Chrysobalanus icaco L. fruits inhibit NADPH oxidase complex and protect DNA against doxorubicin-induced damage in Wistar male rats.

Chrysobalanus icaco L. is an underexplored plant found in tropical areas around the globe. Currently, there is no apparent information regarding the effects C. icaco fruits may exert in vivo or potential role in health promotion. This study aimed at providing evidence regarding the in vivo influence of this fruit on antigenotoxicity, antimutagenicity, and oxidative stress in rats. Male Wistar rats were treated with 100, 200, or 400 mg/kg body weight (bw)/d C. icaco fruit for 14 d. Doxorubicin (DXR, 15 mg/kg bw, ip) was used for DNA damaging and as an oxidant to generate reactive oxygen species (ROS). Genomic instability was assessed by the comet assay and micronucleus (MN) test, while antioxidant activity was determined by oxidative burst of neutrophils. Chrysobalanus icaco fruit polyphenols were quantified and characterized by high-performance liquid chromatography coupled to a diode array detector and tandem mass spectrometer (HPLC-DAD-MS/MS). The concentrations of 19 chemical elements were determined by inductively coupled plasma-mass spectroscopy (ICP-MS). Significant amounts of polyphenols, magnesium, and selenium were found in C. icaco fruit. This fruit displayed in vivo antioxidant activity against DXR-induced damage in rat peripheral blood neutrophils, antigenotoxicity in peripheral blood cells, and antimutagenicity in bone-marrow cells and peripheral blood cells. Correlation analyses between endpoints examined indicated that the mechanism underlying chemopreventive actions of C. icaco fruit was attributed to inhibition of NADPH oxidase complex manifested as low levels of DNA damage in animals exposed to DXR. Data indicate that phytochemicals and minerals in C. icaco fruit protect DNA against damage in vivo associated with their antioxidant properties.

Evaluation of the antihypertensive properties of yellow passion fruit pulp (Passiflora edulis Sims f. flavicarpa Deg.) in spontaneously hypertensive rats.

Various species of the genus Passiflora have been extensively used in traditional medicine as sedatives, anxiolytics, diuretics and analgesics. In the present study, after the identification and quantification of phytochemical compounds from yellow passion fruit pulp by liquid chromatography-photodiode array-mass spectrometry (HPLC-PDA-MS/MS), its antihypertensive effect was investigated on spontaneously hypertensive rats. Additionally, the renal function, evaluated by kidney/body weight, serum creatinine, proteinuria, urinary flow, reduced glutathione (GSH) levels and thiobarbituric acid-reactive substances (TBARS) and mutagenicity in bone marrow cells were assessed to evaluate the safety of passion fruit consumption. Yellow passion fruit pulp (5, 6 or 8 g/kg b.w.) was administered by gavage once a day for 5 consecutive days. HLPC-PDA-MS/MS analysis revealed that yellow passion fruit pulp contains phenolic compounds, ascorbic acid, carotenoids and flavonoids. The highest dose of passion fruit pulp significantly reduced the systolic blood pressure, increased the GSH levels and decreased TBARS. There were no changes in renal function parameters or the frequency of micronuclei in bone marrow cells. In conclusion, the antihypertensive effect of yellow passion fruit pulp, at least in part, might be due to the enhancement of the antioxidant status. The exact mechanisms responsible by this effect need further investigation.

Biomass-to-bio-products application of feruloyl esterase from Aspergillus clavatus.

The structural polysaccharides contained in plant cell walls have been pointed to as a promising renewable alternative to petroleum and natural gas. Ferulic acid is a ubiquitous component of plant polysaccharides, which is found in either monomeric or dimeric forms and is covalently linked to arabinosyl residues. Ferulic acid has several commercial applications in food and pharmaceutical industries. The study herein introduces a novel feruloyl esterase from Aspergillus clavatus (AcFAE). Along with a comprehensive functional and biophysical characterization, the low-resolution structure of this enzyme was also determined by small-angle X-ray scattering. In addition, we described the production of phenolic compounds with antioxidant capacity from wheat arabinoxylan and sugarcane bagasse using AcFAE. The ability to specifically cleave ester linkages in hemicellulose is useful in several biotechnological applications, including improved accessibility to lignocellulosic enzymes for biofuel production.

Further insights on the carotenoid profile of the echinoderm Marthasterias glacialis L.

In this study, the carotenoid profile of the echinoderm Marthasterias glacialis L. was established using HPLC-DAD-APCI-MS/MS equipped with a C(30) column. This approach rendered the identification of 20 compounds, eight of them reported for the first time in this marine organism. Differentiation of carotenoid isomers was also achieved.

Antigenotoxic effects of piquiá (Caryocar villosum) in multiple rat organs.

This study investigated the in vivo genotoxicity of piquiá pulp (Caryocar villosum) and its potential antigenotoxicity on doxorubicin (DXR)-induced DNA damage by comet assay and micronucleus test. In addition, the phytochemicals present in piquiá pulp were determined. Piquiá fruit pulp (75, 150 or 300 mg/kg b.w.) was administered by gavage to Wistar rats for 14 days, and the animals received an injection of saline or DXR (15 mg/kg b.w., i.p.) 24 h before they were euthanized. The phytochemical analysis revealed the presence of carotenoids; phenolic compounds, including flavonoids; tannins and α-tocopherol in piquiá pulp. No statistically significant differences were observed in the evaluated parameters, demonstrating the absence of cytotoxic and genotoxic effects of piquiá pulp at all tested doses. In liver, kidney, cardiac and bone marrow cells, piquiá significantly reduced the DNA damage induced by DXR. Our results showed that the lowest piquiá dose caused the largest decrease in DNA damage and the highest dose caused the smallest decrease, demonstrating an inverse dose-response of piquiá pulp. Furthermore, we observed a difference in the potential antigenotoxic effects in several tissues. In conclusion, our results demonstrated that piquiá pulp was not genotoxic and inhibited the genotoxicity induced by DXR, but some of the protective effects that were observed depended on the doses and experimental conditions. Therefore, further investigations are needed to clarify how piquiá pulp positively affects human health.

An evaluation, using the comet assay and the micronucleus test, of the antigenotoxic effects of chlorophyll b in mice.

We investigated the effects of the dietary pigment chlorophyll b (CLb) on cisplatin (cDDP)-induced oxidative stress and DNA damage, using the comet assay in mouse peripheral blood cells and the micronucleus (MN) test in bone marrow and peripheral blood cells. We also tested for thiobarbituric acid reactive substances (TBARS) and reduced glutathione (GSH) in liver and kidney tissues, as well as catalase (CAT) activity and GSH in total blood. CLb (0.2 and 0.5mg/kg b.w.) was administrated by gavage every day for 13 days. On the 14th day of the experiment, 6 mg/kg cDDP or saline was delivered intraperitoneally. Treatment with cDDP led to a significant decrease in DNA migration and an increase in MN frequency in both cell types, bone marrow and peripheral blood cells. In the kidneys of mice treated with cDDP, TBARS levels were increased, whereas GSH levels were depleted in kidney and liver. In mice that were pre-treated with CLb and then treated with cDDP, TBARS levels maintained normal concentrations and GSH did not differ from cDDP group. The improvement of oxidative stress biomarkers after CLb pre-treatment was associated with a decrease in DNA damage, mainly for the highest dose evaluated. Furthermore, CLb also slightly reduced the frequency of chromosomal breakage and micronucleus formation in mouse bone marrow and peripheral blood cells. These results show that pre-treatment with CLb attenuates cDDP-induced oxidative stress, chromosome instability, and lipid peroxidation.

Brazilian native passion fruit (Passiflora tenuifila Killip) is a rich source of proanthocyanidins, carotenoids, and dietary fiber.

Passiflora tenuifila is a Brazilian native passion fruit consumed by the local population and is a dietary source of bioactive compounds with potential biological activity. The aim of this study is to evaluate the nutritional value of P. tenuifila fruit and its bioactive compounds at two ripening stages. Three batches of fruit were collected at mature-green and ripe stages, and phenolic compounds, carotenoids, and polyamines were analyzed by HPLC-DAD and LC-MS/MS. The fruit is a good source of dietary fiber. Proanthocyanidin dimers are the major phenolic compounds (up to 84%) at both stages, followed by the C-glycosylated luteolin. Lutein and ß-carotene are the major carotenoids, contributing up to 50% of total carotenoids. The OPLS-DA segregates the mature-green and ripe fruits, as carotenoids are responsible for this separation. In conclusion, passion fruit can be consumed at both stages of maturation without losses of bioactive compound contents or nutritional value.

Evaluation of the genotoxic and antigenotoxic effects after acute and subacute treatments with açai pulp (Euterpe oleracea Mart.) on mice using the erythrocytes micronucleus test and the comet assay.

Açai, the fruit of a palm native to the Amazonian basin, is widely distributed in northern South America, where it has considerable economic importance. Whereas individual polyphenolics compounds in açai have been extensively evaluated, studies of the intact fruit and its biological properties are lacking. Therefore, the present study was undertaken to investigate the in vivo genotoxicity of açai and its possible antigenotoxicity on doxorubicin (DXR)-induced DNA damage. The açai pulp doses selected were 3.33, 10.0 and 16.67g/kg b.w. administered by gavage alone or prior to DXR (16mg/kg b.w.) administered by intraperitoneal injection. Swiss albino mice were distributed in eight groups for acute treatment with açai pulp (24h) and eight groups for subacute treatment (daily for 14 consecutive days) before euthanasia. The negative control groups were treated in a similar way. The results of chemical analysis suggested the presence of carotenoids, anthocyanins, phenolic, and flavonoids in açai pulp. The endpoints analyzed were micronucleus induction in bone marrow and peripheral blood cells polychromatic erythrocytes, and DNA damage in peripheral blood, liver and kidney cells assessed using the alkaline (pH >13) comet assay. There were no statistically significant differences (p>0.05) between the negative control and the groups treated with the three doses of açai pulp alone in all endpoints analyzed, demonstrating the absence of genotoxic effects. The protective effects of açai pulp were observed in both acute and subacute treatments, when administered prior to DXR. In general, subacute treatment provided greater efficiency in protecting against DXR-induced DNA damage in liver and kidney cells. These protective effects can be explained as the result of the phytochemicals present in açai pulp. These results will be applied to the developmental of food with functional characteristics, as well as to explore the characteristics of açai as a health promoter.

Lutein improves antioxidant defense in vivo and protects against DNA damage and chromosome instability induced by cisplatin.

Lutein (LT) is the second most prevalent carotenoid in human serum, and it is abundantly present in dark, leafy green vegetables. The objectives of this study were to evaluate the genotoxicity and mutagenicity of LT, and its protective effects in vivo against DNA damage and chromosome instability induced by cisplatin (cDDP). For this purpose, we used the comet assay and micronucleus (MN) test, and we evaluated the antioxidant effects of LT by determination of enzymatic (catalase-CAT) and non-enzymatic (reduced glutathione-GSH) activity. Mice were divided into six groups: cDDP, mineral oil (OM), LT groups and LT + cDDP groups. To perform the MN test on peripheral blood (PB) cells, blood samples were collected before the first treatment (T0), and 36 h (T1) and 14 days (T2) after the first treatment. To perform the comet assay, blood samples were collected 4 h after the first and the last treatment. Oxidative capacity was analyzed in total blood that was collected 24 h after the last treatment, when bone marrow (BM) sample was also collected for the MN test. No genotoxic or mutagenic effects of LT were observed for the doses evaluated. We did find that this carotenoid was able to reduce the formation of crosslinks and chromosome instability induced by cDDP. No differences were observed in CAT levels, and LT treatment increased GSH levels compared with a negative control group, reinforcing the role of this carotenoid as an antioxidant.

Microalgae carotenoids intake: Influence on cholesterol levels, lipid peroxidation and antioxidant enzymes.

The objective of this study was to evaluate the effect of carotenoids intake of Scenedesmus obliquus, on lipid peroxidation, the endogenous antioxidant defense system as well as the serum lipid profile in vivo. Male mice were divided into control groups and supplemented with different doses of microalgae carotenoids: 0.25 (MC1) and 2.5 (MC2) mg·kg-1 bodyweight. The lipid profile (total cholesterol, triglycerides, low and high-density lipoprotein) and markers of hepatic toxicity were determined in serum samples. Antioxidant enzymes and thiobarbituric acid reactive substances were determined in the heart, liver, kidneys, and spleen. Both doses used to treat the animals did not show adverse effects by markers of hepatic toxicity. MC1 did not cause significant changes in the serum lipid profile. In contrast, it created a significant reduction in lipid peroxidation of the spleen (46%) as well as an increase in the GR in the heart (40%) and GPx in the kidneys (79%) activity. The MC2 treatment also increased GR (49%) in the heart and GPx (243%) in the heart and kidneys (58%) activity, however, significantly increased levels of lipid peroxidation in the liver (160%) as well as serum triglycerides (60%). According to results, it is suggested that the consumption of S. obliquus carotenoids at the MC1 dose was safe to the animals and could be explored as an alternative to improve the activity of antioxidant enzymes and reduce lipid peroxidation.

Unraveling the molecular mechanisms underlying interactions between caseins and lutein.

Understanding the food protein binding to bioactive compounds is of utmost importance for the development of efficient protein-based delivery systems. The binding of lutein to sodium caseinate (NaCas) or native casein micelle (PPCN) was investigated at pH 7 to evaluate the effect of casein supramolecular structures on the interaction. Fluorescence quenching, UV-vis spectroscopy, and dynamic light scattering were carried out. Under the medium conditions of interaction analysis (DMSO-water and ethanol-water), lutein exists as H-type aggregates. The investigation of lutein/casein interaction showed a predominantly static mechanism of fluorescence quenching and the presence of two fluorophore populations on NaCas and PPCN, but only one accessible to lutein. Moreover, the Scatchard plot indicated that lutein interacted with both caseins in one binding site. The interaction of lutein with caseins occurred with binding constant Kb of 105 M-1, regardless of casein supramolecular structure.

Bile amount affects both the degree of micellarization and the hydrolysis extent of carotenoid esters during in vitro digestion.

Carotenoid esters are present in considerable amounts in most fruits, such as in citrus. Although the bioavailability of carotenoid esters is similar or even higher compared to that of free carotenoids, these molecules are generally detected only in the free form in human plasma, suggesting that hydrolysis of carotenoid esters occurs in vivo. However, the available in vitro digestion methods were not able to achieve satisfactory carotenoid ester hydrolysis so far. As bile salts play an essential role in the hydrolytic action of lipolytic enzymes from pancreatin, we evaluated the effect of increasing the bile extract/food ratio from 0.045 to 0.12 (g g-1) on the hydrolysis of ß-cryptoxanthin esters from mandarin pulp during in vitro digestion. Additionally, considering the positive effect of lipids on carotenoid bioavailability, the impact of soybean oil addition on carotenoid ester hydrolysis was studied. Finally, bioaccessibility and recovery of 33 carotenoids were assessed by LC-DAD-MS. The hydrolysis extent of ß-cryptoxanthin esters enhanced from 29% to 55% by increasing the bile extract/food ratio, but reduced respectively to 28% and 11% by the addition of 1% and 10% oil (p < 0.05). The bioaccessibility of overall carotenoids improved from 19% to 35% by increasing the bile extract/food ratio, along with that of (all-E)-ß-carotene (from 19 to 31%) and total (all-E)-ß-cryptoxanthin (17% to 49%). Soybean oil addition reduced carotenoid micellarization, regardless of the concentration (p < 0.05). Irrespective of the bile extract amount and oil addition, the bioaccessibility of carotenoids was inversely related to its hydrophobicity, with respect to the following ranking: free xanthophylls > carotenes ≥ xanthophyll esters. Altogether, these results indicate that increasing the bile extract amount is a simple and inexpensive option to improve carotenoid ester hydrolysis in in vitro digestion protocols. Additionally, the constant amounts of bile (and possibly enzymes) of static methods, such as INFOGEST, should be further optimized for experiments involving lipid addition in which carotenoid bioaccessibility is evaluated.

Marigold carotenoids: Much more than lutein esters.

Carotenoids constitute a large group of lipophilic pigments whose health-promoting benefits have been widely recognized. Hydroxy-containing carotenoids can be found in both free form or esterified with fatty acids in several plant matrices, but the native carotenoid profile is overall poorly explored due to the difficulty of analyzing carotenoid esters. One of the main natural sources of carotenoids is the marigold flower, which has been extensively used by the industry for the production of food colorants or supplements, both often manufactured with no saponification process. Although lutein esters are well established as the major compounds naturally found in marigold petals and their products, carotenoid esters other than the lutein ones have not been extensively examined. We carried out a comprehensive identification of carotenoids and carotenoid esters from marigold petals by LC-DAD-(APCI+)MS/MS. Whereas 18 carotenoids were identified in the saponified extract, 56 were identified when no saponification procedure was carried out: 6 free carotenoids, 20 monoesters and 30 diesters. This is the first time that esters of zeaxanthin, violaxanthin, auroxanthin, zeinoxanthin and ß-cryptoxanthin are identified in marigold. The structural information obtained through characteristic fragmentation patterns and diagnostic fragments in MS and MS/MS spectra (APCI+) sustained the differentiation between carotenoid esters with similar characteristics. Therefore, the separation of carotenoids by reversed-phase liquid chromatography using C30 columns in combination with DAD and APCI-MS/MS detection allowed high sensitivity and selectivity for carotenoid ester analysis.

Brazilian Biodiversity Fruits: Discovering Bioactive Compounds from Underexplored Sources.

Large segments of the Brazilian population still suffer from malnutrition and diet-related illnesses. In contrast, many native fruits have biodiversity and are underexploited sources of bioactive compounds and unknown to consumers. The phytochemical composition of nine underexplored Brazilian fruits was determined. Carotenoids and anthocyanins were identified and quantified by high performance liquid chromatography-photodiode array-tandem mass spectrometry (HPLC-PDA-MS/MS), and phenolic compounds and iridoids were identified by flow injection analysis-electrospray-ion trap-tandem mass spectrometry (FIA-ESI-IT-MS/MS); in total, 84 compounds were identified. In addition, the chemical structure and pathway mass fragmentation of new iridoids from jenipapo ( Genipa americana) and jatoba ( Hymenae coubaril) are proposed. The highest level of carotenoids was registered in pequi ( Caryocar brasiliense; 10156.21 µg/100 g edible fraction), while the major total phenolic content was found in cambuci ( Campomanesia coubaril; 221.70 mg GAE/100 g). Anthocyanins were quantified in jabuticaba ( Plinia cauliflora; 45.5 mg/100 g) and pitanga ( Eugenia uniflora; 81.0 mg/100 g). Our study illustrates the chemical biodiversity of underexplored fruits from Brazil, supporting the identification of new compounds and encouraging the study of more food matrixes not yet investigated.

Purple pitanga fruit (Eugenia uniflora L.) protects against oxidative stress and increase the lifespan in Caenorhabditis elegans via the DAF-16/FOXO pathway.

Pitanga, a fruit of the pitangueira tree (Eugenia uniflora L.), is native to Brazil and has a high antioxidant capacity due to the elevated amount of anthocyanins. The present study aimed to investigate the chemical composition of the purple pitanga fruit and to evaluate its antioxidant effect in the nematode Caenorhabditis elegans. We observed that the ethanolic extract of purple pitanga did not cause any toxic effects but notably increased worm lifespan. The extract improved the survival, reproduction and lifespan of the worms in pre- and post-exposure to stressors H2O2 and juglone, as well as improved the lifespan of the oxidative stress hypersensitive strain mev-1. Notably, PPE extract decreased reactive oxygen species by DCF-DA probe and protein carbonyl content from worms stressed with H2O2. The extract also affected the expression of superoxide dismutase SOD-3 and heat shock protein HSP-16.2 levels, daf 16 target genes that modulate lifespan and antioxidant metabolism. In addition, we demonstrate that these effects are dependent on DAF-16, as PPE extract did not provide protection in daf-16 mutants. Therefore, these results suggest that PPE significantly protected against oxidative stress modulating daf-16 target genes.

Impact of in vitro digestion phases on the stability and bioaccessibility of carotenoids and their esters in mandarin pulps.

The composition of carotenoids (carotenes and free and acylated xanthophylls) and their bioaccessibilities were determined for the first time in pulps of mandarins cultivated in Brazil. Two cultivars of mandarin, Citrus reticulata Blanco cv. 'Ponkan' and Citrus reticulata × C. sinensis cv. 'Murcott', showed higher contents of most carotenoids compared to those found in C. deliciosa Tenore cv. 'Rio'. The major carotenoids in mandarin cv. 'Ponkan' and 'Murcott' were (all-E)-ß-cryptoxanthin laurate (19-21%), (all-E)-ß-cryptoxanthin myristate (15-17%) and (Z)-ζ-carotene (7-12%), followed by (all-E)-ß-cryptoxanthin palmitate (4-7%), free (all-E)-ß-cryptoxanthin (5-6%) and (all-E)-ß-carotene (4-5%), while in mandarin cv. 'Rio' (all-E)-ß-cryptoxanthin myristate (22%) was the major compound, followed by (all-E)-ß-cryptoxanthin laurate (16%), (all-E)-ß-cryptoxanthin palmitate (11%), (all-E)-ß-cryptoxanthin (9%) and (all-E)-ß-carotene (6%). After in vitro digestion, the qualitative carotenoid profile of the supernatant containing the micellarized carotenoids was similar to that of fresh fruits, but the contents were significantly lower. Carotenoid and mandarin physico-chemical properties influenced the bioaccessibility of carotenoids. Free (all-E)-ß-cryptoxanthin showed the highest bioaccessibility in all mandarin cultivars (33-42%), while the bioaccessibilities of ß-carotene (16-36%) and the major carotenoid esters (18-33%) were lower. The overall recovery of carotenoids during in vitro digestion was around 98% after the oral phase, 79% after oral + gastric phases and 77% after oral + gastric + duodenal phases, with free (all-E)-ß-cryptoxanthin and (all-E)-ß-carotene being the most stable ones. Besides possible E-Z isomerization and ester hydrolysis, evident losses occurred in total carotenoid contents and also in the most individual carotenoids and they were not compensated for by the former reactions.

Carotenoid esters in foods - A review and practical directions on analysis and occurrence.

Carotenoids are naturally found in both free form and esterified with fatty acids in most fruits and some vegetables; however, up to now the great majority of studies presents data on carotenoid composition only after saponification. The reasons for this approach are that a single xanthophyll can be esterified with several different fatty acids, generating a great number of different compounds with similar chemical and structural characteristics, thus, increasing the complexity of analysis compared to the respective saponified extract. This means that since UV/Vis spectrum does not change due to esterification, differentiation between free and acylated xanthophylls is dependent at least on elution order and mass spectrometry (MS) features. The presence of interfering compounds, especially triacylglycerides (TAGs), in the non-saponified extract of carotenoids can also impair carotenoid ester analyses by MS due to high background noise and ionization suppression since TAGs can be present in much higher concentrations than the carotenoid esters. This leads to the need of development of new and effective clean-up procedures to remove the potential interferents. In addition, only few standards of xanthophyll esters are commercially available, making identification and quantification of such compounds even more difficult. Xanthophyll esterification may also alter some properties of these compounds, including solubility, thermostability and bioavailability. Considering that commonly consumed foods are dietary sources of xanthophyll esters and that it is the actual form of ingestion of such compounds, an increasing interest on the native carotenoid composition of foods is observed nowadays. This review presents a compilation of the current available information about xanthophyll ester analyses and occurrence and a practical guide for extraction, pre-chromatographic procedures, separation and identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

Cooking techniques improve the levels of bioactive compounds and antioxidant activity in kale and red cabbage.

The aim of this study is to investigate the effects of different home cooking techniques (boiling, steaming, and stir-frying) in kale and red cabbage, on the levels of bioactive compounds (carotenoids, anthocyanins and phenolic compounds) determined by high-performance liquid chromatography coupled with photodiode array and mass spectrometry detectors (HPLC-DAD-MS(n)), and on the antioxidant activity evaluated by ABTS, ORAC and cellular antioxidant activity (CAA) assays. The steaming technique resulted in a significant increase in phenolic content in kale (86.1%; p<0.001) whereas in red cabbage it was significantly reduced (34.6%; p<0.001). In the kale, steaming resulted in significant increases in antioxidant activity levels in all of the evaluation methods. In the red cabbage, boiling resulted in a significant increase in antioxidant activity using the ABTS assay but resulted in a significant decrease using the ORAC assay. According to the CAA assay, the stir-fried sample displayed the highest levels of antioxidant activity.

Two-step cleanup procedure for the identification of carotenoid esters by liquid chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry.

Carotenoids are naturally found in both free form and esterified with fatty acids in most fruits; however, up to now the great majority of studies only evaluated their composition after saponification. This fact is easily explained by the difficult to analyze carotenoid esters. Preliminary studies showed that cleanup procedures in the extract are necessary for further analysis by LC-MS/MS since triacylglycerols (TAGs) impair the MS detection. Considering these facts, we developed a new cleanup procedure to remove TAGs and other lipids from carotenoid fruit extracts. This procedure is based on physical removal of solid lipids at low temperature followed by open column chromatography on MgO and diatomaceous earth. Before cleanup, four carotenoid diesters and two free xanthophylls were identified in murici (Byrsonyma crassifolia), corresponding to about 65% of the total chromatogram area. After carrying out the two-step cleanup procedure, 35 carotenoids were identified, being 14 monoesters, six free carotenoids and 15 carotenoid diesters. We can conclude that this two-step procedure was successfully applied to murici, an Amazonian fruit, which contains high amounts of lipids.

An in vitro digestion method adapted for carotenoids and carotenoid esters: moving forward towards standardization.

In vitro digestion methods are a useful approach to predict the bioaccessibility of food components and overcome some limitations or disadvantages associated with in vivo methodologies. Recently, the INFOGEST network published a static method of in vitro digestion with a proposal for assay standardization. The INFOGEST method is not specific for any food component; therefore, we aimed to adapt this method to assess the in vitro bioaccessibility of carotenoids and carotenoid esters in a model fruit (Byrsonima crassifolia). Two additional steps were coupled to the in vitro digestion procedure, centrifugation at 20 000g for the separation of the aqueous phase containing mixed micelles and exhaustive carotenoid extraction with an organic solvent. The effect of electrolytes, enzymes and bile acids on carotenoid micellarization and stability was also tested. The results were compared with those found with a simpler method that has already been used for carotenoid bioaccessibility analysis. These values were in the expected range for free carotenoids (5-29%), monoesters (9-26%) and diesters (4-28%). In general, the in vitro bioaccessibility of carotenoids assessed by the adapted INFOGEST method was significantly higher (p < 0.05) than those assessed by the simplest protocol, with or without the addition of simulated fluids. Although no trend was observed, differences in bioaccessibility values depended on the carotenoid form (free, monoester or diester), isomerization (Z/E) and the in vitro digestion protocol. To the best of our knowledge, it was the first time that a systematic identification of carotenoid esters by HPLC-DAD-MS/MS after in vitro digestion using the INFOGEST protocol was carried out.