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Effect of Lactarius piperatus fruiting body maturity stage on
antioxidant activity measured by several biochemical assays
Lillian Barros, Paula Baptista, Isabel C.F.R. Ferreira *
CIMO-Escola Superior Agra´ria, Instituto Polite´cnico de Braganc¸a, Campus de Sta. Apolo´nia, Apartado 1172, 5301-855 Braganc¸a, Portugal
Received 21 September 2006; accepted 12 March 2007
Abstract
The effects of fruiting body maturity on antioxidant activity and antioxidants production of the wild mushroom, Lactarius piperatus,
were evaluated. Several biochemical assays were used to screen the antioxidant properties: reducing power, 2,2-diphenyl-1-picrylhydrazyl
(DPPH) radical scavenging capacity, inhibition of erythrocytes hemolysis mediated by peroxyl radicals and inhibition of lipid peroxida-
tion using the b-carotene linoleate model system. The amounts of phenols, flavonoids, ascorbic acid, b-carotene and lycopene present in
the immature, mature and degraded fruiting bodies were also determined. The highest antioxidant contents and the lowest EC50 values
for antioxidant activity were obtained in the mature stage with immature spores.
Ó 2007 Elsevier Ltd. All rights reserved.
Keywords: Wild mushroom; Fruiting body maturity; Antioxidant activity; Antioxidant components
1. Introduction
Oxidation is essential to many living organisms for the
production of energy to fuel biological processes, proceed-
ing in lipids with polyunsaturated fatty acids, and generat-
ing reactive oxygen species (ROS) such as hydroxyl radicals
(Halliwell and Gutteridge, 1989). However, oxygen-centred
free radicals and other ROS species, that are continuously
produced in vivo as by products, result in cell death and tis-
sue damage. The oxidative damage caused by free radicals
may be related to aging and diseases, such as atherosclero-
sis, diabetes, cancer and cirrhosis (Halliwell and Gutter-
idge, 1984). Antioxidant compounds reduce the action of
reactive oxygen species (ROS) in tissue damage.
Although humans and other organisms possess antioxi-
dant defences (enzymes, such as superoxide dismutase and
catalase, or compounds such as ascorbic acid, tocopherols
and glutathione) and repair systems that have evolved to
protect them against oxidative damage, these systems are
insufficient to totally prevent the damage (Simic, 1988; Niki
et al., 1994; Mau et al., 2002).
Natural products with antioxidant activity are used to
aid the endogenous protective system, increasing interest
in the antioxidative role of nutraceutic products (Kanter,
1998). Concerning this, the antioxidants in human diets
are of great interest as possible protective agents to help
human body reduce oxidative damage. Recently, a multi-
tude of natural antioxidants have already been isolated
from different kinds of plant materials such as oilseeds, cer-
eal crops, vegetables, fruits, leaves, roots, spices, and herbs
(Ramarathnam et al., 1995). Mushrooms have also become
attractive as a functional food and as source for the devel-
opment of drugs and nutraceuticals (Yanga et al., 2002;
Barros et al., 2007, in press).
Mushrooms accumulate a variety of secondary metabo-
lites, including phenolic compounds, polyketides, terpenes
and steroids. The quality of a nutraceutical is dependent
on the chemical composition of the fruiting body, particu-
larly in relation to the phenols and flavonoids content.
Phenolic compounds were found to have antioxidant
activity in the inhibition of LDL oxidation (Teissedre and
Landrault, 2000). Phenolics are one of the major groups
0278-6915/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2007.03.006
*
Corresponding author. Tel.: +351 273303219; fax: +351 273325405.
E-mail address: iferreira@ipb.pt (I.C.F.R. Ferreira).
www.elsevier.com/locate/foodchemtox
Food and Chemical Toxicology 45 (2007) 1731–1737
Author's personal copy
of nonessential dietary components that have been associ-
ated with the inhibition of atherosclerosis and cancer (Wil-
liams and Iatropoulos, 1997). Flavonoids have been proven
to display a wide range of pharmacological and biochemi-
cal actions, such as antimicrobial, antithrombotic, antimu-
tagenic and anticarcinogenic activities (Cook and Samman,
1996; Kandaswami and Middleton, 1997; Sahu and Green,
1997).
Several studies described by us (Barros et al., 2007, in
press; Ferreira et al., 2007) and also by other authors
(Yen and Hung, 2000; Mau et al., 2002, 2004; Yanga
et al., 2002; Cheung et al., 2003; Cheung and Cheung,
2005; Lo and Cheung, 2005; Turkoglu et al., 2007) report
a correlation between the mushrooms antioxidant activity
and their phenolic content. However, none of the existent
reports on mushrooms antioxidants composition and anti-
oxidant activity indicated the stage of development of the
fruiting bodies selected for the studies. Furthermore, no
studies have been developed to evaluate the antioxidant
activity at different stages of fruiting body maturity for
potential use on the preparation of nutraceutical.
In this study, we examined the evolution of antioxidant
components and antioxidant activity of a Basidiomycete
fungus, L. piperatus, in four stages of fruiting body matu-
rity. The antioxidant properties were evaluated though
reducing power, DPPH (2,2-diphenyl-1-picrylhydrazyl)
radical scavenging activity, inhibition of oxidative hemoly-
sis in erythrocytes induced by 2,20
-azobis(2-amidinopro-
pane)dihydrochloride (AAPH) and inhibition of lipid
peroxidation by b-carotene-linoleate system. These assays
have been extensively studied as models for the peroxida-
tive damage in biomembrane. All these antioxidant activity
parameters were correlated to the phenolic, flavonoidic,
ascorbic acid, b-carotene and lycopene contents.
2. Materials and methods
2.1. Chemicals
Standards BHA (2-tert-butyl-4-methoxyphenol), TBHQ (tert-butyl-
hydroquinone), L-ascorbic acid, a-tocopherol, gallic acid and (+)-catechin
were purchase from Sigma (St. Louis, MO, USA). 2,2-diphenyl-1-pic-
rylhydrazyl (DPPH) was obtained from Alfa Aesar (Ward Hill, MA,
USA). All other chemicals were obtained from Sigma Chemical Co. (St.
Louis, MO, USA). Methanol was obtained from Pronalab (Lisbon, Por-
tugal). Water was treated in a Mili-Q water purification system (TGI Pure
Water Systems, USA).
2.2. Fruiting body selection
Wild fruiting bodies of Lactarius piperatus (L.) Pers. were obtained
under live oak trees (Quercus pyrenaica), in Braganc¸a (northeast of
Portugal), in spring 2006. Taxonomic identification was made according
to several authors (Marchand, 1971-1986; Moser, 1983; Bon, 1988;
Courtecuisse and Duhem, 1995; Courtecuisse, 1999) and a representative
voucher specimen was deposited at the herbarium of Escola Superior
Agra´ria of Instituto Polite´cnico de Braganc¸a. The fruiting bodies were
harvested and dried in different stages of maturity: immature (cap closed)
and mature (cap opened). The mature stage was further characterized into
immature spores and mature spores as seen on Fig. 1.
2.3. Sample preparation
The fruiting bodies were air-dried in a liophylizator (Ly-8-FM-ULE,
Snijders) and powdered before analysis. The dried samples ($5 g) were
extracted by stirring with 100 mL of methanol at 25 °C at 150 rpm for 24 h
and filtered through Whatman No. 4 paper. The residue was then
extracted with two additional 100 mL portions of methanol, as described
earlier. The combined methanolic extracts were evaporated at 40 °C to
Fig. 1. Lactarius piperatus fruiting bodies in different stages of maturity: (SI) immature (cap closed), (SII) mature (cap opened) with immature spores,
(SIII) mature with mature spores, and (SIV) degraded.
1732 L. Barros et al. / Food and Chemical Toxicology 45 (2007) 1731–1737
Author's personal copy
dryness and redissolved in methanol at a concentration of 50 mg/mL, and
stored at 4 °C for further use.
2.4. Determination of total antioxidant components
Phenolic compounds in the mushroom extracts were estimated by a
colorimetric assay, based on procedures described by Singleton and Rossi
with some modifications (Singleton and Rossi, 1965). Briefly, 1 mL of
sample was mixed with 1 mL of Folin and Ciocalteu’s phenol reagent.
After 3 min, 1 mL of saturated sodium carbonate solution was added to
the mixture and adjusted to 10 mL with distilled water. The reaction was
kept in the dark for 90 min, after which the absorbance was read at
725 nm (Analytikijena 200-2004 spectrophotometer). Gallic acid was used
to calculate the standard curve (0.01–0.4 mM; Y = 2.8557X À 0.0021;
R2
= 0.9999) and the results were expressed as mg of gallic acid equiva-
lents (GAEs) per g of extract.
Flavonoid contents in the extracts were determined by a colorimetric
method described by Jia et al. (1999) with some modifications. The
mushroom extract (250 lL) was mixed with 1.25 mL of distilled water and
75 lL of a 5% NaNO2 solution. After 5 min, 150 lL of a 10% AlCl3 Æ H2O
solution was added. After 6 min, 500 lL of 1 M NaOH and 275 lL of
distilled water were added to the mixture. The solution was mixed well and
the intensity of pink colour was measured at 510 nm. (+)-Catechin was
used to calculate the standard curve (0.022–0.34 mM; Y = 0.9629X À
0.0002; R2
= 0.9999) and the results were expressed as mg of (+)-catechin
equivalents (CEs) per g of extract.
Ascorbic acid was determined according to the method of Klein and
Perry (1982). The dried methanolic extract (100 mg) was extracted with
10 ml of 1% metaphosphoric acid for 45 min at room temperature and
filtered through Whatman No. 4 filter paper. The filtrate (1 ml) was mixed
with 9 ml of 2,6-dichlorophenolindophenol and the absorbance was
measured within 30 min at 515 nm against a blank. Content of ascorbic
acid was calculated on the basis of the calibration curve of authentic
L-ascorbic acid (0.020–0.12 mg/ml; Y = 3.4127X À 0.0072; R2
= 0.9905)
and the results were expressed as mg of ascorbic acid/g of extract.
b-Carotene and lycopene were determined according to the method of
Nagata and Yamashita (1992). The dried methanolic extract (100 mg) was
vigorously shaken with 10 ml of acetone–hexane mixture (4:6) for 1 min
and filtered through Whatman No. 4 filter paper. The absorbance of the
filtrate was measured at 453, 505 and 663 nm. Contents of b-carotene and
lycopene were calculated according to the following equations: lycopene
(mg/100 ml) = À0.0458 A663 + 0.372 A505 À 0.0806 A453; b-carotene
(mg/100 ml) = 0.216 A663 À 0.304 A505 + 0.452 A453. The results were
expressed as lg of carotenoid/g of extract.
2.5. DPPH radical-scavenging activity
The capacity to scavenge the ‘‘stable’’ free radical DPPH was moni-
tored according to the method of Hatano et al. (1988). Various concen-
trations of mushroom extracts (0.3 mL) were mixed with 2.7 mL of
methanolic solution containing DPPH radicals (6 · 10À5
mol/L). The
mixture was shaken vigorously and left to stand for 60 min in the dark
(until stable absorption values were obtained). The reduction of the DPPH
radical was determined by measuring the absorption at 517 nm. The
radical scavenging activity (RSA) was calculated as a percentage of DPPH
discolouration using the equation: % RSA = [(ADPPH À AS)/
ADPPH] · 100, where AS is the absorbance of the solution when the sample
extract has been added at a particular level, and ADPPH is the absorbance
of the DPPH solution. The extract concentration providing 50% inhibition
(EC50) was calculated from the graph of RSA percentage against extract
concentration. BHA and a-tocopherol were used as standards.
2.6. Reducing power
The reducing power was determined according to the method of
Oyaizu (1986). Various concentrations of mushroom methanolic extracts
(2.5 mL) were mixed with 2.5 mL of 200 mmol/L sodium phosphate buffer
(pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was
incubated at 50 °C for 2 min. After 2.5 mL of 10% tricloroacetic acid (w/v)
were added, the mixture was centrifuged at 1000 rpm for 8 min (Centorion
K24OR – 2003 refrigerated centrifuge). The upper layer (5 mL) was mixed
with 5 mL of deionised water and 1 mL of 0.1% of ferric chloride, and the
absorbance was measured spectrophotometrically at 700 nm. The extract
concentration providing 0.5 of absorbance (EC50) was calculated from
the graph of absorbance at 700 nm against extract concentration. BHA
and a-tocopherol were used as standards.
2.7. Inhibition of erythrocyte hemolysis mediated by peroxyl free
radicals
The antioxidant activity of the mushroom extracts was measured as
the inhibition of erythrocyte hemolysis (Miki et al., 1987). Blood was
obtained from male ram (churra galega transmontana) of body weight
$67 kg. Eryhrocytes separated from the plasma and the buffy coat were
washed three times with 10 mL of 10 mM phosphate buffer saline (PBS) at
pH 7.4 (prepared by mixing 10 mM of NaH2PO4 and Na2HPO4, and
125 mM of NaCl in 1 L of distilled water) and centrifuged at 1500g for
5 min. During the last washing, the erythrocytes were obtained by
centrifugation at 1500g for 10 min. A 0.1 mL of a 20% suspension of
erythrocytes in PBS was added to 0.2 mL of 200 mM 2,20
-azobis(2-ami-
dinopropane)dihydrochloride (AAPH) solution (in PBS) and 0.1 mL of
mushroom methanolic extracts of different concentrations. The reaction
mixture was shaken gently (30 rpm) while being incubated at 37 °C for 3 h.
The reaction mixture was diluted with 8 mL of PBS and centrifuged at
3000g for 10 min; the absorbance of its supernatant was then read at
540 nm by a spectrophotometer, after filtration with a syringe filter (cel-
lulose membrane 30 mm, 0.20 lm, Titan). The percentage hemolysis
inhibition was calculated by the equation % hemolysis inhibi-
tion = [(AAAPH À AS)/AAAPH] · 100, where AS is the absorbance of the
sample containing the mushroom extract, and AAAPH is the absorbance of
the control sample containing no mushroom extract. The extract con-
centration providing 50% inhibition (EC50) was calculated from the
graph of hemolysis inhibition percentage against extract concentration.
L-Ascorbic acid was used as standard.
2.8. Inhibition of lipid peroxidation using the b-carotene linoleate
model system
The antioxidant activity of mushroom extracts was evaluated by the b-
carotene linoleate model system (Mi-Yae et al., 2003). A solution of b-
carotene was prepared by dissolving 2 mg of b-carotene in 10 mL of
chloroform. Two millilitres of this solution were pipetted into a 100 mL
round-bottom flask. After the chloroform was removed at 40 °C under
vacuum, 40 mg of linoleic acid, 400 mg of Tween 80 emulsifier, and
100 mL of distilled water were added to the flask with vigorous shaking.
Aliquots (4.8 mL) of this emulsion were transferred into different test
tubes containing 0.2 mL of different concentrations of the mushroom
extracts. The tubes were shaken and incubated at 50 °C in a water bath. As
soon as the emulsion was added to each tube, the zero time absorbance
was measured at 470 nm using a spectrophotometer. Absorbance readings
were then recorded at 20-min intervals until the control sample had
changed colour. A blank, devoid of b-carotene, was prepared for back-
ground subtraction. Lipid peroxidation (LPO) inhibition was calculated
using the following equation: LPO inhibition = (b-carotene content after
2 h of assay/initial b-carotene content) · 100. The extract concentration
providing 50% antioxidant activity (EC50) was calculated from the graph
of antioxidant activity percentage against extract concentration. TBHQ
was used as standard.
2.9. Statistical analysis
All the determinations were carried out in triplicate and the results are
expressed as mean values and standard deviations (SD). Differences
among means were done by analysis of variance (ANOVA), using SAS v.
L. Barros et al. / Food and Chemical Toxicology 45 (2007) 1731–1737 1733
Author's personal copy
9.1.3, and averages were compared using Tukey test (p < 0.05). A
regression analysis, using Excel for Windows Software, was established
between phenolic and flavonoid contents of different mushroom samples
and EC50 values obtained by different antioxidant assays.
3. Results and discussion
Table 1 shows phenol, flavonoid, ascorbic acid and
carotenoids concentrations obtained in L. piperatus in dif-
ferent stages of fruiting body maturity. Whereas total phe-
nols were the major antioxidant components found in the
extracts, ascorbic acid was found in small amounts (0.03–
0.16 mg/g), and b-carotene and lycopene were only found
in vestigial amounts (<34 lg/g), which is in agreement with
other reports concerning ascorbic acid and b-carotene
quantification in different mushrooms (Mau et al., 2002;
Barros et al., 2007, in press). Curiously, the stages where
the fruiting bodies presented immature spores (stage I
and II) revealed a higher content in phenol and flavonoid
compounds. The amount found in stages III (with mature
spores) and IV (degraded) significantly (p < 0.05) decreased
when compared with the content found in the first stages.
Probably, the aging process elicits the formation of reactive
oxygen species, which are neutralised by the polyphenolic
compounds, resulting in the lowering of their contents
and antioxidant capacities in the most advanced stages.
The highest content of antioxidant compounds in the first
stages might account for the better results found in their
antioxidant activity (Velioglu et al., 1998; Ferreira et al.,
2007). Furthermore, Cheung et al. (2003) found a direct
correlation between mushrooms antioxidant activity and
total phenolic content, although the antioxidant action is
raised by other substances such as tocopherols and b-caro-
tene. The bioactivity of phenolics may be related to their
ability to chelate metals, inhibit lipoxygenase and scavenge
free radicals (Decker, 1997). Also, in food systems, flavo-
noids can act as free radical scavengers and terminate the
radical chain reactions that occur during the oxidation of
triglycerides. Therefore, they present antioxidative effi-
ciency in oils, fats and emulsions (Madhavi et al., 1996;
Roedig-Penman and Gordon, 1998).
Fig. 2 shows the antioxidant activity of L. piperatus in
different stages of fruiting body maturity, measured by
different biochemical assays: reducing power, scavenging
activity on DPPH radicals, inhibition of the erythrocyte
hemolysis mediated by peroxyl free radicals and LPO inhi-
bition by b-carotene-linoleate system.
The radical scavenging activity (RSA) values were
expressed as the ratio percentage of sample absorbance
decrease and the absorbance of DPPH solution in the
absence of extract at 517 nm. From the analysis of Fig. 2,
we can conclude that the scavenging effects of fruiting body
extracts on DPPH radicals increased with the concentra-
tion increase and were very good for stage II (84.9% at
20 mg/mL), but lower than the scavenging effects of the
standards BHA (96% at 3.6 mg/ml) and a-tocopherol
(95% at 8.6 mg/ml). The RSA values at 20 mg/mL were
moderate for the other stages (stage I – 64.2%, stage III –
49.4%, stage IV – 43.4%) and, particularly stage III and
IV revealed a very similar scavenging activity.
The reducing power also increased with concentration,
and the values obtained for all the extracts were excellent
(Fig. 2); at 20 mg/mL were higher than 1.3 and in the order
of stage II > stage I > stage III > stage IV. Reducing power
of BHA at 3.6 mg/mL and a-tocopherol at 8.6 mg/mL was
only 0.12 and 0.13, respectively. The extracts obtained in
stages III and IV showed similar reducing power values.
It was reported that the reducing properties are generally
associated with the presence of reductones, which have
been shown to exert antioxidant action by breaking the free
radical chain by donating a hydrogen atom (Shimada et al.,
1992). Accordingly, in stage II, it was produced higher
amounts of reductone, which could react with free radicals
to stabilize and block radical chain reactions.
In this study, the protective effect of the fruiting
body extracts on hemolysis by peroxyl radical scavenging
activity was also investigated. AAPH is a peroxyl radical
initiator that generates free radicals by its thermal decom-
position and will attack the erythrocytes to induce the
chain oxidation of lipid and protein, disturbing the mem-
brane organization and eventually leading to hemolysis.
The extracts inhibited hemolysis, as a result of protection
against the oxidative damage of cell membranes of erythro-
cytes from ram induced by AAPH, in a concentration-
dependent manner (Fig. 2). Once more, in stage II there
was a higher protective effect against erythrocytes hemoly-
sis (82.4% at 50 mg/mL) than in other stages (stage I –
57.5%, stage III – 54.2%, stage IV – 41.0%). However,
the inhibition percentage of the standard L-ascorbic acid
on hemolysis of red blood cell was much higher (94.6%
at 1 mg/mL) than those of mushroom extracts.
The lipid peroxidation inhibition (LPO), measured by
the bleaching of b-carotene, is presented in Fig. 2. The
Table 1
Phenols, flavonoids, ascorbic acid, b-carotene and lycopene contents (mean ± SD) of Lactarius piperatus in different stages of maturity
Stage I Stage II Stage III Stage IV
Phenols (mg/g) 5.52 ± 0.14 b 5.76 ± 0.09 a 3.09 ± 0.12 c 2.03 ± 0.11 d
Flavonoids (mg/g) 1.26 ± 0.09 b 1.58 ± 0.02 a 0.35 ± 0.03 c 0.19 ± 0.01 d
Ascorbic acid (mg/g) 0.15 ± 0.01 a 0.16 ± 0.01 a 0.13 ± 0.01 b 0.03 ± 0.01 c
b-carotene (lg/g) 26.08 ± 0.05 b 33.78 ± 0.05 a 17.22 ± 0.00 c 15.11 ± 0.02 d
Lycopene (lg/g) 8.14 ± 0.03 b 13.04 ± 0.02 a 5.80 ± 0.01 c 5.41 ± 0.01 d
In each row different letters mean significant differences (p < 0.05).
1734 L. Barros et al. / Food and Chemical Toxicology 45 (2007) 1731–1737
Author's personal copy
linoleic acid free radical attacks the highly unsaturated b-
carotene models. The presence of different antioxidants
can hinder the extent of b-carotene-bleaching by neutraliz-
ing the linoleate-free radical and other free radicals formed
in the system (Jayaprakasha et al., 2001). Accordingly, the
absorbance decreased rapidly in samples without antioxi-
dant, whereas in the presence of an antioxidant, they
retained their colour, and thus absorbance, for a longer
time. LPO inhibition in the presence of L. piperatus in dif-
ferent stages of fruiting body maturity increased with their
increasing concentration, and the values at 50 mg/mL for
each one of the stages were 58.3%, 61.2%, 52.9% and
49.72%. It is probable that the antioxidative components
in the mushroom extracts can reduce the extent of b-caro-
tene destruction by neutralizing the linoleate free radical
and other free radicals formed in the system. Again, stage
II was the most effective for antioxidant activity. The anti-
oxidant activity of TBHQ standard reached 82.2% at 2 mg/
mL and was more efficient than the samples. Nevertheless,
this and other synthetic antioxidants applied in fat and oily
foods to prevent oxidative deterioration were found to be
anticarcinogenic as well as carcinogenic in experimental
animals (Loliger, 1991).
In Table 2 we present EC50 values obtained in the anti-
oxidant activity assays of L. piperatus in different stages of
fruiting body maturity. Overall, the mushroom in stage II
revealed better antioxidant properties (significantly lower
EC50 values; p < 0.05) than in other stages, which is in
agreement with the higher content of antioxidants found
in the first case. The EC50 values obtained for reducing
power and scavenging effects on DPPH radicals were better
than for LPO inhibition and for hemolysis inhibition med-
iated by peroxyl free radicals.
Significantly negative linear correlations were estab-
lished between the phenols and flavonoids content, and
EC50 values of DPPH scavenging activity (determination
coefficient 0.806 for phenols and 0.868 for flavonoids; p
< 0.001), reducing power (determination coefficient 0.946
Stage I Stage II Stage III Stage IV
0
20
40
60
80
100
0 1 2 3 4 5 10 20
Concentration mg/mL
Scavengingactivity(%)
0
0.5
1
1.5
2
2.5
0 1 2 3 4 5 10 20
Concentration mg/mL
Concentration mg/mL
Absat700nm
0
10
20
30
40
50
60
70
0 1 2 3 4 5 10 20 30 40 50
Concentration mg/mL
LPOinhibition(%)
0
20
40
60
80
100
0 1 5 10 20 40 50
Hemolysisinhibition(%)
Fig. 2. Antioxidant activity of Lactarius piperatus in different stages of maturity: Scavenging activity on DPPH radicals (%), reducing power, hemolysis
inhibition (%) and lipid peroxidation (LPO) inhibition (%). Each value is expressed as mean ± standard error (n = 3).
Table 2
EC50 values obtained in the antioxidant activity assays of Lactarius piperatus in different stages of maturity
EC50 values (mg/L) Stage I Stage II Stage III Stage IV
Scavenging effect 13.92 ± 0.73 c 5.19 ± 0.03 d 20.24 ± 0.78 b 23.44 ± 3.33 a
Reducing power 2.83 ± 0.01 c 2.29 ± 0.02 d 5.40 ± 0.05 b 5.50 ± 0.03 a
Hemolysis inhibition 32.92 ± 0.72 c 12.14 ± 0.03 d 38.91 ± 0.18 b >50.0 a
LPO inhibition 15.26 ± 4.15 c 12.08 ± 3.15 c 34.25 ± 5.46 b 45.06 ± 2.21 a
In each row different letters mean significant differences (p < 0.05).
L. Barros et al. / Food and Chemical Toxicology 45 (2007) 1731–1737 1735
Author's personal copy
for phenols and 0.986 for flavonoids; p < 0.001), hemolysis
inhibition (determination coefficient 0.792 for phenols and
0.798 for flavonoids; p < 0.001) and LPO inhibition (deter-
mination coefficient 0.940 for phenols and 0.914 for flavo-
noids; p < 0.001) (Fig. 3). These negative linear correlations
prove that the sample with highest antioxidant contents
shows higher antioxidant activity and lower EC50 values
(stage II, mature with immature spores), while the sample
with lowest antioxidant contents presents lower antioxi-
dant activity and higher EC50 values (stage IV, degraded).
The correlations also support that the mechanism of action
of the extracts for the antioxidant activity may be identical,
being related with the content in phenols and flavonoids,
and their free-radical scavenging activity.
Over two-thirds of cancer-related death could be pre-
vented through lifestyle modification, particularly through
dietary means and, mushrooms consumption, could con-
tribute to minimize cancer risks trough antioxidants input.
Nevertheless, it is important to know the best maturity
stage to collect wild mushrooms concerning antioxidants
production and, to our best knowledge, the present study
was the first report to demonstrate that the antioxidative
components production by wild mushrooms and their anti-
oxidant properties depends on the stage of fruiting body
maturity.
Acknowledgements
The authors are grateful to Foundation for Science and
Technology (POCI/AGR/56661/2004) for financial sup-
port of this work.
References
Barros, L., Ferreira, M.-J., Queiro´s, B., Ferreira, I.C.F.R., Baptista, P.,
2007. Total phenols, ascorbic acid, b-carotene and lycopene in
Portuguese wild edible mushrooms and their antioxidant activities.
Food Chemistry 100, 413–419.
EC50 = -4.042(Phenols mg/mL) + 32.269
R2
= 0.806***
EC50 = -11.290(Flavonoids mg/mL) + 25.240
R2
= 0.868***
0.0
5.0
10.0
15.0
20.0
25.0
30.0
0 2 4 6
Phenols Flavonoids
ScavengingeffectEC50
Concentration (mg/mL)
EC50 = -0.893(Phenols mg/mL) + 7.665
R2
= 0.946***
EC50 = -2.453(Flavonoids mg/mL) + 6.078
R2
= 0.986***
0
1
2
3
4
5
6
7
0 2 4 6
Phenols Flavonoids
ReducingpowerEC50
Concentration (mg/mL)
EC50 = -9.760(Phenols mg/mL) + 76.271
R2
= 0.792***
EC50 = -26.372(Flavonoids mg/mL) +58.549
R2
= 0.798***
0.0
10.0
20.0
30.0
40.0
50.0
60.0
70.0
0 2 4 6
Phenols Flavonoids
HemolysisinhibitionEC50
Concentration (mg/mL)
EC50 = -8.513(Phenols mg/mL) + 61.550
R2
= 0.940***
EC50 = -22.600(Flavonoids mg/mL) + 45.763
R2
= 0.914***
0.0
10.0
20.0
30.0
40.0
50.0
0 2 4 6
Phenols Flavonoids
LPOinhibitionEC50
Concentration (mg/mL)
Fig. 3. Correlation established between total phenol and flavonoid contents, and scavenging effect on DPPH radicals, reducing power, hemolysis
inhibition, and LPO inhibition.
1736 L. Barros et al. / Food and Chemical Toxicology 45 (2007) 1731–1737
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Ferreira 9

  • 1. This article was published in an Elsevier journal. The attached copy is furnished to the author for non-commercial research and education use, including for instruction at the author’s institution, sharing with colleagues and providing to institution administration. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright
  • 2. Author's personal copy Effect of Lactarius piperatus fruiting body maturity stage on antioxidant activity measured by several biochemical assays Lillian Barros, Paula Baptista, Isabel C.F.R. Ferreira * CIMO-Escola Superior Agra´ria, Instituto Polite´cnico de Braganc¸a, Campus de Sta. Apolo´nia, Apartado 1172, 5301-855 Braganc¸a, Portugal Received 21 September 2006; accepted 12 March 2007 Abstract The effects of fruiting body maturity on antioxidant activity and antioxidants production of the wild mushroom, Lactarius piperatus, were evaluated. Several biochemical assays were used to screen the antioxidant properties: reducing power, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity, inhibition of erythrocytes hemolysis mediated by peroxyl radicals and inhibition of lipid peroxida- tion using the b-carotene linoleate model system. The amounts of phenols, flavonoids, ascorbic acid, b-carotene and lycopene present in the immature, mature and degraded fruiting bodies were also determined. The highest antioxidant contents and the lowest EC50 values for antioxidant activity were obtained in the mature stage with immature spores. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Wild mushroom; Fruiting body maturity; Antioxidant activity; Antioxidant components 1. Introduction Oxidation is essential to many living organisms for the production of energy to fuel biological processes, proceed- ing in lipids with polyunsaturated fatty acids, and generat- ing reactive oxygen species (ROS) such as hydroxyl radicals (Halliwell and Gutteridge, 1989). However, oxygen-centred free radicals and other ROS species, that are continuously produced in vivo as by products, result in cell death and tis- sue damage. The oxidative damage caused by free radicals may be related to aging and diseases, such as atherosclero- sis, diabetes, cancer and cirrhosis (Halliwell and Gutter- idge, 1984). Antioxidant compounds reduce the action of reactive oxygen species (ROS) in tissue damage. Although humans and other organisms possess antioxi- dant defences (enzymes, such as superoxide dismutase and catalase, or compounds such as ascorbic acid, tocopherols and glutathione) and repair systems that have evolved to protect them against oxidative damage, these systems are insufficient to totally prevent the damage (Simic, 1988; Niki et al., 1994; Mau et al., 2002). Natural products with antioxidant activity are used to aid the endogenous protective system, increasing interest in the antioxidative role of nutraceutic products (Kanter, 1998). Concerning this, the antioxidants in human diets are of great interest as possible protective agents to help human body reduce oxidative damage. Recently, a multi- tude of natural antioxidants have already been isolated from different kinds of plant materials such as oilseeds, cer- eal crops, vegetables, fruits, leaves, roots, spices, and herbs (Ramarathnam et al., 1995). Mushrooms have also become attractive as a functional food and as source for the devel- opment of drugs and nutraceuticals (Yanga et al., 2002; Barros et al., 2007, in press). Mushrooms accumulate a variety of secondary metabo- lites, including phenolic compounds, polyketides, terpenes and steroids. The quality of a nutraceutical is dependent on the chemical composition of the fruiting body, particu- larly in relation to the phenols and flavonoids content. Phenolic compounds were found to have antioxidant activity in the inhibition of LDL oxidation (Teissedre and Landrault, 2000). Phenolics are one of the major groups 0278-6915/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2007.03.006 * Corresponding author. Tel.: +351 273303219; fax: +351 273325405. E-mail address: iferreira@ipb.pt (I.C.F.R. Ferreira). www.elsevier.com/locate/foodchemtox Food and Chemical Toxicology 45 (2007) 1731–1737
  • 3. Author's personal copy of nonessential dietary components that have been associ- ated with the inhibition of atherosclerosis and cancer (Wil- liams and Iatropoulos, 1997). Flavonoids have been proven to display a wide range of pharmacological and biochemi- cal actions, such as antimicrobial, antithrombotic, antimu- tagenic and anticarcinogenic activities (Cook and Samman, 1996; Kandaswami and Middleton, 1997; Sahu and Green, 1997). Several studies described by us (Barros et al., 2007, in press; Ferreira et al., 2007) and also by other authors (Yen and Hung, 2000; Mau et al., 2002, 2004; Yanga et al., 2002; Cheung et al., 2003; Cheung and Cheung, 2005; Lo and Cheung, 2005; Turkoglu et al., 2007) report a correlation between the mushrooms antioxidant activity and their phenolic content. However, none of the existent reports on mushrooms antioxidants composition and anti- oxidant activity indicated the stage of development of the fruiting bodies selected for the studies. Furthermore, no studies have been developed to evaluate the antioxidant activity at different stages of fruiting body maturity for potential use on the preparation of nutraceutical. In this study, we examined the evolution of antioxidant components and antioxidant activity of a Basidiomycete fungus, L. piperatus, in four stages of fruiting body matu- rity. The antioxidant properties were evaluated though reducing power, DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity, inhibition of oxidative hemoly- sis in erythrocytes induced by 2,20 -azobis(2-amidinopro- pane)dihydrochloride (AAPH) and inhibition of lipid peroxidation by b-carotene-linoleate system. These assays have been extensively studied as models for the peroxida- tive damage in biomembrane. All these antioxidant activity parameters were correlated to the phenolic, flavonoidic, ascorbic acid, b-carotene and lycopene contents. 2. Materials and methods 2.1. Chemicals Standards BHA (2-tert-butyl-4-methoxyphenol), TBHQ (tert-butyl- hydroquinone), L-ascorbic acid, a-tocopherol, gallic acid and (+)-catechin were purchase from Sigma (St. Louis, MO, USA). 2,2-diphenyl-1-pic- rylhydrazyl (DPPH) was obtained from Alfa Aesar (Ward Hill, MA, USA). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA). Methanol was obtained from Pronalab (Lisbon, Por- tugal). Water was treated in a Mili-Q water purification system (TGI Pure Water Systems, USA). 2.2. Fruiting body selection Wild fruiting bodies of Lactarius piperatus (L.) Pers. were obtained under live oak trees (Quercus pyrenaica), in Braganc¸a (northeast of Portugal), in spring 2006. Taxonomic identification was made according to several authors (Marchand, 1971-1986; Moser, 1983; Bon, 1988; Courtecuisse and Duhem, 1995; Courtecuisse, 1999) and a representative voucher specimen was deposited at the herbarium of Escola Superior Agra´ria of Instituto Polite´cnico de Braganc¸a. The fruiting bodies were harvested and dried in different stages of maturity: immature (cap closed) and mature (cap opened). The mature stage was further characterized into immature spores and mature spores as seen on Fig. 1. 2.3. Sample preparation The fruiting bodies were air-dried in a liophylizator (Ly-8-FM-ULE, Snijders) and powdered before analysis. The dried samples ($5 g) were extracted by stirring with 100 mL of methanol at 25 °C at 150 rpm for 24 h and filtered through Whatman No. 4 paper. The residue was then extracted with two additional 100 mL portions of methanol, as described earlier. The combined methanolic extracts were evaporated at 40 °C to Fig. 1. Lactarius piperatus fruiting bodies in different stages of maturity: (SI) immature (cap closed), (SII) mature (cap opened) with immature spores, (SIII) mature with mature spores, and (SIV) degraded. 1732 L. Barros et al. / Food and Chemical Toxicology 45 (2007) 1731–1737
  • 4. Author's personal copy dryness and redissolved in methanol at a concentration of 50 mg/mL, and stored at 4 °C for further use. 2.4. Determination of total antioxidant components Phenolic compounds in the mushroom extracts were estimated by a colorimetric assay, based on procedures described by Singleton and Rossi with some modifications (Singleton and Rossi, 1965). Briefly, 1 mL of sample was mixed with 1 mL of Folin and Ciocalteu’s phenol reagent. After 3 min, 1 mL of saturated sodium carbonate solution was added to the mixture and adjusted to 10 mL with distilled water. The reaction was kept in the dark for 90 min, after which the absorbance was read at 725 nm (Analytikijena 200-2004 spectrophotometer). Gallic acid was used to calculate the standard curve (0.01–0.4 mM; Y = 2.8557X À 0.0021; R2 = 0.9999) and the results were expressed as mg of gallic acid equiva- lents (GAEs) per g of extract. Flavonoid contents in the extracts were determined by a colorimetric method described by Jia et al. (1999) with some modifications. The mushroom extract (250 lL) was mixed with 1.25 mL of distilled water and 75 lL of a 5% NaNO2 solution. After 5 min, 150 lL of a 10% AlCl3 Æ H2O solution was added. After 6 min, 500 lL of 1 M NaOH and 275 lL of distilled water were added to the mixture. The solution was mixed well and the intensity of pink colour was measured at 510 nm. (+)-Catechin was used to calculate the standard curve (0.022–0.34 mM; Y = 0.9629X À 0.0002; R2 = 0.9999) and the results were expressed as mg of (+)-catechin equivalents (CEs) per g of extract. Ascorbic acid was determined according to the method of Klein and Perry (1982). The dried methanolic extract (100 mg) was extracted with 10 ml of 1% metaphosphoric acid for 45 min at room temperature and filtered through Whatman No. 4 filter paper. The filtrate (1 ml) was mixed with 9 ml of 2,6-dichlorophenolindophenol and the absorbance was measured within 30 min at 515 nm against a blank. Content of ascorbic acid was calculated on the basis of the calibration curve of authentic L-ascorbic acid (0.020–0.12 mg/ml; Y = 3.4127X À 0.0072; R2 = 0.9905) and the results were expressed as mg of ascorbic acid/g of extract. b-Carotene and lycopene were determined according to the method of Nagata and Yamashita (1992). The dried methanolic extract (100 mg) was vigorously shaken with 10 ml of acetone–hexane mixture (4:6) for 1 min and filtered through Whatman No. 4 filter paper. The absorbance of the filtrate was measured at 453, 505 and 663 nm. Contents of b-carotene and lycopene were calculated according to the following equations: lycopene (mg/100 ml) = À0.0458 A663 + 0.372 A505 À 0.0806 A453; b-carotene (mg/100 ml) = 0.216 A663 À 0.304 A505 + 0.452 A453. The results were expressed as lg of carotenoid/g of extract. 2.5. DPPH radical-scavenging activity The capacity to scavenge the ‘‘stable’’ free radical DPPH was moni- tored according to the method of Hatano et al. (1988). Various concen- trations of mushroom extracts (0.3 mL) were mixed with 2.7 mL of methanolic solution containing DPPH radicals (6 · 10À5 mol/L). The mixture was shaken vigorously and left to stand for 60 min in the dark (until stable absorption values were obtained). The reduction of the DPPH radical was determined by measuring the absorption at 517 nm. The radical scavenging activity (RSA) was calculated as a percentage of DPPH discolouration using the equation: % RSA = [(ADPPH À AS)/ ADPPH] · 100, where AS is the absorbance of the solution when the sample extract has been added at a particular level, and ADPPH is the absorbance of the DPPH solution. The extract concentration providing 50% inhibition (EC50) was calculated from the graph of RSA percentage against extract concentration. BHA and a-tocopherol were used as standards. 2.6. Reducing power The reducing power was determined according to the method of Oyaizu (1986). Various concentrations of mushroom methanolic extracts (2.5 mL) were mixed with 2.5 mL of 200 mmol/L sodium phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium ferricyanide. The mixture was incubated at 50 °C for 2 min. After 2.5 mL of 10% tricloroacetic acid (w/v) were added, the mixture was centrifuged at 1000 rpm for 8 min (Centorion K24OR – 2003 refrigerated centrifuge). The upper layer (5 mL) was mixed with 5 mL of deionised water and 1 mL of 0.1% of ferric chloride, and the absorbance was measured spectrophotometrically at 700 nm. The extract concentration providing 0.5 of absorbance (EC50) was calculated from the graph of absorbance at 700 nm against extract concentration. BHA and a-tocopherol were used as standards. 2.7. Inhibition of erythrocyte hemolysis mediated by peroxyl free radicals The antioxidant activity of the mushroom extracts was measured as the inhibition of erythrocyte hemolysis (Miki et al., 1987). Blood was obtained from male ram (churra galega transmontana) of body weight $67 kg. Eryhrocytes separated from the plasma and the buffy coat were washed three times with 10 mL of 10 mM phosphate buffer saline (PBS) at pH 7.4 (prepared by mixing 10 mM of NaH2PO4 and Na2HPO4, and 125 mM of NaCl in 1 L of distilled water) and centrifuged at 1500g for 5 min. During the last washing, the erythrocytes were obtained by centrifugation at 1500g for 10 min. A 0.1 mL of a 20% suspension of erythrocytes in PBS was added to 0.2 mL of 200 mM 2,20 -azobis(2-ami- dinopropane)dihydrochloride (AAPH) solution (in PBS) and 0.1 mL of mushroom methanolic extracts of different concentrations. The reaction mixture was shaken gently (30 rpm) while being incubated at 37 °C for 3 h. The reaction mixture was diluted with 8 mL of PBS and centrifuged at 3000g for 10 min; the absorbance of its supernatant was then read at 540 nm by a spectrophotometer, after filtration with a syringe filter (cel- lulose membrane 30 mm, 0.20 lm, Titan). The percentage hemolysis inhibition was calculated by the equation % hemolysis inhibi- tion = [(AAAPH À AS)/AAAPH] · 100, where AS is the absorbance of the sample containing the mushroom extract, and AAAPH is the absorbance of the control sample containing no mushroom extract. The extract con- centration providing 50% inhibition (EC50) was calculated from the graph of hemolysis inhibition percentage against extract concentration. L-Ascorbic acid was used as standard. 2.8. Inhibition of lipid peroxidation using the b-carotene linoleate model system The antioxidant activity of mushroom extracts was evaluated by the b- carotene linoleate model system (Mi-Yae et al., 2003). A solution of b- carotene was prepared by dissolving 2 mg of b-carotene in 10 mL of chloroform. Two millilitres of this solution were pipetted into a 100 mL round-bottom flask. After the chloroform was removed at 40 °C under vacuum, 40 mg of linoleic acid, 400 mg of Tween 80 emulsifier, and 100 mL of distilled water were added to the flask with vigorous shaking. Aliquots (4.8 mL) of this emulsion were transferred into different test tubes containing 0.2 mL of different concentrations of the mushroom extracts. The tubes were shaken and incubated at 50 °C in a water bath. As soon as the emulsion was added to each tube, the zero time absorbance was measured at 470 nm using a spectrophotometer. Absorbance readings were then recorded at 20-min intervals until the control sample had changed colour. A blank, devoid of b-carotene, was prepared for back- ground subtraction. Lipid peroxidation (LPO) inhibition was calculated using the following equation: LPO inhibition = (b-carotene content after 2 h of assay/initial b-carotene content) · 100. The extract concentration providing 50% antioxidant activity (EC50) was calculated from the graph of antioxidant activity percentage against extract concentration. TBHQ was used as standard. 2.9. Statistical analysis All the determinations were carried out in triplicate and the results are expressed as mean values and standard deviations (SD). Differences among means were done by analysis of variance (ANOVA), using SAS v. L. Barros et al. / Food and Chemical Toxicology 45 (2007) 1731–1737 1733
  • 5. Author's personal copy 9.1.3, and averages were compared using Tukey test (p < 0.05). A regression analysis, using Excel for Windows Software, was established between phenolic and flavonoid contents of different mushroom samples and EC50 values obtained by different antioxidant assays. 3. Results and discussion Table 1 shows phenol, flavonoid, ascorbic acid and carotenoids concentrations obtained in L. piperatus in dif- ferent stages of fruiting body maturity. Whereas total phe- nols were the major antioxidant components found in the extracts, ascorbic acid was found in small amounts (0.03– 0.16 mg/g), and b-carotene and lycopene were only found in vestigial amounts (<34 lg/g), which is in agreement with other reports concerning ascorbic acid and b-carotene quantification in different mushrooms (Mau et al., 2002; Barros et al., 2007, in press). Curiously, the stages where the fruiting bodies presented immature spores (stage I and II) revealed a higher content in phenol and flavonoid compounds. The amount found in stages III (with mature spores) and IV (degraded) significantly (p < 0.05) decreased when compared with the content found in the first stages. Probably, the aging process elicits the formation of reactive oxygen species, which are neutralised by the polyphenolic compounds, resulting in the lowering of their contents and antioxidant capacities in the most advanced stages. The highest content of antioxidant compounds in the first stages might account for the better results found in their antioxidant activity (Velioglu et al., 1998; Ferreira et al., 2007). Furthermore, Cheung et al. (2003) found a direct correlation between mushrooms antioxidant activity and total phenolic content, although the antioxidant action is raised by other substances such as tocopherols and b-caro- tene. The bioactivity of phenolics may be related to their ability to chelate metals, inhibit lipoxygenase and scavenge free radicals (Decker, 1997). Also, in food systems, flavo- noids can act as free radical scavengers and terminate the radical chain reactions that occur during the oxidation of triglycerides. Therefore, they present antioxidative effi- ciency in oils, fats and emulsions (Madhavi et al., 1996; Roedig-Penman and Gordon, 1998). Fig. 2 shows the antioxidant activity of L. piperatus in different stages of fruiting body maturity, measured by different biochemical assays: reducing power, scavenging activity on DPPH radicals, inhibition of the erythrocyte hemolysis mediated by peroxyl free radicals and LPO inhi- bition by b-carotene-linoleate system. The radical scavenging activity (RSA) values were expressed as the ratio percentage of sample absorbance decrease and the absorbance of DPPH solution in the absence of extract at 517 nm. From the analysis of Fig. 2, we can conclude that the scavenging effects of fruiting body extracts on DPPH radicals increased with the concentra- tion increase and were very good for stage II (84.9% at 20 mg/mL), but lower than the scavenging effects of the standards BHA (96% at 3.6 mg/ml) and a-tocopherol (95% at 8.6 mg/ml). The RSA values at 20 mg/mL were moderate for the other stages (stage I – 64.2%, stage III – 49.4%, stage IV – 43.4%) and, particularly stage III and IV revealed a very similar scavenging activity. The reducing power also increased with concentration, and the values obtained for all the extracts were excellent (Fig. 2); at 20 mg/mL were higher than 1.3 and in the order of stage II > stage I > stage III > stage IV. Reducing power of BHA at 3.6 mg/mL and a-tocopherol at 8.6 mg/mL was only 0.12 and 0.13, respectively. The extracts obtained in stages III and IV showed similar reducing power values. It was reported that the reducing properties are generally associated with the presence of reductones, which have been shown to exert antioxidant action by breaking the free radical chain by donating a hydrogen atom (Shimada et al., 1992). Accordingly, in stage II, it was produced higher amounts of reductone, which could react with free radicals to stabilize and block radical chain reactions. In this study, the protective effect of the fruiting body extracts on hemolysis by peroxyl radical scavenging activity was also investigated. AAPH is a peroxyl radical initiator that generates free radicals by its thermal decom- position and will attack the erythrocytes to induce the chain oxidation of lipid and protein, disturbing the mem- brane organization and eventually leading to hemolysis. The extracts inhibited hemolysis, as a result of protection against the oxidative damage of cell membranes of erythro- cytes from ram induced by AAPH, in a concentration- dependent manner (Fig. 2). Once more, in stage II there was a higher protective effect against erythrocytes hemoly- sis (82.4% at 50 mg/mL) than in other stages (stage I – 57.5%, stage III – 54.2%, stage IV – 41.0%). However, the inhibition percentage of the standard L-ascorbic acid on hemolysis of red blood cell was much higher (94.6% at 1 mg/mL) than those of mushroom extracts. The lipid peroxidation inhibition (LPO), measured by the bleaching of b-carotene, is presented in Fig. 2. The Table 1 Phenols, flavonoids, ascorbic acid, b-carotene and lycopene contents (mean ± SD) of Lactarius piperatus in different stages of maturity Stage I Stage II Stage III Stage IV Phenols (mg/g) 5.52 ± 0.14 b 5.76 ± 0.09 a 3.09 ± 0.12 c 2.03 ± 0.11 d Flavonoids (mg/g) 1.26 ± 0.09 b 1.58 ± 0.02 a 0.35 ± 0.03 c 0.19 ± 0.01 d Ascorbic acid (mg/g) 0.15 ± 0.01 a 0.16 ± 0.01 a 0.13 ± 0.01 b 0.03 ± 0.01 c b-carotene (lg/g) 26.08 ± 0.05 b 33.78 ± 0.05 a 17.22 ± 0.00 c 15.11 ± 0.02 d Lycopene (lg/g) 8.14 ± 0.03 b 13.04 ± 0.02 a 5.80 ± 0.01 c 5.41 ± 0.01 d In each row different letters mean significant differences (p < 0.05). 1734 L. Barros et al. / Food and Chemical Toxicology 45 (2007) 1731–1737
  • 6. Author's personal copy linoleic acid free radical attacks the highly unsaturated b- carotene models. The presence of different antioxidants can hinder the extent of b-carotene-bleaching by neutraliz- ing the linoleate-free radical and other free radicals formed in the system (Jayaprakasha et al., 2001). Accordingly, the absorbance decreased rapidly in samples without antioxi- dant, whereas in the presence of an antioxidant, they retained their colour, and thus absorbance, for a longer time. LPO inhibition in the presence of L. piperatus in dif- ferent stages of fruiting body maturity increased with their increasing concentration, and the values at 50 mg/mL for each one of the stages were 58.3%, 61.2%, 52.9% and 49.72%. It is probable that the antioxidative components in the mushroom extracts can reduce the extent of b-caro- tene destruction by neutralizing the linoleate free radical and other free radicals formed in the system. Again, stage II was the most effective for antioxidant activity. The anti- oxidant activity of TBHQ standard reached 82.2% at 2 mg/ mL and was more efficient than the samples. Nevertheless, this and other synthetic antioxidants applied in fat and oily foods to prevent oxidative deterioration were found to be anticarcinogenic as well as carcinogenic in experimental animals (Loliger, 1991). In Table 2 we present EC50 values obtained in the anti- oxidant activity assays of L. piperatus in different stages of fruiting body maturity. Overall, the mushroom in stage II revealed better antioxidant properties (significantly lower EC50 values; p < 0.05) than in other stages, which is in agreement with the higher content of antioxidants found in the first case. The EC50 values obtained for reducing power and scavenging effects on DPPH radicals were better than for LPO inhibition and for hemolysis inhibition med- iated by peroxyl free radicals. Significantly negative linear correlations were estab- lished between the phenols and flavonoids content, and EC50 values of DPPH scavenging activity (determination coefficient 0.806 for phenols and 0.868 for flavonoids; p < 0.001), reducing power (determination coefficient 0.946 Stage I Stage II Stage III Stage IV 0 20 40 60 80 100 0 1 2 3 4 5 10 20 Concentration mg/mL Scavengingactivity(%) 0 0.5 1 1.5 2 2.5 0 1 2 3 4 5 10 20 Concentration mg/mL Concentration mg/mL Absat700nm 0 10 20 30 40 50 60 70 0 1 2 3 4 5 10 20 30 40 50 Concentration mg/mL LPOinhibition(%) 0 20 40 60 80 100 0 1 5 10 20 40 50 Hemolysisinhibition(%) Fig. 2. Antioxidant activity of Lactarius piperatus in different stages of maturity: Scavenging activity on DPPH radicals (%), reducing power, hemolysis inhibition (%) and lipid peroxidation (LPO) inhibition (%). Each value is expressed as mean ± standard error (n = 3). Table 2 EC50 values obtained in the antioxidant activity assays of Lactarius piperatus in different stages of maturity EC50 values (mg/L) Stage I Stage II Stage III Stage IV Scavenging effect 13.92 ± 0.73 c 5.19 ± 0.03 d 20.24 ± 0.78 b 23.44 ± 3.33 a Reducing power 2.83 ± 0.01 c 2.29 ± 0.02 d 5.40 ± 0.05 b 5.50 ± 0.03 a Hemolysis inhibition 32.92 ± 0.72 c 12.14 ± 0.03 d 38.91 ± 0.18 b >50.0 a LPO inhibition 15.26 ± 4.15 c 12.08 ± 3.15 c 34.25 ± 5.46 b 45.06 ± 2.21 a In each row different letters mean significant differences (p < 0.05). L. Barros et al. / Food and Chemical Toxicology 45 (2007) 1731–1737 1735
  • 7. Author's personal copy for phenols and 0.986 for flavonoids; p < 0.001), hemolysis inhibition (determination coefficient 0.792 for phenols and 0.798 for flavonoids; p < 0.001) and LPO inhibition (deter- mination coefficient 0.940 for phenols and 0.914 for flavo- noids; p < 0.001) (Fig. 3). These negative linear correlations prove that the sample with highest antioxidant contents shows higher antioxidant activity and lower EC50 values (stage II, mature with immature spores), while the sample with lowest antioxidant contents presents lower antioxi- dant activity and higher EC50 values (stage IV, degraded). The correlations also support that the mechanism of action of the extracts for the antioxidant activity may be identical, being related with the content in phenols and flavonoids, and their free-radical scavenging activity. Over two-thirds of cancer-related death could be pre- vented through lifestyle modification, particularly through dietary means and, mushrooms consumption, could con- tribute to minimize cancer risks trough antioxidants input. Nevertheless, it is important to know the best maturity stage to collect wild mushrooms concerning antioxidants production and, to our best knowledge, the present study was the first report to demonstrate that the antioxidative components production by wild mushrooms and their anti- oxidant properties depends on the stage of fruiting body maturity. Acknowledgements The authors are grateful to Foundation for Science and Technology (POCI/AGR/56661/2004) for financial sup- port of this work. References Barros, L., Ferreira, M.-J., Queiro´s, B., Ferreira, I.C.F.R., Baptista, P., 2007. Total phenols, ascorbic acid, b-carotene and lycopene in Portuguese wild edible mushrooms and their antioxidant activities. Food Chemistry 100, 413–419. EC50 = -4.042(Phenols mg/mL) + 32.269 R2 = 0.806*** EC50 = -11.290(Flavonoids mg/mL) + 25.240 R2 = 0.868*** 0.0 5.0 10.0 15.0 20.0 25.0 30.0 0 2 4 6 Phenols Flavonoids ScavengingeffectEC50 Concentration (mg/mL) EC50 = -0.893(Phenols mg/mL) + 7.665 R2 = 0.946*** EC50 = -2.453(Flavonoids mg/mL) + 6.078 R2 = 0.986*** 0 1 2 3 4 5 6 7 0 2 4 6 Phenols Flavonoids ReducingpowerEC50 Concentration (mg/mL) EC50 = -9.760(Phenols mg/mL) + 76.271 R2 = 0.792*** EC50 = -26.372(Flavonoids mg/mL) +58.549 R2 = 0.798*** 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 0 2 4 6 Phenols Flavonoids HemolysisinhibitionEC50 Concentration (mg/mL) EC50 = -8.513(Phenols mg/mL) + 61.550 R2 = 0.940*** EC50 = -22.600(Flavonoids mg/mL) + 45.763 R2 = 0.914*** 0.0 10.0 20.0 30.0 40.0 50.0 0 2 4 6 Phenols Flavonoids LPOinhibitionEC50 Concentration (mg/mL) Fig. 3. Correlation established between total phenol and flavonoid contents, and scavenging effect on DPPH radicals, reducing power, hemolysis inhibition, and LPO inhibition. 1736 L. Barros et al. / Food and Chemical Toxicology 45 (2007) 1731–1737
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