Development of an HPLC-PDA Method for the Determination of Capsanthin, Zeaxanthin, Lutein, ß-Cryptoxanthin and ß-Carotene Simultaneously in Chili Peppers and Products.

For the better standardization and widespread application of the determination method of carotenoids in both chili peppers and their products, this work reports for the first time the simultaneous determination of five main carotenoids, including capsanthin, zeaxanthin, lutein, ß-cryptoxanthin and ß-carotene in chili peppers and their products, with optimized extraction and the high-performance liquid chromatography (HPLC) method. All parameters in the methodological evaluation were found to be in good stability, recovery and accuracy compliance with the reference values; the R coefficients for the calibration curves were more than 0.998; and the LODs and LOQs varied from 0.020 to 0.063 and from 0.067 to 0.209 mg/L, respectively. The characterization of five carotenoids in chili peppers and their products passed all the required validation criteria. The method was applied in the determination of carotenoids in nine fresh chili peppers and seven chili pepper products.

Japanese carotenoid database with α- and ß-carotene, ß-cryptoxanthin, lutein, zeaxanthin, lycopene, and fucoxanthin and intake in adult women.

Carotenoid intake is associated with low mortality and cancer risks; data on non-provitamin carotenoid intake is limited especially in Asians. We aimed to estimate carotenoid intake in Japanese adult women. Carotenoid content database comprises 196 food items, including 39 fruits, 87 vegetables and mushrooms, and 11 seaweeds, and was established using data from the literature and analyses of foods available in Japan. We surveyed the intake of these foods in Japanese women aged 21-56 years (n=109). Total intake of 7 carotenoids (mean±SD [range]) was 7,450±3,840 (1,160-21,300) µg/day; α-carotene, ß-carotene, ß-cryptoxanthin, lutein, zeaxanthin, lycopene, and fucoxanthin represented 4.3%, 23%, 3.4%, 15%, 2.0%, 39%, and 13% of total intake, respectively. Lutein intake was 1,132±686 (294-3,490) µg/day; its best sources were spinach, cucumber, chicken egg, green onion, and Chinese chives, representing 51% of total intake. Lutein can be obtained from a variety of sources. Thus, lutein intake levels did not vary widely among individuals and very few individuals consumed insufficient levels of lutein. Intake of zeaxanthin, lycopene, and fucoxanthin was 149±93 (2-479), 2,890±2,970 (0-17,100), and 980±1,230 (0-5,660) µg/day, respectively. Their intake required rich sources including chicken egg for zeaxanthin (52%); tomato products for lycopene (98%), and wakame seaweed for fucoxanthin (76%). The carotenoid content database including all food items consumed in Japan will be helpful for further investigations on carotenoid intake and its health benefits.

Use of stable isotopes to study bioconversion and bioefficacy of provitamin A carotenoids.

Provitamin A carotenoids (pVACs) are important contributors to vitamin A status and in reducing vitamin A deficiency in vulnerable populations. However, there are uncertainties about the effectiveness of dietary pVACs to provide sufficient amounts of vitamin A due to large variations in provitamin A bioefficacy, and if provitamin A carotenoids other than ß-carotene could be utilized in biofortification programs. Although animal studies have compared the bioefficacy of ß-cryptoxanthin and ß-carotene in biofortified maize by measuring liver retinol concentrations after dietary exposure, it is unclear whether the higher bioavailability of ß-cryptoxanthin can be offset by the lower conversion of ß-cryptoxanthin to retinol, and how post-intestinal conversion of ß-cryptoxanthin may contribute to the total bioefficacy of ß-cryptoxanthin. Here, we present a method that, for the first time, uses stable isotope labeled ß-cryptoxanthin and ß-carotene to quantify bioconversion and bioefficacy of both pVACs simultaneously in human volunteers. The paper describes how positioning of the [13C] labels around the centric 15,15' double bond on either the ß-carotene or ß-cryptoxanthin molecule allows quantification of retinoids from both pVACs, and details the procedure for sample preparation and analysis using LC-MS/MS. Finally, we apply and discuss recent approaches to quantify bioconversion and bioefficacy of isotopically labeled ß-carotene and ß-cryptoxanthin in humans.

Preventive action of retinoids in metabolic syndrome/type 2 diabetic rats fed with citrus functional food enriched in ß-cryptoxanthin.

Citrus fruits are known for their beneficial health effects associated with the prevention of metabolic syndrome/type 2 diabetes that is mainly attributed to flavonoids. Few investigations have reported the potential anti-diabetic effects of retinoids from the bioconversion of ß-cryptoxanthin (bcx), a citrus carotenoid. Therefore, the present study explored the anti-diabetic effect of a citrus functional food, obtained by membrane eco-technology of a citrus clementina juice, especially enriched in bcx but also in flavonoids and pectin. We assessed the in vivo effect of citrus bcx absorption and its bioconversion into retinoids in metabolic syndrome/type 2 diabetic fructose rats. Fructose-fed rats were used as a prediabetic control, and a prediabetic group was treated with the citrus concentrate for 8 weeks. The citrus-based food treatment improved glucose tolerance, dyslipidemia and blood pressure, in prediabetic rats. Although these effects were in part due to the synergy between enriched phytonutrients (bcx, hesperidin, pectin) of the citrus matrix, the role of bcx and its bioconversion into retinoids were highlighted. We showed that prediabetic rats absorbed less bcx and the bioconversion was less efficient. Bcx from citrus-based food was able to restore vitamin A status in prediabetic rats suggesting that the absorption/bioconversion of bcx may have a key role in improvement of metabolic syndrome/type 2 diabetes.

Impact of high hydrostatic pressure and thermal treatment on the stability and bioaccessibility of carotenoid and carotenoid esters in astringent persimmon (Diospyros kaki Thunb, var. Rojo Brillante).

The carotenoid and carotenoid ester profile in astringent persimmon (Diospyros kaki Thunb., var. Rojo Brillante) was composed by 13 free xanthophylls, 8 hydrocarbon carotenes and 17 carotenoid esters. The stability and biaoccessibility of these carotenoids was determined by an adaptation of the INFOGEST protocol. Results showed that the stability of persimmon carotenoids ranged from 61 to 74%, depending on the digestion phase, being (all-E)-ß-cryptoxanthin and (all-E)-antheraxanthin 3-O-palmitate the most stable carotenoids. At the final step of the digestion (oral + gastric + duodenal phase), only traces of (all-E)-antheraxanthin, (all-E)-lutein and (all-E)-ß-cryptoxanthin were found in control samples due to the low efficiency of carotenoid micellization, which was affected by the high pectin content naturally present in persimmon tissues. Processing increased the overall carotenoid bioaccessibility to 54% in pressurized samples and to 25% in thermal treated ones. This effect depended on the processing technology as well as on the chemical structure of the carotenoid, being (all-E)-ß-cryptoxanthin and (all-E)-ß-cryptoxanthin laurate the most bioaccessible carotenoids in pressurized samples and (all-E)-ß-cryptoxanthin laurate and (all-E)-antheraxanthin the most bioaccessible ones in pasteurized ones.

Free carotenoids and carotenoids esters composition in Spanish orange and mandarin juices from diverse varieties.

The carotenoid profiles of citrus fruit have been well studied, but knowledge about the xanthophylls esters pattern is more limited. In this study, the native carotenoid profiles of mandarin and orange juices were studied. Xanthophylls in oranges were esterified with lauric, myristic, palmitic and stearic acids. The fatty acids esterifying ß-cryptoxanthin in mandarins were lauric, myristic, palmitic and capric acid. In oranges, the average relationships of total free carotenoids to the sum of mono-esters and di-esters were 0.82 and 1.67, respectively, and the average relationships total mono-esters to total of di-esters was 2.51. In general, the mono-ester fraction was the most significant quantitatively. In mandarins, the average relationship of total free carotenoids to the sum of mono-esters was 0.17, which is in line with the fact that ß-cryptoxanthin was mostly esterified and not free (unesterified).

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.

Esterified carotenoids as new food components in cyanobacteria.

Among the nutritional properties of microalgae, this study is focused in the presence of carotenoid esters in prokaryote microalgae, an event that has not been shown so far. Three carotenoid esters that accumulate in non-stressful culture conditions are identified in Aphanotece microscopica Nägeli and Phormidum autumnale Gomont, what may provide an extra value to the quality attributes of the carotenoid profile in cyanobacteria as functional foods. In addition, new data on the carotenoid characterization added quality criteria for the identification of the esterified metabolites, enabling the monitoring of these food components. Specifically, the metabolomic approach applied to the food composition analysis, has allowed to differentiate between the esters of zeinoxanthin and ß-cryptoxanthin, which were undifferentiated to date during the MS characterization of carotenoids in other food sources. We propose a new qualifier product ion specific for zeinoxanthin ester, which it is not present in the MS2 spectrum of ß-cryptoxanthin esters.

Screening and Assessment of Low-Molecular-Weight Biomarkers of Milk from Cow and Water Buffalo: An Alternative Approach for the Rapid Identification of Adulterated Water Buffalo Mozzarellas.

Adulteration of Mozzarella di Bufala Campana with cow milk is a common fraud because of the high price and limited seasonal availability of water buffalo milk. To identify such adulteration, this work proposes a novel approach based on the use of species-specific, low-molecular-weight biomarkers (LMWBs). Liquid chromatography-tandem mass spectrometry screening analyses identified ß-carotene, lutein, and ß-cryptoxanthin as LMWBs of cow milk, while ergocalciferol was found only in water buffalo milk. Adulterated mozzarellas were prepared in the laboratory and analyzed for the four biomarkers. Combined quantification of ß-carotene and ergocalciferol enabled the detection of cow milk with a sensitivity threshold of 5% (w/w). The method was further tested by analyzing a certificated water buffalo mozzarella and several commercial products. This approach is alternative to conventional proteomic and genomic methods and is advantageous for routine operations as a result of its simplicity, speed, and low cost.

Influence of Agricultural Management on Phytochemicals of Colored Corn Genotypes ( Zea mays L.). Part 2: Sowing Time.

Among the agronomic practices carried out in corn cultivation, the early sowing time is increasingly used by farmers of temperate regions to improve yield and reduce mycotoxin contamination of corn grains. The present study determined the influence of sowing time on the phytochemical content of grains of 10 colored genotypes of corn. There was a significant improvement of both grain yield (+26%), thousand kernel weight (+3%), and test weight (+2%) in plots sown early. The early sowing also significantly influenced the chemical composition of corn grains, with an increase in the concentration of cell-wall-bound phenolic acids (+5%) and ß-cryptoxanthin (+23%) and a decrease in the concentration of lutein (-18%) and total anthocyanins (-21%). Environmental conditions that occurred during grain development significantly influenced the phytochemical content of corn grain, and early spring sowing could impart advantages in terms of both productivity and content of some antioxidants of whole-meal corn flour.

Xanthophyll esters are found in human colostrum.

SCOPE: Carotenoids in human milk are associated with other lipid counterparts in several metabolic processes. One interesting association that has not been demonstrated to date is the presence of xanthophyll esters. Colostrum and mature milk samples were analyzed to determine the occurrence of xanthophyll esters and identify the compounds. Thus, the association of the amounts of these compounds with lactation and whether they are significant contributors to the carotenoid profile of human milk was assessed. METHODS AND RESULTS: Pre-term and term delivering mothers were included in the study to donate colostrum at 3-5 days postpartum and mature milk at 15 days postpartum. Carotenoids extracts were subjected to a clean-up procedure to remove the triacylglycerol fraction and then analyzed by HPLC-MSn . Identification of xanthophyll esters was achieved by considering their chromatographic behaviour, UV-visible characteristics and MSn features. CONCLUSION: Xanthophyll esters are significant contributors to the carotenoid profile in the colostrum, while mature milk does not contain these compounds. Therefore, fatty acid acylation to xanthophylls is activated during the accumulation of carotenoids in the human mammary gland. The sharp decline in the amount of xanthophyll esters in mature milk indicates that the lipophilic components are those recently incorporated in the mammary epithelium.

Identification and quantitative analysis of ß-cryptoxanthin and ß-citraurin esters in Satsuma mandarin fruit during the ripening process.

In this study, to investigate the xanthophyll accumulation in citrus fruits, the major fatty acid esters of ß-cryptoxanthin and ß-citraurin were identified, and changes in their contents were investigated in two Satsuma mandarin varieties, 'Miyagawa-wase' and 'Yamashitabeni-wase', during the ripening process. The results showed that ß-cryptoxanthin and ß-citraurin were mainly esterified with lauric acid, myristic acid, and palmitic acid in citrus fruits. During the ripening process, ß-cryptoxanthin laurate, myristate, and palmitate were accumulated gradually in the flavedos and juice sacs of the two varieties. In the flavedo of 'Yamashitabeni-wase', ß-citraurin laurate, myristate, and palmitate were specifically accumulated, and their contents increased rapidly with a peak in November. In addition, functional analyses showed that CitCCD1 and CitCCD4 efficiently cleaved the free ß-cryptoxanthin, but not the ß-cryptoxanthin esters in vitro. The substrate specificity of CitCCDs towards free ß-cryptoxanthin indicated that ß-cryptoxanthin esters might be more stable than free ß-cryptoxanthin in citrus fruits.

In vitro digestion-assisted development of a ß-cryptoxanthin-rich functional beverage; in vivo validation using systemic response and faecal content.

Bioavailability of carotenoids is low and significant amounts reach the colon where they may be biologically active. We aimed to optimize a previously developed beverage designed to improve cardiovascular and bone remodelling markers in post-menopausal women. By assessing different lipid emulsions (soy lecithin, milkfat globule membrane (MFGM) and olive oil) on the in vitro bioaccessibility of ß-Cryptoxanthin and phytosterols, a MFGM containing beverage was selected and resulted stable over time (recovery >95%) under in vitro digestion and simulated anaerobic conditions. This beverage was tested in a randomized human trial (n=38) by evaluating systemic response and the colonic availability of ß-Cryptoxanthin. Consumption for six weeks provoked an increment in serum ß-Cryptoxanthin of 38.9µg/dl (CI 95%; 31.0, 46.8; p<0.001). In faeces, free ß-Cryptoxanthin, tentatively identified ß-Cryptoxanthin esters and the ratio cis-/trans-ß-carotene approached the profile in the beverage and after in vitro digestion but it was different from serum. In conclusion, in vitro digestion-assisted approach appears adequate to develop functional foods although in vivo validation should consider both systemic response and the availability at the colon.