Lutein bioaccessibility in casein-stabilized emulsions is influenced by the free to acylated carotenoid ratio, but not by the casein aggregation state.

Considering that carotenoids are found acylated to fatty acids in most edible fruits, the influence of the ratio of free to acylated lutein on the hydrolysis extent and bioaccessibility was evaluated by in vitro digestion. For this purpose, for the first time, esterified, free, or a mixture of both carotenoid forms was used in the lipid phase of emulsions stabilized by sodium caseinate (NaCas) and native phosphocaseinate (PPCN). Marigold petals was used as a source of lutein-rich extracts. The emulsions were characterized and the extent of ester hydrolysis, carotenoid recovery, and bioaccessibility were evaluated by LC-DAD-MS/MS. Besides low polydispersity, NaCas and PPCN stabilized emulsions exhibited a constant mean droplet diameter of about 260 and 330 nm, respectively, after 7 days. Caseins were completely digested after the gastric digestion step. Moreover, casein supramolecular structure did not significantly affect carotenoid bioaccessibility. Lutein was majorly found in its free form in all bioaccessible fractions. The carotenoid bioaccessibility increased from 3% to 40% by increasing the percentage of free carotenoids from 0.5 to 100% in the emulsions; but the carotenoid recovery and hydrolysis extent of lutein esters were not affected. In conclusion, emulsion-based systems for carotenoid delivery stabilized either by NaCas or PPCN provided similar carotenoid bioaccessibility. Furthermore, bioaccessibility was inversely dependent on the overall hydrophobicity of the carotenoid extract. Our results suggest that the low bioaccessibility of esterified carotenoids was a consequence of their limited hydrolysis extent. This study provides information that may help design emulsion-based systems stabilized by food protein as a vehicle for carotenoids.

A guide for the evaluation of in vitro bioaccessibility of carotenoids.

Ongoing efforts to improve the nutritional content of carotenoids in food sources to solve global health problems must also consider bioaccessibility and bioavailability of carotenoids. Carotenoid bioaccessibility encompasses carotenoid release from the food matrix, solubilization in lipid emulsion droplets, and transfer to mixed micelles for further absorption into the intestine. Bioavailability refers to the fraction of carotenoids which enters into circulation. To understand the mechanisms controlling bioaccessibility, in vitro digestion methods have been developed and are a useful approach to overcome some of the disadvantages associated with in vivo protocols. The INFOGEST protocol comprises three phases (oral, gastric and intestinal) to simulate adult human digestion of foods, as an international consensus for the conditions of static in vitro digestion. The last steps in any digestion protocol involve isolation of the target food compounds and subsequent analysis, steps not included in the INFOGEST protocol because they may differ depending on specific physical-chemical characteristics and quantification analysis of the target analyte. In this chapter, we describe the INFOGEST protocol which has been adapted for the analysis of carotenoid bioaccessibility, including determination and calculation of enzyme activities according to INFOGEST guidelines, removal of undigested material, collection of mixed micelles containing carotenoids, extraction, storage and subsequent identification and quantification by liquid chromatography analysis of both free and acylated carotenoids.

Addition of either gastric lipase or cholesterol esterase to improve both ß-cryptoxanthin ester hydrolysis and micellarization during in vitro digestion of fruit pulps.

Using the INFOGEST in vitro digestion protocol adapted to carotenoids, the impact of additional rabbit gastric lipase (RGL) on the hydrolysis extent of ß-cryptoxanthin esters was evaluated for the first time, and compared with the addition of porcine cholesterol esterase (CEL). Both the modifications increased the hydrolysis of (all-E)-ß-cryptoxanthin esters from mandarin and peach pulps, although the outcomes were different. Addition of RGL consistently increased the average hydrolysis extent from 55.2% to 59.5% in mandarin pulp and from 22.7% to 48.8% in peach pulp (p < 0.05). The addition of CEL produced lower hydrolysis extents, i.e., 58.5% in mandarin (not statistically significant) and 28.4% in peach (p < 0.05), compared to those obtained with RGL. The hydrolysis extent positively correlated with the carotenoid ester concentration in both matrices. Bioaccessibility values were higher in mandarin pulp (range 32-34%) compared to those in peach pulp (range 16-21%), and were associated with the hydrolysis extent of the carotenoid esters during digestion. Addition of RGL and CEL produced no significant (p < 0.05) effect on the overall carotenoid bioaccessibility values of mandarin, while positively affected those in peach. Altogether these results corroborate that the hydrolysis extent of xanthophyll esters limits bioaccessibility. Additionally, hydrophobicity of the carotenoid inversely correlates with micellarization, as free (all-E)-xanthophylls micellarized in a higher extent compared to (all-E)-ß-carotene and xanthophyll esters. The new information of our results is that the addition of rabbit gastric lipase substantially contributes to the hydrolysis of ß-cryptoxanthin esters from fruit pulps, and consequently, to increase carotenoid bioaccessibility, being even more effective than CEL.

HPLC-PDA-MS/MS as a strategy to characterize and quantify natural pigments from microalgae.

Interest in pigment composition of microalgae species is growing as new natural pigments sources are being sought. However, we still have a limited number of species of microalgae exploited to obtain these compounds. Considering these facts, the detailed composition of carotenoids and chlorophylls of two species of green microalgae (Chlorella sorokiniana and Scenedesmus bijuga) were determined for the first time by high-performance liquid chromatography coupled to diode array and mass spectrometry detectors (HPLC-PDA-MS/MS). A total of 17 different carotenoids were separated in all the extracts. Most of the carotenoids present in the two microalgae species are xanthophylls. C. sorokiniana presented 11 carotenoids (1408.46 µg g-1), and S. bijuga showed 16 carotenoids (1195.75 µg g-1). The main carotenoids detected in the two microalgae were all-trans-lutein and all-trans-ß-carotene. All-trans-lutein was substantially higher in C. sorokiniana (59.01%), whereas all-trans-ß-carotene was detected in higher quantitative values in S. bijuga (13.88%). Seven chlorophyll compounds were identified in both strains with different proportions in each species. Concentrations of chlorophyll representing 7.6% and 10.2% of the composition of the compounds present in the biomass of C. sorokiniana and S. bijuga, respectively. Relevant chlorophyll compounds are reported for the first time in these strains. The data obtained provide significant insights for microalgae pigment composition databases.

Ionic liquid associated with ultrasonic-assisted extraction: A new approach to obtain carotenoids from orange peel.

The aim of this study was to develop a new method for carotenoid extraction from orange peel, using ionic liquid (IL) to replace conventional organic solvents, assisted by ultrasound. Four different IL were tested: 1-butyl-3-methylimidazolium chloride ([BMIM][Cl]), 1-n-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF6]), 1-n-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), and 1-hexyl-3-methylimidazolium chloride ([HMIM][Cl]). Response surface methodology was applied in order to optimize the carotenoid extraction conditions, and Amberlite XAD-7HP resin was used to separate the carotenoids from the IL, allowing their recovery. Determination of carotenoids was carried out by high-performance liquid chromatography coupled to photodiode array and mass spectrometry detectors (HPLC-DAD-MSn). Thermal stability at different temperatures (60 °C and 90 °C) and peroxyl radical scavenging activity of the carotenoid extracts obtained with acetone and IL were evaluated. [BMIM][Cl] was the most effective IL, leading to a total carotenoid content of 32.08 ±â€¯2.05 µg/g, while 7.88 ±â€¯0.59 µg/g of dry matter was obtained by acetone extraction. IL and carotenoid recoveries using XAD-7HP resin were in the range of 59.5-63.8% and 52.2-58.7%, respectively. A carotenoid extract was successfully obtained with IL, finally isolated just by using ethanol, besides being more stable and presenting higher antioxidant activity than that obtained with acetone.

Carotenoid biosynthesis and quality characteristics of new hybrids between tangor (Citrus reticulata x C. sinensis) cv. 'Murcott' and sweet orange (C. sinensis) cv. 'Pêra'.

Phenotypic characteristics, as well as the relation between carotenoid accumulation and gene expression during ripening were determined in fruits of five new hybrids between tangor cv. 'Murcott' and sweet orange cv. 'Pêra'. The genotypes were classified into the orange-like group, showing mainly epoxycarotenoids, oval fruit shape and yellowish color, or in the mandarin-like group, showing mainly ß-cryptoxanthin, flattened shape and deep-orange coloration; although some hybrids presented intermediate characteristics. The diversity in carotenoid composition of hybrids and genitors were mostly explained by patterns of gene expression. High carotenoid (250-426 µg/g dry weight [dw]) and ß-cryptoxanthin (81-125 µg/g dw) contents, observed in the mandarin-like group, were generally associated with high expression of upstream genes (GGPPS1, PSY, PDS). On the other hand, low expression/repression of these genes and high expression of downstream genes (BCHX and ZEP) were associated with low carotenoid (~158 µg/g dw) and ß-cryptoxanthin (5-22 µg/g dw) contents and epoxycarotenoid accumulation, as occurred in the orange-like group. Breeding experiments resulted in hybrids with outstanding higher carotenoid contents than both genitors (up to 426 µg/g dw versus 158-250 µg/g dw in genitors), which was attributed to transgressive segregation. Differences among genotypes have great impact on commercial fruit quality and potential health benefits, such as the provitamin A content.

Carotenoid esters analysis and occurrence: What do we know so far?

Carotenoids possessing hydroxyl groups can be found in nature both as free xanthophylls and as carotenoid esters, i.e. acylated with fatty acids. A single carotenoid can be acylated with different fatty acids, resulting in a high number of possible structures of carotenoid esters. The analysis of carotenoid esters can be challenging; although extraction techniques are essentially the same as those used for free carotenoids, depending on the sample composition, interfering compounds such as triacylglycerides are also present in the extract in significant amounts to cause high background noise and ionization suppression in mass spectrometric analysis. Besides analysis of UV-visible spectrum features, identification of carotenoid esters must include tandem mass spectrometry (MSn) because no changes in the carotenoid molecule chromophore occur due to acylation with fatty acids. Although carotenoid esters are widespread found in foods, especially in fruits, and their bioavailability is equivalent or even higher than that of the free carotenoid, occurrence of xanthophyll esters is very limited in human plasma and tissues. Challenges and further investigations needed on the topics above are given in this review.

Comparison of Two Static in Vitro Digestion Methods for Screening the Bioaccessibility of Carotenoids in Fruits, Vegetables, and Animal Products.

In vitro digestion methods are routinely used to assess the bioaccessibility of carotenoids and other dietary lipophilic compounds. Here, we compared the recovery of carotenoids and their efficiency of micellarization in digested fruits, vegetables, egg yolk, and salmon and also in mixed-vegetable salads with and without either egg yolk or salmon using the static INFOGEST method22 and the procedure of Failla et al.16 Carotenoid stability during the simulated digestion was ≥70%. The efficiencies of the partitioning of carotenoids into mixed micelles were similar when individual plant foods and salad meals were digested using the two static methods. Furthermore, the addition of cooked egg or salmon to vegetable salads increased the bioaccessibility of some carotenoids. Our findings showed that the two methods of in vitro digestion generated similar estimates of carotenoid retention and bioaccessibility for diverse foods.

Identification of chlorophyll molecules with peroxyl radical scavenger capacity in microalgae Phormidium autumnale using ultrasound-assisted extraction.

The identification of chlorophyll molecules with peroxyl radical scavenger capacity in microalgae Phormidium autumnale was determined. The ultrasound-assisted extraction was utilized for obtaining the chlorophyll compounds from biomass. A total of eleven molecules were separated in microalgae chlorophyll extract, with pheophytin a' (371µg·g-1) and chlorophyll a (159.3µg·g-1) as the major ones. The chlorophyll extract was shown to be a potent scavenger of peroxyl radical, being almost 200 times more potent than α-tocopherol. These facts suggest the microalgae Phormidium autumnale as potential source of bioactive tetrapyrrole compounds.

Carotenoid profile of three microalgae/cyanobacteria species with peroxyl radical scavenger capacity.

Carotenoids from cyanobacteria Aphanothece microscopica Nageli and green microalgae Chlorella vulgaris and Scenedesmus obliquus were identified. The total carotenoid content, based on dry weight of biomass, of A. microscopica Nägeli, C. vulgaris and S. obliquus were 1398.88µg/g, 1977.02µg/g and 2650.70µg/g, respectively. A total of 23 different carotenoids were separated in all the extracts, the major ones being all-trans-ß-carotene (29.3%) and all-trans-lutein (28.1%) in Scenedesmus; all-trans-echinenone (22.8%) and all-trans-ß-carotene (17.7%) in Chlorella; all-trans-echinenone (28.3%) and all-trans-ß-carotene (26.2%) in Aphanothece. The carotenoid extracts were shown to be a potent scavenger of peroxyl radical, with values of 31.1 (Chlorella), 14.0 (Scenedesmus) and 7.3 (Aphanothece) times more potent than α-tocopherol.

Effect of Pasteurization on Flavonoids and Carotenoids in Citrus sinensis (L.) Osbeck cv. 'Cara Cara' and 'Bahia' Juices.

Orange juice is considered an excellent dietary source of several bioactive compounds with beneficial properties for human health. Citrus sinensis Osbeck cv. 'Cara Cara' is a bud mutation originated from 'Washington' navel orange, also known as 'Bahia' navel orange. The ascorbic acid, flavonoid, and carotenoid contents in pasteurized and nonpasteurized Bahia and Cara Cara juices using two LC-MS/MS platforms were investigated. Higher ascorbic acid content was observed in Bahia compared to Cara Cara in both pasteurized and nonpasteurized juices. Total flavanones content as well as hesperidin levels were higher in Cara Cara with respect to Bahia pasteurized juice. Cara Cara was also characterized by a significantly higher and diversified carotenoid content compared to Bahia juice with a mixture of (Z)-isomers of lycopene, all-E-ß-carotene, phytoene, and phytofluene isomers accounting for the highest carotenoid proportion. The exceptionally high carotenoid content of Cara Cara may be particularly interesting for nutritional or functional studies of uncommon carotenes in a citrus food matrix.

Composition by LC-MS/MS of New Carotenoid Esters in Mango and Citrus.

Interest in the composition of carotenoid esters of fruits is growing because esterification may affect their bioavailability. Thus, the aim was to provide a detailed identification of carotenoid esters in citrus and mango. Orange cv. 'Valencia' and cv. 'Pera' presented 9 free carotenoids, 38 monoesters, and 60 diesters. Violaxanthin and luteoxanthin derivatives were the major ones, followed by antheraxanthin, lutein, zeaxanthin, ß-cryptoxanthin, and zeinoxanthin esters, many of them reported for the first time in orange pulp. The carotenoid ester composition of tangor cv. 'Murcott', reported for the first time, showed 8 free carotenoids, 34 monoesters, and 33 diesters, with ß-cryptoxanthin esters as major compounds, followed by violaxanthin and zeaxanthin esters. In citrus, carotenoids were acylated mainly with capric, lauric, myristic, myristoleic, palmitic, palmitoleic, and oleic acids. In mango, 5 free carotenoids, 2 monoesters, and 19 diesters were identified, from which many violaxanthin and neoxanthin esters were reported for the first time.

Carotenoids are effective inhibitors of in vitro hemolysis of human erythrocytes, as determined by a practical and optimized cellular antioxidant assay.

ß-Carotene, zeaxanthin, lutein, ß-cryptoxanthin, and lycopene are liposoluble pigments widely distributed in vegetables and fruits and, after ingestion, these compounds are usually detected in human blood plasma. In this study, we evaluated their potential to inhibit hemolysis of human erythrocytes, as mediated by the toxicity of peroxyl radicals (ROO•). Thus, 2,2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH) was used as ROO• generator and the hemolysis assay was carried out in experimental conditions optimized by response surface methodology, and successfully adapted to microplate assay. The optimized conditions were verified at 30 × 10(6) cells/mL, 17 mM of AAPH for 3 h, at which 48 ± 5% of hemolysis was achieved in freshly isolated erythrocytes. Among the tested carotenoids, lycopene (IC(50) = 0.24 ± 0.05 µM) was the most efficient to prevent the hemolysis, followed by ß-carotene (0.32 ± 0.02 µM), lutein (0.38 ± 0.02 µM), and zeaxanthin (0.43 ± 0.02 µM). These carotenoids were at least 5 times more effective than quercetin, trolox, and ascorbic acid (positive controls). ß-Cryptoxanthin did not present any erythroprotective effect, but rather induced a hemolytic effect at the highest tested concentration (3 µM). These results suggest that selected carotenoids may have potential to act as important erythroprotective agents by preventing ROO•-induced toxicity in human erythrocytes.

Changes in antioxidant capacity and colour associated with the formation of ß-carotene epoxides and oxidative cleavage derivatives.

In this study HPLC-DAD-MS/MS was applied for the identification of compounds derived from (all-E)-ß-carotene following epoxidation and oxidative cleavage. The consequences on the CIELAB colour parameters and antioxidant capacity (AC) were also evaluated. Five apocarotenoids, three secocarotenoids, seven Z isomers and two epoxides were detected as a result of the oxidative cleavage. Four epoxides and three Z isomers were detected as a consequence of the epoxidation reaction. Some compounds were detected for the first time as a result of oxidation reactions. Both treatments led to a marked decrease in b(∗) and Cab(∗) values, indicating that these colour parameters can be used for the rapid assessment of ß-carotene oxidation. The oxidative cleavage of ß-carotene resulted in increased capacity to both scavenge ABTS(+) and quench singlet oxygen. These results suggest that the study of the AC of these oxidative derivatives and their possible usefulness as food ingredients deserves further attention.

The Amazonian fruit Byrsonima crassifolia effectively scavenges reactive oxygen and nitrogen species and protects human erythrocytes against oxidative damage.

A hydrophilic extract of murici (Byrsonima crassifolia), a fruit native to the North and Northeast regions of Brazil, was evaluated in relation to its phenolic composition and in vitro antioxidant potential against some physiologically relevant reactive oxygen and nitrogen species. Additionally, the protective effect of murici extract against peroxyl radical (ROO)-induced toxicity to human erythrocytes was also determined. The major phenolic compound, determined by HPLC-DAD-MSn, was quercetin (2.72±0.35µg/mL). The extract was able to scavenge ROO (0.30±0.04µmoltroloxequivalent/mg), hypochlorous acid (IC50=10.0±0.1µg/mL), hydroxyl radical (IC50=7±1µg/mL) and peroxynitrite anion (IC50=21.0±0.6µg/mL and 17.0±1.6µg/mL, respectively, in absence and presence of NaHCO3). Human erythrocytes were subjected to oxidative damage, but murici extract was not able to inhibit hemolysis, even at the highest tested concentration. On the other hand, the extract inhibited hemoglobin oxidation (IC50=271±44µg/mL), lipid peroxidation (1000µg/mL) by 48±5%, depletion of glutathione (100µg/mL) by 49±2% and formation of its oxidized form (100µg/mL) by 96±4%.

Carotenoids and phenolic compounds from Solanum sessiliflorum, an unexploited Amazonian fruit, and their scavenging capacities against reactive oxygen and nitrogen species.

The composition of carotenoids and phenolic compounds from mana-cubiu (Solanum sessiliflorum), a fruit native to Amazonia, was determined by high-performance liquid chromatography coupled to diode array and mass spectrometry detectors (HPLC-DAD-MS(n)). The antioxidant capacities of the hydrophilic and carotenoid extracts against some reactive oxygen (ROO(•), H(2)O(2), HOCl, and HO(•)) and nitrogen (ONOO(-)) species were also determined. Seventeen carotenoids and three phenolic compounds were found in mana-cubiu. The major carotenoids were (all-E)-ß-carotene (7.15 µg/g of dry weight) and (all-E)-lutein (2.41 µg/g of dry weight). The 5-caffeoylquinic acid (1351 µg/g of dry weight) was the major phenolic compound, representing more than 78% (w/w) of the total phenolic compounds. Moreover, two dihydrocaffeoyl spermidines were found in the hydrophilic extract. Both mana-cubiu extracts were able to scavenge all the tested reactive species. The carotenoid extract was shown to be a potent scavenger of peroxyl radical, while the hydrophilic extract was a potent hydrogen peroxide and hypochlorous acid scavenger.

Soybean extracts enriched with free isoflavones promote nitric oxide synthesis and affect the proliferation of breast adenocarcinoma cells

Although soybean isoflavones naturally accumulate in their conjugated forms, the beneficial effects on human health of soybean-containing foods have been credited to their aglycone forms. In the present study we analyzed the effects of a chemical agent, sodium nitroprusside (SNP), in eliciting the exudation of non-conjugated isoflavones from intact soybean seeds, embrionary axes and cotyledons. The isoflavones in the exudates were determined by high performance liquid chromatography and mass spectrometry. The effect of the exudates on the emission of nitric oxide (NO) and on the proliferation of breast carcinoma cells (MCF-7) was also evaluated. SNP elicitation increased the production of the aglycone forms dose- and time-dependently. Exudates of embrionary axes and cotyledons stimulated NO emission and showed biphasic effects on viability of MCF-7 cells. At lower concentrations both extracts presented proliferative effects (10-25%), and at higher concentrations inhibited (15%) cell proliferation. The biphasic effect might be due to the action of isoflavone aglycones in activating estrogen receptors which in turn stimulate the production of NO. Overall, the results suggest that soybean extracts enriched in isoflavone aglycones by elicitation with SNP could be exploited as a functional ingredient in the food industry.

Microcapsules containing antioxidant molecules as scavengers of reactive oxygen and nitrogen species.

The antioxidant capacities of gum arabic and maltodextrin microcapsules containing antioxidant molecules (trolox, α-tocopherol, ß-carotene, apo-8'-carotenal and apo-12'-carotenal) against reactive oxygen and nitrogen species were evaluated. The scavenging capacities were influenced by the wall material, the reactive species, namely ROO(), H(2)O(2), HO(), HOCl and ONOO(-), and the antioxidant molecule. In general, a more pronounced enhancement of the antioxidant capacity due to incorporation of antioxidant molecules was observed in gum arabic microcapsules. The empty microcapsules showed capacity to scavenge all the studied ROS and RNS, being gum arabic a more potent antioxidant than maltodextrin. Apo-8'-carotenal incorporation promoted the highest increase in the scavenging capacities among the evaluated antioxidants, varying from 50% to 132% and from 39% to 85% for gum arabic and maltodextrin microcapsules, respectively, suggesting that this carotenoid presented the best balance between the molecule localization inside the microcapsules and the reactivity against the specific reactive species.

Development of a novel micro-assay for evaluation of peroxyl radical scavenger capacity: application to carotenoids and structure-activity relationship.

A micro-assay was developed and validated, using a microplate reader in 96-well format, C(11)-BODIPY(581/591) as fluorescent probe and AIBN as ROO() generator. The structure-activity relationship was established for 15 carotenoid standards, indicating that the opening of the ß-ionone ring and the increase of chromophore extension in the carotenoid structure were the major factors leading to the increase of ROO() scavenging capacity. The values for ROO() scavenging capacity were calculated using α-tocopherol as reference compound. Among the studied carotenoids, all-trans-lycopene was the most efficient ROO() scavenger (8.67±0.74) followed by all-trans-astaxanthin (6.50±0.62). All the carotenoids showed to be more effective ROO() scavengers than α-tocopherol and some hydrophilic compounds. Finally, the method was successfully applied to assay the ROO() scavenging capacity of carotenoid extracts from two Amazonian fruits, peach palm (7.83±0.21) and mamey (6.90±0.44).

Scavenging capacity of marine carotenoids against reactive oxygen and nitrogen species in a membrane-mimicking system.

Carotenoid intake has been associated with the decrease of the incidence of some chronic diseases by minimizing the in vivo oxidative damages induced by reactive oxygen (ROS) and nitrogen species (RNS). The carotenoids are well-known singlet oxygen quenchers; however, their capacity to scavenge other reactive species, such as peroxyl radical (ROO•, hydroxyl radical (HO•), hypochlorous acid (HOCl) and anion peroxynitrite (ONOO⁻), still needs to be more extensively studied, especially using membrane-mimicking systems, such as liposomes. Moreover, the identification of carotenoids possessing high antioxidant capacity can lead to new alternatives of drugs or nutritional supplements for prophylaxis or therapy of pathological conditions related to oxidative damages, such as cardiovascular diseases. The capacity to scavenge ROO•, HO•, HOCl and ONOO⁻ of seven carotenoids found in marine organisms was determined in liposomes based on the fluorescence loss of a fluorescent lipid (C11-BODIPY58¹/59¹) due to its oxidation by these reactive species. The carotenoid-bearing hydroxyl groups were generally more potent ROS scavengers than the carotenes, whilst ß-carotene was the most efficient ONOO⁻ scavenger. The role of astaxanthin as an antioxidant should be highlighted, since it was a more potent scavenger of ROO•, HOCl and ONOO⁻ than α-tocopherol.