1. No category

Food Chemistry 165 (2014) 140–148
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Psidium cattleianum fruit extracts are efficient in vitro scavengers
of physiologically relevant reactive oxygen and nitrogen species
Alessandra Braga Ribeiro a, Renan Campos Chisté b, Marisa Freitas b, Alex Fiori da Silva c,
Jesuí Vergílio Visentainer a, Eduarda Fernandes b,⇑
a
b
c
Center for Agricultural Sciences, PostGraduate Program of Food Science, State University of Maringá, 87020-900 Maringá, Paraná, Brazil
REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal
PostGraduate Program of Health Sciences, State University of Maringá, 87020-900 Maringá, Paraná, Brazil
a r t i c l e
i n f o
Article history:
Received 14 February 2014
Received in revised form 31 March 2014
Accepted 14 May 2014
Available online 22 May 2014
Keywords:
Strawberry guava
Phenolic compounds
Carotenoids
LC–MS
ROS
RNS
Antioxidant capacity
a b s t r a c t
Psidium cattleianum, an unexploited Brazilian native fruit, is considered a potential source of bioactive
compounds. In the present study, the in vitro scavenging capacity of skin and pulp extracts from
P. cattleianum fruits against reactive oxygen species (ROS) and reactive nitrogen species (RNS) was evaluated by in vitro screening assays. Additionally, the composition of phenolic compounds and carotenoids
in both extracts was determined by LC–MS/MS. The major phenolic compounds identified and quantified
(dry matter) in the skin and pulp extracts of P. cattleianum were ellagic acid (2213–3818 lg/g extracts),
ellagic acid deoxyhexoside (1475–2070 lg/g extracts) and epicatechin gallate (885–1603 lg/g extracts);
while all-trans-lutein (2–10 lg/g extracts), all-trans-antheraxanthin (1.6–9 lg/g extracts) and all-transb-carotene (4–6 lg/g extracts) were the major carotenoids identified in both extracts. P. cattleianum pulp
extract showed higher scavenging capacity than skin extract for all tested ROS and RNS. Considering the
potential beneficial effects to human health, P. cattleianum may be considered as a good source of natural
antioxidants and may be useful for the food and phytopharmaceutical industry.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Psidium cattleianum Sabine, commonly known as strawberry
guava or araçá vermelho (Brazilian name), is a tropical plant
belonging to Myrtaceae family. This plant is native to the Atlantic
coast of Brazil and its unexplored fruits possess high potential to
be commercialised. P. cattleianum can easily adapt to a variety of
climates and has been cultivated in many countries with tropical
climates, such as Hawaii and many Caribbean islands (Biegelmeyer
et al., 2011; Patel, 2012).
The fruits from P. cattleianum have a firm and sweet-acidulous
pulp, noted by an excellent strawberry-like flavour and it
is described as being more aromatic than common guava,
which belongs to the same botanical family (Mccook-Russell,
Muraleedharan, Facey, & Bowen-Forbes, 2012). Additionally,
P. cattleianum possess three to four times more ascorbic acid than
⇑ Corresponding author. Address: REQUIMTE, Department of Chemical Sciences,
Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira, 228,
4050-313 Porto, Portugal. Tel.: +351 220428675; fax: +351 226093483.
E-mail address: [email protected] (E. Fernandes).
http://dx.doi.org/10.1016/j.foodchem.2014.05.079
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
citrus fruits and possess potentially important biological properties,
as the plant is extensively used in Brazilian traditional medicine to
treat several diseases including painful disorders, diarrhoea, dental
caries, diabetes and also as a prophylactic hepatoprotective agent
(Alvarenga et al., 2013; Im et al., 2012; Menezes, Delbem,
Brighenti, Okamoto, & Gaetti-Jardim, 2010).
In this sense, P. cattleianum may be considered a plant with
latent prospects in food and pharmaceutical industry for its
potential application as functional food and phytotherapeutics
(Patel, 2012), most probably due to the presence of bioactive compounds, such as phenolic compounds and carotenoids (Im et al.,
2012; Medina et al., 2011). Phenolic compounds and carotenoids
present in plant extracts are known to contribute for its scavenging
capacity against reactive oxygen species (ROS) and reactive
nitrogen species (RNS) (Almeida, Fernandes, Lima, Costa, & Bahia,
2008a; Chisté, Freitas, Mercadante, & Fernandes, 2012; Chisté
et al., 2011), although this may vary extensively, according to their
inherent antioxidant potency, the chemical interaction among each
other, and the interaction with endogenous antioxidants. In vitro
studies showed that P. cattleianum has higher antioxidant activity
[as determined by ABTS [2,20 -azino-bis(3-ethylbenzothiazoline6-sulfonic acid)] and Ferric Reducing Antioxidant Power (FRAP)
A.B. Ribeiro et al. / Food Chemistry 165 (2014) 140–148
assays] and also higher phenolic contents than many fruits
(Luximon-Ramma, Bahorun, & Crozier, 2003).
The antioxidant activity, antimicrobial and antiproliferative
effects of aqueous and acetone extracts of P. cattleianum were previously correlated to the high levels of phenolic compounds in the
extracts (Medina et al., 2011). Moreover, Im et al. (2012) showed
that the extract of P. cattleianum leaves, which is rich in phenolic
compounds, with major compound identified as quercetin-3-glucuronide, could reduce the proliferation of lung cancer cells and
be used to control the metastatic process.
Despite the potential beneficial effects of P. cattleianum, there
are only few data about the antioxidant capacity of its extracts
and these studies were mostly performed by using stable and
non-physiological radicals, such as ABTS and DPPH (2,2-diphenyl1-picrylhydrazyl) (Luximon-Ramma et al., 2003; Mccook-Russell
et al., 2012; Medina et al., 2011). To the best of our knowledge, data
related to the scavenging capacity of P. cattleianum extracts
against physiologically relevant ROS and RNS are not available
in the literature. Additionally, its phenolic and carotenoid profiles,
as determined by high performance liquid chromatography
coupled to diode array detector and tandem mass spectrometry
(HPLC-DAD–MS/MS), were not reported so far in the literature.
Considering the above, this study aimed to identify and quantify
the phenolic compounds and carotenoids of two extracts of
P. cattleianum (skin and pulp) by HPLC-DAD–MS/MS, as well as to
determine the scavenging capacity of these extracts against the
most common ROS and RNS in biological systems: superoxide
radical (O2), hydrogen peroxide (H2O2), hypochlorous acid (HOCl),
singlet oxygen (1O2), nitric oxide (NO) and peroxynitrite (ONOO).
2. Materials and methods
2.1. Chemicals
Dihydrorhodamine 123 (DHR), 4,5-diaminofluorescein (DAF-2),
30% hydrogen peroxide, sodium hypochlorite solution, with 4%
available chlorine, 3-(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo1-triazene (NOC-5), b-nicotinamide adenine dinucleotide (NADH),
phenazine methosulphate (PMS), nitroblue tetrazolium chloride
(NBT), lucigenin, quercetin, gallic acid, L-ascorbic acid, catechin,
chlorogenic acid, epicatechin, p-coumaric acid, ellagic acid,
ferulic acid, myricetin, quercetin, kaempferol, all-trans-lutein, alltrans-zeaxanthin, all-trans-b-cryptoxanthin, all-trans-b-carotene,
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 ariumÒ pro system (Sartorius, Germany). All phenolic compounds and carotenoids standards showed at least 95% of purity, as
determined by HPLC-DAD.
2.2. P. cattleianum samples and extract preparation
The fruits of P. cattleianum Sabine (5 kg) were collected from a
farm (Sítio Frutas Raras) located in Monte Alegre city, São Paulo
State, Brazil (23°350 3100 S and 48°380 3800 W). The fresh and ripe
fruits were washed with distilled water; the pulp and skin were
manually separated and ground.
Approximately 10 g of the pulp or skin was weighed and absolute ethanol was added in mass:solvent ratio of 1:10 (w/v) and the
extraction was performed under magnetic stirring, for 4 h, at room
temperature (25 °C), and protected against luminosity. The mixture was filtered and the solvent was evaporated under reduced
pressure at 40 °C. The concentrated material was stored under
light-free conditions at 20 °C prior to analysis.
141
2.3. HPLC-DAD–MS/MS analysis of phenolic compounds and
carotenoids
HPLC-DAD analysis of phenolic compounds and carotenoids
was performed in an Accela LC system (Thermo Fisher Scientific,
San Jose, CA) equipped with quaternary pumps (Accela 600), a
DAD detector and an auto-sampler cooled to 5 °C. The equipment
was also connected in series to a LTQ Obritrap™ XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA) with electrospray
ionisation source (ESI), and a hybrid system combining a linear
ion-trap and the Orbitrap mass analyzer. For chromatographic
analysis, samples and solvents were filtered using, respectively,
membranes of 0.22 lm (OlimPeak, TeknokromaÒ, Spain) and
0.45 lm (Billerica, MA, USA).
The contents of carotenoids and phenolic compounds determined by HPLC-DAD, were expressed as lg/g of extract (dry matter), considering three independent extraction procedures (n = 3).
2.3.1. Phenolic compounds
The phenolic compounds of both extracts of skin and pulp from
P. cattleianum fruit were analysed after solubilising 10 mg of each
extract in 500 lL of methanol/water (8:2, v/v) and filtered using
membranes of 0.22 lm. Both identification and quantification of
phenolic compounds by HPLC-DAD–ESI-MS/MS were carried out
on a C18 Synergi Hydro column (4 lm, 250 4.6 mm, Phenomenex) at 0.9 mL/min, column temperature at 29 °C, with a mobile
phase in a linear gradient of water/formic acid (99.5:0.5, v/v) and
acetonitrile/formic acid (99.5:0.5, v/v) (Chisté & Mercadante,
2012). The mass spectra were acquired with a scan range from
m/z 100 to 1000; the MS parameters were set as follows: ESI source
in negative ion mode; the capillary temperature was 275 °C and
the capillary voltage of was set at 2.5 kV. The sheath gas and the
auxiliary gas flow rate were set to 40 and 10, respectively, (arbitrary unit as provided by the software settings) and normalised
collision energy for MS/MS experiments of 35%. The phenolic compounds were tentatively identified based on the following information: elution order, retention time of peaks, and UV–Visible and
mass spectra features as compared to authentic standards (data
not shown) analysed under the same conditions and data available
in the literature (Chisté & Mercadante, 2012; Gordon, Jungfer, Silva,
Maia, & Marx, 2011; Ho et al., 2012; Im et al., 2012; Santos, Vilela,
Freire, Neto, & Silvestre, 2013). The phenolic compounds were
quantified by comparison to external standards using seven-point
analytical curves (in duplicate) for gallic acid, chlorogenic acid,
quercetin (0.5–49.5 lg/mL) and ellagic acid (0.5–33 lg/mL).
2.3.2. Carotenoids
The carotenoids of both extracts of skin and pulp from
P. cattleianum fruit were extracted according to an adaptation of
the procedure described by Chisté et al. (2012) for carotenoids
from extracts of Caryocar villosum fruit. Briefly, 15 mg of each
extract were solubilised in 500 lL of methanol and directed to
liquid–liquid partition in a separation funnel with petroleum
ether/diethyl ether (1:2, v/v) and washed with distilled water.
After partition, the carotenoid extract was saponified overnight
with 10% KOH in methanol (1:0.5, v/v), re-partitioned, evaporated
under N2 flow, re-dissolved in methanol/MTBE (70:30, v/v) and
injected into the chromatographic system. The carotenoids were
separated on a C30 YMC column (5 lm, 250 mm 4.6 mm) using
as mobile phase a linear gradient of methanol/MTBE, with flow rate
of 0.9 mL/min and the column temperature set at 29 °C (Chisté &
Mercadante, 2012). The MS parameters were set as follows: ESI
source in positive ion mode; the capillary temperature was
350 °C and the capillary voltage of was set at 3.1 kV. The sheath
gas and the auxiliary gas flow rate were set to 40 and 10, respectively, (arbitrary unit as provided by the software settings) and
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A.B. Ribeiro et al. / Food Chemistry 165 (2014) 140–148
normalised collision energy for MS/MS experiments of 35%.
The carotenoids were tentatively identified according to the
following combined information: elution order on C30 column,
co-chromatography with authentic standards, and UV–Vis spectrum [(kmax, spectral fine structure (%III/II), peak cis intensity
(%AB/AII)] compared with data available in the literature (Britton,
Liaaen-Jensen, & Pfander, 2004; Chisté & Mercadante, 2012;
Rodrigues, Mariutti, & Mercadante, 2013). The carotenoids were
quantified by HPLC-DAD, using external seven-point analytical
curves (in duplicate) all-trans-lutein, all-trans-zeaxanthin, alltrans-b-cryptoxanthin and all-trans-b-carotene (0.4–30 lg/mL).
All other carotenoids (including epoxy and cis isomers) were estimated using the curve of its all-trans-carotenoid. The NAS-IOM
(2001) conversion factor was used to calculate the vitamin A value,
with 12 lg of dietary all-trans-b-carotene or 24 lg of all-trans-bcryptoxanthin corresponding to 1 lg of retinol activity equivalent
(RAE), and the activity used was 100% for all-trans-b-carotene
and 50% for all-trans-b-cryptoxanthin.
2.4. ROS- and RNS-scavenging assays
All ROS- and RNS-scavenging assays were performed using a
microplate reader (Synergy HT, Biotek, Vermont, USA), for fluorescence, UV/Vis and chemiluminescence measurements, equipped
with a thermostat. Pulp and skin extracts of P. cattleianum fruit
were dissolved in DMSO for all ROS- and RNS-scavenging assays,
except for the extracts in the HOCl assay (dissolved in ethanol).
The standards, quercetin, gallic acid and ascorbic acid, were dissolved in ethanol (0.03 lg/mL to 2000 mg/mL). Each IC50 value
(inhibitory concentration, in vitro, to decrease in 50% the amount
of reactive species in the tested media) corresponds to four experiments using five to seven concentrations in duplicate, calculated
from the curves of percentage of inhibition versus antioxidant concentration, using the GraphPad Prism 5 software, and the comparison graphs were plotted using OriginPro 8 software. Quercetin,
gallic acid and ascorbic acid were used as positive controls in the
scavenging assays of 1O2, HOCl, H2O2, O
and its
2 , NO, ONOO
values were similar to those previously reported by others authors
(Chisté et al., 2012; Gomes et al., 2007). To ensure the results
are not flawed by any interference of solvents or fluorescence/
chemiluminescence/absorbance response of extracts, additional
experiments were performed using P. cattleianum extracts and
the tested standard compounds (data not shown).
2.4.1. Singlet oxygen-scavenging assay
The 1O2-scavenging capacity was measured by monitoring the
oxidation of the non-fluorescent DHR to the fluorescent rhodamine
123 by the reaction with 1O2, generated by thermal decomposition
(37 °C) of a previously synthesised water-soluble endoperoxide
(disodium 3,30 -(1,4-naphthalene) bispropionate, NDPO2) (Costa
et al., 2007). The results were expressed in percentage as the
inhibition of 1O2-induced oxidation of DHR.
2.4.2. Hypochlorous acid-scavenging assay
The HOCl-scavenging capacity was measured as previously
described by Rezk, Haenen, van der Vijgh, and Bast (2004) adapted
to a microplate reader (Gomes et al., 2007). The assay verifies the
effect of the extracts and standards on HOCl-induced oxidation of
DHR to rhodamine 123. HOCl was prepared by adjusting the pH
of a 1% (w/v) solution of NaOCl to 6.2 with dropwise addition
of 10% (v/v) H2SO4. The concentration of HOCl was further
determined spectrophotometrically at 235 nm, using the molar
absorption coefficient of 100 M1 cm1. The results were
expressed, in percentage, as inhibition of HOCl-induced oxidation
of DHR.
2.4.3. Hydrogen peroxide-scavenging assay
The H2O2 scavenging activity was measured using a chemiluminescence methodology, by monitoring the effect of the extracts and
standards on the H2O2-induced oxidation of lucigenin (Chisté et al.,
2011). The results were expressed as the inhibition, in percentage,
of the H2O2-induced oxidation of lucigenin.
2.4.4. Superoxide radical-scavenging assay
The O
2 was generated by a non-enzymatic system NADH/PMS/
O2 and this radical reduces NBT into a purple coloured formazan
(Gomes et al., 2007). The O
2 scavenging capacity was determined
spectrophotometrically, by monitoring the effect of the extracts
and standards on the O
2 -induced reduction of NBT at 560 nm,
after 2 min. The effects were expressed as the inhibition, in
percentage, of the NBT reduction to formazan.
2.4.5. Nitric oxide-scavenging assay
The NO-scavenging capacity was measured by monitoring the
effect of the extracts and standards on NO-induced oxidation of
non-fluorescent DAF-2 to the fluorescent triazolofluorescein
(DAF-2T) (Almeida, Fernandes, Lima, Costa, & Bahia, 2008b). NO
was generated by decomposition of NOC-5. The results were
expressed as the percentage of inhibition of NO-induced oxidation
of DAF-2.
2.4.6. Peroxynitrite-scavenging assay
The ONOO-scavenging capacity was measured by monitoring
the effect of the extracts and standards on ONOO-induced oxidation of non-fluorescent DHR to the fluorescent rhodamine 123
(Almeida et al., 2008a). In a parallel set of experiments, the assays
were performed in the presence of 25 mM NaHCO3, in order to
simulate the physiological CO2 concentrations. The results were
expressed, in percentage, as the inhibition of ONOO induced
oxidation of DHR.
3. Results and discussion
3.1. Phenolic compounds and carotenoids in the extracts of P.
cattleianum fruit
Ethanol was the chosen solvent for obtaining extracts of skin and
pulp from P. cattleianum fruits, this solvent is favourably chosen to
extract bioactive compounds, such as phenolic compounds and
carotenoids from fruit matrix. Moreover, after comparing the most
frequent solvents used for antioxidant activity research (ethanol,
water and methanol), the water has a disadvantage over ethanol,
hence it needs a freeze drying step after extraction and methanol
has its use limited by the highest toxicity (Alam, Bristi, &
Rafiquzzaman, 2013).
Some data are available concerning the identification of phenolic compounds from Psidium guineense (Gordon et al., 2011),
another fruit from the same family (Myrtaceae) and from the same
genus (Psidium). It is also possible to find two studies in the literature related to the identification, by nuclear magnetic resonance
(NMR), of seven flavonoids isolated from leaves of P. cattleianum
(Ho et al., 2012) or by LC–MS (Im et al., 2012). In addition, six
phenolic compounds from extracts of P. cattleianum pulp were
identified based only on the retention time (LC with UV detector)
(Medina et al., 2011). In relation to the carotenoid composition,
only one study was found that reported the carotenoids profile
to the same fruit, in which the identification was based on the
UV–Vis spectra and retention times (Pereira et al., 2012). Another
study reported the conclusive identification of 16 carotenoids
isolated from the flesh of Brazilian red guavas (Psidium guajava
L.) and their structures were established by means of UV–Vis,
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A.B. Ribeiro et al. / Food Chemistry 165 (2014) 140–148
NMR, mass and circular dichroism spectra (Mercadante, Steck, &
Pfander, 1999).
The applied HPLC-DAD–ESI-MS/MS method allowed the separation, quantification and tentative identification of 21 phenolic
compounds (Fig. 1) and 7 carotenoids (Fig. 2). For phenolic compounds, ESI in the negative ion mode and the hybrid m/z analyzer
(linear ion-trap with Orbitrap) provided a very sensitive, selective
method and produced by far the most characteristic data for the
identification of each compound in these extracts. On the other
hand, although ESI in positive ion mode have been used to carry
out the tentative identification of carotenoids (Crupi, Milella, &
Antonacci, 2010; Pop et al., 2014), in our study the ionisation
was not able to allow the observation of protonated molecule
([M+H]+) nor the sodiated molecular ion [M+Na]+. However, since
only seven peaks of carotenoids were observed after HPLC-DAD
analysis, and most of these compounds presented the same
chromatographic behaviour and UV–Vis spectra of the authentic
standards, the tentative identification was performed only
based on the elution order on C30 column, co-chromatography
with authentic standards and UV–Vis spectrum characteristics
[(kmax, spectral fine structure (%III/II) and peak cis intensity
(%AB/AII)].
Phenolic compounds, in nature, generally occur as conjugates
of sugars, especially in the form of O-glycosides, although in the
present study, the identification of the sugar moiety was not be
determined by the applied methodology, for simplification.
According to Table 1, peak 1 was assigned as chlorogenic acid
hexoside with m/z at 515 [MH], and the loss of a hexose moiety
(162 u) exhibited the chlorogenic acid (caffeoylquinic acid)
molecule (m/z 353). Peak 2 presented deprotonated molecule at
m/z 647 and three consecutive losses of galloyl moieties (152 u)
(m/z 495, 343, 191). Peaks 3, 6, 11 and 20 were positively identified
as gallic acid, chlorogenic acid, ellagic acid and quercetin, on the
basis of coelution and comparison of UV–Vis and mass spectra with
authentic standards. Peaks 4 and 5 were assigned as two ellagitannins-like compounds, in which the MS/MS fragments match with
those already reported to P. guineense fruits (Gordon et al., 2011).
Peaks 7, 8, 9, 16, 17 and 19 were tentatively identified ellagic acid
derivatives since MS/MS analysis indicated that cleavage of the
glycosidic linkage with concomitant H rearrangement leads to
elimination of the sugar residue as neutral losses: 162 u (hexose),
132 u (pentose) and 146 u (deoxyhexose) (Chisté & Mercadante,
2012). Peak 10 presented deprotonated molecule at m/z 441 and
was tentatively identified as epicatechin gallate with MS/MS
50
1
2
Absorbance at 450 nm (mAU)
25
6
5
4
3
7
0
skin
50
25
2
1
3
6
5
4
7
0
1000
pulp
Lut
Zea
500
β-Cry
β-Car
standards
0
5
10
15
20
25
30
35
40
45
50
Time (min)
Fig. 2. Chromatogram of carotenoids from extracts of Psidium cattleianum fruit
(skin and pulp) and authentic standard of all-trans-lutein (Lut), all-trans-zeaxanthin
(Zea), all-trans-b-cryptoxanthin (b-Cry) and all-trans-b-carotene (b-Car), obtained
by HPLC-DAD. Chromatographic conditions: see text. Peak characterization is given
in Table 2.
fragment at m/z 289 (epicatechin) after losing a galloyl group
([MH152]). Peaks 12, 13, 14, 15 and 21 were assigned as quercetin derivatives due to the neutral losses of a glucuronide moiety
(176 u, peak 12), hexose (162 u, peak 13), pentose (132 u, peak 14),
deoxyhexose (146 u, peak 15), coumaroyl deoxyhexose (292 u,
peak 21) and after noticing the same fragmentation pattern given
by quercetin (peak 20), which was positively confirmed with
authentic standard. The identification of quercetin 3-glucuronide
was already carried out in a butanol fraction of P. cattleianum leaf
extract by HPLC–ESI-MS/MS (Im et al., 2012), which reinforces the
identification of this compound in our study. Finally, peak 18 was
tentatively identified as cinnamoyl–galloyl hexoside with deprotonated molecule at m/z 461 [MH] and MS/MS fragments at m/z
313 (loss of a cinnamoyl moiety) and m/z 169 ([gallic acidH]),
which MS pattern matches with those previously described in
the literature (Santos et al., 2013).
Therefore, the major phenolic compounds identified in both
extracts were ellagic acid (varying from 2213 to 3818 lg/g extract),
ellagic acid deoxyhexoside (from 1475 to 2070 lg/g extract) and
epicatechin gallate (from 885 to 1603 lg/g extract) and the total
9 11
800
13
Absorbance at 280 nm (mAU)
12
10
15
400
21
14 16
1
2 3
4
5
6
7
8
18
19
17
20
skin
0
5
10
15
20
25
30
1400
35
40
45
50
11
9
12
700
13
15
10
3
1
4
5
7
6
8
14 16
2
18
17
19
20
21
pulp
0
5
10
15
20
25
30
35
40
45
50
Time (min)
Fig. 1. Chromatogram of phenolic compounds from extracts of Psidium cattleianum fruit (skin and pulp), obtained by HPLC-DAD. Chromatographic conditions: see text. Peak
characterisation is given in Table 1.
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A.B. Ribeiro et al. / Food Chemistry 165 (2014) 140–148
Table 1
Chromatographic and spectroscopic characteristics of phenolic compounds from extracts of Psidium cattleianum fruit (skin and pulp).
Peaks tR range (min)a
kmax (nm)b
MS/MS () (m/z)c
[MH](m/z)
Concentration (lg/g extract)d
Compound
Skin
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
7.0–7.1
8.8–9.0
10.4–10.7
14.5–14.7
16.4–16.6
18.5–18.9
21.9–22.1
24.9–25.8
25.7–25.9
26.5–26.6
27.3–27.5
27.9–28.0
28.1–28.3
29.1–29.2
29.8–30.0
30.7–30.8
32.9–33.1
34.9–35.1
35.9–36.1
38.5–38.6
40.2–40.4
264, 300 (sh)
270
272
280
272
283 (sh), 325
345 (sh), 361
300, 345 (sh),
300, 345 (sh),
280
300, 350 (sh),
280, 343
280, 352
271, 349
265 (sh), 300,
300, 350 (sh),
300, 350 (sh),
280
270 (sh), 352,
300 (sh), 371
276, 338, 363
361
361
368
346
365
363
366
515.1242
647.1880
169.0141
933.0624
783.0679
353.0869
463.0504
433.0399
447.0558
441.0818
301.9982
477.0920
463.0870
433.0766
447.0925
461.1074
461.1078
461.0716
503.0821
301.0344
593.2468
353,
495,
125
783,
481,
335,
301,
301,
301,
331,
283,
301,
301,
301,
301,
315,
315,
401,
443,
273,
447,
Chlorogenic acid hexoside1
29 ± 15
Trigalloylquinic acid2
9±2
2
Gallic acid
464 ± 155
2
675, 631, 451, 301, 273 Ellagitannin-like
77 ± 27
301, 275
Di-HHDP-hexoside2
222 ± 64
263, 245, 233, 205, 191 Chlorogenic acid1
69 ± 42
273, 257, 198
Ellagic acid hexoside3
136 ± 5
283, 257, 195, 143
Ellagic acid pentoside3
164 ± 4
283, 257, 245
Ellagic acid deoxyhexoside3
1475 ± 138
2
315, 289, 169
Epicatechin gallate
885 ± 26
273, 257, 229, 185
Ellagic acid3
2213 ± 101
20 ± 9
273, 257, 179, 151
Quercetin glucuronide4
273, 257, 179, 151
Quercetin hexoside4
17.5 ± 0.5
273, 257, 179, 151
Quercetin pentoside4
39 ± 13
273, 257, 179, 151
Quercetin deoxyhexoside4
53 ± 14
3
300, 283, 269
Methyl ellagic acid deoxyhexoside
475 ± 23
3
300, 235, 169
Methyl ellagic acid deoxyhexoside
34 ± 2
573 ± 17
313, 211, 169, 151
Cinnamoyl–galloyl hexoside2
315, 300, 283
Methyl ellagic acid acetyl-deoxyhexoside3
10 ± 1
257, 179, 151
Quercetin4
115 ± 2
301, 273, 257, 193
Quercetin coumaroyl deoxyhexoside4
15 ± 1
Total phenolic (lg/g extract)
7098 ± 280
191, 173, 111
475, 343, 191
Pulp
26 ± 14
85 ± 5
1510 ± 37
1022 ± 13
150 ± 66
121 ± 8
346 ± 15
412 ± 19
2070 ± 216
1603 ± 37
3818 ± 94
18 ± 7
11.4 ± 0.3
27 ± 1
11.2 ± 0.5
646 ± 38
20 ± 1
880 ± 115
2±1
32 ± 5
8.5 ± 0.5
12821 ± 540
a
Retention time on the C18 Synergi Hydro (4 lm) column.
Solvent: gradient of water/formic acid (99.5:0.5, v/v) and acetonitrile/formic acid (99.5:0.5, v/v).
c
In the MS/MS, the most abundant ion is shown in boldface.
d
Mean ± standard deviation (n = 3, dry matter). HHDP = Hexahydroxydiphenoyl. The peaks were quantified as equivalent of chlorogenic acid1, gallic acid2, ellagic acid3 and
quercetin4.
b
phenolic contents found in the pulp extract were almost 2-fold
higher than that found in skin extract. The major phenolic compounds identified and quantified (HPLC-DAD) by Medina et al.
(2011) in water and acetone extracts of 6 different genotypes of
P. cattleianum Sabine were ()-epicatechin and gallic acid in a similar concentration range reported in this study for epicatechin gallate and gallic acid. Although these fruits are from the same species
that we evaluated in this work, the difference in phenolic composition is not surprising, since the extraction procedure was not the
same and the identification carried out by Medina et al. (2011) was
based only on the retention time and UV–Vis spectra in comparison with standards. Considering the total phenolic contents found
in our study, both extracts from skin and pulp presented higher
values than freeze-dried extracts of C. villosum fruit pulp obtained
with water (1745 lg/g) or ethanol/water (5163 lg/g) (Chisté et al.,
2012) and methanol/water extract of Solanum sessiliflorum fruit
(1718 lg/g) (Rodrigues et al., 2013).
In relation to the carotenoids composition of P. cattleianum
extracts (Table 2), peaks 2, 3, 5 and 6 were tentatively identified
as all-trans-lutein, all-trans-zeaxanthin, all-trans-b-cryptoxanthin
and all-trans-b-carotene, respectively, after positive confirmation
with authentic standards. These compounds were previously
reported in the literature for P. cattleianum Sabine fruit (Pereira
et al., 2012). Peaks 1, 4 and 7 were tentatively identified as alltrans-antheraxanthin, 5,6-epoxy-b-cryptoxanthin and 9-cis-b-carotene, respectively, due to the comparison with the same kmax,
spectral fine structure and retention time reported to these compounds at identical HPLC-DAD conditions (Chisté & Mercadante,
2012; Faria et al., 2009; Rodrigues et al., 2013). Peak 4 was
assigned as 5,6-epoxy-b-cryptoxanthin due to 6 nm hypsochromic
shift compared to the kmax of b-cryptoxanthin, indicating the presence of one epoxide group at 5,6 position (Britton et al., 2004; Faria
et al., 2009). The 9-cis isomer of b-carotene (peak 7) was identified
considering that the spectral fine structure (%III/II) decreases and
intensity of the cis-peak (%AB/AII) increases as the cis-double bond
is getting closer to the centre of the molecule (Faria et al., 2009).
Therefore, the major carotenoids identified in both P. cattleianum
extracts were all-trans-lutein, followed by all-trans-antheraxanthin
Table 2
Chromatographic, UV–Vis characteristics (HPLC-DAD) and contents of carotenoids from Psidium cattleianum extracts.
Peak
tR range (min)a
Carotenoid
1
1
All-trans-antheraxanthin
2
All-trans-lutein1
3
All-trans-zeaxanthin2
4
5,6-Epoxy-b-cryptoxanthin3
5
All-trans-b-cryptoxanthin3
6
All-trans-b-carotene4
7
9-cis-b-carotene4
Total carotenoids (lg/g)
Vitamin A value (lg RAE/g extract)
a
10.2–10.6
12.1–12.3
14.3–14.6
17.5–17.8
22.4–22.9
32.3–32.6
34.1–34.5
kmax (nm)b
420,
420,
425,
420,
420,
425,
331,
445,
445,
450,
445,
451,
451,
420,
472
471
476
471
476
477
446, 472
%III/II
61
61
25
58
25
25
20
%AB/AII
0
0
0
0
0
0
9
Concentration (lg/g extract)c
Skin
Pulp
9±1
10 ± 3
<LOQ
7±1
6 ± 0.7
5.9 ± 0.3
3.9 ± 0.3
42 ± 5
1.19
1.6 ± 0.1
1.9 ± 0.1
<LOQ
2.4 ± 0.4
3.4 ± 0.2
3.7 ± 0.5
0.95 ± 0.01
14 ± 1
0.58
Retention time on the C30 column.
Linear gradient of methanol/MTBE.
c
Mean ± standard deviation (n = 3, dry matter). The peaks were quantified as equivalent to lutein1, zeaxanthin2, b-cryptoxanthin3 and b-carotene4. RAE = retinol activity
equivalent. <LOQ, value lower than the limit of quantification.
b
145
A.B. Ribeiro et al. / Food Chemistry 165 (2014) 140–148
and all-trans-b-carotene; and the total carotenoid contents were 3
times higher in skin (42 lg/g) than in pulp extracts (12 lg/g)
(Table 2). The values found for the pulp extracts were similar to
that reported to another Brazilian fruits (Chisté & Mercadante,
2012; Rodrigues et al., 2013). Regarding vitamin A activity, both
extracts presented values lower than those reported for Amazonian fruits (3.44–36.4 lg RAE/g pulp), which are known good
pro-vitamin A sources (De Rosso & Mercadante, 2007). Thus, the
extracts of P. cattleianum fruits obtained in this study should not
be considered as a good pro-vitamin A sources.
3.2. Scavenging of ROS and RNS by P. cattleianum extracts
The endogenous formation of ROS and RNS is involved in important physiological roles, namely during the inflammatory response
and to defend the organism against a microbe invasion (Costa et al.,
2007). However, in the event of a sustained overproduction of prooxidant reactive species, a redox unbalance is generated, which can
lead to cell and/or tissue damage. Additionally, ROS and RNS may
rapidly be interconverted. For example, O2 may be converted to
O
2 by several enzymatic and chemical systems, which is then converted into H2O2, both spontaneously and catalysed by superoxide
dismutases. H2O2 may then be converted into hydroxyl radicals
(OH) by the Fenton or Haber–Weiss reactions and these free radicals may react with Cl to produce hypochlorite or HOCl (Cannizzo,
Clementa, Sahua, Follo, & Santambrogio, 2011). The lethal consequence of NO increases significantly upon reaction with O
2 resulting in the formation of ONOO, a highly reactive specie with
reactivity similar to OH. This reactive specie leads to serious toxic
reactions with biomolecules; thus, the production of NO may be
tightly regulated to minimise the damage (Luo, Sun, Mao, Lu, &
Tan 2004; Matsuda et al., 2000).
The pulp and skin extracts of P. cattleianum showed a notable
capacity to scavenge all the tested ROS and RNS in a concentration-dependent manner, and all IC50 values were found at a low
lg/mL range (Table 3), with special emphasis for the low concentration required for scavenging NO. In all cases, the pulp extract
of P. cattleianum showed higher scavenging capacity against all
tested ROS and RNS than skin extract (Fig. 3), probably due to
the highest content of phenolic compounds (Table 1) found in
the extract obtained from the pulp fruit. Data are noteworthy, since
pulp is the most important edible part of this fruit. The content of
phenolic compounds in skin and pulp extracts were, respectively,
about 170 and 916 times higher than the content of total carotenoids, indicating a close relation between the scavenging capacity
of P. cattleianum extracts and their phenolic compounds content.
Ellagic acid, the major phenolic compound identified in both
extracts, has been shown to elicit interesting biological properties
such as anti-proliferative, and antimicrobial activity. Additionally,
this compound is able to induce apoptosis of some carcinogenic
cells (Puupponen-Pimia et al., 2005; Whitley, Stoner, Darby, &
Walle, 2003).
The pulp extract presented high efficiency in quenching 1O2
with IC50 values being lower than that of lipoic acid (46.15 lg/mL),
an essential cofactor for mitochondrial enzymes and a naturally
occurring antioxidant (Hazra, Sarkar, Biswas, & Mandal, 2010). Furthermore, the skin extract of P. cattleianum showed lower activity
than aqueous extracts of C. villosum fruit pulp (156 lg/mL)
(Chisté et al., 2012), Terminalia chebula, Terminalia belerica and
Emblica officinalis fruit extracts (IC50 from 233.12 to 490.42 lg/mL)
(Hazra et al., 2010).
Fig. 3 shows the behaviour related to HOCl scavenging activity
of pulp and peel of P. cattleianum extracts. For this reactive specie,
the pulp extract exhibited an IC50 similar to that obtained for S. sessiliflorum, an Amazonian fruit (13 lg/mL), and higher efficiency
activity than trolox (134 lg/mL) and 5-caffeoylquinic acid (56 lg/mL),
the major compound of S. sessiliflorum, possibly responsible for
its excellent scavenging capacity against ROS (Rodrigues et al.,
2013).
H2O2 scavenging activities of both extract of P. cattleianum were
higher than quercetin and lower than gallic acid and ascorbic acid
(Table 1), and the scavenging capacity found for the pulp extract
was higher than that found for the skin. In addition, the H2O2 scavenging capacity of P. cattleianum pulp extract was superior to that
described for leaf extracts of Castanea sativa (410 lg/mL) (Almeida
et al., 2008a).
Regarding to O
2 scavenging capacity of both extracts, they
inhibited the formation of formazan in a concentration-dependent
manner (Fig. 3) and the pulp extract showed lower IC50 value
(20.6 ± 0.6 lg/mL) than quercetin (14 ± 1 lg/mL); however, similarly to the most effective extract of Monotheca buxifolia fruit
(22.3 lg/mL) and ascorbic acid (21.1 lg/mL) (Jan, Khan, Rashid, &
Bokhari, 2013). The IC50 value found for skin extract of P. cattleianum (84 ± 2 lg/mL) was lower than the IC50 value previously found
for extracts of Cynodon dactylon (IC50 = 430.06 lg/mL), a plant traditionally used to heal several disorders (Jananie, Priya, &
Vijayalakshmi, 2011).
The ability of P. cattleianum extracts and standards to scavenge
NO is shown in Table 3. The skin extract showed to be three times
less effective than pulp, even though, both parts can be considered
as good scavengers compared with previous results reported by
Hazra et al. (2010) for curcumin (90.82 lg/mL) and fruits used in
traditional Indian medicine (maximum activity of 33.28 lg/mL)
(Hazra et al., 2010).
Table 3
Scavenging activities of extracts of skin and pulp from Psidium cattleianum for superoxide radical (O2), hydrogen peroxide (H2O2), hypochlorous acid (HOCl), nitric oxide (NO),
peroxynitrite (ONOO) and singlet oxygen (1O2).
IC50 (lg/mL) (n = 4)
Reactive species
P. cattleianum extracts
Positive controls
Skin
Pulp
Gallic acid
Quercetin
Ascorbic acid
ROS
1
O2
HOCl
H2O2
O
2
83 ± 1
32 ± 1
431 ± 2
84 ± 2
22.8 ± 0.3
18.7 ± 0.6
378 ± 2
20.6 ± 0.6
1.6 ± 0.1
1.1 ± 0.1
214.8 ± 0.1
3.9 ± 0.1
1.20 ± 0.03
0.12 ± 0.02
526.35 ± 0.04
14 ± 1
5.6 ± 0.4
0.5 ± 0.1
116.50 ± 0.05
NA
RNS
NO
ONOO8
ONOO⁄
6.8 ± 0.2
12.1 ± 0.3
55 ± 1
2.2 ± 0.1
5.6 ± 0.1
26 ± 1
0.1 ± 0.01
0.10 ± 0.06
0.10 ± 0.06
0.27 ± 0.02
0.78 ± 0.05
1.7 ± 0.1
0.20 ± 0.03
0.15 ± 0.09
0.16 ± 0.07
IC50 = inhibitory concentration, in vitro, to decrease in 50% the amount of reactive species in the tested media (mean ± standard error). NA = IC50 no activity was found up to
the highest tested concentration (1 mg/mL).
*
In the presence of 25 mM NaHCO3.
146
A.B. Ribeiro et al. / Food Chemistry 165 (2014) 140–148
75
50
25
Skin
Pulp
(a)
0
0
50
100
150
200
H2 O2 scavenging capacity (%)
100
1
O2 scavenging capacity (%)
100
75
50
25
Skin
Pulp
0
Psidium cattleianum extract concentration ( μg/mL)
500
1000
1500
2000
Psidium cattleianum extract concentration ( μg/mL)
100
80
60
40
20
(c)
0
0
60
120
75
50
25
Skin
Pulp
.-
Skin
Pulp
O2 scavenging capacity (%)
100
HOCl scavenging capacity (%)
(b)
0
250
(d)
0
0
180
60
120
180
Psidium cattleianum extract concentration (μg/mL)
Psidium cattleianum extract concentration (μg/mL)
.NO scavenging capacity (%)
100
75
50
25
Skin
Pulp
(e)
0
0
10
20
30
100
100
ONOO- scavenging capacity (%)
ONOO- scavenging capacity (%) with2Na HCO3
Psidium cattleianum extract concentration ( μg/mL)
75
50
25
Skin
Pulp
0
0
30
(f)
60
90
Psidium cattleianum extract concentration (μg/mL)
75
50
25
Skin
Pulp
(g)
0
0
60
120
180
Psidium cattleianum extract concentration ( μg/mL)
Fig. 3. Scavenging capacity of skin and pulp extracts of P. cattleianum against (a) singlet oxygen (1O2), (b) hydrogen peroxide (H2O2), (c) hypochlorous acid (HOCl), (d)
superoxide radical (O2), (e) nitric oxide (NO) and (f, g) peroxynitrite (ONOO) in the absence and presence of NaHCO3. Each point shows the standard error bars and
represents the values obtained from four experiments, performed in duplicate, in five to seven concentrations.
In relation to ONOO-scavenging capacity, both extracts of
P. cattleianum exhibited a reduction in its scavenging efficiency
(almost 5 times) when this assay was carried out in the presence
of 25 mM NaHCO3 (Fig. 3), which is a disadvantage, since under
physiological conditions the reaction between ONOO and CO2 is
predominant, with a very fast rate constant (k = 3–5.8 104 M1 s1)
(Whiteman, Ketsawatsakul, & Halliwell, 2002). However, gallic acid
and quercetin presented very low IC50 values, both in the absence
A.B. Ribeiro et al. / Food Chemistry 165 (2014) 140–148
or presence of NaHCO3 and a slight increase of IC50 was observed
for ascorbic acid in the presence of NaHCO3, as also described by
Almeida et al. (2008a).
4. Conclusion
The skin and pulp extracts of P. cattleianum showed to be potent
scavengers of ROS and RNS, though more effectively of the latter,
especially NO. Furthermore, the pulp presented to be a potent
scavenger of O2, HOCl and 1O2, which may associated to the high
content of phenolic compounds, especially ellagic acid and epicatechin, well described as potent antioxidants. Noteworthy, this is
the first time that the composition of phenolic compounds in P.
cattleianum extracts (skin and pulp) was reported. Furthermore,
not only the major phenolic compounds were determined by
HPLC–ESI-MS/MS, but more than 15 minor phenolic compounds
were also identified. Thus, P. cattleianum may be considered as a
promising source of bioactive compounds with efficient antioxidant properties and great prospect to commercial growers for the
application in the food and phytopharmaceutical industry.
Acknowledgements
Alessandra Braga Ribeiro acknowledges CAPES Foundation
(Coordenação de Aperfeiçoamento de Pessoal de Nível Superior),
Ministry of Education of Brazil, the financial support for the PDSE
grant. This work received financial support from the European
Union (FEDER funds through COMPETE) and National Funds (FCT,
Fundação para a Ciência e Tecnologia, Portugal) through project
Pest-C/EQB/LA0006/2013. The work also received financial support
from the European Union (FEDER funds) under the framework of
QREN through Project NORTE-07-0124-FEDER-000066. Marisa Freitas acknowledges FCT the financial support for the Post-doc grant
(SFRH/BPD/76909/2011) in the ambit of ‘‘POPH-QREN – Tipologia
4.1-Formação Avançada’’ co-sponsored by FSE and national funds
of MCTES. The authors also thank the Mr. Helton J.T. Muniz, Sítio
Frutas Raras/Campina do Monte Alegre-SP for providing P. cattleianum fruits.
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