A simple,sensitive HPLC-DAD method for simultaneous determination of carotenoids,chlorophylls and α-tocopherol in leafy vegetables

Leafy vegetables are the important components of our diet and are the source of several beneficial phytochemicals. A sensitive, simple analytical method is therefore required to precisely measure the phytochemical composition. A validated reversed phase high performance liquid chromatography with diode array detection (HPLC-DAD) method was developed to determine simultaneously carotenoids, chlorophylls and α-tocopherol composition of six leafy vegetables (B. compestris, B. rapa, B. juncea, M. neglecta, and two spinach varieties). Carotenoids were extracted and efficiently separated using a tertiary mobile gradient system of methanol–water, water and MTBE in 40 min on a reversed phase C18 column. The method was simple, precise, accurate and highly reproducible. Twelve carotenoids namely lutein and its three isomers, β-carotene-5,6-epoxide, neoxanthin, violaxanthin, two cis-isomers of zeaxanthin, 8-apo-carotenal, all-trans-β-carotene and its 13-cis-isomer; one fatty acid ester (β-cryptoxanthin ester); α-tocopherol and chlorophyll a & b were quantified in vegetable leaves. α-Tocopherol, neoxanthin, violaxanthin, lutein, 8-apo-carotenal, chlorophyll a and all-trans-β-carotene were present in higher amounts. Significant variations in the major compounds were observed in the selected vegetables. It was concluded that the developed method was highly sensitive, accurate and can be used to analyze carotenoids, chlorophylls and α-tocopherol simultaneously in leafy vegetables as well as in other plant leaves.     

Simultaneous HPLC–DAD quantification of vitamins A and E content in raw,pasteurized, and UHT cow’s milk and their changes during storage

An improved extraction and HPLC method for the simultaneous extraction and quantitation of retinol, α-tocopherol, α-tocotrienol, and β-carotene was developed to analyze commercial whole/semi-skim/skim samples of raw/pasteurized/UHT milk in transparent plastic/glass bottles and Tetra Brik? containers. The sample preparation method required prior saponification at 40 °C for 15 min followed by n-hexane extraction. An isocratic acetonitrile/methanol (65:35 v/v) mobile phase, C₁₈ analytical column, and UV detector were chosen for HPLC quantification. The liposoluble vitamin content in raw, pasteurized conventional/organic, and UHT milk ranged 0.055–5.540 (retinol), 0.135–1.410 (α-tocopherol), and 0.040–0.850 mg/L (β-carotene). No significant differences (p > 0.05) were observed on losses of retinol, α-tocopherol, and β-carotene content in UHT whole milk after 5 days at 4 °C in the dark. After 14 days at 4 °C in the dark, the contents of retinol, α-tocopherol, and β-carotene remained higher in milk with higher fat content and were higher in unopened containers. In UHT whole milk, samples containing 0.02 % NaN₃, retinol (33 %), and α-tocopherol (11 %) but not β-carotene (2 %) decreased significantly (p < 0.05).     

Major carotenoid composition of Brazilian Valencia orange juice: Identification and quantification by HPLC

The carotenoid composition of Brazilian Valencia orange juice was determined by open column chromatography (OCC) and high-performance liquid chromatography. Carotenoid pigments were extracted using acetone and saponified using 10% methanolic potassium hydroxide. Sixteen pigments were isolated by OCC and identified as α-carotene, ζ-carotene, β-carotene, α-cryptoxanthin, β-cryptoxanthin, lutein-5,6-epoxide, violaxanthin, lutein, antheraxanthin, zeaxanthin, luteoxanthin A, luteoxanthin B, mutatoxanthin A, mutatoxanthin B, auroxanthin B and trollichrome B. Thirteen carotenoid pigments were separated using a ternary gradient (acetonitrile–methanol–ethyl acetate) elution on a C₁₈ reversed-phase column. Among these, violaxanthin, lutein, zeaxanthin, β-cryptoxanthin, ζ-carotene, α-carotene, and β-carotene were quantified. The total carotenoid content was 12 ± 6.7 mg/l, and the major carotenoids were lutein (23%), β-cryptoxanthin (21%), and zeaxanthin (20%).     

Determination of antioxidant activity and antioxidant content in tomato varieties and evaluation of mutual interactions between antioxidants

Major antioxidants and antioxidant activity in eight varieties of tomatoes were determined. Hydrophilic and lipophilic antioxidant activity (HAA and LAA) was determined by the ABTS assay and ascorbic acid and carotenoid contents were determined by HPLC-DAD. The HAA has far more significant impact on total antioxidant activity (83%) as compared with LAA. HAA was increasing during all ripening stages and was strongly correlated with ascorbic acid content (r = 0.83). During the ripening the LAA was increasing till the III. stage of maturity and then decreased. The main carotenoids determined in the red-fruit tomatoes were lycopene, β-carotene and lutein. The content of lycopene has been increasing equally during the ripening. β-Carotene and lutein were intensively synthesized between the I. and II. stage of maturity. Among ascorbic acid, α-tocopherol, lycopene, β-carotene and lutein standards ascorbic acid was determined as the most efficient antioxidant followed by α-tocopherol and β-carotene. Antioxidant activity of ascorbic acid, α-tocopherol, β-carotene and lutein grew equally with increasing concentration, however lycopene was the most effective in its lowest concentration. The analysis of two-component mixtures showed significant synergism between lycopene-lutein, lycopene-β-carotene and α-tocopherol-β-carotene.     

Determination of carotenoids and liposoluble vitamins in sea urchin (Paracentrotus lividus) by high performance liquid chromatography

High-performance liquid chromatography was used to determine carotenoids (lutein, #-carotene, #-isocryptoxanthin, and #-echinenone) and liposoluble vitamins (retinol=vitamin A, and !-tocopherol=vitamin E) in fresh and canned gonads of the edible sea urchin Paracentrotus lividus. #-Carotene, #-isocryptoxanthin, and #-echinenone are vitamin A precursors. The carotenoids were determined with UV-vis detection and the vitamins with fluorescence detection. The sensitivity and precision of the method were good. Retinol was not detected in either fresh or canned material. Mean concentrations in fresh material (mg per 100 g dry weight) were 3.44 for lutein, 0.79 for #-carotene, 1.89 for #-isocryptoxanthin, 11.35 for #-echinenone, and 7.86 for !-tocopherol. Mean concentrations in canned material were 70-80% of concentrations in fresh material, except for #-echinenone, the concentration of which was about 30% higher in canned material.     

Effects of prepartum supplementation of β-carotene on colostrum and calves

Little is known about transfer of dietary β-carotene into colostrum, its absorption by the calf, and its effects on retinol and α-tocopherol in the calf when the dam's dietary vitamin A is adequate. Our objective was to assess the effect of β-carotene supplementation during the close-up dry period on the colostrum and calf. The study was conducted on a large commercial dairy farm in Indiana during early summer of 2015. Ninety-four multiparous Holstein cows were blocked by calving data, parity, and previous production, and then randomly assigned to either control or β-carotene (BC) treatments. While locked in headgates each morning, each cow received a topdress of β-carotene (Rovimix, DSM Nutritional Products, 8 g/d; provided 800 mg β-carotene) or carrier from 21 d before expected calving until calving. Colostrum was collected within 2 h of parturition. Calf blood samples were obtained within 2 h of birth before receiving the dam's colostrum, at 24 h after birth, and at 7 d and 60 d of age. Blood serum was analyzed for β-carotene, retinol, α-tocopherol, and other metabolites and enzymes. Colostrum was analyzed for β-carotene, retinol, α-tocopherol, colorimetry profile, and milk components. Data were analyzed using mixed-effects models in SAS (SAS Institute Inc.). Calf serum β-carotene data were analyzed using the FREQ procedure. Colostrum β-carotene was higher for BC cows. Colostrum from BC cows had increased a* [measures red (positive) to green (negative)] and b* [measures yellow (positive) to blue (negative)] colorimeter values, indicating that β-carotene altered colostrum color toward red and yellow. Supplementation did not affect colostral or calf IgG concentrations. Colostrum color indices were correlated with IgG concentrations as well as concentrations of β-carotene, retinol, and α-tocopherol. Before receiving colostrum, the concentration of β-carotene in calf serum was below the detectable threshold of 0.05 μg/mL. At 24 h of age, the number of calves with detectable β-carotene concentrations increased, with more calves from BC cows (52.1%) having detectable concentrations than calves from cows in the control group (6.1%). No differences in concentrations of retinol or α-tocopherol were observed in calf serum. Supplementation of β-carotene to cows decreased activities of gamma-glutamyl transpeptidase and glutamate dehydrogenase in calf serum. In pregnant cows already receiving adequate vitamin A, supplementation of β-carotene increased concentration of β-carotene in colostrum, altered colostrum color, and increased serum β-carotene in calves at birth.     

An in vitro model to study the intestinal absorption of carotenoids

An in vitro cell culture model to assess the intestinal absorption of carotenoids is described. When supplemented with taurocholate and oleic acid, differentiated Caco-2 cells on membranes are able to produce chylomicrons. Under conditions mimicking the in vivo postprandial state, Caco-2 cells take up carotenoids and secrete them incorporated into chylomicrons; the extents of absorption of all-trans, 9-cis, 13-cis β-carotene, α-carotene, lutein, lycopene were 11%, 2%, 3%, 10%, 7%, and 3%, respectively. Saturation of β-carotene transport through Caco-2 cells occurred at concentrations (>15 μM) higher than “physiological” concentrations. Finally, retinol supplementation resulted in an 1.7-fold increase in β-carotene transport. The data suggest that (1) the intestinal absorption of carotenoids is facilitated by the participation of a specific epithelial transporter and (2) retinol promotes β-carotene incorporation into larger, retinyl ester-enriched chylomicrons. The present in vitro cell culture system is a relevant model to study the intestinal absorption of carotenoids at the molecular level.     

Serum retinol, β-carotene,and α-tocopherol as biomarkers for disease risk and milk production in periparturient dairy cows

The effectiveness of using serum vitamin concentrations as biomarkers to predict diseases in dairy cows during the periparturient period is not well known. The objective of this study was to evaluate the association between serum β-carotene, retinol, and α-tocopherol concentrations and periparturient cow diseases in commercial dairies. We measured serum concentrations of these vitamin-active compounds at dry-off and during close-up (approximately 3 wk before calving) and early lactation (approximately 7 d post-calving), and we examined their association with clinical diseases in the first 30 d in milk. Diseases were diagnosed by trained personnel and recorded using database software. Blood samples were taken from 353 cows from 5 different farms over a 3-yr period. Blood samples were analyzed for β-carotene, retinol, α-tocopherol, and cholesterol. We built separate mixed logistic regression models for each disease outcome: hyperketonuria, lameness, mastitis, uterine diseases (retained placenta or metritis), and an aggregate outcome. For the aggregate outcome, a cow was considered positive if she had one or more of the following: hyperketonuria, lameness, mastitis, uterine disease, pneumonia, milk fever, or displaced abomasum. Concentrations of all 3 fat-soluble vitamins decreased significantly in early lactation relative to the 2 prepartum sampling times. Serum retinol concentrations at close-up and early lactation were negatively associated with odds of developing postpartum hyperketonuria. At early lactation, cows with uterine disease had lower serum retinol concentrations than cows without uterine disease. Similarly, lower serum retinol concentrations were associated with greater odds of having any one disease in the aggregate outcome. First-test 305-d mature-equivalent milk yield was positively correlated with increased serum α-tocopherol and negatively correlated with β-carotene concentrations. This study demonstrates the potential for serum β-carotene, retinol, and α-tocopherol to serve as biomarkers for disease risk.     

MAJOR CAROTENOIDS IN JUICES OF PONKAN MANDARIN AND LIUCHENG ORANGE

The identification and quantitation of major carotenoids in juices of Ponkan mandarin and Liucheng orange and their changes during harvesting were studied. Neoxanthin, violaxanthin, lutein, β-cryptoxanthin, α-carotene and β-carotene are the major carotenoids for Ponkan mandarin. During the harvesting period, the contents of violaxanthin, β-cryptoxanthin and β-carotene increase significantly. For Liucheng orange, neoxanthin, violaxanthin, lutein, antheraxanthin, β-cryptoxanthin, α-carotene and β-carotene are the major carotenoids. Among them, neoxanthin, violaxanthin, lutein, antheraxanthin and β-cryptoxanthin increase significantly during the harvesting period. The β-cryptoxanthin and β-carotene are the major contributors to the color of Ponkan mandarin juice while neoxanthin, violaxanthin, lutein and antheraxanthin are the major ones for the color of Liucheng orange juice.     

Catechin and epicatechin in testa and their association with bioactive compounds in kernels of cashew nut (Anacardium occidentale L.)

Catechins in testa and bioactive compounds in testa-free and testa-containing kernels of cashew nuts were analysed. The cashew nut testa contained (+)-catechin and (−)-epicatechin with concentrations of 5.70 and 4.46 g per kg DM, respectively. Testa-containing kernels revealed significantly higher levels of β-carotene (218 vs. 89.6 μg/kg DM), lutein (525 vs. 292 μg/kg DM), and α-tocopherol (10.1 vs. 2.4 mg/kg DM), similar amounts of zeaxanthin (7.0 vs. 7.1 μg/kg DM), γ-tocopherol (10.6 vs. 10.1 mg/kg DM), stearic acid (41 vs. 43 g/kg DM), oleic acid (214 vs. 219 g/kg DM) and linoleic acid (69 vs. 62 g/kg DM), but a lower concentration of thiamine (3.0 vs. 10.7 mg/kg DM) in comparison to testa-free samples. The testa-containing kernels provide high amounts of catechins and higher concentrations of β-carotene, lutein and α-tocopherol than do testa-free cashew nut kernels. This could have potential health benefits for consumers.     

Carotenoid composition and retinol equivalent in plants of nutritional and medicinal importance: Efficacy of β-carotene from Chenopodiumalbum in retinol-deficient rats

This study aimed to (1) quantify carotenoids in leafy vegetables and plants of nutritional and medicinal importance, (2) evaluate retinol equivalent (RE) of provitamin-A carotenoids and (3) determine efficacy of β-carotene from Chenopodium album and to compare with retinol formed on feeding to retinol-deficient rats for 3 weeks. β-Carotene and lutein contents (mg/100 g dry weight) ranged from 1.5 to 120 and 11.7 to 185 (leafy greens) and 0.4 to 34.7 and 11.8 to 679 (medicinal plants) whereas, α-carotene ranged from 0.3 to 35.6 (leafy greens) and 0.1 to 15.7 (medicinal plants). RE values (mg%) ranged from 0.4 to 20 and 0.42 to 5.8 in leafy greens and medicinal plants. Efficacy of β-carotene (2400 μg/kg diet) from C. album in retinol-deficient rats revealed a 93.6% rise in plasma retinol levels from 0.53 to 8.4 μM. The plants analysed are a good source of retinol precursors and biologically active lutein; therefore can be exploited to meet carotenoid requirements.     

Antioxidant behaviour of carotenoids highly accumulated in HepG2 cells

The antioxidant behaviour of major dietary carotenoids accumulated at high concentrations in human hepatoma HepG2 cells was evaluated, in comparison with α-tocopherol. The cells that accumulated carotenoids and α-tocopherol at levels higher than the values reported in the human liver were exposed to mild oxidative stress with tert-butylhydroperoxide. β-Carotene (>2.6 nmol/mg protein) and astaxanthin (>1.8 nmol/mg protein) significantly suppressed lipid peroxidation, while β-cryptoxanthin and lutein did not. α-Tocopherol remarkably suppressed lipid peroxidation with an IC₅₀ value of 0.16 nmol/mg protein. Neither α-tocopherol nor any of the carotenoids except for lycopene showed pro-oxidant action even at high cellular concentrations. The antioxidant behaviours of carotenoids in a cellular milieu were quite different from those previously found in liposomes and homogeneous solutions. Further studies are required to assess the implications of the antioxidant behaviours found in the cultured cells on human health.     

Tocopherols and total phenolics in 10 different nut types

The study was conducted to assess the content of tocopherols (α-, β-, γ- and δ-) and carotenoids (α- and β-carotene, zeaxanthin, lutein, cryptoxanthin and lycopene) in the unsaponifiable matter as well as the amount of total phenols of 10 different types of nuts. Tocopherols and carotenoids were analysed with HPLC, total phenols photometrically. The mean value of α-tocopherol equivalents ranged from non-detectable (macadamias) to 33.1 mg/100 g extracted oil (hazelnuts). Among all nuts, almonds and hazelnuts had the highest mean α-tocopherol content (24.2 and 31.4 mg/100 g extracted oil, respectively). β- and γ-tocopherols were prevalent in Brazil nuts, cashews, peanuts, pecans, pines, pistachios and walnuts. Mean values oscillated between 5.1 (cashews) and 29.3 (pistachios). Traces of δ-tocopherol (<4 mg/100 g extracted oil) were analysed in cashews, hazelnuts, peanuts, pecans, pines, pistachios and walnuts. There were no carotenoids detected in the tested nuts with the exception of pistachios. The mean content of total phenolics varied between 32 mg gallic acid equivalents/100 g (pines) and 1625 mg (walnuts). The results show the heterogenic amounts of antioxidants in nuts, which emphasises the recommendation of a mixed nuts intake.     

Extraction and quantification of major carotenoids in processed foods and supplements by liquid chromatography

A simple, fast and robust method with minimum steps, small sample size and amounts of solvents was developed to determine major carotenoids contents in processed foods, tablets and gel capsules. The method involves dispersion of the sample in hot water (60 °C) with added butylated hydroxytoluene (BHT) in ethanol to minimize oxidation, followed by extraction with chloroform and analysis by liquid chromatography. Chromatographic parameters were: a C30 column protected with a C18 guard cartridge; gradient elution at the rate of 1.0 mL/min starting with 100% methanol (A) and ending with 40:60 (v/v) methanol/isopropanol (B); detection set at 450 nm for carotenoids, and 325 nm for retinol, retinyl acetate and retinol palmitate. The method exhibited: (i) high degree of repeatability (%rsd); (ii) linear calibration curves (r² ? 0.9998); (iii) low detection; and quantification limits. The method was validated with standard reference material 2383 for trans-β-carotene; and tested for α-, and β-carotenes, lutein, zeaxanthin, cryptoxanthin, trans-retinol in processed foods, tablets and gel supplements.     

Carotenoid composition and vitamin A activity of medicinally important green leafy vegetables

Carotenoid composition of green leafy vegetables (GLVs, n = 30) with medicinal value was analyzed by HPLC; vitamin A activity (as retinol equivalent, RE) of provitamin A carotenoids was calculated. Results show that among GLVs studied, the level of β-carotene (50–130 mg/100 g dry wt) was higher in nine GLVs than other carotenoids while lutein (50–187 mg/100 g dry wt) and zeaxanthin (1–5 mg/100 g dry wt) were higher in 12 GLVs than other xanthophylls. α-Carotene was detected only in nine GLVs, ranging from 1 to 37 mg/100 g dry wt. Interestingly, Chenopodium album, Commelina benghalensis and Solanum nigrum were found to contain higher levels of both lutein and β-carotene in the range of 84–187 and 50–115 mg/100 g dry wt, respectively. The values of retinol equivalents (RE) ranged from 641 to 19101 and were higher (>10,000) in six GLVs of the 30. The results demonstrate that GLVs studied contained higher levels of RE and lutein.     

Antioxidant activity of tomato lipophilic extracts and interactions between carotenoids and α-tocopherol in synthetic mixtures

In the present study we assayed the antioxidant activity of lipophilic extracts obtained from different tomato varieties. The results showed that cherry tomatoes, characterized by a high carotenoid content, had the highest antioxidant activity. A quantitative analysis of lycopene, β-carotene, lutein and α-tocopherol was also performed and the correlation between the antioxidant content and the antioxidant activity was estimated. The highest correlation coefficient was found for lycopene (R² = 0.9236, P ≤ 0.001). The analysis of two-component mixtures containing α-tocopherol and carotenoids showed that significant synergism occurred for all the combinations which contained α-tocopherol and β-carotene mixed together. The highest synergistic effects were detected for α-tocopherol-lycopene mixtures, which were the most efficient combinations tested in the present study. The analysis of the carotenoid combinations indicated that synergism occurred for lycopene-β-carotene, lycopene-lutein and lutein-β-carotene mixtures. The analysis of four-component mixtures did not show statistically significant synergistic effects.     

烘焙类食品中叶黄素酯提取及检测方法建立

为检测烘焙类食品中的叶黄素酯以及筛选无乙醚的萃取溶剂,本文对烘焙产品中的叶黄素酯皂化时间、KOH浓度以及不同萃取溶剂的萃取效果进行研究。结果表明检测烘焙食品中叶黄素酯的皂化最优条件为:2 g样品（面糊/面团）,0.2 g BHT,10 mL无水乙醇,10 mL 60 g/100 mL KOH水溶液,室温振荡3 h。萃取溶剂环己烷:正己烷:乙酸乙酯=1:2:2为最佳萃取剂,回收率为94.31%~103.83%,精密度实验相对标准偏差均小于5%。表明该方法适合于烘焙类食品中叶黄素酯的提取及检测。     

Carotenoid profile and retinol content of baby food products

 The contents of provitamin A carotenoids, non-provitamin A carotenoids and retinol were investigated in two types of 65 commercially available baby food products by reverse-phase high-performance liquid chromatography. The content of β-carotene in vegetable products for babies from 5 months was 57% of the total amount of carotenoids (7.83 mg/100 g); the contents of other carotenoids were lycopene 19%, α-carotene 18%, lutein 4%, cryptoxanthin 1% and zeaxanthin 1%. All together, the content of provitamin A carotenoids was 76% and that of non-provitamin A carotenoids was 24%. A similar carotenoid profile was found in vegetable products for babies from 8 months and from 12 months, although the total carotenoid content was lower (3.07 mg/100 g). In fruit and cereal products the contents of β-carotene and lycopene were 36% and 39% of the total amount of carotenoids (0.44 mg/100 g). The contents of α-carotene, lutein, cryptoxanthin and zeaxanthin ranged from 3% to 9% of the total carotenoids. The contents of provitamin A and non-provitamin A carotenoids were equal for this group (both 50% of all carotenoids). The calculated average content of retinol equivalents (mg RE/100 g) was between 0.33 and 0.88 for the vegetable products and 0.03 for the fruit and cereal products. Received: 27 May 1999 / Revised version: 2 August 1999     

Carotenoid diversity in tropical-adapted yellow maize inbred lines

Maize is a staple food for millions of people in sub-Saharan Africa where a significant number of people suffer from vitamin A deficiency. Yellow maize contains both pro-vitamin A and nonprovitamin A carotenoids with potential health benefits to humans. An improvement in the concentration of these compounds can have a positive impact on dietary intakes in areas where yellow maize is consumed. An essential first step in breeding yellow maize for enhanced carotenoid concentrations involves an assessment of the carotenoid diversity of adapted maize inbred lines. Trials were thus conducted (i) to explore the genetic variation in carotenoid concentrations among tropical-adapted yellow maize inbred lines, (ii) to assess the potential for concurrent improvement of different carotenoids and (iii) to determine the consistency of carotenoid concentrations in different locations. Seed samples of a large set of lines harvested from four trials grown in one location and a fifth trial grown in two locations were analyzed for carotenoid concentrations using HPLC. The analyses of variance revealed that carotenoid concentrations were not strongly affected by the differences in replications or locations. There were large differences among the tropical-adapted yellow maize inbred lines in lutein, zeaxanthin, β-carotene, β-cryptoxanthin, α-carotene and total pro-vitamin A contents. As significant correlations were observed among carotenoids sharing a single branch of the carotenoid biosynthetic pathway, it should be feasible to increase the levels of multiple carotenoids simultaneously. Principal component analysis on the carotenoid composition of the yellow inbred lines identified some lines with higher levels of all carotenoids formed across both major branches of the carotenoid biosynthetic pathway, and other lines having higher levels of those carotenoids formed under a single major branch of the carotenoid biosynthetic pathway. These indicate that the selection of parental lines with diverse carotenoid profiles may possibly be exploited for genetic improvement of carotenoids in tropical maize.     

Characterization of 12 Capsicum varieties by evaluation of their carotenoid profile and pungency determination

In this research 12 different varieties of Capsicum cultivars belonging to three species (Capsicum chinense, Capsicum annuum, Capsicum frutescens) and of various colour, shape, and dimension have been characterised by their carotenoids and capsaicinoids content. The berries were cultivated in the region Emilia-Romagna, in Northern Italy. The native carotenoid composition was directly investigated by an HPLC-DAD-APCI-MS methodology, for the first time. In total, 52 carotenoids have been identified and considerable variation in carotenoid composition was observed among the various cultivars investigated. Among the cultivars with red colour, some Habanero, Naga morich and Sinpezon showed an high β-carotene content, whereas Serrano, Tabasco and Jalapeno showed an high capsanthin content and the absence of β-carotene. Habanero golden and Scotch Bonnet showed a high lutein, α-carotene and β-carotene amounts, and Habanero orange was rich in antheraxanthin, capsanthin and zeaxanthin. Cis-cryptocapsin was present in high amount in Habanero chocolate.