Thermal processing of vegetables increases cis isomers of lutein and zeaxanthin

Carotenoids, lutein and zeaxanthin, found in fruits and vegetables, comprise the macula pigment of the eye. These carotenoids exist in plants as the all-trans geometric form; however, in human plasma, cis isomers of these carotenoids have also been identified. Thermal processing can induce carotenoid trans to cis isomerization. The aim of this research was to determine if thermal processing induces isomerization of lutein and zeaxanthin and to quantify the extent of this reaction. High-performance liquid chromatography was used to separate and quantitate geometric isomers of lutein and zeaxanthin. Isomers were tentatively identified by UV-visible absorbance spectra, comparison of retention times to those of isomerized standards using C(30) chromatography, and mass spectrometry. Thermal processing increased the percent cis isomers of lutein and zeaxanthin up to 22 and 17%, respectively. Further studies are needed to consider the physiological impact of consuming carotenoid isomers in processed vegetables.     

Isolation and structural elucidation of (13Z,13'Z,3R,3'R,6'R)-lutein from marigold flowers, kale, and human plasma

(13Z,13'Z,3R,3'R,6'R)-Lutein has been isolated and purified from extracts of marigold flowers, fresh raw kale (Brassica oleracea var. Acephala), and human plasma and fully characterized by (1)H and (13)C NMR, UV/vis, and MS. While the concentration of (13Z, 13'Z)-lutein in kale and human plasma compared to (all-E,3R,3'R, 6'R)-lutein was found to be quite low, this compound was readily isolated by fractional crystallization of lutein from marigold extracts. Thus, the mother liquors from two consecutive crystallizations of lutein from a saponified extract of marigold flowers were enriched in (13Z,13'Z)-lutein (8.7% of total carotenoids) and employed for the isolation of this compound by HPLC. The identity of the di-Z-lutein in kale and human plasma has been established by comparison of the HPLC-UV/vis-MS profiles of the purified compounds with those of a fully characterized sample, isolated from marigolds. 3-Hydroxy-beta,epsilon-caroten-3'-one and 3'-epilutein have also been identified in extracts from marigolds.     

Phenolic, flavonoid, and lutein ester content and antioxidant activity of 11 cultivars of chinese marigold

This study analyzed 11 Chinese cultivars of marigold to determine their major phytochemical contents and antioxidant activities. Dried marigold flowers were extracted with ethanol, ethyl acetate, and n-hexane and the extracts were analyzed by high-performance liquid chromatography-mass spectrometry and chemical methods to determine their lutein esters, phenolic and flavonoid contents, and antioxidant activity, respectively. The different cultivars of marigold showed considerable variations in their lutein ester contents, ranging from 161.0 to 611.0 mg/100 g of flower (dry basis). The lutein esters in marigolds consisted predominantly of six all trans-diesters, but small amounts of cis isomers of the respective diesters were also present. The different cultivars of marigold also showed marked variations in total phenols and flavonoids, as well as antioxidant and radical-scavenging activities. Ethanol was confirmed to be the best solvent for extracting both phenols and flavonoids from marigold flowers, while n-hexane was the worst. The ethanolic extracts also exhibited the highest antioxidant and radical-scavenging activities. The cultivar Xinhong had the highest phenolic and flavonoid contents and radical-scavenging activity, as well as one of the highest lutein contents and antioxidant activities.     

Differentiation between lutein monoester regioisomers and detection of lutein diesters from marigold flowers (Tagetes erecta L.) and several fruits by liquid chromatography-mass spectrometry.

Liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCIMS) was employed for the identification of eight lutein monoesters, formed by incomplete enzymatic saponification of lutein diesters of marigold (Tagetes erecta L.) by Candida rugosa lipase. Additionally, the main lutein diesters naturally occurring in marigold oleoresin were chromatographically separated and identified. The LC-MS method allows for characterization of lutein diesters occurring as minor components in several fruits; this was demonstrated by analysis of extracts of cape gooseberry (Physalis peruviana L.), kiwano (Cucumis metuliferus E. Mey. ex Naud.), and pumpkin (Cucurbita pepo L.). The assignment of the regioisomers of lutein monoesters is based on the characteristic fragmentation pattern: the most intense daughter ion generally results from the loss of the substituent (fatty acid or hydroxyl group) bound to the epsilon-ionone ring, yielding an allylic cation. The limit of detection was estimated at 0.5 microg/mL with lutein dimyristate as reference compound. This method provides a useful tool to obtain further insight into the biochemical reactions leading to lutein ester formation in plants.     

Thermal degradation kinetics of bixin in an aqueous model system

The kinetics of the thermal degradation of the natural cis carotenoid bixin in a water/ethanol (8:2) solution was studied as a function of temperature (70-125 degrees C), using high-performance liquid chromatography. The curves for the decay of bixin and formation of products (e.g., di-cis and all-trans isomers and a C17 degradation compound) did not adjust well to a first-order rate law, but very good fits were obtained using a biexponential model. This mathematical modeling gave the rate constant values for the formation of the primary products from bixin, and the energy barrier for each step was obtained. The di-cis isomers were formed immediately (15 kcal/mol) together with the decay of bixin, followed by a slow consumption, indicating their role as reaction intermediates. In fact, the di-cis isomers could easily revert to bixin (Ea approximately 3 kcal/mol) or yield the primary C17 degradation product, with an energy barrier of 6.5 kcal/mol. In turn, 24 kcal/mol was necessary for the Bix --> all-trans step, explaining its slower formation.     

Differential incorporation of conjugated linoleic acid isomers into egg yolk lipids

The incorporation pattern of conjugated linoleic acids (CLA) isomers into the egg yolk of hens in relation to that in the diet was studied. Silver-ion high-performance liquid chromatography (Ag-HPLC) was used to separate individual CLA isomers. It was found that the isomeric distribution pattern in the egg yolk lipids was different from that in the dietary fat. Total cis/trans isomers accounted for 81.2% of total CLA incorporated into the egg yolk, which was in contrast to the value of 92.0% of total CLA in the diet. Total cis/cis isomers accounted for 3.8% total CLA in the diet but they were 6.6% of the total CLA in the egg yolk lipids. In contrast, total trans/trans isomers were 12.2% of the total CLA isomers in the egg yolk lipids, whereas they were only 4.2% of total CLA in the diet. The results showed that total trans/trans-CLA was preferentially incorporated into the egg yolk, whereas the incorporation of total cis/trans-CLA isomers was partially discriminated. Within each group, the incorporation of individual isomers into the egg yolk lipids was also selective. cis-9,trans-11/trans-9,cis-11 and cis-10,trans-12/trans-10,cis-12 were the two major isomers in the diet. Ag-HPLC analysis showed that the former was preferentially transferred into the egg yolk compared with the latter. It was observed that supplementation of CLA in the diet of laying hens decreased the concentration of oleic acid (18:1n-9), arachidonic acid (20:4n-6), and docosahexaenoic acid (22:6n-3) but increased that of linolenic acid (18:3n-3), stearic acid (18:0), and palmitic acid (16:0) in the egg yolk, suggesting that CLA may inhibit Delta6 and Delta9 desaturases.     

Carotenoid composition in the fruits of Asparagus officinalis

The carotenoid pigments of the ripe and unripe fruits of Asparagus officinalis were investigated by means of an HPLC technique. Capsanthin, capsorubin, capsanthin 5,6-epoxide, antheraxanthin, violaxanthin, neoxanthin, mutatoxanthin epimers, zeaxanthin, lutein, beta-cryptoxanthin, beta-carotene, and some cis isomers were found. Carotenoids with 3,5,6-trihydroxy and 3,6-epoxy beta-end groups could not be deleted.     

Hydrolysis kinetics of astaxanthin esters and stability of astaxanthin of Haematococcus pluvialis during saponification

The reaction kinetics for the hydrolysis of astaxanthin esters and the degradation of astaxanthin during saponification of the pigment extract from the microalga Haematococcus pluvialis were investigated. Different concentrations of sodium hydroxide in methanol were used for the saponification under nitrogen in darkness at ambient temperature (22 degrees C) followed by the analysis of astaxanthins and other carotenoids using an HPLC method. The concentration of methanolic NaOH solution was important for promoting the hydrolysis of astaxanthin esters and minimizing the degradation of astaxanthin during saponification. With a higher concentration of methanolic NaOH solution, the reaction rate of hydrolysis was high, but the degradation of astaxanthin occurred significantly. The rate constants of the hydrolysis reaction (first order) of astaxanthin esters and the degradation reaction (zero-order) of astaxanthin were directly proportional to the concentration of sodium hydroxide in the saponified solution. Although the concentration of sodium hydroxide in the saponified solution was 0.018 M, complete hydrolysis of astaxanthin esters was achieved in 6 h for different concentrations (10-100 mg/L) of pigment extracts. Results also indicated that a higher temperature should be avoided to minimize the degradation of astaxanthin. In addition, during saponification, no loss of lutein, beta-carotene, and canthaxanthin was found.     

Improved separation method for highly purified lutein from Chlorella powder using jet mill and flash column chromatography on silica gel

We investigated an improved method for the separation of high-purified lutein from a commercially available spray-dried Chlorella powder (CP) using fine grinding by jet mill and flash column chromatography on a silica gel. Saponification and extraction of lutein were enhanced 2.3-2.9-fold in jet mill-treated CP (mean particle size, 20 microm) as compared to untreated CP (mean particle size, 67 microm). The carotenoid extract was dissolved in ether-hexane (1:1 v/v) and subjected to flash column chromatography on silica gel. A mixture of alpha- and beta-carotene was eluted with hexane, followed by elution with hexane-acetone-chloroform (7:2:1 v/v). Lutein (dark-orange band) was collected after the elution of an unknown colorless compound (detected based on UV absorbance). The purity of lutein in this fraction was over 99%, and the yield was 60%. The present study provides key information for obtaining highly purified lutein using flash column chromatography on a silica gel.     

Effects of heating and illumination on trans-cis isomerization and degradation of beta-carotene and lutein in isolated spinach chloroplasts

The influence of thermal treatment and light exposure on degradation and isomerization of the predominant carotenoids (lutein and beta-carotene) occurring in green leafy vegetables was assessed. The effect of lipid addition on carotenoid stability was also evaluated. For the first time, the stabilities of pure carotenoids and chloroplast-bound carotenoids were compared. Besides degradation, heating caused carotenoid isomerization in all samples. Whereas pure carotenoids favor 13-cis isomers, in native chloroplasts and heated chloroplasts 9-cis isomers were predominant. Illumination of freshly prepared chloroplast isolates caused an initial increase in the level of lutein (9.6%) and beta-carotene (29.8%), while pure carotenoids exhibited time-dependent degradation. The addition of lipids to chloroplast preparations had the reverse effects on the retention of both carotenoids after heating; isomerization was not significantly affected. It was demonstrated that carotenoid stability has to be evaluated for every individual pigment in its genuine environment. Stability data based on model systems (e.g., pure carotenoids) may not be transferred to complex food matrices without intensive investigation.     

Analysis and stability of carotenoids in the flowers of daylily (Hemerocallis disticha) as affected by various treatments

The analysis and stability of carotenoids in the flowers of daylily (Hemerocallis disticha) as affected by soaking and drying treatments were studied. The various carotenoids in the flowers of daylily were analyzed using a reversed-phase C(30) HPLC column and a mobile phase of methanol/methylene chloride/2-propanol (89:1:10, v/v/v) with methanol/methylene chloride (45:55, v/v) as sample solvent. Twenty-one pigments were resolved, of which 14 carotenoids were identified, including neoxanthin, violaxanthin, violeoxanthin, lutein-5,6-epoxide, lutein, zeaxanthin, beta-cryptoxanthin, all-trans-beta-carotene, and their cis isomers, based on spectral characteristics and Q ratios. Prior to hot-air-drying (50 degrees C) or freeze-drying, some of the daylily flowers were subjected to soaking in a sodium sulfite solution (1%) for 4 h. Under either the hot-air- or the freeze-drying treatment, the amounts of most carotenoids were higher in the soaked daylily flowers than in those that were not soaked. With hot-air-drying, the amount of cis carotenoids showed a higher yield in soaked samples than in nonsoaked samples. However, with freeze-drying, only a minor change of each carotenoid was observed for both soaked and nonsoaked samples. Also, air-drying resulted in a higher loss of carotenoids than freeze-drying.     

Identification of isolutein (lutein epoxide) as cis-antheraxanthin in orange juice

The carotenoid profile of orange juice is very complex, a common characteristic for citrus products in general. This fact, along with the inherent acidity of the product, which promotes the isomerization of some carotenoids, makes the correct identification of some of these pigments quite difficult. Thus, one of the carotenoids occurring in orange juice has been traditionally identified as isolutein, a term used to refer to lutein epoxide, although enough evidence to support that identification has not been given. In this study, the carotenoid previously identified as isolutein/lutein epoxide in orange juice has been isolated and identified as a 9 or 9'-cis isomer of antheraxanthin as a result of different tests. To support this identification, a mixture of geometrical isomers of lutein epoxide isolated from petals of dandelions was analyzed under the same conditions used for orange juice carotenoids to check that neither their retention times nor their spectroscopic features matched with those of the orange juice carotenoid now identified as a cis isomer of antheraxanthin.     

Supercritical fluid extraction of all-trans-lycopene from tomato

A procedure is proposed for the supercritical fluid extraction of all-trans-lycopene from tomato using carbon dioxide at 40 degrees C without modifier. The present method minimizes the risk of degradation via isomerization and oxidation of health-promoting ingredients, such as lycopene. The effect of different experimental variables on the solvating power of the supercritical fluid was evaluated in terms of both the selectivity achievable in the process and the yield of the extraction of all-trans-lycopene. Satisfactory separations of the all-trans-lycopene isomers from the cis counterparts were achieved using a C(30) column. The obtained extract contained 88% all-trans-lycopene and 12% cis-lycopene.     

Distribution of lutein,zeaxanthin, and related geometrical isomers in fruit,vegetables, wheat,and pasta products

Quantitative data with regard to dietary (3R,3'R,6'R)-lutein, (3R,3'R)-zeaxanthin, and their (E/Z)-geometrical isomers are scarce, and in most cases, only the combined concentrations of these two carotenoids in foods are reported. Lutein and zeaxanthin accumulate in the human macula and have been implicated in the prevention of age-related macular degeneration (AMD). The qualitative and quantitative distributions of lutein, zeaxanthin, and their (E/Z)-isomers in the extracts from some of the most commonly consumed fruits, vegetables, and pasta products were determined by HPLC employing a silica-based nitrile-bonded column. Green vegetables had the highest concentration of lutein (L) and zeaxanthin (Z), and the ratios of these carotenoids (L/Z) were in the range 12-63. The yellow-orange fruits and vegetables, with the exception of squash (butternut variety), had much lower levels of lutein in comparison to greens but contained a higher concentration of zeaxanthin. The ratio of lutein to zeaxanthin (L/Z) in two North American bread varieties of wheat (Pioneer, Catoctin) was 11 and 7.6, respectively, while in a green-harvested wheat (Freekeh) imported from Australia, the ratio was 2.5. Between the two pasta products examined, lasagne and egg noodles, the latter had a much higher concentration of lutein and zeaxanthin. The levels of the (E/Z)-geometrical isomers of lutein and zeaxanthin in these foods were also determined.     

Determination of cis, cis-methylene interrupted polyunsaturated fatty acids in fats and oils by capillary gas chromatography

A capillary gas chromatographic (CGC) method is described for the determination of cis,cis-methylene interrupted polyunsaturated fatty acids (cis-PUFA) in fats and oils. The sample is saponified and the liberated fatty acids are esterified to the corresponding methyl esters. The latter are analyzed by CGC using a 60 M. SP2340 capillary column. Area percent values for 9,12-cis,cis-C18:2 and 9,12,15-cis,cis,cis-C18:3 fatty acid methyl esters are summed to give the total cis-PUFA content. Gas chromatographic results agreed well with those obtained by an enzymatic lipoxygenase method at the 31-48% cis-PUFA levels with a correlation coefficient of 0.98. The method has a precision (relative standard deviation) of 0.33% at a 44.4% cis-PUFA level in margarine oil.     

Evaluation of some carotenoids in grapes by reversed- and normal-phase liquid chromatography: a qualitative analysis

Carotenoids in grapes of three Port winemaking cultivars were investigated. Extracts were obtained with n-hexane/diethyl ether mixtures (0/100; 20/80; 50/50; 100/0) and analyzed by normal and reversed phase HPLC-DAD. Selection and identification of peaks were based on spectroscopic characteristics - lambda(max), (%III/II) and k' values, leading to 28 probable carotenoids. Using pure standards, it was possible to identify seven compounds previously described (neochrome, neoxanthin, violaxanthin, flavoxanthin, zeaxanthin, lutein, and beta-carotene), one more type of neochrome reported here, for the first time, and in addition, two geometrical isomers of lutein and beta-carotene were tentatively described. The remaining 17 need to be further identified. High polarity solvent mixtures lead to qualitatively richer chromatograms. Reversed-phase separations allowed the detection of flavoxanthin and the possible geometrical isomer(s) of beta-carotene. Under normal phase, zeaxanthin was detected, and neochromes were better separated from neoxanthin. Extraction with 50/50 n-hexane/diethyl ether mixtures and reversed-phase conditions was the best combination for analysis of the carotenoids, known as precursors of compounds with high aroma impact in wines.     

Chromoplast morphology and beta-carotene accumulation during postharvest ripening of Mango Cv. 'Tommy Atkins'

Accumulation of beta-carotene and trans-cis isomerization of ripening mango mesocarp were investigated as to concomitant ultrastructural changes. Proceeding postharvest ripening was shown by relevant starch degradation, tissue softening, and a rising sugar/acid ratio, resulting in a linear decrease (R (2) = 0.89) of a ripening index (RPI(KS)) with increasing ripening time. A modest accumulation of all-trans-beta-carotene and its cis isomers resulted in a slight pigmentation of the mango chromoplasts, because ambient temperatures of 18.2-19.5 degrees C provided suboptimal ripening conditions, affecting color development and beta-carotene biosynthesis. The ultrastructures of chromoplasts from mango mesocarp and carrot roots were comparatively studied by means of light and transmission electron microscopy. Irrespective of the ripening stage, mango chromoplasts showed numerous plastoglobuli varying in size and electron density. They comprised the main part of carotenoids, thus supporting the partial solubilization of the pigments in lipid droplets. However, because different pigment-carrying tubular membrane structures were also observed, mango chromoplasts were assigned to the globular and reticulotubular types, whereas the crystalline type was confirmed for carrot chromoplasts. The large portions of naturally occurring cis-beta-carotene in mango fruits contrasted with the predominance of the all-trans isomer characteristic of carrots, indicating that the nature of the structure where carotenoids are deposited and the physical state of the pigments are crucial for the stability of the all-trans configuration.     

Absence of isomerization of retinyl palmitate, retinol, and retinal in chlorinated and nonchlorinated solvents under gold light

Purified solutions of all-trans retinyl palmitate, retinol, and retinaldehyde in chloroform, methylene chloride, or hexane were exposed to white light or gold fluorescent light or were kept in the dark, and the resulting isomer distributions were determined by LC (liquid chromatography). No significant isomerization of any of the retinoids occurred either in the dark or on exposure to gold light in any of the solvents tested. However, a large amount of the 9-cis isomer and only much smaller amounts of other cis isomers were produced when retinol or retinyl palmitate in chloroform or methylene chloride solution was exposed to white light. The isomerization pattern of retinyl palmitate in chloroform was not altered by the addition of free-radical scavengers, addition of an organic base, or substitution of deuterochloroform for chloroform as solvent. Use of other polar solvents such as tetrahydrofuran, acetone, or methanol produced isomer distributions similar to those obtained in chloroform solution. Retinol and retinyl palmitate in hexane solution, on exposure to white light, were isomerized much less extensively than in chloroform or methylene chloride and produced a significant amount of the 13-cis, as well as the 9-cis, isomer. Isomerization of retinaldehyde in chloroform or in methylene chloride solution under white light yielded 13-cis, 11-cis, 9-cis, and 7-cis isomers, in order of decreasing amount, whereas in hexane solution, only the 13-cis and 9-cis isomers were produced in significant quantity.     

Identification and quantification of seed carotenoids in selected wheat species

Selected primitive and modern wheat species were evaluated on the basis of their carotenoid composition and effects of the genotype and environment on lutein using spectrometry and liquid chromatography. Carotenoids in the wheat extracts were identified and confirmed on the basis of their UV/vis and mass spectra compared with those of authentic standards. The protonated molecule (M + 1)+ at m/z 569 was the predominant ion for zeaxanthin compared to the fragment ion at m/z 551 for lutein. A similar carotenoid profile was obtained for the wheat species investigated, but significant differences were observed in the concentration of carotenoids. Einkorn (Triticum monococcum) exhibited the highest level of all-trans-lutein, averaging 7.41 microg/g with small amounts of all-trans-zeaxanthin, cis-lutein isomers, and beta-carotene. Durum, Kamut, and Khorasan (Triticum turgidum) had intermediate levels of lutein (5.41-5.77 microg/g), while common bread or pastry wheat (Triticum aestivum) had the lowest content (2.01-2.11 microg/g). Lutein in einkorn appeared to be influenced significantly by environmental growing conditions.     

Nasunin from eggplant consists of cis-trans isomers of delphinidin 3-[4-(p-coumaroyl)-L-rhamnosyl (1-->6)glucopyranoside]-5-glucopyranoside

Two major anthocyanins were isolated from the acidified methanolic extract of eggplant (Solanum melongena L.) by column chromatography and preparative high-performance liquid chromatography. These anthocyanins were interconvertible under room light illumination condition. By means of tandem time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy, their structures were identified and elucidated as delphinidin 3-[4-(cis-p-coumaroyl)-l-rhamnosyl(1-->6)glucopyranoside]-5-glucopyranoside (compound 1) and delphinidin 3-[4-(trans-p-coumaroyl)-l-rhamnosyl-(1-->6)glucopyranoside]-5-glucopyranoside (compound 2), respectively. The results indicated that nasunin comprised cis and trans isomers of the p-coumaric acid moiety in its structure.