The potential of extracts of Caryocar villosum pulp to scavenge reactive oxygen and nitrogen species

Food Chemistry 135 (2012) 1740–1749

Contents lists available at SciVerse ScienceDirect

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

The potential of extracts of Caryocar villosum pulp to scavenge reactive oxygen and nitrogen species Renan Campos Chisté a, Marisa Freitas b, Adriana Zerlotti Mercadante a, Eduarda Fernandes b,⇑ a b

Department of Food Science, Faculty of Food Engineering, University of Campinas (UNICAMP), 13083-862 Campinas, São Paulo, Brazil REQUIMTE, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal

a r t i c l e

i n f o

Article history: Received 9 December 2011 Received in revised form 4 June 2012 Accepted 19 June 2012 Available online 28 June 2012 Keywords: Piquiá Caryocaraceae ROS RNS Gallic acid Ellagic acid

a b s t r a c t Caryocar villosum (piquiá) is a native fruit from the Amazonian region, considered to be an interesting source of bioactive compounds. In this paper, five extracts of C. villosum pulp were obtained, using solvents with different polarities and their in vitro scavenging capacity against reactive oxygen species (ROS) and reactive nitrogen species (RNS) was determined. Additionally, the phenolic compounds and carotenoids in each extract were identified and quantified by a high performance liquid chromatography coupled to diode array and mass spectrometer detectors (HPLC–DAD–MS/MS). The ethanol/water and water extracts, which presented the highest phenolic contents (5163 and 1745 lg/g extract, respectively), with ellagic acid as the major phenolic compound, proved to have the highest ROS and RNS scavenging potential. Nevertheless, in general, ellagic acid was less effective in scavenging ROS (IC50 from 1.7 to 108 lg/ml) and RNS (IC50 from 0.05 to 0.59 lg/ml), when compared to gallic acid (IC50 from 0.4 to 226 lg/ml for ROS and IC50 from 0.04 to 0.12 lg/ml for RNS). The results obtained in the present study clearly demonstrated that the in vitro antioxidant efficiency of C. villosum extracts was closely related to their contents of phenolic compounds. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Caryocar villosum (Aubl.) Pers (piquiá), a native fruit from the Amazonian region, belongs to the restricted Caryocaraceae family, which comprises 16 species. The pulp of the fruit is used to prepare regional dishes (usually with rice), and homemade soap, and its edible oil can be used as a substitute of butter and also in cosmetic applications (Clement, 1993; Pianovski et al., 2008). The oil products of C. villosum and C. brasiliensis fruits were also reported to possess antifungal properties against dermatophytosis (Grenand, Moretti, Jacquemin, & Prévost, 2004; Passos et al., 2003), as well as in vivo topical anti-inflammatory activity (Xavier et al., 2011). The fruits of C. villosum can be considered an interesting source of bioactive compounds. Actually, piquiá showed the highest values of total phenolic compounds, flavonoids and antioxidant activity when compared to other 18 tropical fruits (nine of them from the Amazonian region) in a previous screening performed by our research group (Barreto, Benassi, & Mercadante, 2009). In a previous study (Chisté & Mercadante, 2012), our research group found that the major phenolic compounds in C. villosum pulp were gallic acid, followed by ellagic acid deoxyhexoside and ellagic acid (Fig. 1). The carotenoid composition was also evaluated and the ⇑ Corresponding author. Tel.: +351 220428675; fax: +351 226093483. E-mail address: [email protected] (E. Fernandes). 0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2012.06.027

main compounds were all-trans-antheraxanthin, all-trans-zeaxanthin, all-trans-neoxanthin (Fig. 1), and a lutein-like carotenoid. Both phenolic compounds and carotenoids are well known to possess scavenging capacity against reactive oxygen species (ROS) and reactive nitrogen species (RNS), as well as quenching properties against singlet oxygen and the triplet state of sensitizers (Chisté et al., 2011; Krinsky, 1994; Rios, Mercadante, & Borsarelli, 2007). The effects of these antioxidants may have primary importance in the prophylaxis or treatment of several pro-oxidant-related chronic degenerative disorders, such as cancer, chronic inflammation, cardiovascular diseases, cataracts, macular degeneration and neurodegenerative diseases (Serdula et al., 1996). Additionally, natural extracts with high levels of bioactive compounds, from accessible natural sources, can also be considered very interesting products for the food, pharmaceutical and cosmetic industries. In spite of the clear antioxidant potential of C. villosum, as predicted by its chemical content, data related to its scavenging capacity against ROS and RNS are not hitherto reported in the literature. Thus, considering the potential of C. villosum pulp as a natural unexploited source of phenolic compounds and carotenoids with antioxidant activity, the aim of this study was to evaluate the capacity of natural extracts obtained with solvents of different polarities as scavengers of superoxide radical (O2), hydrogen peroxide (H2O2), hypochlorous acid (HOCl), singlet oxygen (1O2), nitric oxide (NO) and peroxynitrite (ONOO). These effects were then

1741

R.C. Chisté et al. / Food Chemistry 135 (2012) 1740–1749 R

OH

O

O

Antheraxanthin

HO HO

OH

OH

OH R = OH = Gallic acid R without substituents = Galloyl group

RO

Zeaxanthin

HO

O O

OH

OR

RO

O O OR O

Neoxanthin

R = H = Ellagic acid R also could be glucose, arabinose or rhamnose

OH HO

Fig. 1. Chemical structures of gallic acid (C7H6O5), ellagic acid (C14H6O8), antheraxanthin (C40H56O3), zeaxanthin (C40H56O2) and neoxanthin (C40H56O4) found in Caryocar villosum pulp.

compared to the yields of phenolic compounds and carotenoids attained in each C. villosum extract, determined by high performance liquid chromatography, using the diode array detector (HPLC– DAD).

into halves and the shells manually removed from the pulp and seeds. The yellow-coloured pulp was separated from the seeds, weighed, ground and immediately freeze-dried. The lyophilized material was thoroughly mixed, vacuum-packed and stored under light-free conditions at 36 °C prior to analysis.

2. Materials and methods 2.1. Chemicals Ethanol, ethyl acetate and methanol (PA) were obtained from Synth (São Paulo, Brazil). Methanol and methyl tert-butyl ether (MTBE) of chromatographic grade were obtained from J.T. Baker (Phillipsburg, USA) and ultrapure water from the Millipore system (Billerica, USA). Formic acid was purchased from Merck (Darmstadt, Germany). Dihydrorhodamine 123 (DHR), 4,5-diaminofluorescein (DAF-2), 30% hydrogen peroxide, sodium hypochlorite solution with 4% available chlorine, 3-(aminopropyl)-1-hydroxy3-isopropyl-2-oxo-1-triazene (NOC-5), b-nicotinamide adenine dinucleotide (NADH), phenazine methosulfate (PMS), nitroblue tetrazolium chloride (NBT), histidine, lucigenin, quercetin and bcarotene were obtained from Sigma–Aldrich (St. Louis, USA). Gallic acid and ellagic acid were purchased from Extrasynthèse (Lyon Nord, France). All-trans-zeaxanthin and all-trans-lutein were provided by DSM Nutritional Products (Basel, Switzerland) and 9cis-neoxanthin, all-trans-violaxanthin, all-trans-antheraxanthin acquired from CaroteNature (Lupsingen, Switzerland). All other chemical salts and solvents of analytical grade were purchased from Synth (São Paulo, Brazil) or Merck (Darmstadt, Germany). For chromatographic analysis, samples and solvents were filtered using, respectively, membranes of 0.22 and 0.45 lm, both from Millipore (Billerica, USA). 2.2. C. villosum samples The C. villosum fruits (piquiá) were acquired at the ‘‘Ver-O-Peso’’ market in Belém, Pará State, Brazil (Latitude 01°270 2100 S and Longitude 48°300 1600 W) in March, 2010. All ripe fruits (9 kg) were cut

2.3. Preparation of C. villosum extracts Extractions were performed in triplicate, following a completely randomized design, using the following solvents: water, ethanol/ water (1:1, v/v), ethanol, ethanol/ethyl acetate (1:1, v/v) and ethyl acetate. These solvents were chosen, considering the permissibility of residues in the extracts after evaporation, according to the Commission Directive 95/45/EC from European Communities (1995). The freeze-dried pulp of C. villosum was weighed (5 g for each extract) and the solvents were added at a mass:solvent ratio of 1:8 (w/v), stirred on an orbital shaker MA 140/CFT (Marconi, São Paulo, Brazil) at 168 rpm for 15 h at room temperature (25 °C) and protected against luminosity. After the extraction procedure, the extracts were vacuum-filtered and the residues in the filter were washed with the respective solvent (5 ml). The filtered extracts were transferred to volumetric flasks (50 ml) and filled with the respective solvent. Each liquid extract was frozen with liquid nitrogen and subsequently lyophilised (Liobras, São Paulo, Brazil). The freeze-dried extracts were transferred to amber flasks, sealed under N2 flow and stored at 36 °C prior to analysis.

2.4. Chromatographic analysis of carotenoids and phenolic compounds HPLC–DAD analysis of carotenoids and phenolic compounds was performed in a Shimadzu HPLC (Kyoto, Japan) equipped with quaternary pumps (LC-20AD), a degasser unit (DGU-20A5), a Rheodyne injection valve with a 20 ll loop and a diode array detector (DAD) (SPD-M20A). The equipment was also connected, in series, to a mass spectrometer (MS/MS) from Bruker Daltonics (Esquire 4000 model, Bremen, Germany) with APCI (Atmospheric Pressure

1742

R.C. Chisté et al. / Food Chemistry 135 (2012) 1740–1749

Chemical Ionization), and ESI (Electrospray Ionization) sources, and an ion-trap analyzer. For the quantification and identification of carotenoids, the freeze-dried extracts of C. villosum pulp (0.1 g) were further extracted by agitation with acetone (2 ml) in a vortex for 1 min, followed by centrifugation at 3864g for 5 min at 20 °C. After centrifugation, the supernatant was transferred to another flask. The extraction was exhaustively repeated until the residue was colourless; typically 5–10 extractions were necessary. All supernatants were combined 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 KOH 10% in methanol (1:0.5, v/ v), re-partitioned, evaporated under vacuum (T < 38 °C), re-suspended in MeOH/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 MeOH/MTBE from 95:5 to 70:30 in 30 min, followed by 50:50 in 20 min. The flow rate was 0.9 ml/min and the column temperature was set at 29 °C. The separation and identification of carotenoids from C. villosum pulp extracts by HPLC–DAD– APCI–MS/MS was performed according to the procedure previously described by De Rosso and Mercadante (2007) for carotenoids from Amazonian fruits. The carotenoids were quantified by HPLC–DAD, using external seven-point analytical curves (in duplicate) for 9cis-neoxanthin (0.9–17.1 lg/ml), all-trans-violaxanthin (0.7– 13.6 lg/ml), all-trans-antheraxanthin (0.8–15.9 lg/ml), all-translutein (1.0–59.5 lg/ml), all-trans-zeaxanthin (1.3–59.7 lg/ml) and all-trans-b-carotene (1.1–30.2 lg/ml). All other carotenoids were estimated using the curve of zeaxanthin or using the corresponding all-trans-carotenoid. For all the carotenoids, R2 = 0.99 and the limit of detection was 0.1 lg/ml and the limit of quantification was 0.5 lg/ml, obtained from the analytical curves (standard deviation and the slope) (ICH, 2005). The NAS-IOM (2001) conversion factor was used to calculate the vitamin A value, with 12 lg of dietary all-trans-b-carotene corresponding to 1 lg of retinol activity equivalent (RAE), and the activity used was 100% for all-trans-b-carotene. For the quantification and identification of phenolic compounds, freeze-dried extracts of C. villosum pulp (0.1 g) were further extracted with methanol/water (8:2, v/v) (1 ml) in an ultrasound apparatus (Unique, São Paulo, Brazil) for 5 min at 25 °C. The solution was centrifuged at 2683g for 5 min at 20 °C and the supernatant was transferred to a volumetric flask. The extraction was repeated five times and all supernatants were combined in order to obtain 5 ml as the final volume. The liquid extract was kept in the freezer for 20 min before centrifugation at 290g for 20 min at 20 °C. An aliquot of the centrifuged liquid extract was directly injected into the chromatographic system. The phenolic compounds were separated on a C18 Synergi Hydro column (4 lm, 250  4.6 mm, Phenomenex) at 0.9 ml/min of flow, column temperature set at 29 °C, with a mobile phase consisting of water/ formic acid (99.5:0.5, v/v) (solvent A) and acetonitrile/formic acid (99.5:0.5, v/v) (solvent B) in gradient from A:B 99:1 to 50:50 in 50 min, then from 50:50 to 1:99 in 5 min. This latter ratio (1:99) was maintained for an additional 5 min. The conditions and parameters used for the separation and identification of phenolic compounds from the extracts of C. villosum pulp by HPLC–DAD– ESI-MS/MS were set according to the procedure previously described by Chisté and Mercadante (2012) for C. villosum pulp. Phenolic compounds were quantified by comparison to external standards using seven-point analytical curves (in duplicate) for gallic acid (0.5–51.5 lg/ml), 4-coumaric acid (0.5–49.5 lg/ml), ellagic acid (0.5–52 lg/ml) and methyl quercetin (0.2–19.2 lg/ml). All other phenolic compounds were estimated using the curve of the corresponding aglycone. In all cases, the R2 was 0.99 and the

limit of detection was 0.1 lg/ml and the limit of quantification was 0.4 lg/ml, obtained from the analytical curves (standard deviation and the slope) (ICH, 2005). 2.5. ROS- and RNS-scavenging assays 2.5.1. General A microplate reader (Synergy HT, Biotek, Vermont, USA), for fluorescence, UV/vis and chemiluminescence measurements, equipped with a thermostat, was used for all the assays. Each ROS- and RNS-scavenging result corresponds to four experiments, five concentrations and was performed in duplicate. The C. villosum extracts were dissolved in acetone/DMSO (1:9, v/v) for all ROS- and RNS-scavenging assays, excepting for the extracts in the HOCl assay (dissolved in phosphate buffer or ethanol), while the gallic acid, ellagic acid and zeaxanthin standards were dissolved in ethanol. The IC50 values were calculated from the curves of percentage of inhibition versus antioxidant concentration, using the GraphPad Prism 5 software. Quercetin was used as positive control in the scavenging assays of O2, H2O2, HOCl, 1O2, NO, ONOO and its values were similar to those previously reported by Chisté et al. (2011). No direct effect was observed between the tested solvents and all ROS and RNS under the present assay conditions. In each assay, additional experiments were performed in order to verify the possible interference effects of the C. villosum extracts or the tested chemical compounds (standards) with the methodology used. 2.5.2. Superoxide radical-scavenging assay The O2 was generated by the NADH/PMS/O2 system and the  O2 -scavenging capacity was determined by monitoring the effect of the extracts and standards on the O2-induced reduction of NBT at 560 nm after 2 min (Chisté et al., 2011). The effects were expressed as the inhibition, in percentage, of the NBT reduction to diformazan. 2.5.3. Hydrogen peroxide-scavenging assay The H2O2-scavenging capacity was measured 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. 2.5.4. Hypochlorous acid-scavenging assay The HOCl-scavenging capacity was measured by monitoring the effect of the extracts and standards on HOCl-induced oxidation of DHR to rhodamine 123, as previously described (Chisté et al., 2011). 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. 2.5.5. Singlet oxygen-scavenging assay The 1O2-scavenging capacity was measured by monitoring the effect of the extracts and standards on the oxidation of non-fluorescent DHR to fluorescent rhodamine 123 by this ROS, as previously described (Chisté et al., 2011). 1O2 was generated by the thermal decomposition of a previously synthesized water-soluble endoperoxide NDPO2 (disodium 3,30 -(1,4-naphthalene) bis-propionate) (Costa et al., 2007). The results were expressed in percentage as the inhibition of 1O2-induced oxidation of DHR. 2.5.6. Nitric oxide-scavenging assay The NO-scavenging capacity was measured by monitoring the effect of the extracts and standards on NO-induced oxidation of

1743

R.C. Chisté et al. / Food Chemistry 135 (2012) 1740–1749

non-fluorescent DAF-2 to the fluorescent triazolofluorescein (DAF2T) (Chisté et al., 2011). NO was generated by decomposition of NOC-5. The results were expressed as the percentage of inhibition of NO-induced oxidation of DAF-2. 2.5.7. 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 (Chisté et al., 2011). ONOO was synthesized as previously described by Beckman, Chen, Ischiropoulos, and Crow (1994). 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. This evaluation is important because, under physiological conditions, the reaction between ONOO and bicarbonate is predominant, with a very fast rate constant (k = 3–5.8  104 M1 s1) (Whiteman, Ketsawatsakul, & Halliwell, 2002). The results were expressed in percentage, as the inhibition of ONOO-induced oxidation of DHR. 3. Results and discussion 3.1. Contents of phenolic compounds and carotenoids in the freezedried extracts of C. villosum The HPLC analysis of the freeze-dried extracts of C. villosum, obtained with different solvents of different polarities, allowed the separation and quantification (dry basis) of 17 phenolic compounds and 12 carotenoids. The contents of both total phenolic compounds and carotenoids seem to be dependent on the solvent polarity; i.e., the highest values of phenolic compounds were found in the most polar solvents, ethanol/water and water extracts (5163 and 1704 lg/g, respectively), followed by the less polar solvents, ethanol, ethanol/ethyl acetate and ethyl acetate extracts (Table 1). On the other hand, the highest levels of carotenoid were found in the intermediate polar solvent: ethanol (63.9 lg/g), followed by the less polar solvents: ethanol/ethyl acetate and ethyl acetate; and finally by the most polar ones: water and ethanol/water (Table 2). The profile of phenolic compounds (Fig. 2) showed that ellagic acid was the major phenolic compound found in ethanol/water

(2021 lg/g) and water (376 lg/g) extracts, whilst gallic acid was the major one in ethanol (133 lg/g), ethanol/ethyl acetate (52.8 lg/g) and ethyl acetate (26.5 lg/g) extracts, as can be seen in Table 1. In relation to the profile of carotenoids (Fig. 3), the lutein-like carotenoid was the major one in ethanol (12.2 lg/g) and ethanol/ethyl acetate (5.1 lg/g) extracts, whilst zeaxanthin was the major one found in water (4.5 lg/g), ethyl acetate (4.3 lg/g) and ethanol/water (0.5 lg/g) extracts (Table 2). The structure of lutein-like carotenoid (peak 4, Table 2) could not be identified by the HPLC–DAD–MS/MS analysis. However, some important structural information was obtained; the kmax at 445 nm in MeOH/MTBE, with spectral fine structure (%III/II) of 55 and no cis peak can be attributed to a compound with 10 conjugated double bonds (Chisté & Mercadante, 2012). Moreover, the presence of at least 3 hydroxyl groups was indicated by the MS spectrum features; the protonated molecule ion was not detected, whereas a strong in-source fragmentation was observed at m/z 583 ([M+H–18]+), which corresponds to the neutral loss of H2O, a characteristic attributed to the presence of hydroxyl group. The MS/MS spectrum showed fragments at m/z 565 ([M+H–18–18]+) and at m/z 547 ([M+H–18–18– 18]+), which corresponds to the consecutive neutral losses of two and three H2O molecules, respectively (Chisté & Mercadante, 2012). For vitamin A activity, a carotenoid must have at least one unsubstituted b-ionone ring with an attached polyene side chain of at least eleven carbons and, thus, among the identified carotenoids of C. villosum extracts, only b-carotene possesses vitamin A activity. Since the ethanol extract presented the highest content of b-carotene, its vitamin A value was the highest one (0.13 lg RAE/g), followed by water, ethanol/ethyl acetate and ethyl acetate, both with the same value.

3.2. Scavenging of ROS and RNS by C. villosum extracts The results obtained in this study show that C. villosum extracts present high scavenging potency for ROS and RNS. In a general view, the water and ethanol/water extracts showed the lowest IC50 values, at low concentrations, for HOCl, NO and ONOO (with or without NaHCO3) (Tables 3 and 4). All tested standards of phenolic compounds, namely gallic acid, ellagic acid and quercetin

Table 1 Content (lg/g) of phenolic compounds in the freeze-dried extracts of Caryocar villosum pulp obtained by solvents with different polarities. Peaksa

tR Range (min)b

Compound

[MH] (m/z)c

Caryocar villosum extracts (n = 3) H2O

EtOH/H2O

EtOH

EtOH/EtOAc

EtOAc

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

8.6–8.8 9.9–10.1 12.6–12.8 12.9–13.1 16.3–16.5 17.0–17.2 17.6–17.8 19.9–20.0 21.3–21.5 23.8–23.9 24.5–24.7 25.4–25.6 25.9–26.1 26.4–26.6 28.4–28.6 29.4–29.6 29.9–30.1

Monogalloyl hexoside1 Gallic acid1 HHDP hexoside1 Coumaroyl–galloyl hexoside2 Coumaroyl quinic acid2 HHDP dihexoside1 Di-galloyl hexoside1 Galloyl-HHDP hexoside1 Ellagic acid hexoside3 Ellagic acid pentoside3 Ellagic acid deoxyhexoside3 Not identified1 Ellagic acid3 Methyl ellagic acid pentoside3 Methyl ellagic acid deoxyhexoside3 Methyl quercetin dihexoside4 Di-methyl ellagic acid pentoside3 Total phenolic (lg/g extract)

331 169 481 477 337 625 483 633 463 433 447 495 301 447 461 639 461

162 ± 4.9 176 ± 15.9 109 ± 3.3 25.8 ± 0.2 20.7 ± 1.3 80.4 ± 1.5 80.7 ± 1.1 73.8 ± 2.7 61.7 ± 7.8 nd 276 ± 12.7 141 ± 3.3 376 ± 33.2 nd 74.4 ± 2.4 34.0 ± 0.8 54.8 ± 1.2 1745 ± 42.4

127 ± 26.9 827 ± 100 118 ± 2.4 29.8 ± 0.9 23.2 ± 3.3 nd 89.2 ± 3.4 88.4 ± 3.9 180 ± 3.9 70.4 ± 17.2 1150 ± 20.1 198 ± 5.7 2021 ± 168 nd 85.7 ± 8.3 48.4 ± 0.1 107 ± 5.9 5163 ± 161

40.6 ± 1.7 133 ± 8.3 39.0 ± 0.7 nd 13.1 ± 1.4 nd 38.3 ± 0.8 38.6 ± 0.8 nd nd 41.9 ± 4.2 43.9 ± 1.1 84.0 ± 2.4 nd nd 14.1 ± 0.2 nd 486 ± 25.6

24.8 ± 0.7 52.8 ± 2.5 nd nd nd nd nd nd nd nd 21.6 ± 1.0 26.2 ± 0.6 37.8 ± 2.8 nd nd 8.7 ± 0.2 nd 172 ± 7.7

nd 26.5 ± 0.5 nd nd nd nd nd nd nd nd 17.2 ± 0.4 24.4 ± 0.5 18.5 ± 0.4 nd nd nd nd 86.6 ± 1.7

The peaks were quantified as equivalent of gallic acid1, coumaric acid2, ellagic acid3 and methyl quercetin4. nd, not detected. a Peaks numbered according to the Fig. 2. b Retention time on the C18 Synergi Hydro (4 lm) column. HHDP, hexahydroxydiphenoyl. c The identification of phenolic compounds in the extracts was based on our previous study (Chisté & Mercadante, 2012).

1744

R.C. Chisté et al. / Food Chemistry 135 (2012) 1740–1749

Table 2 Contents (lg/g) of carotenoids in freeze-dried extracts of Caryocar villosum pulp obtained by solvents of different polarities. Peaksa

tR Range (min)b

1 2 3 4 5 6 7 8 9 10 11 12

5.6–5.7 all-trans-Neoxanthin1 6.5–6.6 9-cis-Neoxanthin1 7.2–7.4 all-trans-Violaxanthin2 7.5–7.6 Lutein-like5 8.5–8.6 9’-cis-Neoxanthin1 10.1–10.2 all-trans-Antheraxanthin3 10.6–10.7 9-cis-Violaxanthin2 12.1–12.2 all-trans-Lutein5 12.8–12.9 9-cis-Mutatoxanthin4 14.3–14.4 all-trans-Zeaxanthin4 16.1–16.2 9-cis-Antheraxanthin3 33.6–33.7 all-trans-b-Carotene6 Total carotenoids (lg/g extract) Vitamin A value (lg RAE/g extract)

Compound

[M+H]+ (m/z)c

Caryocar villosum extracts (n = 3) H2O

EtOH/H2O

EtOH

EtOH/EtOAc

EtOAc

601 601 601 583d 601 585 601 551d 585 569 585 537

1.33 ± 0.22 1.38 ± 0.22 0.68 ± 0.03 4.00 ± 0.84 1.14 ± 0.33 1.94 ± 0.24 0.84 ± 0.05 1.87 ± 0.14 2.38 ± 0.24 4.46 ± 0.49 1.41 ± 0.03 0.97 ± 0.05 22.4 ± 2.77 0.08 ± 0.00

0.14 ± 0.02 0.13 ± 0.01 0.23 ± 0.02 0.42 ± 0.01 0.23 ± 0.05 0.40 ± 0.05 0.17 ± 0.00 0.37 ± 0.03 0.41 ± 0.02 0.48 ± 0.05
8.52 ± 0.56 4.46 ± 0.19 1.99 ± 0.07 12.2 ± 0.13 5.76 ± 0.77 7.21 ± 0.49 1.22 ± 0.12 3.92 ± 0.17 4.03 ± 0.11 11.3 ± 0.19 1.91 ± 0.02 1.51 ± 0.22 64.0 ± 0.39 0.13 ± 0.02

3.13 ± 0.18 1.93 ± 0.54 1.73 ± 0.16 5.09 ± 0.27 2.40 ± 0.33 4.72 ± 0.51 0.98 ± 0.03 1.98 ± 0.03 1.51 ± 0.08 4.98 ± 0.33 1.63 ± 0.08 0.53 ± 0.25 30.6 ± 0.76 0.04 ± 0.02

1.82 ± 0.81 1.71 ± 0.71 1.18 ± 0.22 4.18 ± 0.82 1.72 ± 0.66 2.92 ± 0.93 0.82 ± 0.16 1.77 ± 0.41 1.58 ± 0.32 4.26 ± 0.83 1.43 ± 0.16 0.44 ± 0.08 23.8 ± 6.12 0.04 ± 0.01

The peaks were quantified as equivalent of 9-cis-Neoxanthin1, violaxanthin2, antheraxanthin3, zeaxanthin4, lutein5 and b-carotene6. nc, not calculated since the content of bcarotene was lower than the limit of quantification. a Numbered according to the chromatogram shown in Fig. 3. b Retention time on the C30 column. c The identification of carotenoids in the extracts was based on a previous study (Chisté & Mercadante, 2012). d In-source detected fragment [M+H–18]+. RAE, retinol activity equivalent.
60

6 1 2

16

5 3

7

0

Detector response at 271 nm (AU)

11 13

4 8

12

9

H2O extract

15 17

13

250

11 5 7

3

1

0

16

4

2

8 9

10 12

EtOH/H2O extract

15

2

20 1

0 10

1

5 7

3

13 11 12

8

EtOH extract

16

13

2

11 12

0

EtOH/EtOAc extract

16

12

5 2

0 5

10

11 13

15

20

25

EtOAc extract 30

35

40

45

50

Time (min) Fig. 2. HPLC-DAD chromatogram of phenolic compounds for all Caryocar villosum extracts. Chromatographic conditions: see Section 2.4. Processed at 271 nm and the peak numbers correspond to the phenolic compounds of Table 1.

(positive control), scavenged the ROS and RNS in a concentrationdependent manner, also at low concentrations. Possible interferences of solvents, extracts or standards and the reagents used (probe or reactive species generator) were thoroughly evaluated in all ROS and RNS assays. This procedure ensures that the results obtained are not flawed by any interference, such as fluorescence/chemiluminescence/absorbance response of the tested compounds, quenching of probe fluorescence/chemiluminescence or the possible direct redox effect of the tested compounds on the probes. None of the solvents, extracts or phenolic standards showed interference in the results with the applied methodologies. However, the carotenoid zeaxanthin, used as standard, presented several interference issues. First of all, zeaxanthin, a carotenoid with two hydroxyl groups (Fig. 1), did not present good solubility when added to buffer aqueous solutions,

especially at high concentrations. The highest possible dilution was 8.5 lg/ml but, even at this low concentration, zeaxanthin (425, 450, 476 nm) interfered with the assays by quenching fluorescence excitation energy (485 nm) of DHR and the chemiluminescence emission energy (453–455 nm) of lucigenin. Thus, in the systems with zeaxanthin, the values of scavenging were overestimated since the response of fluorescence/chemiluminescence showed a decrease in the signal due to the energy quenching of zeaxanthin and not because of the antioxidant capacity per se. Additionally, when at low concentrations, in which the quenching interference effect was no longer present, the scavenging effect was not detected. Considering that the percentage of interference provided by zeaxanthin was found to be between 15% and 30%, the results of the scavenging capacity of zeaxanthin were not taken into consideration to explain the scavenging behaviour of the C.

1745

R.C. Chisté et al. / Food Chemistry 135 (2012) 1740–1749

30

1 2

H2O extract

7

34 5

6

10

8 9

11

Detector response at 450 nm (AU)

0 4

15

7

0

5

1

8 9 7

3 4

60

6

6 2

EtOH/H2O extract

10

3 1 2

12

EtOH extract

10 89

5

11

0

12

7

30

3 4 1

0 30

2

6

34 1 2 5

11

12

7 6

EtOAc extract

10 89

11

0 5

EtOH/EtOAc extract

10 8 9

5

10

15

12

20

25

30

35

40

Time (min) Fig. 3. HPLC-DAD chromatogram of carotenoids for all Caryocar villosum extracts. Chromatographic conditions: see Section 2.4. Processed at 450 nm and the peak numbers correspond to the carotenoids of Table 2.

Table 3 Superoxide radical (O2)-, hydrogen peroxide (H2O2)-, hypochlorous acid (HOCl)scavenging and singlet oxygen (1O2)-quenching capacities of freeze-dried extracts of Caryocar villosum and standard compounds. Extracts/compounds

IC50 (lg/ml) (n = 4) O2

H2O2

HOCl

1

Extracts H2O EtOH/H2O EtOH EtOH/EtOAc EtOAc

15*± 5a 37*± 8a NA NA NA

19*± 4b 23*± 2b 10*± 4b 9*± 4b 4*± 2b

6.3 ± 2.3 3.6 ± 1.0 199 ± 29 299 ± 49 48*± 5c

156 ± 11 74 ± 8 38* ± 5d 39* ± 3d 12* ± 5d

Standard compounds Gallic acid Ellagic acid Quercetin

13 ± 2 19 ± 3 14 ± 4

226 ± 37 108 ± 9 290 ± 8

0.4 ± 0.1 1.7 ± 0.2 0.10 ± 0.03

1.00 ± 0.06 4.1 ± 0.9 0.8 ± 0.2

Table 4 Nitric oxide (NO)- and peroxynitrite (ONOO)-scavenging capacities of Caryocar villosum extracts and chemical standard compounds. Extracts/ compounds

IC50 (lg/ml) (n = 4) 

NO

O2

IC50, Inhibitory concentration, in vitro, to decrease (by 50%) the oxidative effect of reactive species in the tested media (mean ± standard error). H2O, distilled water; EtOH/H2O, ethanol/water, EtOH, ethanol; EtOH/EtOAc, ethanol/ethyl acetate; EtOAc, ethyl acetate. NA, no activity was found up to the highest tested concentration (833 lg/ml). * Scavenging effect (%) (mean ± standard error) at the highest concentration (a = 833 lg/ml, b = 1000 lg/ml, c = 500 lg/ml and d = 600 lg/ml) which presented inhibition against the reactive specie.

villosum extracts. In fact, the scavenging capacity of all extracts seemed to be closely dependent on the phenolic compound contents. However, all the tested standards of phenolic compounds presented lower IC50 values than did all C. villosum extracts. The ethanol/water extract, which presented the highest phenolic compounds content (5163 lg/g), could inhibit the oxidizing effect of O2 at only 37% at the highest tested concentration (833 lg/ ml) and the IC50 value was not achieved (Table 3). Also, at the same highest concentration, the water extract presented only 15% of inhibition against O2. Both extracts were obtained with the most polar solvents and were characterized to contain ellagic acid as the major phenolic compound, followed by ellagic acid rhamnoside and gallic acid. In fact, the IC50 value of ellagic acid (19.5 lg/ml) was higher than was that of gallic acid (12.9 lg/ml) and that of

Extracts H2O EtOH/H2O EtOH EtOH/EtOAc EtOAc

4.8 ± 0.6 2.8 ± 0.8 82 ± 16 142 ± 2 54 ± 6

Standard compounds Gallic acid 0.12 ± 0.01 Ellagic acid 0.59 ± 0.06 Quercetin 0.05 ± < 0.01

ONOO Absence of NaHCO3

Presence of 25 mM NaHCO3

17.0 ± 0.8 4.8 ± 0.5 181 ± 27 386 ± 94 45* ± 5a

11 ± 1 3.2 ± 0.3 222 ± 61 293 ± 34 30* ± 2a

0.05 ± 0.01 0.05 ± 0.02 0.05 ± < 0.01

0.04 ± 0.01 0.10 ± 0.02 0.07 ± 0.01

IC50, inhibitory concentration, in vitro, to decrease (by 50%) the oxidative effect of reactive species in the tested media (mean ± standard error). H2O, distilled water; EtOH/H2O, ethanol/water; EtOH, ethanol; EtOH/EtOAc, ethanol/ethyl acetate; EtOAc, ethyl acetate. * Scavenging effect (%) (mean ± standard error) at the highest concentration (a = 500 lg/ml) which presented inhibition against the reactive specie.

quercetin (14.4 lg/ml, used as positive control). Moreover, the ethanol, ethanol/ethyl acetate and ethyl acetate extracts, which presented the lowest phenolic compound contents and the highest carotenoid contents, did not present any activity up to the highest tested concentration. In agreement with these results, Chisté et al. (2011) reported no scavenging effect against O2 for annatto extracts obtained with the same solvents (water, ethanol/water, ethanol, ethanol/ethyl acetate and ethyl acetate), and the chemical standard of bixin presented a lower scavenging capacity (IC50 of 26 lg/ml) than did gallic and ellagic acids. O2 is formed enzymatically, and also chemically from triplet oxygen. One of the enzymes that produces O2 is xanthine oxidase, which acts on xanthine or hypoxanthine in the presence of molecular triplet oxygen to produce O2. This ROS is the precursor of a variety of powerful oxidants and is rela-

1746

R.C. Chisté et al. / Food Chemistry 135 (2012) 1740–1749

tively unreactive toward most biological substrates, whereas it is rapidly dismutated into H2O2, either spontaneously, especially at low pH, or through enzyme-catalysis by superoxide dismutase. In relation to the H2O2-scavenging property, the ethanol/water and water extracts were the most effective ones (23% and 19% of inhibition, respectively), against the lucigenin oxidation at the highest tested concentration (1000 lg/ml) (Table 3); however, the IC50 value was not achieved for all the C. villosum extracts. The H2O2-scavenging capacity of ellagic acid (IC50 of 108.3 lg/ml) was higher than that of gallic acid (226 lg/ml) and that of quercetin (290 lg/ml); however, it was lower than that of ascorbic acid (Abreu et al., 2006; Almeida, Fernandes, Lima, Costa, & Bahia, 2008a, 2008b, 2009a; Almeida, Fernandes, Lima, Valentão, et al., 2009). H2O2 alone is practically harmless, whereas it may spread easily through the cell membranes, for example, the membrane of the nucleus, and can react with transition metals (Cu+ and Fe2+) inside cells (Fenton reaction) to generate the most reactive ROS, namely hydroxyl radical (HO). The ethanol/water and water extracts also showed the highest HOCl-scavenging capacity, probably related to their high contents of phenolic compounds. The IC50 values for these extracts (3.6 and 6.3 lg/ml, respectively), were lower than that found for the tincture of Pedilanthus tithymaloides (113 lg/ml) (Abreu et al., 2006) and higher than those of different annatto extracts (0.3– 1.0 lg/ml) (Chisté et al., 2011). The ethanol and ethanol/ethyl acetate extracts presented low activity as compared to ethanol/water and water extracts, and ethyl acetate extract (with the lowest phenolic compounds content) could inhibit 48% of the oxidizing effect

of HOCl on DHR at the highest tested concentration (500 lg/ml) (Fig. 4). HOCl is the most bactericidal oxidant known to be produced by neutrophils in the event of an inflammatory process, being approximately 100–1000 times more toxic than O2 and H2O2 (Conner & Grisham, 1996). The standards of phenolic compounds showed scavenging capacity against HOCl at very low concentration; i.e., gallic acid presented an IC50 value of 0.4 lg/ml, ellagic acid of 1.7 lg/ml and quercetin (positive control) of 0.1 lg/ml. These values are similar to that reported for lipoic acid, an essential cofactor for mitochondrial enzymes and a naturally occurring antioxidant (IC50 value of 1.2 lg/ml) (Abreu et al., 2006) and bixin standard (0.3 lg/ml) (Chisté et al., 2011). Concerning the quenching capacity of 1O2, the C. villosum extract obtained with ethanol/water was the most effective, with an IC50 value of 73.5 lg/ml, followed by the water extract (156 lg/ml) (Fig. 4). However, the IC50 values of these extracts indicated lower efficiency as 1O2 quenchers than extracts from Castanea sativa, Quercus robur (Almeida et al., 2008b) and Hypericum androsaemum (Almeida, Fernandes, Lima, Costa, et al., 2009). Considering the IC50 values of gallic acid (1.0 lg/ml) and ellagic acid (4.1 lg/ml), they can be considered good 1O2 quenchers, since they presented values similar to the quercetin (0.8 lg/ml, used as positive control), ascorbic acid (3.4 lg/ml) (Almeida et al., 2008a; Almeida, Fernandes, Lima, Costa, et al., 2009) and the carotenoid bixin (1.0 lg/ml) (Chisté et al., 2011). The ethanol, ethanol/ethyl acetate and ethyl acetate extracts could inhibit 38%, 39% and 12%, respectively, the oxidizing effect of 1O2 against DHR, at the highest tested concentration (600 lg/ml). Since these extracts

100

75

50

25 H2O EtOH/H2O

0

0

5

10

15

HOCl scavenging capacity (%)

HOCl scavenging capacity (%)

100

75

50

25

0

20

EtOH EtOH/EtOAc EtOAc

0

150

300

450

600

Caryocar villosum extract concentration (µg/mL)

Caryocar villosum extract concentration (µg/mL)

75

50

25 H2O

1

O2 quenching capacity (%)

100

EtOH/H2O

0

0

50

100

150

200

250

300

Caryocar villosum extract concentration (µg/mL) Fig. 4. Hypochlorous acid (HOCl)-scavenging capacity and singlet oxygen (1O2)-quenching activity of Caryocar villosum extracts. Each point shows the standard error bars and represents the values obtained from four experiments, in five concentrations, performed in triplicate.

1747

R.C. Chisté et al. / Food Chemistry 135 (2012) 1740–1749

2009) and annatto seed extracts (Chisté et al., 2011). Additionally, the IC50 values of ethanol/water and water extracts, for NO-scavenging properties, were similar to the other plant extracts, such as Juglans regia (Almeida et al., 2008a), C. sativa, Q. robur (Almeida et al., 2008b), H. androsaemum (Almeida, Fernandes, Lima, Costa, et al., 2009) and Eucalyptus globulus (Almeida, Fernandes, Lima, Valentão, et al., 2009). On the other hand, the extracts of C. villosum obtained with ethanol, ethanol/ethyl acetate and ethyl acetate, which presented the highest carotenoid contents and the lowest phenolic levels, were the least effective in scavenging NO (Fig. 5). The standards of phenolic compounds showed higher scavenging capacity than did all the C. villosum extracts, with IC50 values of 0.1 lg/ml for gallic acid, 0.6 lg/ml for ellagic acid and 0.05 lg/ml for quercetin (positive control). NO, which is endogenously produced by nitric oxide synthase (NOS) through the conversion of L-arginine to L-citrulline, possesses a short life time and rapidly reacts with surrounding molecules, in the order of seconds. The sustained NO generation can cause tissue injury. The C. villosum extracts obtained with ethanol/water and water also showed the highest efficiency as ONOO scavengers, with and without NaHCO3, probably again due to the high phenolic contents, with lower IC50 values than those of ethanol, ethanol/ethyl acetate and ethyl acetate extracts (Table 4). The ethyl acetate extract, which presented the lowest phenolic content, elicited 45% and 30% of inhibition against ONOO (absence and presence of NaHCO3, respectively), at the highest tested concentration (500 g/ml) (Fig. 5). The ethanol/water and water extracts showed lower IC50 values than did the medicinal tincture from Pedilanthus tithymalo-

were characterized by the highest carotenoid content among the other extracts, and the major carotenoid found was the unidentified compound and zeaxanthin (10 and 11 conjugated double bonds, respectively), this behaviour was not expected. Carotenoids with more than seven conjugated double bonds are able to quench 1 O2 by an efficient energy transfer process, and this ability increases with the increasing number of conjugated double bonds (Di Mascio, Kaiser, & Sies, 1989). These unexpected results are probably due to the applied methodology, which was developed to evaluate extracts and/or chemical compounds with more polar characteristics. 1O2 is a very reactive, diffusing and long-lived, electronically excited state of molecular oxygen with two paired electrons and seems to be responsible for some of the damage inflicted by phagocytes on their target sites, since it reacts with a high number of biological molecules, including membrane lipids to initiate peroxidation. Regarding the RNS-scavenging properties, the higher the phenolic compound contents in C. villosum extracts, the higher is the efficiency in the inhibition of the oxidizing effect of NO and ONOO. The ethanol/water and water extracts, which presented the highest phenolic contents, showed the highest scavenging capacity against NO and ONOO (in NaHCO3 presence or absence) with IC50 values at very low concentrations (Table 4). The efficiency of ethanol/water and water extracts in the scavenging capacity against NO was higher than that of Pedilanthus tithymaloides medicinal tincture (Abreu et al., 2006), extracts of seeds, stems, leaves and petals of Catharanthus roseus (Ferreres et al., 2008), extracts of Sesamum indicum seeds (Visavadiya, Soni, & Dalwadi,

•NO scavenging capacity (%)

100

75

50 H2O EtOH/H2O EtOH EtOH/EtOAc EtOAc

25

0

0

20

150

300

450

600

Caryocar villosum extract concentration (µg/mL) 100

50

H2O

25

EtOH/H2O EtOH EtOH/EtOAc EtOAc

0 0

15

30

150

300

450

600

Caryocar villosum extract concentration (µg/mL)

-

75

ONOO scavenging capacity (%)

Without NaHCO3

-

ONOO scavenging capacity (%)

100

with NaHCO3

75

50 H2O EtOH/H2O

25

EtOH EtOH/EtOAc EtOAc

0

0

15

30

150

300

450

600

Caryocar villosum extract concentration (µg/mL)

Fig. 5. Nitric oxide (NO), peroxynitrite (ONOO) without NaHCO3 and peroxynitrite (ONOO), with NaHCO3, scavenging capacities of Caryocar villosum extracts. Each point shows the standard error bars and represents the values obtained from four experiments, in five concentrations, performed in triplicate.

1748

R.C. Chisté et al. / Food Chemistry 135 (2012) 1740–1749

ides (Abreu et al., 2006). However, other plant extracts, such as J. regia (Almeida et al., 2008a), C. sativa, Q. robur (Almeida et al., 2008b), H. androsaemum (Almeida, Fernandes, Lima, Costa, et al., 2009) and E. globulus (Almeida, Fernandes, Lima, Valentão, et al., 2009), were reported to present higher scavenging capacity against ONOO (in the presence or absence of NaHCO3) than did all the C. villosum extracts. The standards of phenolic compounds presented very low IC50 values and showed higher efficiency than did ebselen, a mimetic compound of glutathione peroxidase, considered as a potent scavenger of ONOO (Abreu et al., 2006), cysteine (Almeida et al., 2008a, 2008b) and bixin (Chisté et al., 2011). The scavenging capacity against ONOO can be considered a very important ability to be attributed to a natural antioxidant, since NO reacts rapidly with O2 to form ONOO, whose reactivity is similar to that of HO and readily damages biomolecules. Furthermore, C. villosum is also expected to be an effective scavenger of NO2 and CO3 species, since the scavenging effectiveness is maintained in the presence of HCO3. It has been reported that physiological concentrations of CO2 can modulate the ONOO reactivity due to the fast reaction between these two compounds, yielding NO2 and CO3, which are the main radicals responsible for the nitration and oxidation reactions that are usually observed in vivo. Thus, a scavenger can directly trap ONOO only if it reacts faster with the latter than does CO2. On the other hand, a putative scavenging effect on NO2 and CO3 may increase the efficiency of the compounds (Gomes, Costa, Lima, & Fernandes, 2006). The results obtained in the present study clearly demonstrate that the efficiency of C. villosum extracts was closely related to the contents of phenolic compounds, in which the ethanol/water and water extracts are the most effective scavengers against the tested ROS and RNS. In general, the IC50 values for ROS and RNS showed that the C. villosum extracts presented better scavenging capacity for the RNS (NO and ONOO) than for the ROS (O2, H2O2, HOCl and 1O2). The chemical standards of phenolic compounds, namely gallic acid and ellagic acid, which were the major phenolic compounds found in all the C. villosum extracts, were better scavengers when compared to all extracts. Gallic acid and its derivatives have also been found in many phytomedicines with a number of biological and pharmacological activities, including scavenging of free radicals, such as HO (Dwibedy, Dey, Naik, Kishore, & Moorthy, 1999), inducing apoptosis of cancer cells (Saeki et al., 2000). According to Hwang, Cho, Kim, and Lee (2010), ellagic acid exhibits protective properties against oxidative stress-induced hepatocyte damage by preventing vitamin k3-induced ROS production, apoptotic and necrotic cellular damage and mitochondrial depolarization, which is a main cause of ROS production. Moreover, the antioxidant activity (TEAC) of C. villosum pulp was reported to present the highest value among other 18 tropical fruits (Barreto et al., 2009). Magid, Voutquenne-Nazabadioko, Harakat, Moretti, and Lavaud (2008) reported the mushroom tyrosinase inhibitory activity of two phenol-rich fractions of C. villosum with IC50 values varying from 0.98 to 1.86 lg/ml. Furthermore, Almeida et al. (2012) suggested that C. villosum pulp has protective effects against doxorubicin-induced DNA damage in rats, caused by several mechanisms, such as free radicals production, and the antioxidant activity of bioactive compounds found in the pulp could be a response for these protective effects. In summary, ethanol/water and water are the most promising solvents for obtaining C. villosum extracts with the highest efficiency as antioxidants, and the extracts can be used as an easily accessible source of natural antioxidants. This study shows that the scavenging capacities of C. villosum extracts against all tested ROS and RNS were closely related to the content of phenolic compounds. In a perspective for the future, accurate analytical methods to evaluate the scavenging capacity of coloured and lipophilic com-

pounds must be developed in order to understand all aspects related to the antioxidant knowledge of carotenoids against the main ROS and RNS available in physiological systems. Acknowledgement The authors would like to thank FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) for the financial support. References Abreu, P., Matthew, S., González, T., Costa, D., Segundo, M. A., & Fernandes, E. (2006). Anti-inflammatory and antioxidant activity of a medicinal tincture from Pedilanthus tithymaloides. Life Sciences, 78, 1578–1585. Almeida, I. F., Fernandes, E., Lima, J. L. F. C., Costa, P. C., & Bahia, M. F. (2008a). Walnut (Juglans regia) leaf extracts are strong scavengers of pro-oxidant reactive species. Food Chemistry, 106, 1014–1020. Almeida, I. F., Fernandes, E., Lima, J. L. F. C., Costa, P. C., & Bahia, M. F. (2008b). Protective effect of Castanea sativa and Quercus robur leaf extracts against oxygen and nitrogen reactive species. Journal of Photochemistry and Photobiology B-Biology, 91, 87–95. Almeida, I. F., Fernandes, E., Lima, J. L. F. C., Costa, P. C., & Bahia, M. F. (2009). In vitro protective effect of Hypericum androsaemum extract against oxygen and nitrogen reactive species. Basic & Clinical Pharmacology & Toxicology, 105, 222–227. Almeida, I. F., Fernandes, E., Lima, J. L. F. C., Valentão, P., Andrade, P. A., Seabra, R. M., et al. (2009). Oxygen and nitrogen reactive species are effectively scavenged by Eucalyptus globulus leaf water extract. Journal of Medicinal Food, 12, 175–183. Almeida, M. R., Darin, J. D. C., Aissa, A. F., Gomes, T. D. U. H., Mercadante, A. Z., Antunes, L. M. G., & Bianchi, M. L. (2012). Antigenotoxic effects of piquiá (Caryocar villosum) in multiple rat organs. Plant Foods for Human Nutrition, 67, 171–177. Barreto, G. P. M., Benassi, M. T., & Mercadante, A. Z. (2009). Bioactive compounds from several tropical fruits and correlation by multivariate analysis to free radical scavenger activity. Journal of the Brazilian Chemical Society, 20, 1856–1861. Beckman, J. S., Chen, J., Ischiropoulos, H., & Crow, J. P. (1994). Oxidative chemistry of peroxynitrite. Methods in Enzymology, 223, 229–240. Chisté, R. C., Mercadante, A. Z., Gomes, A., Fernandes, E., Lima, J. L. F. C., & Bragagnolo, N. (2011). In vitro scavenging capacity of annatto seed extracts against reactive oxygen and nitrogen species. Food Chemistry, 127, 419–426. Chisté, R. C., & Mercadante, A. Z. (2012). Identification and quantification, by HPLC– DAD–MS/MS, of carotenoids and phenolic compounds from the Amazonian fruit Caryocar villosum. Journal of Agricultural and Food Chemistry, 60, 5884–5892. Clement, C. R. (1993). Piquiá. In J. W. Clay, & C. R. Clement (Eds.), Selected species and strategies to enhance income generation from Amazonian forests (pp. 108–114). Rome: FAO. Commission Directive 95/45/EC (1995) p. 19. Laying down specific purity criteria concerning colours for use in foodstuffs (L226). Official Journal of the European Community. Conner, E. M., & Grisham, M. B. (1996). Inflammation, free radicals and antioxidants. Nutrition, 12, 274–277. Costa, D., Fernandes, E., Santos, J. L. M., Pinto, D. C. G. A., Silva, A. M. S., & Lima, J. L. F. C. (2007). New noncellular fluorescence microplate screening assay for scavenging activity against singlet oxygen. Analytical and Bioanalytical Chemistry, 387, 2071–2081. De Rosso, V. V., & Mercadante, A. Z. (2007). Identification and quantification of carotenoids, by HPLC–PDA–MS/MS, from Amazonian fruits. Journal of Agricultural and Food Chemistry, 55, 5062–5072. Di Mascio, P. D., Kaiser, S., & Sies, H. (1989). Lycopene as the most efficient biological carotenoid singlet oxygen quencher. Archives of Biochemistry and Biophysics, 274, 532–538. Dwibedy, P., Dey, G. R., Naik, D. B., Kishore, K., & Moorthy, P. N. (1999). Pulse radiolysis studies on redox reaction of gallic acid: One electron oxidation of gallic acid by hallic acid OH adduct. Physical Chemistry Chemical Physics, 1, 1915–1918. Ferreres, F., Pereira, D. M., Valentão, P., Andrade, P. B., Seabra, R. M., & Sottomayor, M. (2008). New phenolic compounds and antioxidant potential of Catharanthus roseus. Journal of Agricultural and Food Chemistry, 56, 9967–9974. Gomes, A., Costa, D., Lima, J. L. F. C., & Fernandes, E. (2006). Antioxidant activity of bblockers: An effect mediated by scavenging reactive oxygen and nitrogen species? Bioorganic & Medicinal Chemistry, 14, 4568–4577. Grenand, P., Moretti, C., Jacquemin, H., & Prévost, M. F. (2004). Pharmacopées traditionnelles en guyane, créoles, wayasi, palikur. Paris: IRD, pp. 293–298. Hwang, J. M., Cho, J. S., Kim, T. H., & Lee, Y. I. (2010). Ellagic acid protects hepatocytes from damage by inhibiting mitochondrial production of reactive oxygen species. Biomedicine & Pharmacotherapy, 64, 264–270. International Conference on Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use (ICH) (2005) p. 7-10. Guidance for industry, Q2B validation of analytical procedures: Methodology. Geneva: ICH Secretariat, c/o IFPMA. Krinsky, N. I. (1994). The biological properties of carotenoids. Pure and Applied Chemistry, 66, 1003–1010.

R.C. Chisté et al. / Food Chemistry 135 (2012) 1740–1749 Magid, A. A., Voutquenne-Nazabadioko, L., Harakat, D., Moretti, C., & Lavaud, C. (2008). Phenolic glycosides from the stem bark of Caryocar villosum and C. glabrum. Journal of Natural Products, 71, 914–917. NAS-IOM (2001). Dietary reference intakes for vitamin A, vitamin K, arsenic, boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc p. 92. Washington: National Academy Press. Passos, X. S., Castro, A. C. M., Pires, J. S., Garcia, A. C.-F., Campos, F.-C., Fernandes, O. F. L., et al. (2003). Composition and antifungal activity of the essential oils of Caryocar brasiliensis. Pharmaceutical Biology, 41, 319–324. Pianovski, A. R., Vilela, A. F. G., Silva, A. A. S., Lima, C. G., Silva, K. K., Carvalho, V. F. M., et al. (2008). Use of pequi oil (Caryocar brasiliense) in cosmetics emulsions: Development and evaluate of physical stability. Brazilian Journal of Pharmaceutical Sciences, 44, 249–259. Rios, A. O., Mercadante, A. Z., & Borsarelli, C. D. (2007). Triplet state energy of the carotenoid bixin determined by photoacoustic calorimetry. Dyes and Pigments, 74, 561–565.

1749

Saeki, K., You, A., Isemura, M., Abe, I., Seki, T., & Noguchi, H. (2000). Apoptosis inducing activity of lipid derivatives of gallic acid. Biological & Pharmaceutical Bulletin, 23, 1391–1394. Serdula, M. K., Byers, T., Mokdad, A. H., Simões, E., Mendlein, J. M., & Coates, R. J. (1996). The association between fruit and vegetable intake and chronic disease risk factors. Epidemiology, 7, 161–165. Visavadiya, N. P., Soni, B., & Dalwadi, N. (2009). Free radical scavenging and antiatherogenic activities of Sesamum indicum seed extracts in chemical and biological model systems. Food and Chemical Toxicology, 47, 2507– 2515. Whiteman, M., Ketsawatsakul, U., & Halliwell, B. (2002). A reassessment of the peroxynitrite scavenging activity of uric acid. Annals of the New York Academy of Sciences, 962, 242–259. Xavier, W. K. S., Junior Medeiros, B., Lima, C. S., Favacho, H. A., Andrade, E. H. A., et al. (2011). Topical anti-inflammatory action of Caryocar villosum oil (Aubl) Pers. Journal of Applied Pharmaceutical Science, 01, 62–67.