Supercritical CO(2) and subcritical propane extraction of pungent paprika and quantification of carotenoids, tocopherols, and capsaicinoids.

Ground paprika (Capsicum annuum L.) was extracted with supercritical carbon dioxide (SC-CO(2)) and subcritical propane at different conditions of pressure and temperature to estimate the yield and variation in carotenoid, tocopherol, and capsaicinoid contents and composition. The yield of paprika extract was found to be affected by the extraction conditions with SC-CO(2) but fairly constant at different conditions with subcritical propane. The maximum yields of oleoresin were 7.9 and 8.1% of ground paprika by SC-CO(2) and subcritical propane, respectively. The quantitative distribution of carotenoids, tocopherols, and capsaicinoids between paprika extract and powder was influenced by extraction conditions. SC-CO(2) was inefficient in the extraction of diesters of xanthophylls even at 400 bar and 55 degrees C, whereas tocopherols and capsaicinoids were easy to extract at these conditions. Under mild conditions subcritical propane was superior to SC-CO(2) in the extraction of carotenoids and tocopherols but less efficient in the extraction of capsaicinoids.     

Association of carotenoids, tocopherols and vitamin C in plasma with allergic rhinitis and allergic sensitisation in adults

OBJECTIVES: Antioxidant nutrients like carotenoids, tocopherols and vitamin C have been suggested to protect against allergic rhinitis and allergic sensitisation but scientific evidence is scarce. The aims of the study were to measure the plasma concentration of six carotenoids, alpha- and gamma-tocopherol and vitamin C as biomarkers of the intake, absorption and subsequent metabolism of these nutrients, and to assess their association with allergic rhinitis and sensitisation. METHOD: Data from a cross-sectional study on representative dietary and lifestyle habits of the population of Bavaria, Germany, were analysed. The plasma levels of six carotenoids (alpha-carotene, beta-carotene, lycopene, lutein/zeaxanthin, canthaxanthin and cryptoxanthin) as well as of alpha-tocopherol, gamma-tocopherol and vitamin C were measured in 547 adults aged between 19 and 81 years. Participants with specific serum immunoglobulin E > or =700 U l(-1) were categorised as sensitised. The association of plasma antioxidant levels, allergic rhinitis and allergic sensitisation was assessed by means of unconditional logistic regression models. RESULTS: We observed a negative association between plasma total carotenoids and the prevalence of allergic rhinitis, with odds ratio (95% confidence interval) of 1.13 (0.54-2.39) for the second, 0.72 (0.33-1.58) for the third and 0.44 (0.19-1.03) for the fourth quartile of total carotenoids concentration (P for trend=0.0332); results for lycopene failed to reach statistical significance (P=0.0608). Other single carotenoids, tocopherols and vitamin C were unrelated to allergic rhinitis. Allergic sensitisation was negatively associated with plasma gamma-tocopherol, with odds ratio (95% confidence interval) of 0.92 (0.51-1.65) for the second, 1.00 (0.56-1.80) for the third and 0.45 (0.23-0.88) for the fourth quartile of plasma gamma-tocopherol concentration (P for trend=0.0410). No other antioxidant was significantly related to allergic sensitisation. CONCLUSIONS: High plasma carotenoid concentrations reflecting a diet high in various fruits and vegetables might have a protective effect on allergic rhinitis in adulthood.     

Simultaneous determination of all polyphenols in vegetables,fruits, and teas

Polyphenols, which have beneficial effects on health and occur ubiquitously in plant foods, are extremely diverse. We developed a method for simultaneously determining all the polyphenols in foodstuffs, using HPLC and a photodiode array to construct a library comprising retention times, spectra of aglycons, and respective calibration curves for 100 standard chemicals. The food was homogenized in liquid nitrogen, lyophilized, extracted with 90% methanol, and subjected to HPLC without hydrolysis. The recovery was 68-92%, and the variation in reproducibility ranged between 1 and 9%. The HPLC eluted polyphenols with good resolution within 95 min in the following order: simple polyphenols, catechins, anthocyanins, glycosides of flavones, flavonols, isoflavones and flavanones, their aglycons, anthraquinones, chalcones, and theaflavins. All the polyphenols in 63 vegetables, fruits, and teas were then examined in terms of content and class. The present method offers accuracy by avoiding the decomposition of polyphenols during hydrolysis, the ability to determine aglycons separately from glycosides, and information on simple polyphenol levels simultaneously.     

Mechanism of deactivation of triplet-excited riboflavin by ascorbate, carotenoids, and tocopherols in homogeneous and heterogeneous aqueous food model systems

Tocopherols (alpha, beta, gamma, and delta) and Trolox were found to deactivate triplet-excited riboflavin in homogeneous aqueous solution (7:3 v/v tert-butanol/water) with second-order reaction rates close to diffusion control [k2 between 4.8 x 10(8) (delta-tocopherol) and 6.2 x 10(8) L mol(-1) s(-1) (Trolox) at 24.0 +/- 0.2 degrees C] as determined by laser flash photolysis transient absorption spectroscopy. In aqueous buffer (pH 6.4) the rate constant for Trolox was 2.6 x 10(9) L mol(-1) s1 and comparable to the rate constant found for ascorbate (2.0 x 10(9) L mol(-1) s(-1)). The deactivation rate constant was found to be inferior in heterogeneous systems as shown for alpha-tocopherol and Trolox in aqueous Tween-20 emulsion (approximately by a factor of 4 compared to 7:3 v/v tert-butanol/water). Neither beta-carotene (7:3 v/v tert-butanol/water and Tween-20 emulsion), lycopene (7:3 v/v tert-butanol/water), nor crocin (aqueous buffer at pH 6.4, 7:3 v/v tert-butanol/water, and Tween-20 emulsion) showed any quenching on the triplet excited state of riboflavin. Therefore, all carotenoids seem to reduce the formation of triplet-excited riboflavin through an inner-filter effect. Activation parameters were based on the temperature dependence of the triplet-excited deactivation between 15 and 35 degrees C, and the isokinetic behavior, which was found to include purine derivatives previously studied, confirms a common deactivation mechanism with a bimolecular diffusion-controlled encounter with electron (or hydrogen atom) transfer as rate-determining step. DeltaH for deactivation by ascorbic acid, Trolox, and homologue tocopherols (ranging from 18 kJ mol(-1) for Trolox in Tween-20 emulsion to 184 kJ mol(-1) for ascorbic acid in aqueous buffer at pH 6.4) showed a linear dependence on DeltaS (ranging from -19 J mol(-1) K(-1) for Trolox in aqueous buffer at pH 6.4 to +550 J mol(-1) K(-1) for ascorbic acid in aqueous buffer pH 6.4). Among photooxidation products from the chemical quenching, lumicrome, alpha-tocopherol quinones and epoxyquinones, and alpha-tocopherol dimers were identified by ESI-QqTOF-MS.     

Identification of chlorophylls and carotenoids in major teas by high-performance liquid chromatography with photodiode array detection

The separation and identification of pigments, chlorophylls, and carotenoids of seven teas and fresh leaf of tea (Camellia sinensis) by high-performance liquid chromatography (HPLC) are described. HPLC was carried out using a Symmetry C(8) column with a photodiode array detector. Pigments were eluted with a binary gradient of aqueous pyridine solution at a flow rate of 1.0 mL/min at 25 degrees C. HPLC analyses achieved the separation of more than 100 pigment peaks, and 79 pigment species, 41 chlorophylls, and 38 carotenoids were detected. The presence of degraded chlorophylls was a common feature, and the number and the variety of pigments differed with tea species. Generally, the numbers of chlorophyll species tended to increase with processing steps, while carotenoid species were decreased, especially by heating. Particularly in green teas, a change of carotenoid structure, conversion of violaxanthin to auroxanthin, occurred. In hot water extracts of teas, both chlorophylls and carotenoids were also detected, but the concentration of chlorophylls was less than 2% as compared with acetone extracts. The pigment compositions were compared between tea species, and they are discussed in terms of the differences in their manufacturing processes.     

Bioaccessibility of tocopherols, carotenoids, and ascorbic acid from milk- and soy-based fruit beverages: influence of food matrix and processing

A study was made of the effect of high-pressure processing (HPP) and thermal treatment (TT) on plant bioactive compounds (tocopherols, carotenoids, and ascorbic acid) in 12 fruit juice-milk beverages and of how the food matrix [whole milk (JW), skimmed milk (JS), and soy milk (JSy)] modulates their bioaccessibility (%). HPP (400 MPa/40 °C/5 min) produced a significant decrease in carotenoid and ascorbic acid bioaccessibility in all three beverages and maintained the bioaccessibility of tocopherols in JW and JS while decreasing it in JSy. TT (90 °C/30 s) produced a significant decrease in tocopherol and carotenoid bioaccessibility in all three beverages and increased the bioaccessibility of ascorbic acid. With regard to the food matrix, α-tocopherol and ascorbic acid bioaccessibility was greatest in JW beverages and lowest in JSy beverages, whereas no significant differences were found among the three beverages in terms of carotenoid bioaccessibility. HPP-treated samples showed higher tocopherol and carotenoid bioaccessibility than TT-treated samples, thus indicating that HPP combined with a milk matrix positively modulates the bioaccessibility of certain types of bioactive components of food, mainly those of a lipophilic nature.     

Simple saponification method for the quantitative determination of carotenoids in green vegetables

A simple, reliable, and gentle saponification method for the quantitative determination of carotenoids in green vegetables was developed. The method involves an extraction procedure with acetone and the selective removal of the chlorophylls and esterified fatty acids from the organic phase using a strongly basic resin (Ambersep 900 OH). Extracts from common green vegetables (beans, broccoli, green bell pepper, chive, lettuce, parsley, peas, and spinach) were analyzed by high-performance liquid chromatography (HPLC) for their content of major carotenoids before and after action of Ambersep 900 OH. The mean recovery percentages for most carotenoids [(all-E)-violaxanthin, (all-E)-lutein epoxide, (all-E)-lutein, neolutein A, and (all-E)-beta-carotene] after saponification of the vegetable extracts with Ambersep 900 OH were close to 100% (99-104%), while the mean recovery percentages of (9'Z)-neoxanthin increased to 119% and that of (all-E)-neoxanthin and neolutein B decreased to 90% and 72%, respectively.     

Relationship of carotenoids and tocopherols in a sample of carrot root-color accessions and carrot germplasm carrying Rp and rp alleles

Carotenoids and tocopherols are powerful antioxidants synthesized in plants from a common precursor. They may offer significant health benefits to humans. Seed oils have been shown to possess high levels of tocopherols, but little is known about their levels in the edible portions of most vegetable crops. A two-year field experiment was conducted at two locations to assess levels of major carotenoids and tocopherols in carrot (Daucus carota) root and leaf tissue. Levels of compounds in root tissue reported on a dry weight basis were as follows: alpha-tocopherol, 0.04-0.18 ppm; lycopene, 0.00-52.94 ppm; alpha-carotene, 10.63-1504.76 ppm; and beta-carotene, 26.69-1673.76 ppm. Higher levels of all carotenoids were measured in phloem tissue than in xylem. Leaf tissue levels of tocopherols measured on a dry weight basis ranged from 0.02 to 0.85 ppm, whereas levels of carotenoids ranged from 12.81 to 411.66 ppm. In xylem tissue, alpha-tocopherol was significantly (P < or =0.001) positively correlated with alpha-carotene (r = 0.65) and with beta-carotene (r = 0.52). This positive correlation indicates it may be possible to select for both increased alpha-tocopherol and carotenoids in carrot. The reduced pigment (rp) mutation of carrot exhibited a 96% reduction in levels of alpha- and beta-carotene and a 25-43% reduction in alpha-tocopherol when compared to a near-isogenic line. In plants homozygous for rp, a substantial increase was observed in phytoene, a precursor to carotenoids, suggesting the location of the rp lesion in the carotenoid synthesis pathway.     

Resonance Raman quantification of nutritionally important carotenoids in fruits, vegetables, and their juices in comparison to high-pressure liquid chromatography analysis

A rapid nondestructive estimation of carotenoid levels in intact fruits and vegetables and their juices could have great value when selecting nutritionally valuable crops for further propagation and commercial use. Carotenoid levels of a variety of agricultural products and juices were measured using resonance Raman spectroscopy and compared to levels determined by extraction and high-pressure liquid chromatography. A strong correlation was observed between the two methods when evaluating juices and when comparing different strains of intact tomatoes at the same stage of ripening.     

Determination of major carotenoids in a few Indian leafy vegetables by high-performance liquid chromatography

Leafy vegetables [Basella rubra L., Peucedanum sowa Roxb., Moringa oleifera Lam., Trigonella foenum-graecum L., Spinacia oleracea L., Sesbania grandiflora (L.) Poir., and Raphanus sativus L.] that are commonly used by the rural population in India were evaluated in terms of their main carotenoid pattern. The extracted carotenoids were purified by open column chromatography (OCC) on a neutral alumina column to verify their identity by their characteristic UV-visible absorption spectra. Reverse-phase high-performance liquid chromatography (HPLC) on a C18 column with UV-visible photodiode array detection under isocratic conditions was used for quantification of isolated carotenoids. Acetonitrile/methanol/dichloromethane (60:20:20 v/v/v) containing 0.1% ammonium acetate was used as a mobile phase. The major carotenoids identified by both methods were lutein, beta-carotene, violaxanthin, neoxanthin, and zeaxanthin. Among the carotenoids identified, lutein and beta-carotene levels were found to be higher in these leafy vegetables. Results show that P. sowa and S. oleracea are rich sources of lutein (77-92 mg/100 g of dry wt) and beta-carotene (36-44 mg/100 g of dry wt) compared with other leafy vegetables. The purity of carotenoids eluted by OCC was clarified by HPLC, and they were found to be 92% +/- 3% for neoxanthin, 94% +/- 2% for violaxanthin, 97% +/-2% for lutein and zeaxanthin, and 90% +/- 3% for beta-carotene. It could be recommended to use P. sowa and S. oleracea as rich sources of lutein and beta-carotene for health benefits. The OCC method proposed is relatively simple and provides purified carotenoids for feeding trials.     

Structure determination of partially deuterated carotenoids from intrinsically labeled vegetables by HPLC-MS and 1H NMR

The structures of biosynthetic deuterated carotenoids in labeled vegetables were investigated: (all-E)-lutein and (all-E)-beta-carotene from spinach, and (all-E)-beta-carotene and (all-E)-alpha-carotene from carrots. The vegetables were grown hydroponically using a nutrient solution enriched with deuterium oxide (D(2)O) and were extracted using matrix solid-phase dispersion (MSPD). Deuterium enrichment in the carotenoid molecules was determined by liquid chromatography-mass spectrometry (LC-MS). (all-E)-Lutein and (all-E)-beta-carotene in spinach showed partial deuteration from (2)H(1) to (2)H(12), with the abundance maximum at (2)H(5). (all-E)-beta-Carotene and (all-E)-alpha-carotene from carrots showed partial deuteration from (2)H(1) to (2)H(17), with the abundance maximum at (2)H(11). The (1)H NMR spectra of the four deuterated carotenoids showed additional signals for all methyl groups and decreased signal intensity for the olefinic protons and the methylene protons in the ring. These differences are due to isotopic effects and are based on the substitution of protons by deuterium atoms. The deuteration was distributed randomly throughout the carotenoid molecules.     

Simultaneous fluorometric determination of chlorophylls a and B and pheophytins a and B in olive oil by partial least-squares calibration

The resolution of quaternary mixtures of chlorophylls a and b and pheophytins a and b has been accomplished by partial least-squares (PLS) multivariate calibration, applied to the fluorescence signals of these pigments. The total luminescence information of the compounds has been used to optimize the spectral data set to perform the calibration. After preliminary studies, a method is described in acetone media, to avoid emulsions with the olive oil samples. Different scanning paths have been selected for each method. For the simultaneous determination of the pigments in olive oil samples, a comparative study of the results found by using excitation, emission, and synchronous spectral data, as analytical signal, was performed. The excitation spectra were selected as the better analytical signals for the determination of the pigments in olive oil samples. The optimum wavelength range to record the excitation spectra (lambda(em) = 662 nm) was selected to minimize the contribution of pheophytin a and to maximize the contribution of the other pigments, which are the minor constituents in olive oil. Determination of these pigments in olive oil samples was effected from the excitation spectra of dissolutions o suitable aliquots in acetone. Recovery values from olive oil, spiked with chlorophylls a and b and pheophytins a and b, were in the ranges of 70-112, 71-111, 76-105, and 82-109%, respectively.     

Vitamin C,provitamin A carotenoids,and other carotenoids in high-pressurized orange juice during refrigerated storage

Vitamin C, provitamin A carotenoids, and other carotenoids were measured in freshly squeezed juices from oranges (Citrus sinensis L. var. Valencia late) that were subjected to high-pressure (HP) treatment. Also, the stability of these compounds was studied during refrigerated storage at 4 degrees C. HP treatment is an alternative to heat preservation methods for foods; therefore, it is essential to assess the impact of HP on bioactive compounds. Several processes that combine HP treatment with heat treatment for various time periods were assayed: T0, fresh juice (without treatment); T1, 100 MPa/60 degrees C/5 min; T2, 350 MPa/30 degrees C/2.5 min; T3, 400 MPa/40 degrees C/1 min. Fresh and treated samples were kept refrigerated (4 degrees C) over 10 days. After application of HP and during the refrigeration period, the qualitative and quantitative determination of vitamin C, provitamin A carotenoids (beta- and alpha-carotene; beta- and alpha-cryptoxanthin), and the xanthophylls zeaxanthin and lutein was achieved by high-performance liquid chromatography. T1 and T3 juices showed a decrease in ascorbic acid and total vitamin C just after HP treatment (D0) compared with T0 juices. On the contrary, T2 juices, just after HP treatment (D0), had the same levels of both compounds compared to untreated juices. T1, T2, and T3 treatments led to an increase in the extraction of carotenoids and provitamin A carotenoids. Total carotenoid content after the 10-day refrigerated storage period resulted in no significant quantitative changes in T1 juices, whereas in T2 and T3 juices small losses were found at the end of the storage period (20.56 and 9.16%, respectively). These losses could be influenced by the depleted protection of vitamin C toward carotenoid oxidation during the same period. A similar trend was found in provitamin A carotenoids for the different treated juices.     

Enzyme-mediated solvent extraction of carotenoids from marigold flower (Tagetes erecta)

Marigold flowers are the most important source of carotenoids for application in the food industry. However, the extraction gives almost 50% losses of the carotenoids depending on conditions for silaging, drying, and solvent extraction. In the past decades, macerating enzymes have been successfully applied to improve the extraction yield of valued compounds from natural products. In this work, an alternative extraction process for carotenoids is proposed, consisting of a simultaneous enzymatic treatment and solvent extraction. The proposed process employs milled fresh flowers directly as raw material, eliminating the inefficient silage and drying operations as well as the generation of hard to deal with aqueous effluents present in traditional processes. The process developed was tested at the 80 L scale, where under optimal conditions a carotenoid recovery yield of 97% was obtained.     

Isotopic labeling and LC-APCI-MS quantification for investigating absorption of carotenoids and phylloquinone from kale (Brassica oleracea)

The ability to study bioavailability of nutrients from foods is an important step in determining the health impact of those nutrients. This work describes a method for studying the bioavailability of nutrients from kale (Brassica oleracea var. Acephala) by labeling the nutrients with carbon-13, feeding the kale to an adult volunteer, and analyzing plasma samples for labeled nutrients. Results showed that conditions for producing atmospheric intrinsically labeled kale had no detrimental effect on plant growth. Lutein, beta-carotene, retinol, and phylloquinone were analyzed using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. Analysis of plasma samples showed that labeled lutein peaked in plasma at 11 h (0.23 microM), beta-carotene peaked at 8 (0.058 microM) and 24 h (0.062 microM), retinol peaked at 24 h (0.10 microM), and phylloquinone peaked at 7 h (3.0 nM). This method of labeling kale with (13)C was successful for producing clearly defined kinetic curves for (13)C-lutein,(13)C-beta-carotene, (13)C-retinol, and (13)C-phylloquinone.     

Optimization of the trienzyme extraction for the microbiological assay of folate in vegetables

Response surface methodology was applied to optimize the trienzyme digestion for the extraction of folate from vegetables. Trienzyme extraction is a combined enzymatic digestion by protease, alpha-amylase, and conjugase (gamma-glutamyl hydrolase) to liberate the carbohydrate and protein-bound folates from food matrices for total folate analysis. It is the extraction method used in AOAC Official Method 2004.05 for assay of total folate in cereal grain products. Certified reference material (CRM) 485 mixed vegetables was used to represent the matrix of vegetables. Regression and ridge analysis were performed by statistical analysis software. The predicted second-order polynomial model was adequate (R2 = 0.947), without significant lack of fit (p > 0.1). Both protease and alpha-amylase have significant effects on the extraction. Ridge analysis gave an optimum trienzyme digestion time: Pronase, 1.5 h; alpha-amylase, 1.5 h; and conjugase, 3 h. The experimental value for CRM 485 under this optimum digestion was close to the predicted value from the model, confirming the validity and adequacy of the model. The optimized trienzyme digestion condition was applied to five vegetables and yielded higher folate levels than the trienzyme digestion parameters employed in AOAC Official Method 2004.05.     

Comparison and analysis of fatty acids, sterols, and tocopherols in eight vegetable oils

The similarities and differences of eight vegetable oils produced in China were investigated in terms of their fatty acid, sterol, and tocopherol compositions and subsequent data processing by hierarchical clustering analysis and principal component analysis. The lipid profiles, acquired by analytical techniques tailored to each lipid class, revealed great similarities among the fatty acid profiles of corn and sesame oil as well as few differences in their sterol profiles. It turns out that not only was there great similarity between the fatty acid profiles of corn oil and sesame oil but also there were not too many differences for the sterol profiles. Sunflower and tea-seed oil showed similar sterol compositions, while the tea-seed oil tocopherol was very similar to palm oil. The results demonstrated that the use of only one of these profiles was unreliable for indentifying oil origin and authenticity. In contrast, the use of the sterol or tocopherol profile together with the fatty acid profile more accurately discriminates these oils.