánchez-Moreno, Larrauri, & Saura-Calixto, phenazine methosulphate (PMS) and sodium salicylate were pur-
1998) was used for screening antiradical activity of sample ex- chased from Fluka (Buchs, Switzerland).
tracts, based on decolorization of the DPPH radical upon single
electron uptake from antioxidants (Koleva, Van Beek, Linssen,
2.2. Samples
Groot, & Evstatieva, 2002). On the other hand, the measurement
of the scavenging activity of reactive oxygen species (ROS) is also
Wild edible mushrooms (T. boudieri Chatin, B. edulis, L. vole-
important for plant foods containing antioxidants. Although ROS
mus):were purchased from local markets in different regions of
cause adverse health effects such as lipid and protein oxidation,
Anatolia-Turkey. The mushrooms were lyophilized (Telstar
DNA strand break and base modification leading to tissue damage,
LyoQuest, Terrassa, Spain), and kept at +4 °C in hermetically vac-
they may have benign functions, including the activation of nuclear
uum-sealed plastic bags prior to analysis. The yields of T. boudieri
transcription factors, gene expression, cell signalling and regula-
Chatin, B. edulis and L. volemus were 26.1%, 14.9% and 15.3%,
tion, and a defence mechanism to target tumour cells and micro-
respectively.
bial infections (Lee, Koo, & Min, 2004). Thus, in this work, the
antioxidant/antiradical activities of the wild mushroom sample ex-
tracts were determined for the first time by a variety of protocols 2.3. Sample preparation
including the measurement of the scavenging activities of DPPH
radical, hydroxyl radical, hydrogen peroxide, and superoxide anion The fine dried mushroom samples were extracted with the aid
radicals, the latter three ROS being the partially reduced and highly of a microwave-assisted extraction system (Milestone ETHOS
reactive metabolites of molecular oxygen. ONE, Shelton, CT, USA). MAE was utilized in twelve Teflon closed
Several extraction techniques and solvents are used for obtain- vessels with an automatic fibre optic temperature control system,
ing phenolic extracts from different food sources. Extraction of dried sample (0.2 g) was extracted with 20 mL methanol/water
phenolics from mushrooms has been abundantly investigated in (80:20, v/v) at 80 °C for 5 min after 3 min temperature balancing
the last decades, focusing mainly on conventional solvent extrac- time, and the microwave power (0–1500 W) was adjusted auto-
tion. Interest in microwave-assisted extraction (MAE) has recently matically according to temperature. The obtained methanolic ex-
increased due to its special advantages (reduction in extraction tracts were filtered through a filter paper, then through 0.45 lm
time, solvent volume, and better extraction efficiency) over PTFE syringe filters (Whatman), and kept at +4 °C until use. All
conventional solid–liquid extraction techniques (Ballard, the following spectrophotometric assays on the extracts were car-
Mallikarjunan, Zhou, & O’Keefe, 2010), because localized heating ried out in n = 5 replicates.
by microwaves increases the temperature of the solvent above
its boiling point to enhance the extraction efficiency. This high 2.4. Determination of total phenolic content (TPC)
temperature does not give rise to thermal degradation, as MAE
with hexane–acetone (1:1, v/v) solvent mixture at 115 °C and for Total phenolic content (TPC) of the methanolic extract was
10 min was shown earlier not to cause any degradation of the 14 determined using the Folin–Ciocalteau (FC) method as described
tested phenols, recoveries ranging between 80% and 111% by Singleton, Orthofer, and Lamuela-Raventos (1999). The solu-
(Lopez-Avila & Young, 1994). In MAE, all parameters of the extrac- tions used in the assay were prepared as follows: Lowry A: 2%
tion can be achieved by a precise software-based control. The aqueous Na2CO3 in 0.1 M NaOH; Lowry B: 0.5% CuSO4 aqueous
M. Özyürek et al. / Food Chemistry 157 (2014) 323–331 325

solution in 1% NaKC4H4O6 solution; Lowry C: a mixture of 50 mL H2O2 rapidly in this order. The mixture in a total volume of 5 mL
Lowry A and 1 mL Lowry B solutions. The FC reagent was diluted was incubated for 10 min in a water bath kept at 37 °C. After incu-
with H2O at a volume ratio of 1:3 prior to use. The methanolic ex- bation, the reaction was stopped with adding 0.5 mL of 268 U mL 1
tract (0.5 mL) was mixed with 1.5 mL of distilled water and 2.5 mL catalase solution, and mixed for 30 s. Final mixtures (0.5 mL of the
of Lowry C solution in a test tube. Then, 0.25 mL of FC reagent was incubation solution) were subjected to the HRS-CUPRAC method.
added and mixed. After 30 min, the absorbance of the reaction The HRS activity (%) of methanolic extract was calculated using
solution was measured against blank at 750 nm. Since FC assay the equation: HRS activity (%) = [(A0 A)/A0] 100, where A0 and
simultaneously measures total phenolics and antioxidant, the re- A are the CUPRAC absorbances of the system in the absence and
sult was converted to trolox equivalent (lmol TR/g of sample) unit presence of scavenger, respectively. The assays were carried out
based on the standard curve obtained with trolox. The assays were in triplicate and the results expressed as mean value ± standard
carried out in triplicate and the results expressed as mean val- deviation. The extract concentration providing 50% inhibition
ues ± standard deviations. (EC50) was calculated from the curve of HRS inhibition percentage
against extract concentration.
2.5. Determination of total antioxidant capacity (TAC)

The total antioxidant capacity (TAC) of the methanolic extract

was evaluated by the optical sensor-based CUPRAC method de- 2.8. Determination of hydrogen peroxide scavenging (HPS) activity
scribed by Bener et al. (2010). This method involves the reduction
of the CUPRAC chromogenic reagent (Cu(II)–Neocuproine (Nc)) to The ability of methanolic extract to scavenge hydrogen perox-
the Cu(I)–Nc chelate by antioxidants, indirectly measured by virtue ide was determined according to the method of Özyürek et al.
of the light absorption of the cuprous chelate electrostatically ad- (2010). To a test tube were added 0.7 mL of phosphate buffer (pH
sorbed on the Nafion membrane. Nafion, a perfluorosulfonate ion 7.4), 0.4 mL of 1 mM H2O2, 0.4 mL of 0.1 mM CuCl22H2O in this or-
exchange membrane having R-{-O-CF2-CF(CF3)-}x-O-(CF2)2SO3H der (H2O2 incubation solution, used as reference). To the other two
functional groups, was cut into 4.5 0.5 cm pieces and dipped into test tubes were added 0.5 mL of phosphate buffer (pH 7.4), 0.4 mL
a tube that contained 8.2 mL of CUPRAC reagent mix solution (2 mL of 1.0 mM H2O2, 0.2 mL methanolic extract, and 0.4 mL of 0.1 mM
of 10 mM CuCl2, 2 mL of 1.0 M NH4Ac, 2 mL of 7.5 mM Nc fresh CuCl22H2O solution rapidly in this order (named as scavenger
solution, and 2.2 mL of distilled water). After the tube was shaken solutions-I and II). The mixtures in a total volume of 1.5 mL were
for 30 min, the reagent saturated membrane (Nafion–Cu(II)–Nc) incubated for 30 min in a water bath kept at 37 °C. At the end of
was immersed in a tube containing 0.4 mL sample solution and this period, to both reference and scavenger solution-I was added
7.8 mL of ethanol. After agitating for 30 min, the absorbance of 0.4 mL H2O, and to scavenger solution-II was added 0.4 mL of
the coloured membrane (Nafion–Cu(I)–Nc) was measured at 268 U mL 1 catalase solution, and mixed for 30 s. Final mixtures
450 nm against a blank membrane prepared under identical (1.0 mL of the incubation solution) were subjected to the HPS-CU-
conditions excluding analyte (using a Varian CARY Bio 100 UV– PRAC method. The HPS activity (%) of methanolic extract was cal-
Vis spectrophotometer (Mulgrave, Victoria, Australia)). The total culated using the equation: HPS activity (%) = [(A0 (A1 A2))/
antioxidant capacity was expressed using trolox equivalent A0] 100, where A0 is the CUPRAC absorbance of reference H2O2
(lmol TR/g of sample) unit based on the standard curve obtained incubation solution, A1 and A2 are the CUPRAC absorbances of scav-
with trolox. The assays were carried out in triplicate and the re- enger solutions-I and -II, respectively. The assays were carried out
sults expressed as mean value ± standard deviation. in triplicate and the results expressed as mean value ± standard
deviation. The extract concentration providing 50% inhibition
2.6. Determination of free radical scavenging (FRS) activity (EC50) was calculated from the curve of HPS inhibition percentage
against extract concentration.
The scavenging activity of the methanolic extract on DPPH rad-
icals was measured according to the method of Sánchez-Moreno
et al. (1998) with minor modifications. The methanolic extract
(0.35 mL) was mixed with 2.65 mL of methanol and 1 mL of 2.9. Determination of superoxide anion radical scavenging (SARS)
0.1 mM DPPH solution in a test tube. The tubes were stoppered, activity
and after 30 min, the absorbance at 515 nm was recorded against

methanol. The free radical scavenging (FRS) activity was calculated The superoxide anion radicals O2 were generated in vitro in a
as the percentage of DPPH decolorization using the equation: FRS non-enzymatic system (PMS–NADH) and determined spectropho-
activity (%) = [(ADPPH AS)/ADPPH] 100, where ADPPH is the absor- tometrically by nitroblue tetrazolium (NBT) reduction method de-
bance of DPPH solution without sample, AS is the absorbance of scribed by Yu et al. (2005). To a test tube were added (2.5-) mL
the solution when the sample extract has been added at a particu- DMSO, 0.2 mL of methanolic extract, 2 mL of 468 lM NADH,
lar level. The assays were carried out in triplicate and the results 1 mL of 300 lM NBT, in this order. The reaction was started by add-
expressed as mean value ± standard deviation. The extract concen- ing 1 mL of 60 lM PMS solution to the incubation mixture. The
tration providing 50% inhibition (EC50) was calculated from the mixture in a total volume of 6.5 mL was incubated for 5 min in a
curve of FRS inhibition percentage against extract concentration. water bath kept at 25 °C, and the absorbance was read at 560 nm
against DMSO. Decreased absorbance of the incubation reaction
2.7. Determination of hydroxyl radical scavenging (HRS) activity mixture indicated increased superoxide anion radical scavenging
activity. The SARS activity (%) of methanolic extract was calculated
The hydroxyl radicals (OH) in aqueous media were generated using the equation: SARS activity (%) = [(A0 A)/A0] 100, where
through the Fenton system and spectrophotometrically deter- A0 and A are the absorbances of the incubation reaction mixture
mined -via hydroxylation of a probe- by the modified CUPRAC in the absence and presence of scavenger, respectively. The assays
method (Özyürek et al., 2008). To a test tube were added 1.5 mL were carried out in triplicate and the results expressed as mean
of phosphate buffer (pH 7.0), 0.5 mL of 10 mM sodium salicylate, value ± standard deviation. The extract concentration providing
0.25 mL of 20 mM Na2–EDTA, 0.25 mL of 20 mM FeCl2 solution, 50% inhibition (EC50) was calculated from the curve of SARS inhibi-
1.9 mL H2O, 0.1 mL methanolic extract, and 0.5 mL of 10 mM tion percentage against extract concentration.
326 M. Özyürek et al. / Food Chemistry 157 (2014) 323–331

2.10. UPLC–ESI-MS/MS analyses

Ultra performance liquid chromatography (UPLC) analysis was

performed using a Waters Acquity (Milford, MA, USA) ultra
performance liquid chromatographic system equipped with a bin-
ary solvent manager, sample manager, column thermostat were
used for chromatographic measurements. The analyses were per-
formed using Acquity BEH C18 analytical column (100 2.1 mm,
1.7 lm) (Milford, MA, USA). All injected solutions were stored at
4 °C in the auto-sampler. For direct injection analyses, an Acquity
UPLC system coupled to a Xevo TQD (triple quadrupole analyzer)
mass spectrometer (Waters) was employed, equipped with Z-spray
electrospray ionization source (ESI) operating in negative mode.
The ionization working conditions were as follows: capillary volt-
age, 3 kV; collision energy, 20 V; cone gas flow rate, 50 L/h; desolv-
ation temperature, 400 °C; desolvation gas flow rate, 1000 L/h. To
analyze phenolic compounds, the mobile phase consisted of two
solvents, i.e., 0.1% of formic acid in bidistilled water (A) and meth-
anol (B). The polyphenolic antioxidants were analyzed using gradi-
ent elution: (Flow rate = 0.45 mL/min; column temperature:
30 °C): 0 min 90% A – 10% B; 2 min 85% A – 15% B (slope 1.0);
3 min 80% A – 20% B (slope 1.0); 4 min 70% A – 30% B (slope
1.0); 6 min 60% A – 40% B (slope 1.0); 8–10 min isocratic elution
(50% A – 50% B (slope 1.0)); 12 min 90% A – 10% B (slope 1.0). Using
the above working mode, these polyphenolic compounds were
identified by matching retention times and mass spectral data with
those of calibration standards.

2.11. Statistical analysis

Descriptive statistical analyses were performed using Excel

software (Microsoft Office 2007) for calculating the means and
the standard error of the mean. Results were expressed within
95% confidence interval as {mean ± (t.05 s/n1/2)}, where t.05 is the
Student’s table t-value for 4 degrees of freedom and s is the stan-
dard deviation of n = 5 measurements. In related figures, these val-
ues were depicted within error bars.

3. Results and discussion

3.1. Optimization of microwave-assisted extraction (MAE) conditions

Before the analysis of antioxidant properties of wild edible Fig. 1. Effect of microwave-assisted extraction conditions on the total antioxidant
mushroom extracts, MAE conditions were optimized with the aid capacity of Boletus edulis methanolic extract: (a) Solvent concentration effect, (b)
MAE time effect, and (c) MAE temperature effect.
of total antioxidant capacity (TAC) measurements. B. edulis was
chosen as a representative sample for MAE optimization study.
Selecting the right solvent affects the amount and rate of polyphe- extract (Fig. 1b) indicate that the TAC of methanolic extract in-
nols extracted. In particular, methanol has been generally found to creased with time of extraction and reached its maximal value
be more efficient in extraction of lower molecular weight polyphe- within 5 min. So, a MAE time of 5 min was used in further experi-
nols (Dai & Mumper, 2010). Generally, the antioxidant capacities of ments. The investigation of MAE temperature on the TAC of extract
phenolic compounds measured in MeOH were found to be higher (Fig. 1c) showed that TAC increased with temperature up to a tem-
than those in EtOH, probably due to facilitated electron-transfer perature of 80 °C, above which a slight decrease in TAC was ob-
in ionizing solvents capable of anion (phenolate) solvation, because served, probably due to partial thermal degradation of heat-
MeOH is the alcohol that best supports ionization (Litwinienko & sensitive antioxidant constituents. This temperature was well
Ingold, 2003). The effect of methanol concentration on the TAC of above the boiling point of 80% (v/v) methanol (i.e., 64.5 °C), a
extract was investigated. Fig. 1a shows that the TAC of extract known feature of MAE increasing the extraction yield. Presumably,
was greatly influenced by the methanol concentration in water. the cell rupture accelerating effect of microwave radiation gives
When the methanol volume percentage in water was P50% rise to a sudden temperature rise and internal pressure increase in-
(v/v), TAC of extract was found high, possibly due to increased side the mushroom cells, thereby causing maximal extraction at
extraction of partly polar phenolics, previously identified as pheno- 80 °C, which was used in further experiments.
lic acids and flavonoids (Wong & Chye, 2009), with methanol
enrichment in the solvent mixture. Maximal TAC recovery was ob- 3.2. Total phenolic content (TPC)
tained with 80% (v/v) methanol concentration in water, and there-
fore this concentration was used in the following experiments. The A great many studies in the literature reported that the antiox-
results showing the effect of MAE time on the TAC of methanolic idant capacity/activity of food materials is well correlated with
M. Özyürek et al. / Food Chemistry 157 (2014) 323–331 327

their total phenolic content: TPC (Apak et al., 2007; Apak, Güçlü,
Özyürek, Karademir, & Erçağ, 2006; Velioglu, Mazza, Gao, &
Oomah, 1998). TPC is an important nutritional parameter closely
related to TAC, and phenolics are the major naturally occurring
antioxidant constituents of a wide variety of mushrooms (Yang,
Lin, & Mau, 2002). Although the Folin–Ciocalteu (FC) method is
not a genuine antioxidant test, it is an alternative assay for indicat-
ing the total quantity of oxidizable substances (Wangensteen,
Samuelsen, & Malterud, 2004), because all antioxidant compounds
as well as phenols donate electrons to molybdotungstophosphate
heteropolyanion reagent (3H2O–P2O5–13WO3–5MoO3–10H2O) in
a complex redox reaction, producing the FC chromophore (Single-
ton et al., 1999). Table 1 shows the total phenolic contents of the
mushroom extracts, expressed as lmol of trolox (TR) equivalents
per g of extract. These values were higher than the corresponding Fig. 2. FRS activity (%) of the mushroom methanolic extract (values depicted within
TAC values (Table 1) because of the indefinitely high redox error bars).
potential of the FC reagent capable of oxidizing a variety of organic
compounds besides antioxidants (Apak et al., 2007). The order of
TPC was B. edulis (357.7 ± 3.9 lmol TR/g) > L. volemus (230.2 ±
2.6 lmol TR/g) > T. boudieri Chatin (182.2 ± 2.1 lmol TR/g). The lausen, 1979). The DPPH radical scavenging effect of methanolic
highest content of total phenolics in the B. edulis extract might extracts from wild edible mushrooms increased with concentra-
account for the better results found for its antioxidant/antiradical tion (Fig. 2). As seen in Fig. 2, DPPH radical scavenging abilities
properties in general. of these three methanolic extracts sharply increased from 24.5%,
35.2%, and 40.1% to 81.1%, 84.4%, and 99.2% when the concentra-
3.3. Total antioxidant capacity (TAC) tion was increased from 1.5 to 15.0 mg/mL for T. boudieri Chatin,
L. volemus, and B. edulis, respectively. Thus, the free radical scav-
Total antioxidant capacity (TAC) in food extracts and biological enging activities of methanolic extracts from wild edible mush-
fluids considers a cumulative action of all antioxidants present in room species decreased in the order of B. edulis > L. volemus >
these samples, providing an integrated parameter rather than a T. boudieri Chatin at the concentration 15.0 mg/mL, showing a
simple sum of measurable antioxidants (Ghiselli, Serafini, Natella, parallelism with that of either TPC and TAC. Since the DPPH meth-
& Scaccini, 2000). Table 1 shows the total antioxidant capacities od is a mixed-mode assay (of hydrogen atom transfer: HAT and
of the mushroom extracts, expressed as lmol of trolox (TR) equiv- electron-transfer: ET mechanisms), it is natural that this order
alents per g of extract. The order of antioxidant capacities of mush- was similar to those found by the ET-based FC and CUPRAC assays.
rooms was: B. edulis > L. volemus > T. boudieri Chatin, with TAC
values of 122.4 ± 1.3, 74.1 ± 1.3, and 61.7 ± 0.5 lmol TR/g, respec- 3.5. Hydroxyl radical scavenging (HRS) activity
tively (Table 1). This order was parallel to that of TPC, pointing
out to the fact that phenolics were the main antioxidant constitu- Among reactive oxygen species, OH exhibits the strongest oxi-
ents in mushroom extracts (Yang et al., 2002). TPC values of mush- dative activity in terms of its very high redox potential and extre-
room extracts were much higher than TAC in trolox equivalents, mely fast kinetics. Hydroxyl radical is the most toxic radical
because the molybdotungstophosphate heteropolyanion reagent known, as it can non-specifically oxidize all classes of biological
of Folin–Ciocalteu method had a higher redox potential (compared macromolecules at virtually diffusion-limited rates (Imlay & Linn,
to that of the optical sensor-based CUPRAC method) in the alkaline 1988). Thus, hydroxyl radical scavenging activity is very important
medium of the FC assay where most phenolic compounds are for evaluating the antioxidant activity of food extracts. With the
deprotonated and easily oxidized (Apak et al., 2007). aid of a modified CUPRAC method using salicylate hydroxylation
from a Fenton reaction, a kinetic approach was adopted to assess
3.4. Free radical scavenging (FRS) activity the hydroxyl radical scavenging properties of wild edible mush-
room extracts. Fig. 3 indicates that the T. boudieri Chatin, B. edulis
Antioxidant activity, especially free radical scavenging activity, and L. volemus extracts exhibited a quite strong concentration-
has a great importance due to the deleterious role of free radicals dependent inhibition of hydroxyl radicals at a pretty low concen-
in foods and in biological systems. One of the most common meth- tration. The scavenging activities of methanolic extracts on the

ods to evaluate antioxidant activity of specific compounds or ex- OH radical decreased in the order of B. edulis > L. volemus > T. bou-
tracts is the DPPH free radical scavenging activity assay which dieri Chatin, and were 93.1%, 75.3%, and 71.4% at the concentration
relies on the reduction of DPPH solution in the presence of hydro- of 15 mg/mL, respectively. Although hydroxyl radical scavenging
gen donating antioxidant compounds (Baumann, Wurn, & Bruch- activities of polyphenols do not necessarily correlate with their

Table 1
TPC (lmol TR/g), TAC (lmol TR/g), and EC50 values (mg/mL) of mushroom extracts.

Samples TPC (lmol TR/g) TAC (lmol TR/g) FRS activity (EC50a) HRS activity (EC50a) HPS activity (EC50a) SARS activity (EC50a)
Terfezia boudieri Chatin 182.2 ± 2.1 61.7 ± 0.5 6.8 ± 0.3 8.3 ± 0.4 11.4 ± 0.6 7.9 ± 0.5
Boletus edulis 357.7 ± 3.9 122.4 ± 1.3 2.9 ± 0.1 4.9 ± 0.2 4.6 ± 0.2 5.9 ± 0.3
Lactarius volemus 230.2 ± 2.6 74.1 ± 1.3 4.6 ± 0.2 7.1 ± 0.4 7.5 ± 0.4 7.2 ± 0.6

Each value is expressed within 95% confidence interval as {mean ± (t.05 s/n1/2)}, where t.05 is the Student’s table t-value for 4 degrees of freedom and s is the standard deviation
of n = 5 measurements.
a
EC50 (mg/mL): effective concentration at which 50% of radicals are scavenged.
328 M. Özyürek et al. / Food Chemistry 157 (2014) 323–331

of catechol and/or pyrogallol groups of phenolic acids as well as on

the number and position of hydroxyl groups of flavonoids (Özyürek
et al., 2010), a similar hypothesis -to that of HRS- may be drawn on
the possible presence of such groups in the phenolic profile of the
studied mushroom extracts.

3.7. Superoxide anion radical scavenging (SARS) activity

Reactive oxygen species (ROS), including superoxide anion

radical O2 , have gained great attention due to their important role
in the progression of a number of human diseases and carcinogene-
sis. So it is important to eliminate excessive O2 in vivo to prevent
important oxidative stress originated diseases (Bekdesßer, Özyürek,
Güçlü, & Apak, 2011). SARS activities of wild edible mushroom
Fig. 3. HRS activity (%) of the mushroom methanolic extract (values depicted extracts were evaluated with the use of the NBT method where O2
within error bars). reduces the yellow dye (NBT2+) to produce the blue formazan, and
antioxidants inhibit formazan formation (Yu et al., 2005). Scaveng-
ing effects of methanolic extracts of the mushrooms on the O2 rad-
icals increased with concentration (Fig. 5). Mushroom extracts had
TAC values, the parallel order of HRS with either TPC or TAC/FRS of strong superoxide anion radical scavenging activities. The SARS
mushroom extracts leads to hypothesize that catechol and/or pyro- activity of polyphenols was reported to show a higher correlation
gallol groups as the most active sites of hydroxyl radical attack with total flavonoids than with total phenolics, which indicated that
(Özyürek et al., 2008) may be expected to be abundant in the phe- flavonoids might contribute to the total SRSA more directly than
nolic profile of the studied mushrooms. In fact, pyrogallol and gal- other polyphenols (Chun, Kim, & Lee, 2003). This may be connected
lic acid were identified as the second and third phenolic to the antioxidant flavonoid content of the studied mushrooms. As
compounds in occurrence (after homogentisic acid) in edible seen in Fig. 5, the scavenging activities of methanolic extracts
mushrooms (Palacios et al., 2011). against O2 radical decreased in the order of B. edulis > L. volemus >
T. boudieri Chatin, and were 86.1%, 75.6%, and 70.7%, respectively,
3.6. Hydrogen peroxide scavenging (HPS) activity at the concentration of 15 mg/mL. This trend is parallel to those
found in other antioxidant activity tests, indicating that the same
Hydrogen peroxide (H2O2) is a relatively stable non-radical oxi- structural features of phenolic acids and flavonoids are responsible
dizing species that may be formed in tissues through oxidative for the observed antioxidant/antiradical effects (see Fig. 6).
processes. The HPS-CUPRAC method, replacing the older UV meth-
od working at 230 nm prone to interference by many organic sub- 3.8. EC50 values in antioxidant activity
stances, was used to determine the hydrogen peroxide scavenging
activity of wild edible mushroom extracts. A concentration-depen- Table 1 shows the EC50 values (mg extract per mL) for the anti-
dent assay was carried out with the mushroom extracts (Fig. 4) to oxidant activity assays obtained from each mushroom methanolic
provide a direct comparison of the HPS activities. Fig. 4 illustrates extract. Antioxidant effectiveness inversely correlated with their
that the methanolic extracts possessed significant scavenging EC50 values. The three wild edible mushrooms were comparable
activities on H2O2 in the presence of Cu(II) catalyst, and this scav- in antioxidant activity of their methanolic extracts. Consistently
enging effect increased with concentration. In addition, the meth- in scavenging activity of DPPH radicals, hydroxyl radicals, hydro-
anolic extract of B. edulis, at the concentration of 15 mg/mL, gen peroxide, and superoxide anion radicals, effectiveness was in
showed the highest HPS activity (94.5%), followed by L.volemus the descending order: B.edulis > L. volemus > T. boudieri Chatin. A
extract (78.7%), andT. boudieri Chatin extract (58.1%) at the same relationship among the FRS activity, HRS activity, HPS activity,
concentration. So, the H2O2 scavenging activities were in the order: and SARS activity was found, indicating that the mechanisms of
B. edulis > L. volemus > T. boudieri Chatin, parallel to the trend ob- action of the methanolic extracts for antioxidant activity may be
served in other antioxidant activity tests. Since high HPS activity similar, being related to TPC and TAC. Overall, B.edulis had the
of polyphenols was earlier shown to be dependent on the presence

Fig. 4. HPS activity (%) of the mushroom methanolic extract (values depicted Fig. 5. SARS activity (%) of the mushroom methanolic extract (values depicted
within error bars). within error bars).
M. Özyürek et al. / Food Chemistry 157 (2014) 323–331 329

Fig. 6. Chromatograms of the microwave-assisted methanolic extracts of (a) Boletus edulis, (b) Terfezia boudieri Chatin, and (c) Lactarius volemus (1. Gallic acid; 2.
Protocatechuic acid; 3. Vanillic acid; 4. Syringic acid; 5. Rutin; 6. Hesperidin; 7. Rosmarinic acid; 8. Quercetin; 9. Naringenin; 10. Kaempferol; 11. Apigenin).

strongest antioxidant/antiradical activity of the tested species, in 3.9. Analysis of polyphenols by UPLC–ESI-MS/MS
accordance with the findings of plant scientists working on Taiwan
wild mushrooms (Tsai et al., 2007) and Anatolian wild mushrooms Since all polyphenols contain one or more hydroxyl and some
(Sarikurkcu, Tepe, & Yamac, 2008). Based on the results obtained, carboxylic acid groups, MS data were acquired in negative ioniza-
the methanolic extracts from these three wild edible mushrooms tion mode. The phenolic compounds were tentatively identified on
contained effective antioxidants. the basis of their deprotonated ions [M H] and the fragments
330 M. Özyürek et al. / Food Chemistry 157 (2014) 323–331

Table 2 Acknowledgements
Concentration of phenolic compounds found in three wild edible mushrooms.

Boletus Terfezia boudieri Lactarius The authors express their gratitude to T. R. Ministry of Develop-
edulis Chatin volemus ment for the Advanced Research Project of Istanbul University
Gallic acid 0.19 ± 0.012 0.06 ± 0.003 n.d. (2011K120320).
Protocatechuic 0.21 ± 0.09 1.03 ± 0.070 10.82 ± 0.51
acid
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