Isolation and Characterization of an Antioxidant Aryl Polyene Pigment from Antarctic Bacterium Lysobacter sp. A03.

Lysobacter is known as a bacterial genus with biotechnological potential, producing an array of enzymes, antimicrobial metabolites, and bioactive antioxidant compounds, including aryl polyene (APE) pigments that have been described as protecting substances against photooxidative damage and lipid peroxidation. In this study, the pigment extracted from keratinolytic Lysobacter sp. A03 isolated from Antarctic environment was characterized. The results of KOH test, UV-vis spectroscopy, CIELAB color system, 1H-NMR, and FTIR-ATR spectroscopy suggest the pigment is a yellow xanthomonadin-like pigment. The in vitro antioxidant activity of the pigment was confirmed by the scavenging of ABTS and DPPH radicals. In silico analysis of the genome through antiSMASH software was also performed and the secondary metabolite gene clusters for APE and resorcinol synthesis were identified, suggesting that proteins responsible for the pigment biosynthesis are encoded in Lysobacter A03 genome.

Acid hydrolysis conditions do affect the non-extractable phenolic compounds composition from grape peel and seed.

This study aimed to evaluate the effect of hydrolysis conditions on non-extractable phenolic compounds (NEPC) composition of grape peel and seed powder. The effect of temperature (50-90 °C), hydrochloric acid concentration (0.1-15.0 %), and time (5-20 min) were evaluated to understand their impact on NEPC release/extraction and degradation. The use of 1.0 and 8.0 % of HCl concentrations (v/v) and temperatures of 65 and 80 °C produced extracts with higher concentrations and a larger set of compounds. These conditions promoted a balance between release/extraction and degradation processes, thereby maximizing the NEPC content in the extracts. Furthermore, the results suggest that hydrolysis conditions can be set to modulate the release of specific classes. Non-extractable proanthocyanidins showed higher concentrations when intermediate values of temperature and acid concentration were applied. Hydrolysable tannins and hydroxybenzoic acids, on the other hand, were better extracted using higher acid concentrations and higher temperatures. The results suggest that the concentration and composition of NEPC are influenced by the hydrolysis conditions and the type of matrix. Hence, it is crucial to account for this compositional variation when conducting research on the biological effects of NEPC and when using this fraction as supplements or food ingredients.

Uncovering the phenolic diversity of Guabiju fruit: LC-MS/MS-based targeted metabolomics approach.

The comprehensive composition of phenolic compounds (PC) from seven genotypes of guabiju were analyzed by high-performance liquid chromatography coupled to a diode array detector and mass spectrometry (HPLC-ESI-qTOF-MS/MS), and a targeted metabolomic approach was utilized to explore the PC-related similarities among the genotypes. Sixty-seven phenolic compounds were annotated and twenty-four were quantified in all genotypes of guabiju. The phenolic acids and anthocyanins were the major PC, representing more than 63% (w/w) of the total PC. Di-O-galloylquinic and tri-O-galloylquinic acids and ellagitannins were reported for the first time in guabiju. The results of hierarchical clustering and principal components analysis (PCA) suggested seven groups as suitable clusters to be formed according to phenolic composition. Eleven PC were selected as relevant for sample clustering, and six of them were highlighted as the most informative (in decreasing order of importance): epicatechin, catechin, (epi)gallocatechin gallate II, di-O-galloylquinic acid I, tri-O-galloylquinic acid and delphinidin 3-O-glucoside. To the best of our knowledge, this study contributes to the literature with the most complete phenolic profile of guabiju genotypes up to date. Moreover, guabiju susceptibility to fungal infestation related to PC composition was briefly discussed based on a parallel study using the same genotypes.

Influence of sprouting on the bioaccessibility and bioactivity of benzoxazinoids, phenolic acids, and flavonoids of soft and hard wheat cultivars.

Grain germination increases the contents of benzoxazinoids and the antioxidant capacity of wheat and differentially affects the phytochemical composition of hard and soft wheat cultivars. It was investigated whether wheat cultivars (sprouted or not) with distinct grain texture characteristics (BRS Guaraim, soft vs. BRS Marcante, hard texture) would have different behavior in relation to changes in phytochemical compounds, bioaccessibility and antioxidant capacity during simulated gastrointestinal digestion of a tabbouleh preparation. Sprouting increased the nominal amount of phytochemicals in tabbouleh resulting in increased release of phenolic acids (up to 7.5-fold) and benzoxazinoids (up to 12.5-fold) during all digestion phases besides higher bioaccessibility (up to 2.8-fold). Sprouting caused greater increase in the bioaccessibility of phenolic acids for the soft wheat cultivar (4.5-fold) than for the hard cultivar (1.9-fold) and it increased the colon available index of phenolic acids only for the soft cultivar (1.8-fold). Flavonoids, mainly represented by apigenin glycosides, were marginally increased after sprouting but underwent relative increase along digestion being the major phytochemicals found in the bioaccessible fraction obtained after intestinal digestion (73-94% of total phytochemicals). The increase in apigenin glycosides was associated to the increase of in vitro and intracellular antioxidant capacity of tabbouleh along digestion. Sprouting increased the peroxyl radical removal capacity of tabbouleh in the gastric phase and in the non-bioaccessible fraction regardless of the cultivar. The highest hydroxyl radical removal capacities were found in non-sprouted cultivars, especially in the soft texture cultivar in the undigested and bioaccessible fractions. The bioaccessible fraction obtained after wheat digestion was more efficient to scavenge intracellular ROS than undigested samples, the highest scavenging potency being observed for the hard texture cultivar with no effect of sprouting. These findings confirm the hypothesis that the phytochemicals of hard and soft wheat cultivars (sprouted or not) have different behavior during digestion in terms of biotransformation, bioaccessibility and ability to remove reactive species and indicate that tabbouleh produced from sprouted wheat results in increased release of bioactive phytochemicals during digestion.

Theoretical estimation of nitrates and nitrites intake from food additives by the Brazilian population.

Sodium and potassium nitrates and nitrites are preservatives widely used in meat products and some cheese. They are important toxicologically but there is a lack of data on the exposure of the Brazilian population to these additives. This study aimed to verify the frequency of the use of nitrates and nitrites in processed foods in Brazil and to estimate their theoretical intake by the Brazilian population. A database was built of supermarket products containing nitrates, nitrites, and antioxidants. The Theoretical Maximum Daily Intake (TMDI) and TMDI balanced by the prevalence of food consumption (TMDI BPFC) were determined using consumption data from the Household Budget Surveys (2008/2009 and 2017/2018). The TMDI for nitrates and nitrites was lower than the Acceptable Daily Intake (ADI) for all population groups. Considering the prevalence of food consumption (consumers only), the TMDI BPFC values were lower than the ADI for nitrates (between 0.4 and 0.9 times the ADI) but very high values were obtained for nitrites (between 10 and 24 times the ADI). Our results suggest that the Brazilian population, especially some population groups, may be consuming unsafe amounts of nitrite. As a consequence, their health may be at risk.

MS-FINDER and SIRIUS for phenolic compound identification from high-resolution mass spectrometry data.

LC-HR-MS/MS is the predominant analytical technique in phenolic compound (PC) research. However, the manual interpretation of mass spectra is a heavy nontrivial time-consuming task and depends on mass spectrometry and phenolic compounds fragmentation deep knowledge. We think this manual approach should be partially translated into a practical software that allows users to perform such complicated analyses. In silico fragmentation software have been tested for small molecule identification, MS-FINDER and SIRIUS stood out at identification contests and challenges. We evaluated both software to identify PC from two data categories: 1st MS/MS spectra from 18 phenolic compound standards (PCS) and 2nd phenolic compounds from 8 food samples (FPC) (coffee, green tea, cranberry juice, grape juice, orange juice, apple juice, soy extract and parsley extract). MS-FINDER and SIRIUS were able to correctly identifymore than 90% of the PCS by LC-HR-MS/MS. The main FPC were also correctly identified by MS-FINDER (70%) and SIRIUS (38%). We highlight that these software were unable to differentiate PC isomers. This task is only possible by using additional information, such as chromatographic behavior and manual analysis of the relative intensity of fragments in the MS/MS spectra. Therefore, the combination of initial screening by using MS-FINDER and SIRIUS with manual analyses of additional information is a powerful and efficient approach for identifying phenolic compounds.

Grain Germination Changes the Profile of Phenolic Compounds and Benzoxazinoids in Wheat: A Study on Hard and Soft Cultivars.

Pre-harvest sprouting is a frequent problem for wheat culture that can be simulated by laboratory-based germination. Despite reducing baking properties, wheat sprouting has been shown to increase the bioavailability of some nutrients. It was investigated whether wheat cultivars bearing distinct grain texture characteristics (BRS Guaraim, soft vs. BRS Marcante, hard texture) would have different behavior in terms of the changes in phytochemical compounds during germination. Using LC-Q-TOF-MS, higher contents of benzoxazinoids and flavonoids were found in the hard cultivar than in the soft one. Free phytochemicals, mainly benzoxazinoids, increased during germination in both cultivars. Before germination, soft and hard cultivars had a similar profile of matrix-bound phytochemicals, but during germination, these compounds have been shown to decrease only in the hard-texture cultivar, due to decreased levels of phenolic acids (trans-ferulic acid) and flavonoids (apigenin) that were bound to the cell wall through ester-type bonds. These findings confirm the hypothesis that hard and soft wheat cultivars have distinct behavior during germination concerning the changes in phytochemical compounds, namely the matrix-bound compounds. In addition, germination has been shown to remarkably increase the content of benzoxazinoids and the antioxidant capacity, which could bring a health-beneficial appeal for pre-harvested sprouted grains.

New insights into non-extractable phenolic compounds analysis.

Most of the studies regarding phenolic compounds (PC) have been focused only on one fraction of PC, named extractable phenolic compounds (EPC). As the name suggests, EPC can be directly extracted from the food matrix by using an appropriate solvent. Otherwise, non-extractable phenolic compounds (NEPC) remain in the food matrix after the conventional extraction, and their analysis depends on a hydrolysis process. NEPC is a relevant fraction of PC that acts in the colon, where they are extensively fermented by the action of the microbiota. To understand the health effects associated with the NEPC intake, it is necessary to know which types of compounds are present and their content in foods. In this review, 182 studies published in the last five years about NEPC in foods were evaluated, focusing on critical points of the NEPC analysis. First, EPC exhaustive extraction should be performed before the hydrolysis processes to avoid overestimation of the NEPC fraction. NEPC hydrolysis by aggressive methods modifies their original structure and makes their complete elucidation difficult. These methods must be optimized considering the research objective, as different conditions may result in different amounts and profiles of compounds. Concerning quantification, the widely used spectrophotometric Folin-Ciocalteu method should be avoided as it leads to overestimation. Liquid chromatography coupled to a diode array detector is the most appropriate technique for this purpose. Although pure standard compounds are unavailable in most cases, standards representative of a PC family can be used, and results can be expressed as equivalent. The best approach for NEPC identification is liquid chromatography coupled to a diode array detector and tandem high-resolution mass spectrometry, which generates information regarding chromatographic behavior, UV-vis absorption, accuracy mass and fragmentation pattern. The identification process should associate manual data handling with the bioinformatics-assisted approach.

Grape UV-C irradiation in the postharvest period as a tool to improve sensorial quality and anthocyanin profile in 'Cabernet Sauvignon' wine.

Anthocyanins are important compounds in grapes and wine and significantly influence their characteristics. Ultraviolet light-C (UV-C) can be used as a tool to induce secondary metabolism, and in this study, it was used in 'Cabernet Sauvignon' grapes in the postharvest period to improve the anthocyanin profile and sensory attributes in wine produced with irradiated grapes. Grapes in the postharvest period were exposed to 0, 2, or 3 kJ m-2 UV-C radiation. After winemaking and storage time (6 months), physicochemical and color analyses, anthocyanin quantification and identification (HPLC-PDA-MS/MS), and sensory analyses were carried out. 'Cabernet Sauvignon' wine produced with grapes irradiated with 3 kJ m-2 UV-C showed increased spectrophotometric color, which is likely due to a 22.5% increase in total anthocyanin monomers, 59.3% of pyranoanthocyanins, 92.3% of direct condensation products, and 62.8% of acetaldehyde-mediated condensation products. In addition, this irradiated dose presented higher perception scores for visual color, aroma, taste, and was preferred by the tasters over the wine produced with non-irradiated grapes. This study is the first of its kind to show that the UV-C radiation treatment of grapes in the postharvest period can be a promising tool to improve the anthocyanin profile and sensorial quality of 'Cabernet Sauvignon' wine. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-021-05191-5.

Influence of cultivar and season on carotenoids and phenolic compounds from red lettuce influence of cultivar and season on lettuce.

This paper presents complete HPLC profiles and MS spectrometric data of bioactive compounds from four cultivars of red lettuce produced in winter and summer and their antioxidant capacity. The experiment was carried out in a greenhouse, where red curly lettuce was cultivated: Mila, Maira, Carmin and Scarlet. The cultivar and season have not influenced the qualitative profile of carotenoids (CAR) and phenolic compounds (PC) of red lettuce. Instead, the season influenced the concentration of these components in all cultivars. The levels of phenolic compounds were significantly higher in winter, while the levels of carotenoids were higher in summer. Ten anthocyanins were identified (cyanidins and delphinidins). The main carotenoid found was the all-trans-ß-carotene (45-48%), followed by lutein (13-20%) and zeaxanthin (11-15%). Major phenolic compounds include 5-caffeoylquinic acid, rutin and amentoflavone. Red lettuce cultivars have their main bioactive compounds described and compared within the variety and within the growing season. Different season and different lettuce cultivars may differ in the content of their bioactive compounds and in their antioxidant capacity.

Metabolomics: An analytical technique for food processing evaluation.

This review aimed to retrieve the most recent research with strong impact concerning the application of metabolomics analysis in food processing. The literature reveals the high capacity of this methodology to evaluate chemical and organoleptic transformations that occur during food production. Current and potential applications of metabolomics analysis will be addressed, focusing on process-composition-function relationships. The use of the metabolomics approach to evaluate transformations in foods submitted to minimal processes, heat or cold treatments, drying, fermentation, chemical and enzymatic treatments and processes using innovative technologies will be discussed. Moreover, the main strategies and advantages of metabolomics-based approaches are reviewed, as well as the most used analytical platforms. Overall, metabolomics can be seen as an important tool to support academia and industry on pursuing knowledge about the transformation of raw animal or plant materials into ready-to-eat products.

Antimycobacterial activity of Achyrocline flaccida (Asteraceae) aqueous extract from Southern Brazil.

Achyrocline flaccida aqueous extract was obtained by macerating wildflowers. The phytochemical profile present in the A. flaccida aqueous extract was elucidated by HPLC-ESI-MS/MS. Toxicity was evaluated in vitro by comet assay in peripheral blood mononuclear cells (PBMCs) and in vivo using Caenorhabditis elegans as a model. The antioxidant activity was also evaluated, and antimycobacterial activity was assessed by the broth microdilution method. The compounds present in the aqueous extract mainly belonged to the flavonoid class (89%). The concentrations that showed protective effects in C. elegans against oxidative stress and antimycobacterial activity had no toxic effects. The antimycobacterial activity test demonstrated that the concentration of 1,560 µg mL-1 inhibited the growth and eradication of the mycobacterial tested strains. Based on our findings, the A. flaccida aqueous extract presents a viable potential in developing new phytotherapeutic drugs against mycobacteria of clinical relevance.

Enhancement of the Functional Properties of Mead Aged with Oak (Quercus) Chips at Different Toasting Levels.

Consumers increasingly prefer and seek functional beverages, which, given their characteristics, provide important bioactive compounds that help prevent and treat chronic diseases. Mead is a traditional fermented alcoholic beverage made from honey solution. The aging process of mead with oak chips is innovative and bestows functional characteristics to this beverage. Thus, in this study, we sought to develop and characterize a novel functional beverage by combining the health benefits of honey with the traditional aging process of alcoholic beverages in wood. Phenolic compounds, flavonoids, and antioxidant capacity were analyzed in mead using oak chips at different toasting levels and aged for 360 days. LC-ESI-QTOF-MS/MS was used to analyze the chemical profile of different meads. Over time, the aging process with oak chips showed a higher total phenolic and flavonoid content and antioxidant capacity. Eighteen compounds belonging to the classes of organic acids, phenolic acids, flavonoids, and tannins were identified in meads after 360 days. Our findings revealed that the addition of oak chips during aging contributed to p-coumaric, ellagic, abscisic, and chlorogenic acids, and naringenin, vanillin, and tiliroside significantly impacted the functional quality of mead.

Antiproliferative Effect of Colonic Fermented Phenolic Compounds from Jaboticaba (Myrciaria trunciflora) Fruit Peel in a 3D Cell Model of Colorectal Cancer.

Jaboticaba is a Brazilian native berry described as a rich source of phenolic compounds (PC) with health promoting effects. PC from jaboticaba peel powder (JPP) have low intestinal bio-accessibility and are catabolized by gut microbiota. However, the biological implication of PC-derived metabolites produced during JPP digestion remains unclear. This study aimed to evaluate the antiproliferative effects of colonic fermented JPP (FJPP) in a 3D model of colorectal cancer (CRC) composed by HT29 spheroids. JPP samples fermented with human feces during 0, 2, 8, 24 or 48 h were incubated (10,000 µg mL-1) with spheroids, and cell viability was assessed after 72 h. Chemometric analyses (cluster and principal component analyses) were used to identify the main compounds responsible for the bioactive effect. The antiproliferative effect of FJPP in the CRC 3D model was increased between 8 h and 24 h of incubation, and this effect was associated with HHDP-digalloylglucose isomer and dihydroxyphenyl-γ-valerolactone. At 48 h of fermentation, the antiproliferative effect of FJPP was negligible, indicating that the presence of urolithins did not improve the bioactivity of JPP. These findings provide relevant knowledge on the role of colonic microbiota fermentation to generate active phenolic metabolites from JPP with positive impact on CRC.

Natural deep eutectic solvent (NADES): A strategy to improve the bioavailability of blueberry phenolic compounds in a ready-to-use extract.

This study investigated whether a ready-to-use extract obtained using a natural deep eutectic solvent (NADES) affects the pharmacokinetic profile of blueberry phenolic compounds compared to organic solvent (SORG)-extracted compounds. SORG extract was administered as an aqueous solution after solvent removal. Wistar rats received a single dose of crude extract of blueberry obtained using NADES (CE-NADES) or SORG (CE-SORG), followed by LC-DAD-MS/MS analysis of blood and cecal feces. Non-compartmental pharmacokinetic analysis revealed that CE-NADES increased the bioavailability of anthocyanins by 140% compared to CE-SORG. CE-NADES increased the stability of phenolic compounds during in vitro digestion by delaying gastric chyme neutralization. These results suggest that besides being an eco-friendly solvent for the extraction of phytochemicals, choline chloride:glycerol:citric acid-based NADES can be used as a ready-to-use vehicle for increasing oral absorption of bioactive compounds such as anthocyanins.

Effects of indoor, greenhouse, and field cultivation on bioactive compounds from parsley and basil.

BACKGROUND: Aromatic herbs are an important source of bioactive compounds. Different cultivation systems should give each plant a specific amount of those compounds, which should be of a particular quality. In this study, the effects of three cultivation systems (indoor, greenhouse, and organic field) on the composition of bioactive compounds in parsley (Petroselinum crispum cv. 'Flat Leaf'), green basil (Ocimum basilicum var. minimum cv. 'Greek'), and purple basil (Ocimum basilicum cv. 'Red Rubin') were evaluated. RESULTS: ß-Carotene and lutein were the carotenoids with the highest concentration in the three plants in all the cultivation systems. Overall, parsley proved to be a source of flavonoids. The major phenolic compound found in basil plants was rosmarinic acid, whereas most anthocyanins were derived from cyanidin aglycone. Among the three plants studied, the highest vitamin C content was found in parsley from the field. This was 2.6 and 5.4 times higher than the indoor and greenhouse cultivation, respectively. CONCLUSION: The results suggest that different cultivation systems influence and modulate the concentration of bioactive compounds in plants differently, varying according to their class, and that, above all, an indoor system is an effective cultivation system for the production of bioactive compounds. © 2021 Society of Chemical Industry.

Production of antimicrobial metabolites against pathogenic bacteria and yeasts by Fusarium oxysporum in submerged culture processes.

The antimicrobial activity of the metabolites produced by Fusarium oxysporum PR-33 in submerged culture was evaluated against Gram-positive and Gram-negative bacteria and yeasts. Metabolites were determined by HPLC-DAD-MS/MS. An extract was obtained following the removal of mycelium by centrifugation and lyophilisation of the supernatant. The compounds in this extract demonstrated broad-spectrum antimicrobial action, with rates of inhibition between 60 and 80%, depending on the species and extract tested. The major compounds of the extracts were identified as fusarinolic acid and its isomer [56.9% flask extract (FE)] and 59.2% bioreactor extract (BE), dehydrofusaric acid (35.7% FE and 31.6% BE), and fusaric acid (6.5% FE and 1.1% BE). Fusaric acid has been shown to be responsible for antimicrobial activity. The cytotoxicity of the extracts was evaluated in culture of HEK-293 and SH-SY5Y animal cells and toxicity of these extracts was verified even in the lowest tested concentrations. Therefore, our results indicate that the compounds identified exhibit potential as antimicrobial agents.

QuEChERS-LC-QTOFMS for the simultaneous determination of legislated and emerging mycotoxins in malted barley and beer using matrix-matched calibration as a solution to the commercial unavailability of internal standards for some mycotoxins.

The combination of QuEChERS-LC-QTOFMS and matrix-matched calibration (MMC) to simultaneously determinate legislated and emerging mycotoxins in malt and beer was evaluated for the first time. The method performance was satisfactory displaying suitable linearity (R2 >0.99) and recovery (71-102%). The lowest values (in µg kg-1) of LOD (0.01) and LOQ (0.05) were found for enniatins, while the highest LOD (15) and LOQ (50) were reported for fumonisin B1. Precision and sensitivity (RSD <10%) were in accordance with the different guidelines of method validation. MMC was important to avoid inaccurate quantification of all mycotoxins due to signal enhancement or suppression. Another advantage was the enhanced throughput, requiring 1.2 min of analysis per analyte. The detection of legislated (aflatoxins, ochratoxin A, deoxynivalenol, fumonisin B1, zearalanone, T-2 and HT-2 toxin) and emerging mycotoxins (enniatins, beauvericin, moniliformin and sterigmatocystin) allowed verifying compliance with legislation and generating data to support the establishment of limits for emerging mycotoxins.

Chymase inhibition: A key factor in the anti-inflammatory activity of ethanolic extracts and spilanthol isolated from Acmella oleracea.

ETHNOPHARMACOLOGICAL RELEVANCE: Acmella oleracea (L.) R. K. Jansen (Asteraceae), known as jambú in Brazil, is used in traditional medicine as analgesic and for inflammatory conditions, characterized by the presence of N-alkylamides, mainly spilanthol. This bioactive compound is responsible for the above-described pharmacological properties, including sialagogue and anesthetic. AIM OF THE STUDY: This study aimed to characterize the anti-inflammatory effects of A. oleracea leaves (AOEE-L) and flowers (AOEE-F) extracts, including an isolated alkylamide (spilanthol), using in vitro and in vivo models. The mechanism underlying this effect was also investigated. MATERIALS AND METHODS: Extracts were analyzed by HPLC-ESI-MS/MS in order to characterize the N-alkylamides content. AOEE-L, AOEE-F (25-100 µg/mL) and spilanthol (50-200 µM) were tested in vitro on VSMC after stimulation with hyperglycemic medium (25 mM glucose). Their effects over nitric oxide (NO) generation, chymase inhibition and expression, catalase (CAT), superoxide anion (SOD) radical activity were evaluated. After an acute administration of extracts (10-100 mg/mL) and spilanthol (6.2 mg/mL), the anti-inflammatory effects were evaluated by applying the formalin test in rats. Blood was collected to measure serum aminotransferases activities, NO activity, creatinine and urea. RESULTS: A number of distinct N-alkylamides were detected and quantified in AOEE-L and AOEE-F. Spilanthol was identified in both extracts and selected for experimental tests. Hyperglycemic stimulation in VSMC promoted the expression of inflammatory parameters, including chymase, NO, CAT and SOD activity and chymase expression, all of them attenuated by the presence of the extracts and spilanthol. The administration of extracts or spilanthol significantly inhibited edema formation, NO production and cell tissue infiltration in the formalin test, without causing kidney and liver toxicity. CONCLUSION: Taken together, these results provide evidence for the anti-inflammatory activity of leaves and flowers extracts of jambú associated distinctly with their chemical profile. The effects appear to be associated with the inhibition of chymase activity, suppression of the proinflammatory cytokine NO and antioxidant activities.

Natural deep eutectic solvent (NADES)-based blueberry extracts protect against ethanol-induced gastric ulcer in rats.

Blueberry is a polyphenol-rich fruit bearing great bioactive potential. Natural deep eutectic solvents (NADES) emerged as putatively biocompatible solvents that could substitute for toxic organic solvents in the extraction of fruit phenolic compounds for developing nutraceuticals or functional foods. Therefore, the aim of this study was to investigate the gastroprotective effects and the biocompatibility of a blueberry crude extract (CE) obtained using NADES and of the extract fractions (anthocyanin-rich fraction - ARF; non-anthocyanin phenolic fraction - NAPF) in a model of ethanol-induced gastric ulcer in rats. CE was the NADES-containing, ready-to-use extract that was obtained using choline chloride:glycerol:citric acid NADES (0.5:2:0.5 M ratio). ARF and NAPF were the NADES-free fractions obtained by solid phase purification of CE and were investigated to identify the bioactive fraction responsible for the effects of CE. Animals were treated for 14 days with water, NADES vehicle, CE, ARF, NAPF or lansoprazole (intragastric) and then received ethanol to induce gastric ulcer. CE decreased ulcer index and preserved the integrity of gastric mucosa. The pretreatment with CE or ARF reduced glutathione depletion and the inflammatory response. All treatments, including NADES vehicle reduced protein oxidation and nitric oxide overproduction in ethanol-treated rats. Additionally, ARF increased short-chain fatty acids in feces. These findings suggest that NADES can be used to obtain biocompatible extracts of blueberry that exhibit gastroprotective effects with no need of solvent removal. The gastroprotective effects were mainly associated to ARF but NAPF and even NADES vehicle also contributed to some protective effects.