Food and Chemical Toxicology 47 (2009) 1076–1079

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Food and Chemical Toxicology

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Phenolic acids determination by HPLC–DAD–ESI/MS in sixteen different

Portuguese wild mushrooms species
Lillian Barros a,b, Montserrat Dueñas a, Isabel C.F.R. Ferreira b,*, Paula Baptista b, Celestino Santos-Buelga a,*
a
Unidad de Nutrición y Bromatología, Facultad de Farmacia, Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain
b
CIMO-Escola Superior Agrária de Bragança, CIMO-ESAB, Instituto Politécnico de Bragança, Campus de Sta. Apolónia, 1172, 5301-855 Bragança, Portugal

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

Article history: Analysis of phenolic compounds in sixteen Portuguese wild mushrooms species has been carried out by
Received 15 September 2008 high-performance liquid chromatography coupled to photodiode array detector and mass spectrometer
Accepted 27 January 2009 (HPLC–DAD–ESI/MS). No flavonoids were detected in the analysed samples, but diverse phenolic acids
namely protocatechuic, p-hydroxybenzoic and p-coumaric acids, and two vanillic acid isomers were
found and quantified. A related non-phenolic compound, cinnamic acid, was also detected in some sam-
Keywords: ples, being the only compound found in Cantharellus cibarius (14.97 mg/kg, dry matter), Lycoperdon perl-
Wild mushrooms
atum (14.36 mg/kg) and Macrolepiota procera (21.53 mg/kg). p-Hydroxybenzoic acid was found in the
Phenolic compounds
HPLC–DAD–ESI/MS
majority of the samples, being the most abundant compound in Agaricus silvicola (238.7 mg/kg). Ramaria
botrytis showed the highest phenolic acids concentration (356.7 mg/kg) due to the significant contribu-
tion of protocatechuic acid (342.7 mg/kg).
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction 2003, 2005; Soobrattee et al., 2005). Many of their biological effects
have been attributed to free radical scavenging and antioxidant
Natural phenolics are compounds possessing one or more aro- activity (Middleton et al., 2000).
matic rings with one or more hydroxyl groups and can range from The use of mushrooms extracts as antioxidants is becoming
simple molecules (phenolic acids, phenylpropanoids, flavonoids) to increasingly popular (Mau et al., 2002; Lo and Cheung, 2005;
highly polymerised compounds (lignins, melanins, tannins) (Bravo, Elmastas et al., 2007; Tsai et al., 2007) and our research group pub-
1998). Particularly, phenolic acids can be subdivided into two ma- lished several studies reporting the antioxidant properties of wild
jor groups, hydroxybenzoic acids and hydroxycinnamic acids edible mushrooms, particularly their free radical scavenging activ-
(Fig. 1). Hydroxybenzoic acids include p-hydroxybenzoic, proto- ity and lipid peroxidation inhibition in animal erythrocytes and in
catechuic, vanillic, syringic, and gallic acids. They are commonly brain cells membranes (Barros et al., 2007a, 2008a, 2008b). The
present in the bound form and are typically a component of a com- antioxidant properties were correlated to different antioxidant
plex structure like lignins and hydrolyzable tannins. They can also components such as tocopherols, carotenoids, ascorbic acid and to-
be found linked to sugar derivatives and organic acids in plant tal phenolics (Barros et al.,2007b, 2008c). However, little is known
foods. Hydroxycinnamic acids include p-coumaric, caffeic, ferulic, about the individual phenolic compounds present in mushroom
and sinapic acids. In natural sources they are mainly found esteri- species. A few studies concerning the analysis of the phenolic com-
fied with small molecules like, e.g., quinic or tartaric acids, as well ponents of Portuguese wild mushrooms can be found in the litera-
as bound to cell-wall structural components such as cellulose, lig- ture, particularly for Cantharellus cibarius (Valentão et al., 2005),
nin, and proteins through ester bonds (Liu, 2004). Suillus bellini, Tricholomopsis rutilans, Hygrophorus agathosmus,
Phenolic compounds, commonly found in vegetables, fruits and Amanita rubescens, Russula cyanoxantha, Boletus edulis, Tricholoma
many plant-derived foods that form a significant portion of our equestre, Suillus luteus, Suillus granulatus (Ribeiro et al., 2006), and
diet, are among the most potent and therapeutically useful bioac- Fistulina hepatica (Ribeiro et al., 2007). Nevertheless, being the
tive substances, providing health benefits associated with reduced Northeast of Portugal one of the European regions with higher wild
risk of chronic and degenerative diseases (Luximon-Ramma et al., edible mushroom diversity, it is important to characterize the phe-
nolic composition of other species also important and with gastro-
nomic relevance. In this study, individual profiles of phenolic
compounds in sixteen Portuguese wild mushrooms were charac-
* Corresponding authors. Tel.: +351273303219; fax: +351273325405 (I.C.F.R.
terised by high-performance liquid chromatography coupled to
Ferreira), tel.: +34 923 294537; fax: +34 923 294515 (C. Santos-Buelga).
E-mail addresses: [email protected] (I.C.F.R. Ferreira), [email protected] (C. Santos- photodiode array detector and mass spectrometer (HPLC–DAD–
Buelga). ESI/MS).

0278-6915/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2009.01.039
 L. Barros et al. / Food and Chemical Toxicology 47 (2009) 1076–1079 1077

1 1 (Analytikijena 200-2004 spectrophotometer, Jena, Germany). Gallic acid was used

R R
to prepare the standard curve (0.01–0.4 mM; y = 2.8557x 0.0021; R2 = 0.9999)
and the results were expressed as mg of gallic acid equivalents (GAEs) per g of
extract.
2 2
R COOH R CH=CH-COOH
2.4. DPPH radical scavenging activity

Various concentrations of mushroom extracts (0.3 mL) were mixed with 2.7 mL
3 3 of methanolic solution containing DPPH radicals (6  10 5 mol/L). The mixture was
R R
shaken vigorously and left to stand for 60 min in the dark (until stable absorption
Hydroxybenzoic acids Cinnamic acids derivatives values were obtained). The reduction of the DPPH radical was determined by mea-
suring the absorption at 517 nm. The radical scavenging activity (RSA) was calcu-
Fig. 1. Chemical structure of the identified phenolic acids in the wild mushroom lated as a percentage of DPPH discolouration using the equation: % RSA =
species. Benzoic acids: p-hidroxybenzoic (R1@R3@H, R2@OH), protocatechuic [(ADPPH AS)/ADPPH]  100, where AS is the absorbance of the solution when the
(R1@H, R2@R3@OH), vanillic (R1@CH3O, R2@OH, R3@H). Cinnamic acid (R1@ sample extract has been added at a particular level, and ADPPH is the absorbance
R2@R3@H) and derivatives: p-coumaric (R1@R3@H, R2@OH). of the DPPH solution. The extract concentration providing 50% of radical scavenging
activity (EC50) was calculated from the graph of RSA percentage against extract con-
2. Materials and methods centration. BHA and a-tocopherol were used as standards.

2.1. Samples 2.5. Phenolic compounds identification and quantification

Sixteen mushrooms species were collected from different places in Trás-os- 2.5.1. Sample preparation
Montes region in the Northeast of Portugal (Table 1). The morphological identifica- Each mushroom sample (3 g) was extracted with acetone:water (80:20;
tion of the wild macrofungi was made till species according to macro and micro- 50 mL) mixture at 20 °C for 6 h. The extract was put in an ultrasonic bath for
scopic characteristics, and following several authors (Moser, 1983; Courtecuisse 15 min, centrifuged at 4000g for 10 min, and filtered through Whatman No. 4 paper.
and Duhem, 1995) and representative voucher specimens were deposited at the The residue was then extracted with three additional 50 mL portions of the ace-
herbarium of Escola Superior Agrária of Instituto Politécnico de Bragança. After taxo- tone:water mixture. The combined extracts were evaporated at 30 °C to remove
nomic identification, the mushrooms were immediately lyophilized (Ly-8-FM-ULE, acetone. The aqueous phase was washed with n-hexane, and then submitted to a
Snijders, Holland), and kept in the dark in hermetically sealed plastic bags up to liquid–liquid extraction with diethyl ether (3  50 mL) and ethyl acetate
analysis. (3  50 mL). The organic phases were evaporated at 30 °C to dryness, redissolved
in water:methanol (80:20), and filtered through a 0.22 lm disposable LC filter disk
2.2. Standards and reagents for HPLC analysis.

Acetonitrile 99.9% was of HPLC-grade from Lab-Scan (Lisbon, Portugal). All the 2.5.2. HPLC–DAD–ESI/MS analyses
other reagents (methanol, n-hexane, ethyl acetate and diethyl ether) were of ana- The phenolic extracts were analysed using a Hewlett-Packard 1100 series liquid
lytical grade purity and were also supplied by Lab-Scan. Gallic acid was from Supe- chromatograph (Agilent Technologies, Waldbronn, Germany). Separation was
lco (Bellefonte, PA, USA) and the rest of phenolic standards were from Sigma achieved on a Spherisorb (Phenomenex, Torrance, CA) reverse phase C18 column
Chemical Co. (St. Louis, MO, USA). The Folin and Ciocalteu’s reagent was purchased (3 lm, 150 mm  4.6 mm i.d.) thermostatted at 25 °C. The solvents used were:
from Merck (Darmstadt, Germany). Water was treated in a Milli-Q water purifica- (A) 2.5% acetic acid in water, (B) acetic acid 2.5%/acetonitrile (90:10), and (C)
tion system (TGI Pure Water Systems, USA). 100% HPLC-grade acetonitrile. The gradient employed was: isocratic 100% A for
10 min, 50% A and 50% B for 10 min, isocratic 100% B for 15 min, 90% B and 10%
2.3. Analysis of total phenolics C for 10 min, 70% B and 30% C for 10 min, 50% B and 50% C for 5 min, 20% B and
80% C for 5 min, 100% A for 5 min, at a flow rate of 0.5 mL/min. Detection was car-
2.3.1. Sample preparation ried out in a diode array detector (DAD), using 280 nm as the preferred wavelength,
A fine dried mushroom powder (20 mesh) sample (3 g) was extracted by stir- and in a mass spectrometer (MS) connected to the HPLC system via the DAD cell
ring with 100 mL of methanol at 25 °C at 150 rpm for 24 h and filtered through outlet.
Whatman No. 4 paper. The residue was then extracted with two additional LC–MS analyses were performed using a FinninganTM LCQ MS detector (Thermo-
100 mL portions of methanol, as described earlier. The combined methanolic ex- quest, San Jose, CA, USA) equipped with an API source, using an electrospray ioni-
tracts were evaporated at 40 °C to dryness and redissolved in a known concentra- sation (ESI) interface. Both the sheath gas and the auxiliary gas were nitrogen at
tion of methanol. flow rates of 1.2 and 6 L/min, respectively. The capillary and source voltage were
10 V and 3.5 kV, respectively, and the capillary temperature was 175 °C. Spectra
2.3.2. Folin–Ciocalteu’s assay were recorded in negative ion mode between m/z 80 and 620. The MS was pro-
Briefly, the methanolic extract solution (1 mL) was mixed with the Folin–Ciocal- grammed to carry out a series of three consecutive scans: a full mass from 150 to
teu reagent (1 mL). After 3 min, saturated sodium carbonate solution (1 mL) was 1500 amu, a zoom scan of the most abundant ion in a ±5 amu range, and an MS–
added to the mixture and adjusted to 10 mL with distilled water. The reaction MS scan of the most abundant ion in the full mass using a normalised energy of col-
was kept in the dark for 90 min, after which the absorbance was read at 725 nm lision of 45%.

Table 1
Collection information of the wild mushroom samples, and their total phenols (by Folin–Ciocalteu’s assay) and antioxidant activity (by DPPH assay). In each column different
letters mean significant differences (p < 0.05).

Species Origin Orchard Date of collection Total phenols (mg/g extract) Antioxidant activity (EC50 value, mg/mL)
Agaricus arvensis Carrazeda de Ansiães Pinus pinaster October 2006 2.75 ± 0.17 f 15.85 ± 0.27 d
Agaricus bisporus Bragança Grassland October 2006 4.49 ± 0.16 e 9.61 ± 0.07 e
Agaricus romagnesii Vinhais Pinus pinaster October 2006 6.18 ± 0.44 d 6.22 ± 0.10 gf
Agaricus silvicola Bragança Quercus pyrenaica October 2006 6.40 ± 0.17 d 6.39 ± 0.16 f
Cantharellus cibarius Vinhais Quercus pyrenaica June 2007 1.75 ± 0.50 g 19.65 ± 0.28 b
Hypholoma fasciculare Bragança Quercus pyrenaica October 2006 17.67 ± 0.27 b 1.13 ± 0.03 l
Lactarius deliciosus Bragança Pinus pinaster November 2005 3.40 ± 0.18 f 16.31 ± 0.24 c
Lactarius piperatus Bragança Quercus pyrenaica June 2006 3.09 ± 0.12 f 20.24 ± 0.78 a
Lepista nuda Cova de Lua Pinus pinaster November 2006 6.31 ± 0.13 d 4.41 ± 0.01 i
Leucopaxillus giganteus Cova de Lua Pinus pinaster October 2005 6.29 ± 0.20 d 1.44 ± 0.09 l
Lycoperdon molle Vinhais Quercus pyrenaica October 2006 11.48 ± 0.52 c 3.23 ± 0.09 k
Lycoperdon perlatum Vinhais Quercus pyrenaica October 2006 10.57 ± 0.17 c 3.95 ± 0.04 j
Macrolepiota procera Carrazeda de Ansiães Quercus pyrenaica November 2006 3.17 ± 0.92 f 5.38 ± 0.50 h
Ramaria botrytis Vinhais Quercus pyrenaica October 2006 20.32 ± 1.87 a 0.66 ± 0.00 m
Sarcodon imbricatus Vinhais Pinus pinaster November 2006 3.06 ± 0.10 f 5.82 ± 0.06 g
Tricholoma acerbum Vinhais Quercus pyrenaica October 2006 5.53 ± 0.63 d 3.60 ± 0.08 kj
1078 L. Barros et al. / Food and Chemical Toxicology 47 (2009) 1076–1079

The phenolic compounds present in the samples were characterised according In previous studies of our group (Barros et al., 2007a, 2007b,
to their UV–vis spectra and identified by their mass spectra and retention times
2007c, 2008a, 2008b), antioxidant activity assessed in mushroom
in comparison with those of commercial standards. For the quantitative analysis
of phenolic compounds, a calibration curve was obtained by injection of different
extracts by different chemical and biochemical assays was corre-
concentration of protocatechuic acid, p-hydroxybenzoic acid, vanillic acid, p-cou- lated with their contents of phenolic compounds as measured by
maric acid, and cinnamic acid standards. the Folin–Ciocalteu method. However, no analyses of individual
phenolics were made and, therefore, the compounds responsible
2.6. Statistical analysis for that antioxidant activity were unknown. In the present study
we aimed to identify and quantify individual compounds that
The analysis of phenolic compounds contents in each mushroom species was
carried out in triplicate and the results expressed as mean ± standard deviation may contribute to the bioactive properties already found for these
(SD). Data were analysed by one-way analysis of variance (ANOVA) followed by Tu- Portuguese wild mushroom species.
key’s HSD Test with a = 0.05, using SPSS v. 16.0 program. Three phenolic acids (protocatechuic, p-hydroxybenzoic and p-
coumaric acids) and a related compound (cinnamic acid) could be
3. Results and discussion positively identified and quantified in some samples (Table 2) by
comparison of their chromatographic characteristics and absorp-
Total phenolic compounds in the analyzed Portuguese wild tion spectra with the standards compounds and confirmed by mass
mushrooms species were determined by the Folin–Ciocalteu’s as- analysis. In Fig. 2 a representative chromatogram obtained for one
say. Table 1 presents those results as also the EC50 values (extract of the mushroom extracts analysed is shown as an example. Other
concentration correspondent to 50% of radical scavenging activity) two compounds were also detected in the samples of L. molle and T.
obtained in the assessment of the antioxidant activity of mush- acerbum whose UV spectra, molecular ion (m/z [M H] at 167) and
rooms measured by the DPPH (2,2-diphenyl-1-picrylhydrazyl) MS2 spectra (one fragment at m/z 123, [M 44] , loss of a CO2 res-
assay. idue) coincided with those of vanillic acid (4-hydroxy-3-methoxy-
Phenolic compounds include different subclasses (flavonoids, benzoic acid), but that showed higher retention times (40.5 and
phenolic acids, stilbenes, lignans, tannins, oxidized polyphenols) 44.0 min, respectively, in comparison with 30.1 min for vanillic
displaying a large diversity of structures, some of which may es- acid). Thus, these compounds were tentatively associated to vanil-
cape the usual methodologies of analysis, commonly carried out lic acid isomers like, e.g., o-vanillic (i.e., 2-hydroxy-3-methoxy-
by HPLC (high-performance liquid chromatography) coupled to benzoic acid) or isovanillic acid (i.e., 3-hydroxy-4-methoxybenzoic
distinct detection devices. Various reasons exist for that, like the acid), for which no standards were available.
existence of isomers, difficulty for chromatographic separation of No phenolic acids were detected in six mushroom species: H.
some compounds, lack of commercial standards, or structure not fasciculare, L. piperatus, L. giganteus, C. cibarius, L. perlatum and M.
yet elucidated (Georgé et al., 2005). The method of Folin–Ciocal- procera, although the presence of cinnamic acid was found in the
teu’s is, therefore, largely used to evaluate total phenolics despite three latter. No peaks were found in the extracts whose UV spectra
all the interferences of this assay since the reagent (mixture of could be associated to hydroxycinnamic acids or their tartaric or
phosphotungstic acid and phosphomolibdic acid) also reacts with quinic esters (i.e., chlorogenic acids). Further, no detection of those
other non-phenolic reducing compounds leading to an overvalua- compounds was made when the full mass chromatograms of the
tion of the phenolic content. For instance, ascorbic acid is a wide- samples were screened for their molecular ions. Similarly, no peaks
spread reducing agent that can interfere in the Folin–Ciocalteu whose UV spectra or mass characteristics could be associated to
reaction (Georgé et al., 2005) and that was, in fact, reported to be flavonoids were found. This fact should not be surprising since,
present in the studied species (Barros et al., 2007a, 2007b, 2008a, in general, it is assumed that only plants possess the biosynthetic
2008b). Other reducing substances such as some sugars and amino ability to produce flavonoids and not animals and fungi (Iwashina,
acids could also interfere. In addition, the results have to be ex- 2000), even if some flavonoids have exceptionally been reported
pressed in equivalents of a particular standard compound (like cat- from fungi Aspergillus candidus and Phallus impudicus (reviewed
echin, gallic acid or tannin acid). All these aspects make the results in Iwashina, 2000) and more recently in the edible beefsteak fun-
obtained for different authors difficult to compare. gus F. hepatica (Ribeiro et al., 2007).

Table 2
Phenolic acids found in the mushroom samples. In each column different letters mean significant differences (p < 0.05).

Phenolic compounds (mg/kg, dry matter) Other compounds

(mg/kg, dry matter)
Protocatechuic acid p-Hydroxybenzoic Vanillic acid isomer p-Coumaric Vanillic acid isomer Total phenolic Cinnamic acid (51.4 min)
(15.1 min) acid (22.9 min) (40.5 min) acid (41.7 min) (44.1 min) compounds
A. arvensis n.d. 70.13 ± 1.20 n.d. 48.67 ± 3.40 n.d. 118.8 ± 4.6 c 49.10 ± 8.03
A. bisporus n.d. 25.59 ± 1.55 n.d. n.d. n.d. 25.59 ± 1.55 e 8.72 ± 0.71
A. silvicola n.d. 238. 7 ± 12.4 n.d. 45.72 ± 1.19 n.d. 284.4 ± 11.2 b 68.37 ± 11.32
A. romagnesii n.d. 32.40 ± 0.83 n.d. n.d. n.d. 32.40 ± 0.83 e 49.22 ± 3.90
C. cibarius n.d. n.d. n.d. n.d. n.d. n.d. 14.97 ± 0.40
L. deliciosus n.d. 22.66 ± 0.36 n.d. n.d. n.d. 22.66 ± 0.36 e n.d.
L. giganteus n.d. n.d. n.d. n.d. n.d. n.d. n.d.
L. nuda 33.47 ± 0.50 29.31 ± 1.54 n.d. 3.75 ± 0.56 n.d. 66.53 ± 2.62 d n.d.
L. molle n.d. 41.66 ± 0.33 35.97 ± 6.16 n.d. 4.02 ± 0.55 81.65 ± 7.04 d n.d.
L. perlatum n.d. n.d. n.d. n.d. n.d. n.d. 14.36 ± 1.27
L. piperatus n.d. n.d. n.d. n.d. n.d. n.d. n.d.
M. procera n.d. n.d. n.d. n.d. n.d. n.d. 21.53 ± 1.65
H. fascicular n.d. n.d. n.d. n.d. n.d. n.d. n.d.
S. imbricatus n.d. 33.19 ± 1.92 n.d. n.d. n.d. 33.19 ± 1.92 e n.d.
R. botrytis 342.7 ± 10.2 14.00 ± 0.77 n.d. n.d. n.d. 356.7 ± 9.4 a n.d.
T. acerbum n.d. 29.66 ± 0.26 4.92 ± 0.72 n.d. 7.81 ± 0.56 42.38 ± 1.53 e n.d.

n.d. – not detected.

 L. Barros et al. / Food and Chemical Toxicology 47 (2009) 1076–1079 1079

1500 3 Conflict of interest statement

1400
The authors declare that there are no conflicts of interest.
1200
1
1000 Acknowledgement
2
800
AU

The authors are grateful to Foundation for Science and

600 Technology (PPCDT/AGR/56661/2004) for financial support of this
400 work.

200
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