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Improved normal-phase high-performance liquid chromatography procedure for the determination of carotenoids in cereals.

Besides the health benefits associated with whole-grain consumption, cereals are recognized sources of health-enhancing bioactive components such as carotenoids, which are a group of yellow pigments involved in the prevention of many degenerative diseases and which have been used for a long time as indicators of the color quality of durum wheat and pasta products. This work reports a fast, sensitive, and selective procedure for the extraction and determination of carotenoids from cereals and cereal byproducts. The method involves sample saponification and extraction followed by normal-phase high-performance liquid chromatography, allowing the separation of the main carotenoids pigments of cereals, especially lutein and zeaxanthin. An application of the established method to various species of cereals and cereal byproducts is also shown. The highest carotenoid levels were found in maize (approximately 11.14 mg/kg of dry weight), which contains high amounts of beta-cryptoxanthin (2.40 mg/kg of dry weight), and, among the cereals considered, has the highest content of zeaxanthin (6.43 mg/kg of dry weight) and alpha+beta-carotene (1.44 mg/kg of dry weight). With the exception of maize, lutein is the main compound found (from 0.23 to 2.65 mg/kg of dry weight in oat and durum wheat, respectively). Moreover, whereas alpha+beta-carotene and zeaxanthin are principally localized in the germ, lutein is equally distributed along the kernel.

Carotenoids↗

Carotenoids from new apricot (Prunus armeniaca L.) varieties and their relationship with flesh and skin color.

Thirty-seven apricot varieties, including four new releases (Rojo Pasión, Murciana, Selene, and Dorada) obtained from different crosses between apricot varieties and three traditional Spanish cultivars (Currot, Mauricio, and Búlida), were separated according to flesh color into four groups. The L*, a*, b*, hue angle, and chroma color measurements on the skin and flesh as well as other quality indices including flesh firmness, soluble solids, titratable acidity, and pH were plotted against the total carotenoid content measured by HPLC. Among the 37 apricot varieties, the total carotenoid content ranged from 1,512 to 16,500 microg 100 g(-1) of edible portion, with beta-carotene as the main pigment followed by beta-cryptoxanthin and gamma-carotene. The wide range of variability in the provitamin A content in the apricot varieties encouraged these studies in order to select the breeding types with enhanced carotenoid levels as the varieties with a higher potential health benefit. The carotenoid content was correlated with the color measurements, and the hue angle in both flesh and peel was the parameter with the best correlation (R = 0.92 and 0.84, respectively). An estimation of the carotenoid content in apricots could be achieved by using a portable colorimeter, as a simple and easy method for field usage applications.

Carotenoids↗

Effects of blood orange juice intake on antioxidant bioavailability and on different markers related to oxidative stress.

Orange juice is a source of antioxidants that might afford in vivo protection from oxidative stress. To test this hypothesis, we carried out a human intervention study with blood orange juice containing high amounts of vitamin C, anthocyanins, and carotenoids. Sixteen healthy female volunteers were enrolled in a crossover study and were given 600 mL/day of blood orange juice or a diet without juice for 21 days. Before and after each intervention period, plasma vitamin C, cyanidin-3-glucoside, and carotenoids were quantified. Furthermore, plasma antioxidant capacity, malondialdehyde concentration in plasma, 11-dehydrotromboxane B(2) urinary excretion, and lymphocyte DNA damage were evaluated as biomarkers of oxidative stress. Blood orange juice consumption determined a significant increase in plasma vitamin C, cyanidin-3-glucoside, beta-cryptoxanthin, and zeaxanthin. Also, lymphocyte DNA resistance to oxidative stress was improved whereas no effect was observed on the other markers that we analyzed. In turn, these results suggest that blood orange juice is a bioavailable source of antioxidants, which might moderately improve the antioxidant defense system; however, the long-term effects of its consumption are to be further investigated.

Adult↗

Fatty acid composition and antioxidant properties of cold-pressed marionberry, boysenberry, red raspberry, and blueberry seed oils.

Cold-pressed marionberry, boysenberry, red raspberry, and blueberry seed oils were evaluated for their fatty acid composition, carotenoid content, tocopherol profile, total phenolic content (TPC), oxidative stability index (OSI), peroxide value, and antioxidant properties. All tested seed oils contained significant levels of alpha-linolenic acid ranging from 19.6 to 32.4 g per 100 g of oil, along with a low ratio of n-6/n-3 fatty acids (1.64-3.99). The total carotenoid content ranged from 12.5 to 30.0 micromoles per kg oil. Zeaxanthin was the major carotenoid compound in all tested berry seed oils, along with beta-carotene, lutein, and cryptoxanthin. Total tocopherol was 260.6-2276.9 mumoles per kg oil, including alpha-, gamma-, and delta-tocopherols. OSI values were 20.07, 20.30, and 44.76 h for the marionberry, red raspberry, and boysenberry seed oils, respectively. The highest TPC of 2.0 mg gallic acid equivalents per gram of oil was observed in the red raspberry seed oil, while the strongest oxygen radical absorbance capacity was in boysenberry seed oil extract (77.9 micromol trolox equivalents per g oil). All tested berry seed oils directly reacted with and quenched DPPH radicals in a dose- and time-dependent manner. These data suggest that the cold-pressed berry seed oils may serve as potential dietary sources of tocopherols, carotenoids, and natural antioxidants.

Antioxidants↗

Identification and aroma impact of norisoprenoids in orange juice.

Four norisoprenoids, alpha-ionone, beta-ionone, beta-cyclocitral, and beta-damascenone, along with their putative carotenoid precursors, were identified in Valencia orange juice using time-intensity GC-O, GC-MS, and photodiode array HPLC. alpha-Ionone and beta-cyclocitral are reported in orange juice for the first time. GC-O aroma peaks were categorized into seven groups with similar sensory qualities: citrus/minty, metallic/mushroom/geranium, roasted/cooked/meaty/spice, fatty/soapy/green, sulfury/solventy/medicine, floral, and sweet fruity. The four norisoprenoids contributed approximately 8% of the total aroma intensity and 78% of the total floral aroma category. The putative carotenoid norisoprenoid precursors, alpha- and beta-carotene, alpha- and beta-cryptoxanthin, and neoxanthin, were identified in the same orange juice using photodiode array HPLC retention times and spectral characteristics.

Beverages↗

Phytochemicals and antioxidant properties in wheat bran.

Bran samples of seven wheat varieties from four different countries were examined and compared for their phytochemical compositions and antioxidant activities. Phenolic acid composition, tocopherol content, carotenoid profile, and total phenolic content were examined for the phytochemical composition of wheat bran, whereas the measured antioxidant activities were free radical scavenging properties against 2,2-diphenyl-1-picrylhydrazyl radical, radical cation 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, peroxide radical anion O(2)(.-), and oxygen radical and chelating capacities. The results showed that the tested wheat bran samples differed in their phytochemical compositions and antioxidant properties. Ferulic acid, with a concentration range of 99-231 microg/g, was the predominant phenolic acid in all of the tested bran samples and accounted for about 46-67% of total phenolic acids on a weight basis. The concentrations for alpha-, delta-, and gamma- tocopherols were 1.28-21.29, 0.23-7.0, and 0.92-6.90 microg/g, respectively. In addition, lutein and cryptoxanthin were detected in all of the tested bran samples with levels of 0.50-1.80 and 0.18-0.64 microg/g, respectively. Zeaxanthin was detected in the six bran samples, and the greatest zeaxanthin concentration of 2.19 microg/g was observed in the Australian general purpose wheat bran. Beta-carotene was detected in four of the tested bran samples at a range of 0.09-0.40 microg/g. These data suggest that wheat and wheat bran from different countries may differ in their potentials for serving as dietary sources of natural antioxidants.

Antioxidants↗

Biochemical and molecular analysis of carotenoid biosynthesis in flavedo of orange (Citrus sinensis L.) during fruit development and maturation.

Changes in carotenoid content and composition and expression of carotenoid biosynthetic genes were analyzed in the flavedo of sweet orange (Citrus sinensis L. Osbeck, cv. Navelate) fruit during development and maturation. Lutein and all-E-violaxanthin were the major carotenoids in chloroplast-containing tissues. During fruit coloration, phytoene, beta-cryptoxanthin, zeaxanthin, and mainly (9Z)-violaxanthin progressively accumulated, and a large proportion of apocarotenoids was also found in the flavedo of full-colored fruits. We have cloned partial and full-length cDNAs corresponding to genes involved in early condensation and desaturase reactions [phytoene synthase (PSY), phytoene desaturase (PDS), and zeta-carotene desaturase (ZDS)], coupled redox reaction (plastid terminal oxidase), cyclizations [beta-lycopene cyclase (beta-LCY) and epsilon-lycopene cyclase (epsilon-LCY)], hydroxylation [beta-carotene hydroxylase (beta-CHX)], and epoxidation [zeaxanthin epoxidase (ZEP)] and analyzed their mRNA accumulation in the flavedo of fruits during development and ripening as compared with those of leaves. Collectively, the results indicated that PDS gene expression correlated with carotenoid content in developing fruit and that up-regulation of PSY and ZDS genes at the onset of fruit coloration would enhance the production of linear carotenes and the flux into the pathway. The shift from the beta,epsilon-branch to the beta,beta-branch of the pathway that originates the changes in carotenoid composition during fruit coloration may be explained by a down-regulation of epsilon-LCY and by the increase of the beta-CHX transcript.

Carotenoids↗

Bioavailability and antioxidant effects of orange juice components in humans.

Seven healthy females and six males consumed daily 256 mg of vitamin C, 271 mg of flavanones (mainly as glycosides), 6 mg of carotenoids (mainly xanthophylls and cryptoxanthins), and 0.16 mg of folate by incorporation of daily three times 236 mL of not from concentrate orange juice (OJ) into their habitual diet. At the end of 3 weeks, mean vitamin C, folate, carotenoid, and flavanone plasma concentrations increased significantly relative to baseline by 59% (p < 0.001), 46% (p = 0.018), and 22% (p < 0.001), and 8-fold (p = 0.045), respectively. Flavanones were excreted in urine 9-fold more at the end of the intervention (p = 0.01) but returned to baseline 2 days after study completion. After the 3 week intervention, plasma concentrations of vitamins A and E did not change. 8-Hydroxydeoxyguanosine in white blood cells declined by 16% (p = 0.38; n = 11), and in individuals with high baseline concentrations by 29% (p = 0.36; n = 7), respectively. Low-density lipoprotein (LDL)-/high-density lipoprotein (HDL)-cholesterol ratios decreased but cholesterol (HDL, LDL, total) and thiobarbituric acid reactive substance plasma concentrations did not change significantly. We conclude from this pilot study that OJ is an excellent food source to enhance circulating concentrations of valuable hydrophilic as well as lipophilic phytochemicals.

Antioxidants↗

Supercritical CO2 extraction of beta-carotene from a marine strain of the cyanobacterium Synechococcus species.

Dynamic extraction of carotenoids from a marine strain of Synechococcus sp. (Cyanophyceae) with supercritical CO2 (SC-CO2) was investigated with regard to operation pressure and temperature effects on extraction efficiency. Extraction yield (milligrams of pigment per gram of dry weight) for SC-CO2) was compared with the extraction yield for dimethylformamide (DMF). Carotenoids extracted with SC-CO2 were beta-carotene (Ct), zeaxanthin (Z), beta-cryptoxanthin (Cr), and equinenone; chlorophyll a was poorly extracted, whereas myxoxanthophyll, another major carotenoid, was not extracted under any experimental condition. The highest relative yield, which is defined here as y(r) = [(mg of pigment(SC-CO2)/mg of pigment(DMF))] x 100, was 76.1 +/- 8.6% for Ct, but it rose to 87.0 +/- 3.4% when 15% ethanol was used as cosolvent. The pressure effect on y(r) was found to be significant (p < 0.05) for both Cr and Z, along with total carotenoids, whereas the effect of square T (TT) was significant for only Ct. From empirical correlations, pairwise pressure (bar) and temperature (degrees C), respectively, for optimal extraction were determined to be (358, 50) for Ct, (454, 59) for Cr, and (500, 60) for Z. Cell disruption by sonication or detergent treatment of the biomass did not improve the extraction efficiency. Matrix structure together with material state could explain the low carotenoid extraction yield obtained with SC-CO2 as compared to DMF in Synechococcus sp. However, the process can be applied to selective extraction of different carotenoids.

Carbon Dioxide↗

Carotenoid diversity in cultivated citrus is highly influenced by genetic factors.

Citrus fruits are complex sources of carotenoids with more than 100 kinds of pigments reported in this genus. To understand the origin of the diversity of carotenoid compositions of citrus fruit, 25 genotypes that belong to the 8 cultivated Citrus species were analyzed. Juice extracts of mature fruit were analyzed by high-performance liquid chromatography using a C30 column. The 25 citrus genotypes presented different carotenoid profiles with 25 distinct compounds isolated. Statistical analyses revealed a strong impact of genotype on carotenoid compositions. Two kinds of classifications of genotypes were performed: on qualitative data and on quantitative data, respectively. The results showed that variability in carotenoid compositions was more interspecific than intraspecific. Two carotenoids, cis-violaxanthin and the beta-cryptoxanthin, strongly determined the classification on qualitative data, which was also in agreement with previous citrus variety classifications. These findings provide evidence that, as for other phenotypical traits, the general evolution of cultivated Citrus is the main factor of the organization of carotenoid diversity among citrus varieties. To the authors' knowledge this is the first study that links the diversity of carotenoid composition to the citrus genetic diversity. These results lead to the proposed major biosynthetic steps involved in the differential carotenoid accumulation. Possible regulation mechanisms are also discussed.

Carotenoids↗

Carotenoids in three species of Corbicula Clams, Corbicula japonica, Corbicula sandai, and Corbicula sp. (Chinese Freshwater Corbicula Clam).

Carotenoids were investigated in three species of corbicula clams, Corbicula japonica, Corbicula sandai, and Corbicula sp. (Chinese freshwater corbicula clam). Forty-three carotenoids were isolated. Among them, 7,8-didehydro-beta-cryptoxanthin (12), peridininol 5,8-furanoxide (38), pyrrhoxanthin 5,8-furanoxide (40), and pyrrhoxanthinol 5,8-furanoxide (43) are newly reported as naturally occurring carotenoids. Their structures were characterized on the basis of UV-vis, FAB-MS including MS/MS experiments, and 1H NMR spectroscopic data. The total carotenoid contents in C. japonica, C. sandai, and Chinese freshwater corbicula clam were found to be 5.3, 2.6, and 0.3 mg/100 g in the edible part (wet weight), respectively. Peridinin (34) and its derivatives were found to be major carotenoids in C. japonica, which inhabits brackish water. On the other hand, lutein (13) was found to be the major carotenoid in C. sandai and Chinese corbicula clams, which inhabit freshwater. These patterns well reflected the carotenoids in their dietary algae. 7',8'-Didehydrodeepoxyneoxanthin (19), corbiculaxanthin (21), corbiculaxanthin 3'-acetate (22), and 6-epiheteroxanthin (24) were found in all three species of corbicula clams and have not previously been found in other shellfishes. They were assumed to be peculiar carotenoids in corbicula clams.

Animals↗

Regulation of color break in citrus fruits. Changes in pigment profiling and gene expression induced by gibberellins and nitrate, two ripening retardants.

Citrus clementina fruits were repeatedly treated on-tree from mature green until breaker stages with either nitrate or gibberellin, two retardants of external ripening. The natural color break was characterized by a reduction in chlorophyll concentration, a decrease in beta,epsilon-carotenoids, beta-carotene, neoxanthin, and all-E-violaxanthin, and an increase in beta,beta-xanthophylls [mainly (9Z)-violaxanthin and beta-cryptoxanthin]. The two retardants delayed both chlorophyll depletion and total carotenoid accumulation and in addition altered carotenoid composition. Treated fruits maintained longer the typical carotenoid composition of green fruits and reduced beta,beta-xanthophyll accumulation. Natural degreening was accompanied by a marked decrease in transcript levels of 1-deoxy-d-xylulose 5-phosphate synthase (DXS) and geranylgeranyl reductase (CHL P) while, conversely, pheophorbide a oxygenase (PaO) and phytoene synthase (PSY) gene expression increased. Gibberellin and nitrate delayed the reduction of DXS expression and the induction of PaO and PSY transcript accumulation, while no differences in CHL P were observed. The data indicate that both ripening retardants repressed natural PaO and PSY expression, suggesting a mechanistic basis for the elevated levels of chlorophyll and lower carotenoid concentration resulting from the gibberellin and nitrogen treatments and the consequent color break delay in citrus fruit peels.

Carotenoids↗

Evaluation of analytical methods for carotenoid extraction from biofortified maize (Zea mays sp.).

Biofortification of maize with beta-carotene has the potential to improve vitamin A status in vitamin A deficient populations where maize is a staple crop. Accurate assessment of provitamin A carotenoids in maize must be performed to direct breeding efforts. The objective was to evaluate carotenoid extraction methods and determine essential steps for use in countries growing biofortified maize. The most reproducible method based on coefficient of variation and extraction efficiency was a modification of Kurilich and Juvik (1999). Heat and saponification are required to release carotenoids from biofortified maize and remove oils interfering with chromatographic analysis. For maize samples with high oil content, additional base may be added to ensure complete saponification without compromising results. Degradation of internal standard before carotenoids were released from the maize matrix required the addition of internal standard after heating to prevent overestimation of carotenoids. This modified method works well for lutein, zeaxanthin, beta-cryptoxanthin, alpha-carotene, and beta-carotene.

Carotenoids↗

Carotenoids from guava (Psidium guajava l.): isolation and structure elucidation.

Sixteen carotenoids were isolated from the flesh of Brazilian red guavas (Psidium guajava L.). Their structures were established by means of UV-visible, 400 and 500 MHz (1)H NMR, 120 and 125 MHz (13)C NMR, mass, and circular dichroism spectra. The carotenoids were identified as phytofluene, (all-E)-, (9Z)-, (13Z)-, and (15Z)-beta-carotene, (all-E)-gamma-carotene, (all-E)-, (9Z)-, (13Z)-, and (15Z)-lycopene, (all-E,3R)-beta-cryptoxanthin, (all-E, 3R)-rubixanthin, (all-E,3S,5R,8S)-cryptoflavin, (all-E,3R,3'R, 6'R)-lutein, (all-E,3S,5R,6R,3'S,5'R,8'R)-, and (all-E,3S,5R,6R,3'S, 5'R,8'S)-neochrome. Thirteen of the carotenoids identified are reported as guava carotenoids for the first time.

Carotenoids↗

Quantification of carotenoid and tocopherol antioxidants in Zea mays.

Recent investigations into carotenoid and tocopherol biological activity in mammalian systems indicate that these antioxidants are associated with the prevention of degenerative diseases. Both carotenoids and tocopherols can be found in corn kernel tissue. A replicated survey of 44 sweet and dent corn lines was conducted to determine qualitative and quantitative variability of lutein, zeaxanthin, beta-cryptoxanthin, alpha-carotene, and beta-carotene, as well as the alpha-, delta-, and gamma- forms of tocopherol. The primary carotenoids in fresh market sweet corn were found to be lutein and zeaxanthin, with the gamma form dominating among the tocopherols. Mean values among the genotypes were observed to range from 0 to 20.0 and 2.4 to 63.3 microg/g dry weight for lutein and gamma-tocopherol, respectively, indicating variability among genotypes in genes regulating the metabolism of these compounds. The observed genetic variability suggests profound differences in potential health promotion among genotypes and supports the feasibility of developing germplasm with enhanced levels of these antioxidant compounds at dosages that could promote health among the consuming public.

Antioxidants↗

Paprika (Capsicum annuum) oleoresin extraction with supercritical carbon dioxide.

Paprika oleoresin was fractionated by extraction with supercritical carbon dioxide (SCF-CO(2)). Higher extraction volumes, increasing extraction pressures, and similarly, the use of cosolvents such as 1% ethanol or acetone resulted in higher pigment yields. Within the 2000-7000 psi range, total oleoresin yield always approached 100%. Pigments isolated at lower pressures consisted almost exclusively of beta-carotene, while pigments obtained at higher pressures contained a greater proportion of red carotenoids (capsorubin, capsanthin, zeaxanthin, beta-cryptoxanthin) and small amounts of beta-carotene. The varying solubility of oil and pigments in SCF-CO(2) was optimized to obtain enriched and concentrated oleoresins through a two-stage extraction at 2000 and 6000 psi. This technique removes the paprika oil and beta-carotene during the first extraction step, allowing for second-stage oleoresin extracts with a high pigment concentration (200% relative to the reference) and a red:yellow pigment ratio of 1.8 (as compared to 1.3 in the reference).

Capsicum↗

Effect of high-pressure treatment on the carotenoid composition and the radical scavenging activity of persimmon fruit purees.

The carotenoid composition of persimmon fruit purees of two cultivars, cvs. Rojo Brillante and Sharon, grown in Spain was determined by HPLC to assess the effects of high-pressure processing on some sensory (carotenoids), nutritional (provitamin A value), and health-related (radical-scavenging capacity) parameters. Total carotenoid content was higher in untreated Rojo Brillante puree (22. 11 microg g(-)(1)) than in untreated Sharon puree (15.22 microg g(-)(1)). Purees of both untreated cultivars showed similar carotenoid patterns after saponification with beta-cryptoxanthin, beta-carotene, and zeaxanthin as the main pigments. A high content of lycopene was quantified in Rojo Brillante (5.34 microg g(-)(1)), whereas only traces were detected in Sharon. The provitamin A value, reported as retinol equivalents (RE), was in untreated Rojo Brillante puree (77 RE/100 g) similar to that of Sharon (75 RE/100 g). Scavenging free radical capacity, measured as antiradical efficiency (AE), showed in untreated Rojo Brillante puree a value (12.14 x 10(-)(3)) 8.5 times higher than that in untreated Sharon (1. 42 x 10(-)(3)). Nonuniform behavior of high-pressure treatment was detected. Pressure treatments at 50 and 300 MPa/15 min/25 degrees C for Rojo Brillante and at 50 and 400 MPa/15 min/25 degrees C for Sharon increased the amount of extractable carotenoids (9-27%), which are related with the increase of vitamin A value (75-87 RE/100 g). No correlation with the increase of AE (from 1.42 x 10(-)(3) to 16.73 x 10(-)(3) and 19.58 x 10(-)(3)) after some pressure treatments (150 and 300 MPa/15 min/25 degrees C) was found.

Carotenoids↗

Carotenoid biosynthesis changes in five red pepper (Capsicum annuum L.) cultivars during ripening. Cultivar selection for breeding.

Changes in the biosynthesis of individual carotenoid pigments have been investigated during fruit ripening of five cultivars of red pepper (Capsicum annuum L.): Mana, Numex, Belrubi, Delfin, and Negral (a chlorophyll-retaining mutant when ripe). The study was carried out throughout the ripening process, and with special emphasis on the ripe stage, to discover possible differences between cultivars and to characterize these by their carotenoid pattern and content for selecting the best varieties for breeding programs. Ripening fruit of the five cultivars showed the typical and characteristic pattern of carotenoid biosynthesis for the Capsicum genus. In the five cultivars, lutein and neoxanthin, both characteristic chloroplast pigments, decreased in concentration with ripening and eventually disappeared. beta-Carotene, antheraxanthin, and violaxanthin increased in concentration, and other pigments were biosynthesized de novo: zeaxanthin, beta-cryptoxanthin, capsanthin, capsorubin, capsanthin-5,6-epoxide, and cucurbitaxanthin A. A pool of zeaxanthin stands out of the rest of pigment during ripening, which reveals the importance of this pigment as a branching point in the carotenoid biosynthesis in Capsicum. Quantitatively, Negral cultivar showed the highest increase in total carotenoid content (48. 39-fold), followed by Mana and Delfin with 38.03- and 36.8-fold, respectively, and by Belrubi and Numex with 28.03- and 23.48-fold, respectively. In all the red varieties, there was an inverse relationship between total carotenoid content and the red to yellow isochromic pigment fraction ratio (R/Y) and the capsanthin-to-zeaxanthin ratio (Caps/Zeax). This seems to be related to the carotenogenic capacity of the cultivar, and thus selection and breeding should not only seek a higher total carotenoid content but also attempt to increase these ratios. In the present study, the cultivar Mana had the highest total carotenoid content (13 208 mg/kg dwt), but the lowest R/Y (1.25) and Caps/Zeax (3.38) ratios, which are therefore the parameters to improve. The cultivar Negral had a high carotenoid content (8797 mg/kg dwt) and high R/Y and Caps/Zeax ratios and could be used for transfer of these characters in direct crosses with the cultivar Mana. The cultivar Numex had the highest Caps/Zeax ratio (7.17) and is thus an ideal progenitor for this character.

Capsicum↗