Antioxidant Interactions between Citrus Fruit Carotenoids and Ascorbic Acid in New Models of Animal Cell Membranes.

The regular consumption of citrus fruits by humans has been associated with lower incidence of chronic-degenerative diseases, especially those mediated by free radicals. Most of the health-promoting properties of citrus fruits derive from their antioxidant content of carotenoids and ascorbic acid (ASC). In the current work we have investigated the scavenging (against hydroxyl radical) and quenching capacities (against singlet oxygen) of four different carotenoid extracts of citrus fruits in the presence or absence of ASC (µM range) in organic solvent, aqueous solution, micelles and in an innovative biomimicking liposomal system of animal cell membrane (AML). The fruits of four varieties of citrus were selected for their distinctive carotenoid composition (liquid chromatography characterization): 'Nadorcott' mandarin and the sweet oranges 'Valencia late', 'Ruby Valencia' and 'Pinalate' mutant. The quenching activity of citrus carotenoids strongly depended on the biological assemblage: freely diffusible in organic solvent, 'Ruby Valencia' carotenoids (containing lycopene) showed the highest quenching activity, whereas 'Nadorcott' mandarin extracts, rich in ß-cryptoxanthin, prevailed in micellar systems. Interestingly, the addition of 10 µM ASC significantly increased the quenching activity of all citrus extracts in micelles: 'Valencia' orange (+53%), 'Pinalate' (+87%), 'Ruby' (4-fold higher) and 'Nadorcott' mandarins (+20%). Accurate C11-BODIPY581/591 fluorescence assays showed solid scavenging activities of all citrus extracts against AML oxidation: 'Valencia' (-61%), 'Pinalate' (-58%) and 'Ruby' oranges (-29%), and 'Nadorcott' mandarins (-70%). Indeed, all four citrus extracts tested here have balanced antioxidant properties; extracts from the 'Nadorcott' mandarin slightly prevailed overall, due, at least in part, to its high content of ß-cryptoxanthin. This study depicts some of the antioxidant interactions between citrus fruit carotenoids and ascorbic acid in models of animal cell membranes and reinforces the contribution of them in promoting health benefits for humans.

Juices and By-Products of Red-Fleshed Sweet Oranges: Assessment of Bioactive and Nutritional Compounds.

The content of nutrients and bioactive compounds, and antioxidant capacity were assessed in the juices from two red-fleshed oranges, Cara Cara and Kirkwood, and compared with that of a standard Navel orange. Two juice extraction procedures, hand-squeezing and industrial, and two treatments, pasteurization (85 °C/30 s) and high-pressure homogenization (HPH, 150 MPa/55 °C/1 min), were evaluated. For most of the nutrients and bioactive compounds, the hand and industrial juice squeezing rendered similar extraction efficiency. Individual composition of carotenoids in the juices were differentially affected by the extraction procedure and the treatments, but the red-fleshed orange juices contained between 3- to 6-times higher total carotenoids than the standard Navel juices, being phytoene and phytofluene the main carotenoids. The industrial and treated juices of both red-fleshed oranges contained 20-30% higher amounts of tocopherols but about 20% lower levels of vitamin C than Navel juices. Navel juices exhibited higher hydrophilic antioxidant capacity, while the red-fleshed orange juices showed an improved lipophilic antioxidant capacity. The main distinctive characteristic of the industrial juice by-product of the red-fleshed oranges was a higher content of carotenoids (×10) and singlet oxygen antioxidant capacity (×1.5-2) than the Navel by-product.

A comprehensive analysis of carotenoids metabolism in two red-fleshed mutants of Navel and Valencia sweet oranges (Citrus sinensis).

Kirkwood Navel and Ruby Valencia are two spontaneous bud mutations of the respective parental lines of sweet orange (Citrus sinensis) Palmer Navel and Olinda Valencia, showing an atypical red pigmentation of the pulp. These red-fleshed varieties are commercially available and highly attractive for consumers but their carotenoid metabolism and the basis of the mutation have not been investigated. The red colour of Kirkwood and Ruby pulp was observed from the very early stages of fruit development until full maturity and associated with an altered carotenoid profiling. The red-fleshed varieties accumulated from 6- up to 1000-times more total carotenoids compared to the standard oranges. Specifically, the pulp of Kirkwood and Ruby accumulated large amounts of phytoene and phytofluene, and moderate contents of lycopene. Moreover, the red-fleshed oranges contained other unusual carotenes as δ-carotene, and lower concentrations of downstream products such as ß,ß-xanthophylls, abscisic acid (ABA) and ABA-glucosyl ester. This peculiar profile was associated with chromoplasts with lycopene crystalloid structures and round vesicles likely containing colourless carotenes. The flavedo and leaves of Kirkwood and Ruby showed minor changes in carotenoids, mainly limited to higher levels of phytoene. The carotenoid composition in Kirkwood and Ruby fruits was not explained by differences in the transcriptional profile of 26 genes related to carotenoid metabolism, covering the main steps of biosynthesis, catabolism and other processes related to carotenoid accumulation. Moreover, sequence analysis of the lycopene cyclase genes revealed no alterations in those of the red-fleshed oranges compared to the genes of the standard varieties. A striking event observed in Kirkwood and Ruby trees was the reddish coloration of the inner side of the bark tissue, with larger amounts of phytoene, accumulation of lycopene and lower ABA content. These observation lead to the conclusion that the mutation is not only manifested in fruit, affecting other carotenogenic tissues of the mutant plants, but with different consequences in the carotenoid profile. Overall, the carotenoid composition in the red-fleshed mutants suggests a partial blockage of the lycopene ß-cyclization in the carotenoid pathway, rendering a high accumulation of carotenes upstream lycopene and a reduced flow to downstream xanthophylls and ABA.

Bioactive Compounds, Nutritional Quality and Antioxidant Capacity of the Red-Fleshed Kirkwood Navel and Ruby Valencia Oranges.

Kirkwood Navel and Ruby Valencia are two spontaneous bud-mutations of the ordinary Washington Navel and Valencia late oranges characterized by the red coloration of their flesh. The purpose of this study was to analyze the physiological features, internal fruit quality, contents of relevant bioactive compounds and antioxidant capacity in the pulps of the red-fleshed fruits compared with the ordinary oranges during late development and maturation. In general, the content of sugars, organic acids, vitamin C, tocopherols, total phenolics and flavonoids, the hydrophilic antioxidant capacity and their changes during maturation were similar in the red-fleshed oranges and in the corresponding blond oranges. However, the mature Ruby fruits contained lower concentrations of sugars, malic and succinic acid and higher levels of citric acid than the ordinary Valencia. The major difference between the pulps of the Kirkwood and Ruby oranges and those of the ordinary oranges was the higher lipophilic antioxidant capacity and SOAC (singlet oxygen absorption capacity) of the former. Together, the high and unique content and composition of carotenoids in Kirkwood and Ruby may contribute to an enhanced antioxidant capacity without any detrimental effects on other fruit-quality attributes, making these varieties good sources of phytochemicals for the fresh-fruit and juice-processing citrus industries.

Characterization of the Pale Yellow Petal/Xanthophyll Esterase gene family in citrus as candidates for carotenoid esterification in fruits.

In orange-pigmented citrus fruits, the xanthophyll esters are the predominant carotenoids, but their biosynthetic origin is currently unknown. In this work, seven PYP/XES (Pale Yellow Petal/ Xanthophyll esterase) genes were identified in Citrus genomes, but only PYP1-4 and 6 contained the structural domains essential for activity. The PYP/XES expression profiles in sweet orange and in other Citrus species such as lemon, mandarin and pummelo with marked differences in fruit pigmentation and content of xanthophylls esters, showed the upregulation of PYP1,2 and 6 genes during ripening only in orange-pigmented fruits. Moreover, transcript levels of PYP1, 2 and 6 genes in peel and pulp of sweet orange were accompanied by the accumulation of xanthophyll esters during ripening. This work reports for the first time the PYP/XES gene family in Citrus and strongly suggests its involvement in xanthophyll esterification in citrus fruit tissues and its influence in carotenoid accumulation and fruit pigmentation.

Antioxidant capacity in fruit of Citrus cultivars with marked differences in pulp coloration: Contribution of carotenoids and vitamin C.

The purpose of this study was to evaluate the specific contribution of carotenoids and vitamin C to the lipophilic and hydrophilic antioxidant capacity, respectively, of the pulp of citrus fruits using the genetic diversity in pigmentation and in the carotenoid complement. To this end, six citrus varieties were selected: two mandarins, Clemenules (Citrus clementina) and Nadorcott (C. reticulata); two grapefruits (C. paradisi), Marsh and Star Ruby; and two sweet oranges (C. sinensis), Valencia late and Valencia Ruby. Total carotenoid content and composition in the pulp of fruits were very different, in relation to their color singularities. Valencia Ruby and Nadorcott had the highest carotenoid content, accumulating the former large amounts of linear carotenes (phytoene, phytofluene, and lycopene) and Nadorcott of ß-cryptoxanthin. Orange fruits contained the highest amount of vitamin C while in Nadorcott mandarin it was substantially lower. Analysis of antioxidant capacity, evaluated by 2,2'-azino-di-(3-ethylbenzthiazoline sulfonate) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) assays, in the pulp of the different fruit varieties indicated a high and positive correlation between vitamin C content and hydrophilic antioxidant capacity. Nevertheless, a weak correlation was observed between carotenoids content and lipophilic antioxidant capacity in the pulp extracts assayed by ABTS. Overall, vitamin C in the pulp of citrus fruit had an important contribution to the hydrophilic antioxidant capacity, whereas that of carotenoids to lipophilic antioxidant capacity was very variable, being the highest that of Valencia Ruby orange, with large concentrations of lycopene and phytoene, followed by Nadorcott mandarin, with high ß-cryptoxanthin content.

Transcriptional Analysis of Carotenoids Accumulation and Metabolism in a Pink-Fleshed Lemon Mutant.

Pink lemon is a spontaneous bud mutation of lemon (Citrus limon, L. Burm. f) characterized by the production of pink-fleshed fruits due to an unusual accumulation of lycopene. To elucidate the genetic determinism of the altered pigmentation, comparative carotenoid profiling and transcriptional analysis of both the genes involved in carotenoid precursors and metabolism, and the proteins related to carotenoid-sequestering structures were performed in pink-fleshed lemon and its wild-type. The carotenoid profile of pink lemon pulp is characterized by an increased accumulation of linear carotenoids, such as lycopene, phytoene and phytofluene, from the early stages of development, reaching their maximum in mature green fruits. The distinctive phenotype of pink lemon is associated with an up-regulation and down-regulation of the genes upstream and downstream the lycopene cyclase, respectively. In particular, 9-cis epoxycarotenoid dioxygenase genes were overexpressed in pink lemon compared with the wild-type, suggesting an altered regulation of abscisic acid biosynthesis. Similarly, during early development of the fruits, genes of the carotenoid-associated proteins heat shock protein 21, fibrillin 1 and 2 and orange gene were overexpressed in the pulp of the pink-fleshed lemon compared to the wild-type, indicating its increased capacity for sequestration, stabilization or accumulation of carotenes. Altogether, the results highlighted significant differences at the transcriptomic level between the pink-fleshed lemon and its wild-type, in terms of carotenoid metabolism and the capacity of stabilization in storage structures between the two accessions. Such changes may be either responsible for the altered carotenoid accumulation or in contrast, a metabolic consequence.