Food Research International 77 (2015) 236–243

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Bioactive compounds and scavenging capacity of pulp, peel and seed

extracts of the Amazonian fruit Quararibea cordata against ROS and RNS
Alessandra Berto a, Alessandra Braga Ribeiro b, Nilson Evelázio de Souza a,
Eduarda Fernandes b, Renan Campos Chisté b,⁎
a
Postgraduate Program of Chemistry, State University of Maringá (UEM), 87020-900 Maringá, Paraná, Brazil
b
UCIBIO-REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, (FFUP), 4050-313 Porto, Portugal

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

Article history: Quararibea cordata, an unexploited Amazonian fruit was investigated regarding the identification of phenolic
Received 3 March 2015 compounds and the scavenging capacity of its pulp, peel and seed extracts against reactive oxygen (ROS) and ni-
Received in revised form 9 June 2015 trogen species (RNS). The major phenolic compound in the pulp extract was epicatechin (320 μg/g extract), while
Accepted 17 June 2015
A-type proanthocyanidin dimer was the major one in the peel and seed extracts. Regarding the carotenoid com-
Available online 19 June 2015
position, all-trans-zeaxanthin (2.5–42 μg/g extract), all-trans-lutein and all-trans-β-carotene were the major
Keywords:
compounds in all extracts. The seed extract, which presented the highest content of phenolic compounds and ca-
Phenolic compounds rotenoids, was the most effective scavenger against all RNS (IC50 from 19.5 to 37.6 μg/mL), while the peel extract
Carotenoids presented the highest efficiency against ROS, especially HOCl. Thus, Q. cordata fruit may present an excellent po-
Reactive oxygen species tential to be used as a bioactive compound source with high antioxidant capacity.
Reactive nitrogen species © 2015 Elsevier Ltd. All rights reserved.
Antioxidant capacity

1. Introduction inundated areas in the Amazon region of Peru, Colombia, Venezuela,

The Amazon is the largest reserve of biodiversity in the world and is
also the largest Brazilian biome, occupying almost half of Brazil and in-
cludes a great number of edible fruits with great nutritional potential
(Andrade and Andrade, 2014; De Rosso, 2013; Oliveira, Yamada, Fagg,
and Brandão, 2012). In the last decades, the search for functional
foods has increased the interest on the properties of Amazonian fruits,
whereas most of them remain unknown both in Brazil and elsewhere
in the world. Thus, the study of such fruit species, their bioactive com-
pounds characterization and the evaluation of its functional properties
are challenges to overcome in order to ensure their effective apprecia-
tion in agribusiness, generating high quality raw material for the food,
pharmaceutical and cosmetic industries.
Our study approaches the antioxidant potential of Quararibea
cordata (Bonpl.) Vischer (synonym: Matisia cordata; Brazilian name:
“sapota-do-Solimões”) and also the bioactive compounds characteriza-
tion of its fruit extracts. Q. cordata, which belongs to the Malvaceae fam-
ily, is a large-sized tree (up to 40 m high) originating from Occidental
Amazon, found in its wild form in the forests of mainlands and non-
⁎ Corresponding author at: UCIBIO-REQUIMTE, Department of Chemical Sciences,
Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313
Porto, Portugal.
E-mail address: rcchiste@ff.up.pt (R.C. Chisté).
Ecuador and Brazil. In the Brazilian Amazon, Q. cordata is cultivated in
the region of “Alto Solimões” in open environments and agroecological
systems, reaching up to 20 m high (Carvalho, Damiani, and Asquieri,
2014; Murillo et al., 2013). The fruit pulp is consumed in nature; howev-
er, some studies have reported the use of pulp in the preparation of
juices, refreshments, salads, candies, jams, ice creams and as flavouring
of beverages. Furthermore, the inner part of the peel can also be used in
the preparation of sweet syrup (Carvalho, Damiani, Asquieri, Orsi, and
Nishi, 2012).
Scientific evidences suggest that bioactive compounds found in
plants, such as phenolic compounds and carotenoids, have the property
of scavenging reactive oxygen (ROS) and nitrogen species (RNS) to in-
hibit or decrease the rate of oxidative reactions at different levels
(Chisté, Freitas, Mercadante, and Fernandes, 2012; González et al.,
2013; Ribeiro et al., 2014). Furthermore, the intake of these bioactive
compounds from food has been associated with the improvement of
the immune system and decrease of the risk of development of chronic
degenerative diseases, such as cardiovascular diseases, cataract, macular
degeneration and certain types of cancer (Lavecchia, Rea, Antonacci,
and Giardi, 2013). Additionally, they may interact synergistically
among them to scavenge ROS and RNS in a more efficient way than
they do separately (Rossetto et al., 2002; Trombino et al., 2004).
Concerning the bioactive compounds of Q. cordata, few studies have
reported the contents of total phenolic compounds (21–265 mg/100 g,

http://dx.doi.org/10.1016/j.foodres.2015.06.018
0963-9969/© 2015 Elsevier Ltd. All rights reserved.
 A. Berto et al. / Food Research International 77 (2015) 236–243
expressed as equivalent of gallic acid) (Carvalho et al., 2012; Cerón, Ng,
El-Halwagi, and Cardona, 2014) and, in both studies, the antioxidant po-
tential was determined by the scavenging capacity against DPPH (1,1-
diphenyl-2-picrylhydrazyl), a stable and non-physiological radical. Ad-
ditionally, several carotenoids were detected in Q. cordata fruits from
Panama, including β-carotene and different esters of zeaxanthin
(Murillo et al., 2013). Nevertheless, no data related to the phenolic com-
pounds composition or the scavenging capacity of Q. cordata fruits
against ROS and RNS were reported yet in the literature.
Therefore, the goal of this study is to report, for the first time, the an-
tioxidant potential of Q. cordata extracts obtained from different parts of
the fruit (peel, pulp and seed) against physiologically relevant ROS and
RNS, namely superoxide radical (O•− 2 ), hydrogen peroxide (H2O2),
hypochlorous acid (HOCl), nitric oxide (•NO) and peroxynitrite
(ONOO−). In addition, the phenolic compounds and carotenoids of the
Q. cordata extracts were tentatively identified, quantified and associated
with the scavenging capacities found in each extract.
2. Materials and methods
2.1. Chemicals
Nitroblue tetrazolium chloride (NBT), β-nicotinamide adenine dinu-
cleotide (NADH), phenazine methosulphate (PMS), lucigenin, 30% hy-
drogen peroxide, sodium hypochlorite solution (with 4% available
chlorine), dihydrorhodamine 123 (DHR), 4,5-diaminofluorescein
(DAF-2), 3-(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene
(NOC-5), quercetin, epicatechin, cafeic acid, kaempferol, ferulic acid,
all-trans-lutein, all-trans-zeaxanthin, all-trans-β-carotene, dimethyl
sulfoxide (DMSO), ethanol, methanol, methyl tert-butyl ether (MTBE),
acetonitrile and all other chemical salts and solvents of analytical
grade were obtained from Sigma-Aldrich (St. Louis, USA). Ultrapure
water was obtained from the arium® pro system (Sartorius,
Germany). All phenolic compounds and carotenoids standards showed
at least 95% of purity, as determined by HPLC–DAD.
2.2. Q. cordata samples and extract preparation
Twelve fresh and ripe fruits of Q. cordata, varying from 500 to 950 g
each, were acquired in three street markets in the city of Manaus, Ama-
zonas, Brazil (03°06′07″S and 60°01′30″W). The fruits were washed
with distilled water, manually separated in three parts (peel, pulp and
seeds), ground in a food processor and immediately directed to extract
preparation. Approximately 100 g of each part of the fruit were subject-
ed to extraction with absolute ethanol (1:10, w/v), at room temperature
(25 °C) and protected from light exposure. After 4 h of extraction in a
magnetic stirrer, the extracts were filtered and the solvent was evapo-
rated using low pressure at T b 40 °C in a rotary evaporator. All concen-
trated extracts were lyophilized, transferred to amber bottles and stored
at −20 °C until analysis. For this extraction procedure, the yield of peel,
pulp and seed extracts were 7%, 14.5% and 4%, respectively.
2.3. Analyses of phenolics compounds and carotenoids
The identification and quantification of phenolic compounds in all
extracts was performed in an Accela LC system (Thermo Fisher Scientif-
ic, San Jose, CA) equipped with quaternary pumps (Accela 600), a DAD
detector and automatic sampler cooled to 5 °C, connected in series to
a LTQ Orbitrap XL™ Hybrid Ion Trap–Orbitrap Mass Spectrometer
(Thermo Fisher Scientific, San Jose, CA) with an electrospray ionization
(ESI) source. The tentative identification and quantification of caroten-
oids was carried out in a LaChrom HPLC system (D-700, Merck Hitachi
Ltd., Tokyo, Japan) equipped with quaternary pumps (L-7100) and
DAD detector (L-7455). Samples and solvents used in chromatographic
analysis were previously filtered through 0.22 μm (OlimPeak,
Teknokroma®, Spain) and 0.45 μm (Billerica, MA, USA) membranes,
237
respectively. The content of phenolics and carotenoids determined by
HPLC–DAD were expressed in μg/g extract (dry basis), considering
three independent analyses (n = 3).
2.3.1. Phenolic compounds
The phenolic compounds were analysed by HPLC–DAD-ESI-MSn
after solubilizing 40–100 mg of each freeze-dried extract in 1 mL of
methanol/water (8:2, v/v), filtered and injected into the chromato-
graphic system. The identification and quantification of phenolic com-
pounds were performed as described by Chisté and Mercadante
(2012). Briefly, the compounds were separated in a C18 Synergi Hydro
column (4 μm, 250 × 4.6 mm, Phenomenex), at 0.9 mL/min, column
temperature of 29 °C and mobile phase consisting of formic acid/
water (99.5:0.5, v/v) and acetonitrile/formic acid (99.5:0.5, v/v) in a lin-
ear gradient. The column eluate was split to allow only 0.3 mL/min to
enter the ESI interface. Mass spectra were obtained after ionization in
the ESI source in the negative ion mode, with a scan range from m/z
100 to 1000, and the MS parameters were set at the same conditions
as described in our previous work (Ribeiro, Chisté, et al., 2014). The phe-
nolic compounds were tentatively identified based on the following
data: elution order, retention time of peak and UV–Visible and mass
spectra characteristics in comparison with authentic standards analysed
under the same conditions (data not shown) and data available in the
literature (Chisté and Mercadante, 2012; Mariutti, Rodrigues, Chisté,
Fernandes, and Mercadante, 2014; Rodrigues, Mariutti, and
Mercadante, 2013; Souza, Cipriani, Iacomini, Gorin, and Sassaki, 2008).
Phenolic compounds were quantified by comparison to external stan-
dards using analytical curves with six-point (in duplicate) for caffeic
acid, epicatechin, ferulic acid and kaempferol (1–100 μg/mL, r2 N 0.99).
2.3.2. Carotenoids
For the carotenoid analysis, approximately 50 mg of freeze-dried ex-
tract of each part of Q. cordata fruits were solubilized with 2 mL of ace-
tone/methanol (1:1, v/v). The solubilized extracts were individually
transferred to separation funnels and subjected to liquid–liquid parti-
tion using petroleum ether/diethyl ether (1:1, v/v) and washed with dis-
tilled water. After partition, the extracts were saponified overnight with
5% KOH in methanol (1:1, v/v), partitioned once more and the solvent
was evaporated at N2 flow rate. The dried extracts were resuspended
in methanol/MTBE (70:30 v/v) and injected into the chromatographic
system. For the chromatographic separation of carotenoids, a C30 YMC
column (5 μm, 250 mm × 4.6 mm) was used, mobile phase consisted
of methanol and MTBE in a linear gradient at a flow rate of 0.9 mL/min
and column temperature at 29 °C (Chisté and Mercadante, 2012). The
UV–Vis spectra were recorded between 200 and 600 nm and the chro-
matograms were processed at 450 nm. The carotenoids were tentatively
identified using the following combined information: elution order, re-
tention time, co-chromatography with authentic standards and UV–Vis-
ible spectrum [λmax, spectral fine structure (%III/II), peak cis intensity
(%AB/AII)] as compared with data available in the literature (Chisté and
Mercadante, 2012; De Rosso and Mercadante, 2007; Murillo et al.,
2013). The carotenoids were quantified by HPLC–DAD by comparison
to external standards using five-point analytical curves (0.5–30 μg/mL,
in duplicate) for all-trans-lutein, all-trans-zeaxanthin and all-trans-β-
carotene. All other carotenoids (including epoxy and cis isomers) were
estimated using the curve of its all-trans-carotenoid.
2.4. ROS and RNS scavenging assays
All extracts of Q. cordata fruits were dissolved in DMSO for the
H2O2, O•− •
2 , NO and ONOO
−
scavenging assays. The ethanol was used
to solubilize quercetin standard and extracts for the HOCl-scavenging
assay. For each test, four independent experiments were performed,
in duplicate, using 5 different concentrations. All ROS and RNS scaveng-
ing assays were performed in a microplate reader (Synergy HT, BioTek,
Vermont, USA) with fluorescence, UV/Vis and chemiluminescence
238 A. Berto et al. / Food Research International 77 (2015) 236–243
measurements and equipped with thermostat. The GraphPad Prism 6
software was used to calculate all IC50 values (in vitro inhibitory concen-
tration of extract to reduce, by 50%, the oxidizing effect of ROS/RNS in
the tested media), obtained from curves of percentage inhibition versus
antioxidant concentration. Quercetin was used as positive control in all
assays and its IC50 values were similar to those already reported by our
research group (Chisté et al., 2012; Ribeiro, Chisté, et al., 2014). In each
assay, additional experiments were performed in order to check possi-
ble interference effects of the extracts and standard with the used meth-
odology (data not shown).
2.4.1. O•−
2 -scavenging assay
The non-enzymatic system NADH/PMS/O2 was used to generate O•− 2 trations and ONOO− (600 nM). The fluorescence signal (emission at
, which promotes the reduction of NBT into a purple coloured
diformazan compound. The reaction mixtures in the sample wells
contained the following reactants at the indicated final concentrations
(in a final volume of 300 μL): NADH (166 μM), NBT (43 μM), aliquots
of each extract (31–1000 μg/mL) and the positive control at five concen-
trations, and PMS (2.7 μM). NADH, NBT, and PMS were dissolved in
19 mM phosphate buffer, pH 7.4. Thus, the capacity of each extract
and the positive control to inhibit the reduction of NBT was monitored
spectrophotometrically at 560 nm for 2 min at 37 °C (Chisté et al.,
2011). The scavenging capacities were expressed as the percentage of
inhibition of the NBT reduction to diformazan.
2.4.2. H2O2-scavenging assay
The assay was carried out in reaction mixtures containing the fol-
lowing reagents at the indicated final concentrations (final volume of
250 μL): 50 mM Tris–HCl buffer, pH 7.4, lucigenin (0.8 mM), dissolved
in the buffer solution, aliquots of each extract (31–1000 μg/mL) and
the positive control at five concentrations and H2O2 (1%). The capacity
of each extract and the positive control to inhibit the H2O2-induced ox-
idation of lucigenin was monitored by chemiluminescence at 37 °C,
after immediate detection of the signal obtained by the microplate read-
er (Chisté et al., 2011). The scavenging capacities were expressed as the
percent of inhibition of the H2O2-induced oxidation of lucigenin.
2.4.3. HOCl-scavenging assay
The capacity of each extract (0.9–62.5 μg/mL) and the positive con-
trol to scavenge HOCl was monitored by fluorescence based on the
HOCl-induced oxidation of DHR (non-fluorescent) to Rhodamine 123
(fluorescent). The HOCl was prepared immediately before the assay,
using a 1% NaOCl solution (w/v), dropwise addition of 10% H2SO4 to
pH 6.2 and the concentration of HOCl was determined by spectropho-
tometry at 235 nm and molar absorptivity of 100 M−1 cm−1 (Gomes
et al., 2007). The assay was carried out in reaction mixtures containing
the following reactants at the indicated final concentrations (final vol-
ume of 300 μL): 100 mM phosphate buffer solution at pH 7.4, aliquots
of each extract and the positive control at five concentrations, DHR
(5 μM) and HOCl (5 μM). The fluorescence signal of Rhodamine 123
was measured immediately after the plate introduction (emission
wavelength at 528 ± 20 nm and excitation at 485 ± 20 nm) (Chisté
et al., 2011). The results were expressed as the percentage of inhibition
of HOCl-induced oxidation of DHR.
2.4.4. •NO-scavenging assay
The decomposition of the NOC-5 generates •NO that induces
the oxidation of the non-fluorescent DAF-2 to the fluorescent
triazolofluorescein (DAF-2T). The scavenging capacity of each extract
(3.9–1000 μg/mL) and the positive control was determined by monitor-
ing the oxidation of DAF-2 to DAF-2T in reaction mixtures in the sample
wells containing the following reagents at the indicated final concentra-
tions (final volume of 300 μL): DAF-2 (5 μM), aliquots of each extract
and the positive control at five concentrations and NOC-5 (10 μM).
The fluorescence signal (emission at 528 ± 20 nm and excitation at
485 ± 20 nm) was detected in the microplate reader after a 30 min
period at 37 °C and the results were expressed as the percentage of in-
hibition of •NO-induced oxidation of DAF-2.
2.4.5. ONOO−-scavenging assay
The ONOO−-scavenging capacities of each extract (3.9–500 μg/mL)
and the positive control were carried out by monitoring the ONOO−-
induced oxidation of DHR to Rhodamine 123. The ONOO− was previ-
ously synthesized according to the methodology described by
Fernandes, Gomes, Costa, and Lima (2005). The assays was performed
at 37 °C with reaction mixtures containing the following reactants at
the indicated final concentrations (final volume of 300 μL): DHR
(5 μM), aliquots of each extract and the positive control at five concen-
528 ± 20 nm and excitation at 485 ± 20 nm) was detected in the micro-
plate reader after 2 min of incubation period (Chisté et al., 2011). Paral-
lel experiments simulating physiological concentrations of CO2 were
performed using 25 mM NaHCO3. The results are expressed as the per-
centage of inhibition of ONOO−-induced oxidation of DHR.
2.5. Statistical analysis
The IC50 values (mean ± standard error of the mean, SEM) were
submitted to analyses of variance using one-way ANOVA and the
means were classified by Tukey's test at the level of 95% of significance,
using the Statistica 7.0 software (Statsoft Inc.).
3. Results and discussion
3.1. Phenolic compounds
According to Table 1, the HPLC–DAD-ESI-MSn allowed the separa-
tion (Fig. 1), tentative identification and quantification of 13 phenolic
compounds. To the best of our knowledge, the profile of phenolic com-
pounds from Q. cordata fruits was not reported until this moment. Peak
1 could not be identified, whereas presented a deprotonated molecule
([M–H]−) at m/z 115 and was detected only in pulp extract. Peaks 2–6
were also detected only in the pulp extract and they were assigned as
caffeic acid derivative compounds due to the presence of characteristic
fragments at m/z 179 (caffeic acid) or at m/z 161 (caffeoyl moiety) in
both the MS2 or MS3 spectra. Two spermidines (polyamines com-
pounds) were tentatively identified in Q. cordata extracts: one derived
from caffeic acid at m/z 294 [M–H]− (peak 7) with a high intense frag-
ment at m/z 179 (caffeic acid) (MS2), followed by the characteristics
fragments (MS3) of an authentic standard of caffeic acid; and the second
one was assigned as a derivative compound from ferulic acid, with [M–
H]− at m/z 308 (peak 10) and a high intense fragment at m/z 193
(ferulic acid) (MS2) with the same fragmentation pattern (MS3) exhib-
ited by the authentic standard of ferulic acid. These kinds of spermidine
hydroxycinnamic acid conjugate compounds were already identified in
Solanum sessiliflorum (“mana-cubiu”), another Amazonian fruit
(Rodrigues et al., 2013). -(-)-Epicatechin (peak 9), which was found
only in the pulp extract, was positively identified based on comparison
with UV–Vis and MS spectrum characteristics of authentic standards
and by co-chromatography. The peaks 8, 11 and 13 were tentatively
identified as proanthocyanidin dimers (condensed tannins) of A and
B-types based on the [M–H]− at m/z 577 (B-type, peak 8, found only
in the seed extracts) and m/z 575 (A-type, peaks 11 and 13), with
known fragments (Mariutti et al., 2014; Souza et al., 2008) in the MS2
spectra that correspond to the neutral loss of 152 u (m/z 425 for the
B-type and m/z 423 for the A-type) due to retro-Diels– Alder fission. Ad-
ditionally, peaks 8, 11 and 13 also showed fragments at m/z 289 (MS2),
which confirms the presence of catechin or epicatechin molecules. Peak
12, which was found only in the peel extracts, was assigned as
kaempferol rutinoside (m/z 593) due to the presence of an intense frag-
ment at m/z 285 [M– H-rutinose]− after losing 308 u, and the MS3
 A. Berto et al. / Food Research International 77 (2015) 236–243 239

Table 1
Chromatographic, spectroscopic characteristics (HPLC–DAD-ESI-MSn) and contents of phenolic compounds of pulp, peel and seed extracts obtained from Quararibea cordata fruits.

Peaks tR range λmax [M–H]−(m/z) Fragments (m/z)c Compounds Extract concentration

(min)a (nm)b (μg/g extract, dry basis)

Pulp Peel Seed

2 1
1 7.6–7.8 260(sh), 305 115.0043 MS [115]: 97, 71, 69 Not identified 94 ± 4 nd nd
MS3 [115 → 71]: nd
2 8.8–8.9 274, 310(sh) 225.0628 MS2 [225]: 179, 161, 119, 89 Caffeic acid derivative 1
83 ± 4 nd nd
MS3 [225 → 179]: 161, 143, 119, 89
3 9.8–9.9 284 387.1168 MS2 [387]: 369, 341, 179, 161 Di-caffeoyl acid derivative 1
7±1 nd nd
MS3 [387 → 341]: 179, 161, 143, 113
4 10.3–10.5 292 387.1165 MS2 [387]: 369, 341, 179, 161 Di-caffeoyl acid derivative1 4±2 nd nd
MS3 [387 → 341]: 179, 161, 143, 113
5 14.3–14.5 272 317.0870 MS2 [317]: 299, 203, 179, 155, 113 Caffeic acid derivative1 46 ± 5 nd nd
MS3 [317 → 113]: 95, 57
6 15.5–15.7 264, 309 387.1165 MS2 [387]: 341, 179, 161 Di-caffeoyl acid derivative 1
19 ± 2 nd nd
MS3 [387 → 341]: 179, 161, 143, 113
7 17.0–17.1 275, 320 294.0631 MS2 [294]: 276, 250, 179, 131 Caffeoyl polyamine derivative1 86 ± 6 112 ± 5 669 ± 9
MS3 [294 → 179]: 135
8 19.9–20.1 284 577.1347 MS2 [577]: 577, 559, 425, 407, 289 Proanthocyanidin dimer (B-type)2 nd nd 4000 ± 18
MS3 [577 → 577]: 407, 289.
9 21.0–21.1 278, 324 289.0727 MS2 [289]: 271, 245, 231, 205, 179 (−)-Epicatechin 2
320 ± 9 nd nd
MS3 [289 → 245]: 227, 203, 187, 161
10 21.6–22.0 279, 320 308.0764 MS2 [308]: 290, 264, 246, 193 Feruloyl polyamine derivative 3
nd 35 ± 2 118 ± 9
MS3 [308 → 193]: 178, 149, 134, 117
11 27.5–27.7 280 575.1190 MS2 [575]: 575, 529, 289 Proanthocyanidin dimer (A-type)2 186 ± 5 623 ± 9 18714 ± 42
MS3 [575 → 529]: nd.
12 28.6–28.7 280, 316, 348 593.1530 MS2 [593]: 447, 285 Kaempferol rutinoside4 nd 13 ± 1 nd
MS3 [593 → 285]: 257, 241, 197, 151
13 29.4–29.6 282 575.1178 MS2 [575]: 575, 449, 289 Proanthocyanidin dimer (A-type) 2
56 ± 4 373 ± 8 nd
MS3 [575 → 575]: 575, 449, 289
Total sum 901 ± 42 1156 ± 26 23501 ± 77
1 2 3 4
sh = shoulder. nd = not detected. The peaks were quantified as equivalent to caffeic acid , epicatechin , ferulic acid and kaempferol .
a
Retention time on the C18 Synergi Hydro (4 μm) column.
b
Solvent: gradient of 0.5% formic acid in water and acetonitrile with 0.5% formic acid.
c
In the MS2 and MS3, the most abundant ion is shown in boldface.

spectrum of m/z 285 showed the same fragmentation pattern exhibited
by the authentic standard of kaempferol.
The seed extract presented higher content of phenolic compounds
(23501 μg/g extract, dry basis) than the pulp and peel extracts (901 μg/
g and 1156 μg/g, respectively). Moreover, the seed extract of Q. cordata
showed higher phenolic compound contents than the extracts of
S. sessiliflorum fruits (1718 μg/g) (Rodrigues et al., 2013), freeze-dried ex-
tracts of Caryocar villosum pulp (86.6–5163 μg/g) (Chisté et al., 2012) and
pulp and peel extracts of Psidium cattleianum fruits (7098–12821 μg/g)
(Ribeiro, Chisté, et al., 2014). Furthermore, some authors reported the
total phenolic compound contents of pulp extracts of Q. cordata fruits, as
determined by the colorimetric method with Folin–Ciocalteau reagent,
and the values vary from 6 to 15 mg gallic acid equivalent (GAE)/100 g
(ethanol and aqueous extracts) (Carvalho et al., 2014) to 244–265 mg
GAE/100 g (fresh pulp) (Cerón et al., 2014).
3.2. Carotenoids
As can be seen in Table 2, the HPLC–DAD technique allowed the sep-
aration (Fig. 2), tentative identification and quantification of 12

60
13
1 2
40 6
5 7 9 11
20 34
Detector response at 320 nm (mAU)

pulp
0

300
7
200

100 10
11 12 13
peel
0

1200
11
800 7

400 8
10
seed
0
5 10 15 20 25 30 35
Time (min)

Fig. 1. HPLC–DAD chromatogram of phenolic compounds of pulp, peel and seed extracts obtained from Quararibea cordata fruits. Peak characterization is given in Table 1.
240 A. Berto et al. / Food Research International 77 (2015) 236–243

carotenoids in the freeze-dried extracts of Q. cordata fruits. The identified 7

0.02
carotenoids were the usual compounds already identified in other Ama-

Detector response at 450 nm (AU)

6 9
zonian fruits, at the same experimental conditions (Chisté and 0.01
Mercadante, 2012; De Rosso and Mercadante, 2007), with the predomi- 45
0.00 pulp
nance of xanthophylls (peaks 1–8). The identification of all-trans-lutein
(peak 6), all-trans-zeaxanthin (peak 7) and all-trans-β-carotene (peak
0.08
9) was positively confirmed through co-elution with authentic standards, 6 7
and based on the comparison of their UV–Vis spectra features. Peaks 1 23
0.04
and 2 were assigned as all-trans-neochrome and 9-cis-neochrome 1 8 9
45
based on the decrease of ≈ 25–30 nm in the λmax of these peaks 0.00
10 peel
(421 nm) in comparison to β-carotene (450 nm, peak 9), which indi-
cates the presence of a 5,8-furanoid group. Peaks 3 and 4 were tenta- 0.16 7
tively identified as all-trans-violaxanthin and all-trans-antheraxanthin,
0.08 6 8 9
respectively, due to the decrease of ≈15 nm in the λmax of violaxanthin 4
5 10 11 12
(435 nm) in relation to β-carotene, indicating the presence of a seed
0.00
5,6:5′,6′-epoxy group, as well as the decrease of ≈6 nm in the λmax of
antheraxanthin (444 nm) due to the presence of one 5,6-epoxy group 5 10 15 20 25 30 35 40 45 50
in the carotenoid structure. All these peaks showed high intensity of Time (min)
%III/II, which is a characteristic behaviour of epoxy-carotenoids (De
Rosso and Mercadante, 2007). The 5,6:5′,6′-diepoxy-β-carotene (peak Fig. 2. HPLC–DAD chromatogram of carotenoids of pulp, peel and seed extracts obtained
8) and the all-trans-δ-carotene (peak 12) showed UV–Vis spectra and from Quararibea cordata fruits. Peak characterization is given in Table 2.

retention times on C30 column similar to those reported in the literature

to Amazonian fruits (De Rosso and Mercadante, 2007; Faria et al., 2009).
The assignments of all cis-carotenoids [9-cis-neochrome (peak 2), cis-
lutein (peak 5), 9-cis-β-carotene (peak 10) and cis-δ-carotene (peak
11)] were done considering that the %III/II decreases and the intensity
of cis-peak (%AB/AII) increases (Table 2) as the cis-double bond is get-
ting closer to the centre of the molecule as compared to its parent all-
trans-carotenoid and data available in the literature (De Rosso and
Mercadante, 2007).
In our study, the seed extract presented higher carotenoid contents
(143 μg/g extract) than the peel (33 μg/g) and pulp (6 μg/g) extracts
and the major identified carotenoids were all-trans-zeaxanthin and
all-trans-β-carotene in both the seed and pulp extracts and lutein and
zeaxanthin in the peel extracts. All-trans-zeaxanthin accounted for
about 29% and 42% of the carotenoids identified in seed and pulp ex-
tracts, respectively, while all-trans-β-carotene was about 26% and 36%,
respectively. The carotenoid composition of fresh pulp of Q. cordata
fruits from Panama were already investigated by Murillo et al. (2013)
and they reported the identification of different di-esters of zeaxanthin
as the major carotenoids (≈ 43% of total carotenoids) followed by β-
carotene (≈23%), which are in agreement with our results. However,
the carotenoid composition of peel and seed extracts of Q. cordata was
not yet reported in the literature until now.
3.3. Scavenging capacity of Q. cordata extracts against ROS
Among the extracts, the peel extract of Q. cordata fruit was the
most efficient scavenger of O •− 2 with IC 50 value of 385 μg/mL,
while the pulp extract was able to scavenge O •− 2 by 18.5%, at the
highest tested concentration (1000 μg/mL) (Table 3). On the
other hand, the seed extract did not show any scavenging capacity
against O •−
2 , up to the highest tested concentration (Table 3), and
similar behaviour was also reported to annatto seed extracts
(Chisté et al., 2011) and some pulp extracts of Caryocar villosum
fruit, another Amazonian fruit (Chisté et al., 2012). The Q. cordata
extracts demonstrated low scavenging capacity as compared to
quercetin (14 μg/mL) (Table 3) and peel extract of P. cattleianum
fruits (84 μg/mL) (Ribeiro, Chisté, et al., 2014). Although O•− 2 itself
is not considered as a potent pro-oxidant reactive species, it is con-
sidered an important precursor to the formation of other reactive
species in physiological system, such as hydroxyl radical ( • OH),
singlet oxygen ( 1 O2 ) and hydrogen peroxide (H2 O 2 ) (Valko et al.,
2007).
Concerning the H2O2-scavenging capacities, all extracts of Q. cordata
fruits were able to decrease the oxidizing effect of H2O2 by 25–35% at
the highest tested concentration of 1000 μg/mL and the IC50 values

Table 2
Chromatographic, UV–Vis characteristics (HPLC–DAD) and contents of carotenoids of pulp, peel and seed extracts obtained from Quararibea cordata fruits.

Peaks Carotenoids HPLC–DAD characteristics Concentration (μg/g extract, dry basis)c

a b
tR range (min) λmax (nm) %III/II %AB/AII Pulp Peel Seed

1 All-trans-Neochrome1 6.4–6.7 399, 421, 448 90 0 nd 3.0 ± 0.1 nd

2 9-cis-Neochrome1 7.1–7.3 304, 398, 419, 448 89 7 nd 2.1 ± 0.1 nd
3 All-trans-Violaxanthin1 7.3–7.5 405, 435, 460 85 0 nd 2.4 ± 0.1 nd
4 All-trans-Antheraxantin1 10.1–10.3 415, 444, 470 65 0 0.30 ± 0.01 1.8 ± 0.1 6.8 ± 0.4
5 cis-Lutein2 11.6–11.8 405, 425, 455 nc nc 0.37 ± 0.01 1.9 ± 0.1 5.4 ± 0.4
6 All-trans-Lutein2 12.2–12.5 420, 444, 472 50 0 0.39 ± 0.01 8.2 ± 0.5 8.0 ± 1
7 All-trans-zeaxanthin1 14.3–14.6 420, 450, 476 12 10 2.5 ± 0.2 7.8 ± 0.4 42 ± 2
8 5,6:5′6′-Diepoxy-β-carotene3 15.1–15.3 410, 440, 465 44 0 nd 0.96 ± 0.04 21 ± 1
9 All-trans-β-carotene3 31.4–31.9 420, 450, 477 25 0 2.2 ± 0.1 4.0 ± 0.1 37 ± 1
10 9-cis-β-carotene3 33.0–33.7 321, 420, 446, 470 nc 13 nd 1.11 ± 0.04 7.0 ± 0.2
11 -δ-carotene3 35.7–35.9 345, 430, 450, 480 20 32 nd nd 7.5 ± 0.2
12 All-trans-δ-carotene3 40.1–40.3 430, 453, 480 50 0 nd nd 9±1
Total (μg/g extract) 6±1 33 ± 2 143 ± 12

nc = not calculated. nd = not detected. The peaks were quantified as equivalent to zeaxanthin1, lutein2 and β-carotene3.
a
Retention time on the C30 column.
b
Linear gradient of methanol/MTBE.
c
Mean ± standard deviation (n = 3).
 A. Berto et al. / Food Research International 77 (2015) 236–243 241

Table 3 et al., 2012), Cocos nucifera (20%), Citrullus vulgaris (27%) (Vissotto,
Scavenging capacities of extracts of pulp, peel and seeds obtained from Quararibea cordata Rodrigues, Chisté, Benassi, and Mercadante, 2013), but lower than
against superoxide radical (O•−
2 ), hydrogen peroxide (H2O2), hypochlorous acid (HOCl),
nitric oxide (•NO) and peroxynitrite (ONOO−).
pulp extracts of Theobroma grandiflorum (IC50 = 700 μg/mL), Spondias
lutea (IC50 = 526 μg/mL) (Vissotto et al., 2013) and quercetin (IC50 =
Reactive species IC50 (μg/mL) (n = 4) 509 μg/mL) (Table 3).
Quararibea cordata extracts Positive control Another important ROS to be considered in the oxidative stress im-
Pulp Peel Seed Quercetin plications, HOCl is frequently associated with a series of pathologies
resulting from chronic inflammation, such as atherosclerosis, cardiovas-
ROS
O•− 18.5⁎ ± 0.1c 385 ± 2b NA 14.2 ± 0.4a
cular diseases and degenerative diseases, such as Alzheimer's, sclerosis
2
H2O2 32.4⁎ ± 0.6b 25.2⁎ ± 0.5a 35⁎ ± 1b 509 ± 6c and various types of cancer (Ho, Galougahi, Liu, Bhindi, and Figtree,
HOCl 22 ± 1d 4.8 ± 0.3b 7.1 ± 0.2c 0.10 ± 0.01a 2013; Malle, Marsche, Arnhold, and Davies, 2006). Furthermore, HOCl
RNS
is considered a potent oxidizing agent and its toxicity surpasses
•
NO 369 ± 1d 230 ± 1c 19.5 ± 0.6b 0.15 ± 0.01a 100–1000 times the toxicity of O•− 2 and H2O2 (Conner and Grisham,
ONOO− 152 ± 1d 97 ± 1c 22.7 ± 0.2b 0.122 ± 0.004a 1996). In our study, all extracts of Q. cordata fruits showed high scaveng-
ONOO−⁎⁎ 104 ± 2d 49.4 ± 0.3c 37.6 ± 0.2b 0.121 ± 0.005a ing capacity against HOCl (Table 3 and Fig. 3) and the peel extract was
IC50 = inhibitory concentration, in vitro, to decrease in 50% the oxidizing effect of the re- the most effective one with the lowest IC50 value (4.8 μg/mL). In general,
active species in the tested media (mean ± standard error of the mean, SEM). NA = No the extracts were more efficient than the ethanol extract of C. villosum
activity was found up to the highest tested concentration (1000 μg/mL).
pulp (199 μg/mL) (Chisté et al., 2012), extract of branches of Cytisus
Means with different superscript letters at the same line are significantly different (p b 0.05).
⁎ Scavenging effect (%) (mean ± standard error of the mean, SEM) for the highest scoparius (58 μg/mL) (González et al., 2013), extracts of peel of
tested concentration (1000 μg/mL). P. cattleianum fruit (32 μg/mL) (Ribeiro, Chisté, et al., 2014) and 5-
⁎⁎ Experiment carried out in the presence of 25 mM NaHCO3 to simulate the physiological caffeoylquinic acid (56 μg/mL) (Rodrigues et al., 2013). However, the
concentrations of CO2. scavenging capacity of extracts of Q. cordata fruits against HOCl were
less efficient than that described to well-known antioxidant com-
were not achieved (Table 3). H2O2 alone is very little reactive; neverthe- pounds, such as quercetin (0.10 μg/mL) (Table 3), gallic acid
less, it may cross cell membranes and react with transition metals (1.1 μg/mL) and ascorbic acid (0.5 μg/mL) (Ribeiro, Berto, et al., (2015).
(Fenton reaction), producing hydroxyl radicals (•OH), which is consid-
ered on of the most reactive species. It was already reported that the 3.4. Scavenging capacity of Q. cordata extracts against RNS
IC50 values for inhibiting the harmful effect of H2O2 are generally greater
than those shown for other ROS (Pistón et al., 2014). However, the The reactivity of RNS may have profound effects on the biological ac-
H2O2-scavenging capacity of these extracts were higher than those re- tivity of numerous molecules. In the physiological system, •NO is pro-
ported for different fruit extracts, such as C. villosum (4–23%) (Chisté duced by nitric oxide synthase through the conversion of L-arginine to
Scavenging capacity of peel extract (%)
Scavenging capacity of pulp extract (%)

100 100
ONOO-
ONOO-
(with NaHCO3) HOCl ONOO- - (without NaHCO3)
75 ONOO- 75 (with NaHCO3)
HOCl (without NaHCO3) •NO

O2•-
50 50
•NO

25 25

(a) (b)
0 0
0 100 200 300 400 500 0 100 200 300 400 500
Quararibea cordata extract (µg/mL) Quararibea cordata extract (µg/mL)
Scavenging capacity of seed extract (%)

100 HOCl

•NO
75

ONOO-
(without NaHCO3)
50

ONOO-
(with NaHCO3)
25

(c)
0
0 30 60 90 120 150
Quararibea cordata extract (µg/mL)

Fig. 3. Scavenging capacities of extracts of pulp (a), peel (b) and seeds (c) obtained from Quararibea cordata against superoxide radical (O•− •
2 ), hypochlorous acid (HOCl), nitric oxide ( NO)
and peroxynitrite (ONOO−) in a concentration dependent-manner. Each point shows the standard error of the mean (SEM) bars and represents the values obtained from four experi-
ments, in duplicate.
242 A. Berto et al. / Food Research International 77 (2015) 236–243
L-citrulline. When present at low concentrations, •NO is essential to
many physiological processes. Nevertheless, high rates may trigger
harmful effects, such as severe inflammatory conditions and cytotoxici-
ty (Pacher, Beckman, and Liaudet, 2007). Our results clearly demon-
strated that the extracts of Q. cordata fruits are effective scavengers of
RNS, namely •NO and ONOO−. The seed extract of Q. cordata fruits,
which presented the highest carotenoid and phenolic compound con-
tents, showed the highest ability to scavenge with IC50 of 19.5 μg/mL
(Table 3; Fig. 3). Such extract was more effective in scavenging •NO
than extracts from the seeds of Sesamun indicum (98–238 μg/mL)
(Visavadiya, Soni, and Dalwadi, 2009) and different extracts of
C. villosum pulp (54–142 μg/mL) (Chisté et al., 2012), but less efficient
than extracts obtained from C. scoparius (10 μg/mL) (González et al.,
2013), Vismia cauliflora (0.9–3.6 μg/mL) (Ribeiro, Berto et al., 2015),
P. catlleianum (2.2–6.8 μg/mL) (Ribeiro, Chisté et al., 2014) and some
well-known antioxidants, such as quercetin (0.15 μg/mL) (Table 3),
bixin (3 μg/mL) (Chisté et al., 2011) and gallic acid (0.20 μg/mL)
(Ribeiro, Berto et al., 2015).
Although •NO has been found to play a critical role in numerous phys-
iological processes, its toxicity is not only related to the levels of •NO gen-
eration, but also to the formation of ONOO− from the reaction between
• Cerón, I.X., Ng, R.T.L., El-Halwagi, M., & Cardona, C.A. (2014). Process synthesis for antiox-
NO and O•− −
2 . ONOO has been shown to oxidize a variety of biomolecules
including thiols, lipids, proteins, carbohydrates, and DNA among others
(Pacher et al., 2007). In our study, all extracts of Q. cordata fruits were
able to scavenge ONOO− in a concentration-dependent manner (Fig. 3),
with IC50 values ranging from 22.7 to 152 μg/mL (Table 3). Again, the
seed extract was the most efficient one, followed by the peel and pulp ex-
tracts with scavenging capacity higher than that described for the pulp
extract of another Amazonian fruit (C. villosum) (Chisté et al., 2012), but
lower than extracts of V. cauliflora fruits (Ribeiro, Berto et al., 2015), leaf
extracts of artichoke (Pistón et al., 2014) and also known antioxidants,
such as quercetin (0.12 μg/mL) (Table 3) and chlorogenic acid
(0.4 μg/mL) (Rodrigues et al., 2013). In physiologic conditions, CO2 mod-
ulates ONOO− reactivity due to the fast reaction with ONOO−, generating
• by HPLC-PDA-MS/MS, from Amazonian fruits. Journal of Agricultural and Food
NO2 and CO•− 3 , which are the main responsible species for the in vivo ni-
tration and/or oxidation reactions (Freitas, Lima, and Fernandes, 2009).
Thus, according to our results, all the tested extracts of Q. cordata fruits
may be also considered as efficient scavengers of •NO2 and CO•− 3 , since
they kept their scavenging efficacy both in the absence and in the pres-
ence of NaHCO3 (Table 3). Interestingly, although the seed extract pre-
sented the highest scavenging capacity against ONOO−, its antioxidant
efficiency decreased by about 1.6 times in the presence of NaHCO3,
while the scavenging capacity of the peel and pulp extracts increased by
approximately 2 and 1.5 times in the experiments without NaHCO3.
Therefore, based on our results, the scavenging efficiency of extracts of
Q. cordata fruits may be strongly influenced by their high phenolic and ca-
rotenoid contents. The seed extract, which presented the highest yield of
phenolic compounds and carotenoids was the most efficient one to inhib-
it the oxidizing effect of the tested reactive species, especially against RNS.
Moreover, this is the first report concerning the scavenging capacity of
Q. cordata extracts against the most physiologically relevant ROS and
RNS. All extracts were able to scavenge the tested ROS and RNS in a
concentration-dependent manner (Fig. 3), except for the seed extract in
the O•−2 -scavenging assay. Importantly, extracts of Q. cordata may be con-
sidered more selective to scavenge HOCl than H2O2 and O•− 2 , since they
showed IC50 values at the lowest μg/mL range (4.8–22 μg/mL).
Conflict of interest
The authors have declared no conflicts of interest.
Acknowledgements
This work received financial support from the European Union
(FEDER funds through COMPETE) and Fundação para a Ciência e
Tecnologia (FCT, Portugal) through project Pest-C/EQB/LA0006/
2013 and also under the framework of QREN through Project
NORTE-07-0124-FEDER-000066. A.B. Ribeiro acknowledges CNPq
(Conselho Nacional de Desenvolvimento Científico e Tecnológico,
Brazil) the financial support for the Post-doc grant (Proc. 248656/
2013-9).
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.foodres.2015.06.018.
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