Food Chemistry 126 (2011) 610–616

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

Chemical composition of wild edible mushrooms and antioxidant properties

of their water soluble polysaccharidic and ethanolic fractions
Josiana A. Vaz a,b,c,d, Lillian Barros a,e,f, Anabela Martins e, Celestino Santos-Buelga f,
M. Helena Vasconcelos b,c, Isabel C.F.R. Ferreira a,e,⇑
a
Mountain Research Centre (CIMO), Instituto Politécnico de Bragança, Campus de Santa Apolónia, Apartado 1172, 5301-855 Bragança, Portugal
b
Faculty of Pharmacy, University of Porto, Portugal
c
Cancer Biology Group, IPATIMUP, Institute of Molecular Pathology and Immunology of the University of Porto, Portugal
d
CEQUIMED-UP, Center of Medicinal Chemistry, University of Porto, Portugal
e
Escola Superior Agrária, Instituto Politécnico de Bragança, Campus de Santa Apolónia, Apartado 1172, 5301-855 Bragança, Portugal
f
Grupo de Investigación en Polifenoles (GIP-USAL), Facultad de Farmacia, Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain

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

Article history: Mushrooms have become attractive as functional foods and as a source of physiologically beneficial bio-
Received 18 June 2010 active compounds. Herein, we describe and compare the chemical constituents (phenolic compounds,
Received in revised form 4 October 2010 macronutrients, sugars, fatty acids, tocopherols and ascorbic acid) of four wild edible mushrooms widely
Accepted 11 November 2010
appreciated in gastronomy: Armillaria mellea (Vahl) P. Kumm., Calocybe gambosa (Fr.) Donk, Clitocybe
odora (Fr.) P. Kumm., Coprinus comatus (O.F. Müll.) Pers. Furthermore, the antioxidant activity of their
water soluble polysaccharidic and ethanolic fractions was studied by three different in vitro assays. C.
Keywords:
comatus revealed the highest concentrations of sugars (43.23/100 g dry weight), PUFA (77.46%), phenolic
Wild edible mushrooms
Bioactive compounds
compounds (45.02 mg/kg), tocopherols (301.03 lg/100 g) and, among all of the fractions tested, its eth-
Phenolic compounds anolic fraction showed the highest antioxidant activity (EC50 < 2.6 mg/ml). C. odora revealed one of the
Polysaccharides highest ascorbic acid (172.65 mg/100 g) contents and its water soluble polysaccharidic fraction showed
Chemical composition the best antioxidant properties (EC50 < 3.6 mg/ml) among the polysaccharidic fractions. The studied
mushrooms species could potentially be used in well-balanced diets and as a source of bioactive
compounds.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Armillaria mellea is an edible, medicinal mushroom also known

as honey fungus. It has been reportedly used in treating geriatric
Reactive oxygen species (ROS) production occurs during normal patients with palsy, dizziness, headache, neurasthenia, insomnia,
cell metabolism, both in animals and plants. Excess of ROS leads to numbness in limbs and infantile convulsion, while also reportedly
oxidative stress, resulting in oxidative DNA damage which is impli- exerting neuroprotective effects (Colak, Sahin, Yildirim, & Sesli,
cated in the pathogenesis of numerous disorders, e.g. cardiovascular, 2007; Gao, Yang, Wang, & Liu, 2001; Kim et al., 2010). The sub-
atherosclerosis, reperfusion injury, cataractogenesis, rheumatoid stance distilled from A. mellea has a similar pharmacological activ-
arthritis, inflammatory disorders and cancer (Halliwell & Gutteridge, ity and clinical curative effect to that of Rhizoma Gastrodiae and has
1999; Valko et al., 2007). Mushrooms contain many different dietary no poisonous or side effects. The substance has shown anti-tumor,
nutrients with strong antioxidant capacities, such as phenolic com- anti-inflammation, anti-radiation and immunomodulation func-
pounds and vitamins (Ferreira, Barros, & Abreu, 2009). tions (Sun, Liang, Zhang, Tong, & Liu, 2009).
In recent years, increased interest in human health, nutrition Calocybe gambosa, commonly known as St. George’s mushroom,
and disease prevention has enlarged consumer demand for func- can be eaten raw, dry or pickled. It has shown antibacterial activity
tional foods. In fact, mushrooms have become attractive as func- towards Bacillus subtilis and Escherichia coli (Keller, Maillard, Keller,
tional foods and as a source of bioactive compounds (Chang, & Hostettmann, 2002) and an ability to reduce blood sugar levels
1996; Lindequist, Niedermeyer, & Julich, 2005; Poucheret, Fons, & (Brachvogel, 1986).
Rapior, 2006; Wasser, 1999; Zhang, Cui, Cheung, & Wang, 2007). Clitocybe odora, also known as the aniseed toadstool, is an edible
mushroom and its caps can be dried, used as a condiment, or used
fresh for flavouring. Polysaccharides extracted from the mycelial
⇑ Corresponding author at: Mountain Research Centre (CIMO), Instituto Polité-
cnico de Bragança, Campus de Santa Apolónia, Apartado 1172, 5301-855 Bragança,
culture of C. odora and administered intraperitoneally into white
Portugal. Tel.: +351 273 303219; fax: +351 273 325405. mice at a dosage of 300 mg/kg have been shown to inhibit the
E-mail address: iferreira@ipb.pt (I.C.F.R. Ferreira). growth of Sarcoma 180 and Ehrlich solid cancers by 70% and

0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.11.063
 J.A. Vaz et al. / Food Chemistry 126 (2011) 610–616 611

60%, respectively (Ohtsuka et al., 1973). Coprinus comatus (Shaggy re-dissolved in ethanol, at final concentrations of 50 mg/ml, for
mane) is a delicious, highly nutritious edible fungus and is also the antioxidant activity assays.
considered a source of valuable medicinal compounds. It has been
reported to have various bioactive effects such as immunomodula- 2.3.2. DPPH radical-scavenging activity
tion, hypoglycaemic, hypolipidemic, antitumor and antibacterial This assay was performed in 96-well microtiter plates using an
(Yu et al., 2009). ELX800 Microplate Reader (Bio-Tek Instruments, Inc). The reaction
The aim of the present work was to obtain quantitative data on mixture in each of the 96-wells of the plate consisted of one of the
the chemical constituents of the mentioned mushrooms widely different concentrations of the extracts (30 ll) and aqueous meth-
appreciated in gastronomy, including on their antioxidants, such anolic solution (80:20, v/v, 270 ll) containing DPPH radicals
as phenolic compounds, reducing sugars, tocopherols and ascorbic (6  10 5 M). The mixture was left to stand for 60 min in the dark.
acid. Furthermore, the antioxidant activity of two different frac- Reduction of the DPPH radical was determined by measuring the
tions (water soluble polysaccharidic and ethanolic fractions) ob- absorption at 515 nm. Radical scavenging activity (RSA) was calcu-
tained from each species was compared. lated as a percentage of DPPH discolouration using the equation:%
RSA = [(ADPPH AS)/ADPPH]  100, where AS is the absorbance of the
solution when the sample extract has been added at a particular le-
2. Material and methods
vel and ADPPH is the absorbance of the DPPH solution. The extract
concentration providing 50% of radicals scavenging activity (EC50)
2.1. Mushroom species
was calculated from the graph of RSA percentage against extract
concentration.
Samples of Armillaria mellea (Vahl) P. Kumm., Calocybe gambosa
(Fr.) Donk, Clitocybe odora (Fr.) P. Kumm. and Coprinus comatus
2.3.3. Reducing power
(O.F. Müll.) Pers. were collected in Bragança (Northeast Portugal)
This assay was also performed using microtiter plates and the
in the autumn of 2009. Taxonomic identification of sporocarps
Microplate Reader described above. Different extract concentra-
was made according to other authors (Bon, 1988; Courtecuisse &
tions (0.5 ml) were mixed with sodium phosphate buffer
Duhem, 2005) and online keys (http://www.mycokey.com/) and
(200 mM, pH 6.6, 0.5 ml) and potassium ferricyanide (1% w/v,
representative voucher specimens were deposited at the herbar-
0.5 ml). The mixture was incubated at 50 °C for 20 min and trichlo-
ium of Escola Superior Agrária of Instituto Politécnico de Bragança.
roacetic acid (10% w/v, 0.5 ml) added. This mixture (0.8 ml) was
All samples were lyophilised (Ly-8-FM-ULE, Snijders, Holland) and
then poured into the wells of a 48-well microplate, also containing
reduced to a fine dried powder (20 mesh).
deionised water (0.8 ml) and ferric chloride (0.1% w/v, 0.16 ml) and
the absorbance was measured at 690 nm. The extract concentra-
2.2. Standards and reagents tion providing 0.5 of absorbance (EC50) was calculated from the
graph of absorbance at 690 nm against extract concentration.
Acetonitrile 99.9%, n-hexane 95% and ethyl acetate 99.8% were
of HPLC grade from Lab-Scan (Lisbon, Portugal). The fatty acids 2.3.4. Inhibition of b-carotene bleaching
methyl ester (FAME) reference standard mixture 37 (standard A solution of b-carotene was prepared by dissolving b-carotene
47885-U) was purchased from Sigma (St. Louis, MO, USA), as well (2 mg) in chloroform (10 ml). Two millilitres of this solution were
as other individual fatty acid isomers, ascorbic acid, tocopherols, pipetted into a round-bottom flask. The chloroform was removed
sugars and phenolic standards (gallic, protocatechuic, p-hydroxy- at 40 °C under vacuum and linoleic acid (40 mg), Tween 80 emul-
benzoic, p-coumaric, and cinnamic acids). Racemic tocol, 50 mg/ sifier (400 mg) and distilled water (100 ml) were added to the flask
ml, was purchased from Matreya (PA, USA). 2,2-Diphenyl-1-pic- with vigorous shaking. Aliquots (4.8 ml) of this emulsion were
rylhydrazyl (DPPH) was obtained from Alfa Aesar (Ward Hill, MA, transferred into test tubes containing different concentrations of
USA). All other chemicals and solvents were of analytical grade the extracts (0.2 ml). The tubes were shaken and incubated at
and purchased from common sources. Water used in the studies 50 °C in a water bath. As soon as the emulsion was added to each
was treated in a Milli-Q water purification system (TGI Pure Water tube, the zero time absorbance was measured at 470 nm (Analy-
Systems, USA). tikjena 200–2004 spectrophotometer). A blank, devoid of b-caro-
tene, was prepared for background subtraction. b-Carotene
2.3. Evaluation of antioxidant activity bleaching inhibition was calculated using the following equation:
(b-carotene content after 2 h of assay/initial b-carotene con-
2.3.1. Preparation of the fractions tent)  100. The extract concentration providing 50% antioxidant
The water soluble polysaccharidic and ethanolic fractions were activity (EC50) was calculated by interpolation from the graph of
prepared following the procedure described by Cheng, Lin, Lur, b-carotene bleaching inhibition percentage against extract
Chen, and Lu (2008) with some modifications. Polysaccharides concentration.
were extracted from lyophilised mushrooms (1.5 g) with boiling
water (50 ml) for 2 h under agitation (150 rpm; Velp Are magnetic 2.4. Phenolic compounds
stirrer) before being subsequently filtered through Whatman No. 4
paper. The residue was then extracted with two further portions of Each sample (3 g) was extracted with acetone:water (80:20, v/
boiling water over a total 6 h extraction. The combined extracts v; 30 ml) at 20 °C for 6 h. After sonication for 15 min, the extract
were lyophilised before 95% ethanol (10 ml) was added and poly- was centrifuged at 4000 g for 10 min, and filtered through What-
saccharides were precipitated overnight at 4 °C. The precipitated man n° 4 paper. The residue was then extracted with two addi-
polysaccharides were collected after centrifugation (Centorion tional 30 ml portions of the acetone:water mixture. The
K24OR-2003 refrigerated centrifuge) at 3100g for 40 min followed combined extracts were evaporated at 40 °C under reduced pres-
by filtration, before being lyophilised, resulting in a crude polysac- sure to remove acetone. The aqueous phase was washed with n-
charidic sample. The ethanolic supernatant was evaporated at hexane, and then submitted to a liquid–liquid extraction with
40 °C under reduced pressure (rotary evaporator Büchi R-210), diethyl ether (3  30 ml) and ethyl acetate (3  30 ml). The organic
giving the ethanolic fraction. The crude polysaccharidic samples phases were evaporated at 40 °C to dryness, re-dissolved in
were re-dissolved in water while the ethanolic extracts were water:methanol (80:20, v/v; 1 ml), followed by filtering through
612 J.A. Vaz et al. / Food Chemistry 126 (2011) 610–616

a 0.22 lm disposable LC filter disk for HPLC analysis. The analysis 160 rpm; to obtain phase separation 3 ml of deionised water were
was performed using a Hewlett–Packard 1100 series liquid chro- added; the fatty acids methyl esters (FAME) were recovered by
matograph (Agilent Technologies) as previously described (Barros, shaking in a vortex with 3 ml of diethyl ether, and the upper phase
Dueñas, Ferreira, Baptista, & Santos-Buelga, 2009). Separation was was passed through a micro-column of anhydrous sodium sulphate
achieved on a Spherisorb S3 ODS-2 (Waters) reverse phase C18 col- to eliminate the water. The sample was recovered in a vial with Tef-
umn (3 lm, 150  4.6 mm) thermostated at 25 °C. The solvents lon and filtered through a 0.2 lm Whatman nylon filter. The fatty
used were: (A) 2.5% acetic acid in water, (B) 2.5% acetic acid:aceto- acid profile was analysed with a DANI model GC 1000 instrument
nitrile (90:10, v/v), and (C) 100% HPLC-grade acetonitrile. The gra- equipped with a split/splitless injector, a flame ionisation detector
dient employed was: isocratic 100% A for 10 min, 50% A and 50% B (FID), and a Macherey–Nagel column (30 m  0.32 mm ID  0.25
for 10 min, isocratic 100% B for 15 min, 90% B and 10% C for 10 min, lm df). The oven temperature program followed was an initial col-
70% B and 30% C for 10 min, 50% B and 50% C for 5 min, 20% B and umn temperature of 50 °C, held for 2 min, followed by a 10 °C/min
80% C for 5 min, 100% A for 5 min, at a flow rate of 0.5 ml/min. ramp to 240 °C for 11 min. The carrier gas (hydrogen) flow-rate
Detection was carried out in a diode array detector (DAD), using was 4.0 ml/min (0.61 bar), measured at 50 °C. Split injection
280 nm as the preferred wavelength. The phenolic compounds (1:40) was carried out at 250 °C. For each analysis 1 ll of the sam-
were quantified by comparison of the area of their peaks recorded ple was injected in GC. Fatty acid identification was made by com-
at 280 nm with calibration curves obtained from commercial stan- paring the relative retention times of FAME peaks from samples
dards of each compound. The results were expressed as mg per kg with standards. The results were recorded and processed using
of dry weight (dw). CSW 1.7 software (DataApex 1.7) and expressed as a relative per-
centage of each fatty acid.
2.5. Macronutrients
2.8. Tocopherols
The samples were analysed for chemical composition (moisture,
protein, fat, carbohydrates and ash) using the AOAC (1995) proce- The tocopherol content was determined following a procedure
dures. Protein content (N  4.38) of the samples was estimated by previously described by the authors (Barros et al., 2008). Butylated
the macro-Kjeldahl method; fat was determined by extracting a hydroxytoluene, BHT solution in hexane (10 mg/ml; 100 ll) and IS
known weight of powdered sample with petroleum ether, using solution in hexane (tocol; 50 lg/ml; 400 ll) were added to the
a Soxhlet apparatus; ash content was determined by incineration sample prior to the extraction procedure. Samples (500 mg) were
at 600 ± 15 °C. Carbohydrates were calculated by difference: Car- homogenised with methanol (4 ml) by vortex mixing (1 min). Sub-
bohydrates = 100 (g protein + g fat + g ash). Energy was calcu- sequently, hexane (4 ml) was added and again vortex mixed for
lated according to the following equation: Energy (kcal) = 4  (g 1 min. Saturated NaCl aqueous solution (2 ml) was added, the mix-
protein + g carbohydrate) + 9  (g lipid). ture was homogenised (1 min), centrifuged (5 min, 4000g) and the
clear upper layer was carefully transferred to a vial. The sample
2.6. Sugars was re-extracted twice with n-hexane. The combined extracts
were taken to dryness under a nitrogen stream, redissolved in
Free sugars were determined by high performance liquid chro- 2 ml of n-hexane, dehydrated with anhydrous sodium sulphate
matography coupled to a refraction index detector (HPLC-RI) as and filtered through 0.2 lm nylon filters and transferred into a
previously described by the authors (Barros, Cruz, Baptista, Estev- dark injection vial. The analyses were performed by the HPLC sys-
inho, & Ferreira, 2008). The equipment consisted of an integrated tem (described above) connected to a fluorescence detector (FP-
system with a pump (Knauer, Smartline System 1000), degasser 2020; Jasco) programmed for excitation at 290 nm and emission
system (Smartline Manager 5000), auto-sampler (AS-2057 Jasco) at 330 nm. Chromatographic separation was achieved with a Poly-
and RI detector (Knauer Smartline 2300). Dried sample powder amide II (250  4.6 mm) normal-phase column from YMC Waters
(1.0 g) was spiked with raffinose as internal standard (IS, 5 mg/ operating at 30 °C. The mobile phase used consisted of a mixture
ml) and was extracted with 40 ml of 80% aqueous ethanol at of n-hexane and ethyl acetate (70:30, v/v) at a flow rate of 1 ml/
80 °C for 30 min. The resulting suspension was centrifuged at min, with an injection volume of 20 ll. The compounds were iden-
15,000g for 10 min. The supernatant was concentrated at 60 °C un- tified by chromatographic comparisons with authentic standards.
der reduced pressure and defatted three times with 10 ml of ethyl Quantification was based on the fluorescence signal response,
ether, successively. After concentration at 40 °C, the solid residues using the IS method. Tocopherol contents in the samples were ex-
were dissolved in water to a final volume of 5 ml and filtered pressed in lg per 100 g of dry weight.
through 0.2 lm nylon filters. Chromatographic separation was
achieved with a Eurospher 100–5 NH2 column (4.6  250 mm,
5 lm, Knauer) operating at 30 °C (7971 R Grace oven). The mobile 2.9. Ascorbic acid
phase was acetonitrile:deionized water, 7:3 (v/v) at a flow rate of
1 ml/min. Sugar identification was made by comparing the relative A fine dried powder (20 mesh; 150 mg) was extracted with
retention times of sample peaks with standards. Data was analysed metaphosphoric acid (1%, 10 ml) for 45 min at room temperature
using Clarity 2.4 Software (DataApex). Quantification was made by and filtered through a Whatman N° 4 filter paper. The filtrate
internal normalisation of the chromatographic peak area and the (1 ml) was mixed with 2,6-dichloroindophenol (9 ml) and the
results expressed in g per 100 g of dry weight. absorbance measured within 30 min at 515 nm against a blank.
The ascorbic acid content was calculated on the basis of the cali-
2.7. Fatty acids bration curve of authentic L-ascorbic acid (0.006–0.1 mg/ml) and
the results expressed as mg per 100 g of dry weight.
Fatty acids were determined by gas chromatography with flame
ionisation detection (GC-FID) as described previously by the 2.10. Statistical analysis
authors (Barros et al., 2008) and after the following trans-esterifi-
cation procedure: fatty acids (obtained after Soxhlet extraction) All assays were carried out in triplicate. The results are ex-
were methylated with 5 ml of methanol:sulphuric acid 95%:tolu- pressed as mean values and standard deviation (SD). The results
ene 2:1:1 (v/v/v), for at least 12 h in a bath at 50 °C and were analysed using one-way analysis of variance (ANOVA) fol-
 J.A. Vaz et al. / Food Chemistry 126 (2011) 610–616 613

lowed by Tukey’s HSD Test with a = 0.05. This treatment was car- concentration of phenolic acids (80.33 mg/kg, dry weight), due to
ried out using SPSS v. 16.0 program. the contribution of p-hydroxybenzoic and p-coumaric acids,
whereas C. gambosa revealed the highest concentration of cin-
3. Results and discussion namic acid (17.69 mg/kg). Several phenolic compounds were iden-
tified and quantified in wild mushrooms from Finland, India, Korea
Some literature refers to a number of bioactive chemical constit- and Portugal (Ferreira et al., 2009) but not in the studied species.
uents of A. mellea from the Czech Republic (Kalac, 2009), Turkey However, to the best of our knowledge this is the first report on
(Ouzouni, Petridis, Koller, & Riganakos, 2009; Yilmaz, Solmaz, Turk- individual phenolic compounds of A. mellea, C. gambosa, C. odora
ekul, & Elmastas, 2006) and US (Cox, Scherm, & Riley, 2006) and to and C. comatus. Nonetheless, Signore, Romeo, and Giaccio (1997)
antioxidant properties of A. mellea from China (Yu et al., 2009) and had previously reported total phenolics in A. mellea and C. odora
C. comatus from Taiwan (Tsai, Tsai, & Mau, 2007) but there are no from Italy, measured by the Folin Ciocalteu assay.
such reports on Portuguese samples of these mushrooms. The results of the moisture, macronutrients composition, indi-
A sequential extraction with boiling water and ethanol was per- vidual sugars and estimated energetic value obtained for the four
formed in order to obtain extracts with high molecular weight wild edible mushrooms are shown in Table 3. Significant differ-
compounds, such as polysaccharides and low molecular weight ences (p < 0.05) were observed in the moisture (90.92/100 g) and
compounds, such as phenolic compounds. Both kinds of com- ash (13.89/100 g dry weight) contents between the different
pounds play important roles in mushrooms, including medicinal mushrooms, with C. gambosa revealing the highest values, whilst
functions (Ferreira, Vaz, Vasconcelos, & Martins, 2010). The extrac- no differences were found for carbohydrates and proteins (70/
tion yields obtained for ethanolic fractions were lower than the 100 g and 16/100 g, expressed in dry weight, respectively), which
yields for water soluble polysaccharidic fractions (Table 1). were the most abundant macronutrients. Fat was the less abun-
To evaluate the antioxidant properties of both fractions, three dant macronutrient, being lower than 5.6/100 g, dw. A. mellea
different assays were carried out: scavenging activity on DPPH rad- showed the highest energetic contribution (400.68 kcal/100 g,
icals, reducing power and inhibition of lipid peroxidation. The dw) mainly due its higher fat values. A sample of this same mush-
water soluble polysaccharidic fractions revealed a higher antioxi- room species from Greece (Ouzouni et al., 2009) revealed similar
dant activity than ethanolic fractions, unless for C. comatus (Ta- moisture (87.17/100 g, dw) and ash (7.95/100 g, fw) contents, but
ble 1). Moreover, the ethanolic fraction of this species showed higher protein concentration (24.47/100 g, dw), and lower fat
the highest DPPH radical scavenging activity (EC50 value 2.56 mg/ (2.10/100 g, dw) and carbohydrate (65.47/100 g, dw) levels. A A.
ml). This observation is in agreement with its higher content in mellea sample from Poland (Kalac, 2009) showed a quite different
phenolic compounds compared to the other mushrooms (Table 2). macronutrient composition from the sample from Greece and the
The ethanolic fraction studied here gave better results (reducing sample here studied, particularly regarding carbohydrate content
power 1.61 at 5 mg/ml; DPPH scavenging activity 79.92% at (16.4/100 g, dw). Regarding C. gambosa, a commercial sample pre-
5 mg/ml, data not shown) than the ethanolic (reducing power viously analysed by our group (Barros et al., 2008) revealed higher
0.45 at 5 mg/ml; DPPH scavenging activity 84.5% at 5 mg/ml) and protein (47.22/100 g, dw) and lower carbohydrate (43.01/100 g,
hot water (reducing power 0.25 at 5 mg/ml; DPPH scavenging dw) contents than the wild sample studied here. The variability
activity 58.9% at 20 mg/ml) extracts from C. comatus from Taiwan among samples of different origin might be related to the environ-
(Tsai et al., 2007). mental temperature, the relative humidity during growth and the
The water soluble polysaccharidic fraction of C. odora showed relative amount of metabolic water produced or utilised during
the lowest EC50 value for reducing power (0.94 mg/ml) and b-car- storage, as well as to the industrial processes to which the com-
otene bleaching inhibition (0.27 mg/ml). The ethanolic fraction of mercial mushrooms are submitted (Ouzouni et al., 2009). To the
C. gambosa showed higher EC50 values (i.e., lower antioxidant best of our knowledge, this is the first report on C. odora and C.
activity) than a commercial sample previously studied by us comatus macronutrient composition.
(7.14 mg/ml, 4.31 mg/ml and 2.77 mg/ml for DPPH scavenging In relation to the sugar composition (Table 3), the edible mush-
activity, reducing power and b-carotene bleaching inhibition, rooms yielded trehalose as the main sugar. The C. comatus sample
respectively). Nevertheless, that sample was a crude methanolic showed the highest total sugars concentration (43.23/100 g, dw),
extract obtained at room temperature (Queirós, Barreira, Sarmen- mostly due to trehalose (42.82/100 g, dw). The highest values of
to, & Ferreira, 2009) and was not produced following the fraction- mannitol (5.45/100 g, dw) were found in the A. mellea sample, and
ated procedure with boiling water and ethanol like the one used in arabinose was only detected in this sample (0.78/100 g, dw). The su-
the present work. gar composition of the wild C. gambosa was very similar to the results
Up to three phenolic acids (protocatechuic, p-hydroxybenzoic obtained for a commercial sample (mannitol 0.27/100 g, trehalose
and p-coumaric acids) and a related compound (cinnamic acid) 8.01/100 g and total sugars 9.13/100 g, expressed in dry weight ba-
could be identified and quantified in the different samples sis), although in that case melezitose was also detected, but could
analysed by HPLC–DAD (Table 2). C. comatus showed the highest not be found in the wild sample (Barros et al., 2008).

Table 1
Extraction yields and antioxidant activity (EC50 valuesa) of two fractions obtained from wild edible mushrooms (mean ± SD; n = 3). In each row different letters imply significant
differences (p < 0.05).

Species Armillaria mellea Calocybe gambosa Clitocybe odora Coprinus comatus

Fraction Ethanolic Water soluble Ethanolic Water soluble Ethanolic Water soluble Ethanolic Water soluble
polysaccharidic polysaccharidic polysaccharidic polysaccharidic
Extraction yield (g/100 g dry weight) 10.91 ± 0.39 c 39.66 ± 1.49 a 14.89 ± 0.51 c 37.92 ± 2.09 a 1.26 ± 0.10 e 18.68 ± 0.23 b 5.56 ± 0.18 d 12.54 ± 0.61 c
DPPH scavenging activity (mg/ml) 17.13 ± 0.67 b 3.95 ± 0.16 d 34.60 ± 0.44 a 7.08 ± 0.12 c 6.77 ± 0.05 c 3.56 ± 0.13 d 2.56 ± 0.31 e 7.31 ± 0.22 c
Reducing power (mg/ml) 7.53 ± 0.10 b 0.98 ± 0.00 g 11.46 ± 0.18 a 2.38 ± 0.02 e 3.63 ± 0.14 d 0.94 ± 0.01 g 1.47 ± 0.01 f 4.67 ± 0.04 c
b-Carotene bleaching inhibition (mg/ml) 8.94 ± 0.02 a 0.87 ± 0.01 e 7.57 ± 0.09 c 8.17 ± 0.34 b 1.36 ± 0.08 d 0.27 ± 0.00 f 1.26 ± 0.01 d 7.43 ± 0.02 c
a
Concentration of extract providing 50% of antioxidant activity in DPPH scavenging activity and b-carotene bleaching inhibition assays, and 0.5 of absorbance in reducing
power assay.
614 J.A. Vaz et al. / Food Chemistry 126 (2011) 610–616

Table 2
Contents of phenolic compounds and cinnamic acid (mg/kg of dry weight) measured by HPLC in the four wild edible mushroom species (mean ± SD; n = 3). In each row different
letters imply significant differences (p < 0.05).

Armillaria mellea Calocybe gambosa Clitocybe odora Coprinus comatus

Protocatechuic acid nd 2.58 ± 0.49 nd nd
p-Hydroxybenzoic acid 4.00 ± 0.72 c 38.40 ± 3.14 b 27.93 ± 2.79 b 61.53 ± 1.19 a
p-Coumaric acid nd 4.04 ± 0.29 b 1.81 ± 0.09 c 18.79 ± 0.92 a
Total phenolic acids 4.00 ± 0.72 d 45.02 ± 3.34 b 28.83 ± 1.51 c 80.33 ± 0.27 a
Cinnamic acid 8.67 ± 0.03 c 17.69 ± 0.21 a 13.77 ± 0.59 b 12.58 ± 0.12 b

nd, not detected.

Table 3
Moisture (g/100 g of fresh weight), macronutrients (g/100 g of dry weight) and energetic value (kcal/100 g of dry weight) in the four wild edible mushroom species analysed
(mean ± SD; n = 3). In each row different letters imply significant differences (p < 0.05).

Armillaria mellea Calocybe gambosa Clitocybe odora Coprinus comatus

Moisture 88.27 ± 0.60 c 90.92 ± 1.08 a 88.49 ± 3.03 b 85.19 ± 0.50 d
Ash 6.78 ± 1.28 c 13.89 ± 1.41 a 9.55 ± 0.68 b 12.85 ± 0.42 a
Proteins 16.38 ± 1.34 a 15.46 ± 0.24 a 17.33 ± 1.37 a 15.67 ± 0.23 a
Fat 5.56 ± 0.53 a 0.83 ± 0.11 c 2.46 ± 0.04 b 1.13 ± 0.05 c
Carbohydrates 71.28 ± 1.06 a 69.83 ± 1.22 a 70.66 ± 1.09 a 70.36 ± 0.26 a
Energy 400. 68 ± 5.50 a 348.58 ± 3.58 c 374.12 ± 1.81 b 354.27 ± 1.18 c
Mannitol 5.45 ± 0.04 a 0.29 ± 0.01 c 0.59 ± 0.02 b 0.40 ± 0.04 d
Trehalose 9.33 ± 0.04 b 7.96 ± 0.28 b 7.77 ± 0.30 b 42.82 ± 2.59 a
Arabinose 0.78 ± 0.04 nd nd nd
Total sugars 15.66 ± 0.04 b 8.26 ± 0.29 c 8.36 ± 0.32 c 43.23 ± 2.62 a

nd, not detected.

Table 4
Relative percentages of fatty acids in the four wild edible mushroom species analysed (mean ± SD; n = 3). In each row different letters imply significant differences (p < 0.05).

Armillaria mellea Calocybe gambosa Clitocybe odora Coprinus comatus

C6:0 nd 0.27 ± 0.03 0.04 ± 0.00 0.05 ± 0.00
C8:0 0.11 ± 0.01 0.25 ± 0.02 0.03 ± 0.00 0.05 ± 0.00
C10:0 0.09 ± 0.00 0.13 ± 0.01 0.02 ± 0.00 0.09 ± 0.00
C12:0 0.65 ± 0.00 0.15 ± 0.01 0.07 ± 0.00 0.16 ± 0.00
C14:0 0.27 ± 0.00 0.39 ± 0.02 0.20 ± 0.01 0.41 ± 0.02
C15:0 0.27 ± 0.02 0.36 ± 0.02 0.54 ± 0.01 0.35 ± 0.01
C16:0 11.04 ± 0.06 13.57 ± 0.50 12.46 ± 0.25 10.56 ± 0.44
C16:1 6.36 ± 0.01 0.65 ± 0.03 0.17 ± 0.03 0.59 ± 0.02
C17:0 0.03 ± 0.00 0.26 ± 0.05 0.10 ± 0.00 0.20 ± 0.02
C18:0 3.53 ± 0.01 3.24 ± 0.12 3.46 ± 0.26 1.90 ± 0.13
C18:1n9c 47.74 ± 0.35 32.54 ± 1.37 46.07 ± 0.17 6.27 ± 0.03
C18:2n6c 27.71 ± 0.32 43.88 ± 0.31 34.90 ± 0.68 74.86 ± 0.95
C18:3n3 0.04 ± 0.00 0.93 ± 0.01 0.06 ± 0.00 1.90 ± 0.14
C20:0 0.15 ± 0.00 0.47 ± 0.06 0.39 ± 0.02 0.11 ± 0.00
C20:1c 0.10 ± 0.01 0.13 ± 0.02 0.05 ± 0.00 0.10 ± 0.00
C20:2c 0.01 ± 0.05 0.06 ± 0.00 0.02 ± 0.00 0.37 ± 0.02
C20:3n3 + C21:0 nd 0.07 ± 0.01 0.03 ± 0.00 0.32 ± 0.03
C20:5n3 0.01 ± 0.00 0.14 ± 0.04 0.04 ± 0.00 nd
C22:0 0.26 ± 0.01 0.72 ± 0.04 0.46 ± 0.03 0.43 ± 0.01
C23:0 0.02 ± 0.00 0.69 ± 0.03 0.24 ± 0.01 0.21 ± 0.03
C24:0 0.81 ± 0.02 1.04 ± 0.18 0.57 ± 0.00 0.91 ± 0.02
C24:1 0.81 ± 0.01 0.07 ± 0.00 0.09 ± 0.00 0.16 ± 0.02
SFA 17.23 ± 0.07 c 21.54 ± 1.62 a 18.57 ± 0.55 b 15.42 ± 0.55 c
MUFA 55.01 ± 0.36 a 33.38 ± 1.42 c 46.39 ± 0.14 b 7.12 ± 0.02 d
PUFA 27.76 ± 0.29 d 45.07 ± 0.20 b 35.04 ± 0.70 c 77.46 ± 0.57 a

Caproic acid (C6:0); caprylic acid (C8:0); capric acid (C10:0); lauric acid (C12:0); myristic acid (C14:0); pentadecanoic acid (C15:0); palmitic acid (C16:0); palmitoleic acid
(C16:1); heptadecanoic acid (C17:0); stearic acid (C18:0); oleic acid (C18:1n9c); linoleic acid (C18:2n6c); a-linolenic acid (C18:3n3); arachidic acid (C20:0); eicosenoic acid
(C20:1c); eicosadienoic acid (C20:2c); eicosatrienoic acid + heneicosanoic acid (C20:3n3 + C21:0); eicosapentaenoic acid (C20:5n3); behenic acid (C22:0); tricosanoic acid
(C23:0); lignoceric acid (C24:0); nervonic acid (C24:1). nd, not detected.

The results for fatty acid composition, total saturated fatty acids contributing to the prevalence of PUFA in the latter two species.
(SFA), monounsaturated fatty acids (MUFA) and polyunsaturated The studied species also contained palmitic acid (C16:0) as a major
fatty acids (PUFA) of the wild edible mushrooms are given in fatty acid. Linoleic acid was also described as a major fatty acid in a
Table 4. Up to 22 fatty acids were detected and quantified. The ma- sample of C. comatus from Turkey (25.8% in fruit body and 59.5% in
jor fatty acid found was oleic acid (C18:1n9) for A. mellea and stem; Yilmaz et al., 2006) but at a lower percentage than that
C. odora, and linoleic acid (C18:2n6) for C. gambosa and C. comatus, found in the present study (74.86%). The major fatty acid found
 J.A. Vaz et al. / Food Chemistry 126 (2011) 610–616 615

Table 5
Vitamin contents in the four wild edible mushroom species analysed (mean ± SD; n = 3). In each row different letters imply significant differences (p < 0.05).

Armillaria mellea Calocybe gambosa Clitocybe odora Coprinus comatus

a-Tocopherol 9.06 ± 0.15 c 15.81 ± 0.01 b 18.24 ± 1.03 b 22.75 ± 2.67 a
c-Tocopherol 57.96 ± 0.60 b 119.42 ± 0.03 a 116.66 ± 6.37 a 124.27 ± 0.74 a
d-Tocopherol nd 24.70 ± 5.78 b 54.46 ± 2.82 b 154.01 ± 23.30 a
Total tocopherols (lg/100 g dry weight) 67.02 ± 0.46 c 159.93 ± 5.74 b 189.36 ± 2.53 b 301.03 ± 26.71 a
Ascorbic acid (mg/100 g dry weight) 148.02 ± 6.64 b 180.47 ± 23.96 a 172.65 ± 19.77 a 132.88 ± 11.35 b

nd, not detected.

in samples of A. mellea from Turkey (49.6% in fruit body and 2.56% Consolider-Ingenio 2010 Programme (FUN-C-FOOD, CSD2007-
in stem; Yilmaz et al., 2006) and USA (39.6%; Cox et al., 2006) was 00063), and Junta de Castilla y León (Grupo de Investigación de
linoleic acid and not oleic acid as here obtained. A commercial Excelencia, GR133).
sample of C. gambosa revealed a similar profile to that obtained
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