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.

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.

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.

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.

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.

Virgin Coconut Oil Associated with High-Fat Diet Induces Metabolic Dysfunctions, Adipose Inflammation, and Hepatic Lipid Accumulation.

Obesity reaches an epidemic level worldwide, and this condition is associated with chronic low-grade inflammation and secondary comorbidities, largely driven by global changes in lifestyle and diet. Various dietary approaches are proposed for the obesity treatment and its associated metabolic disorders. Good taste, antioxidant functions, and vitamins have been attributed to virgin coconut oil (VCO). However, VCO contains a large amount of saturated fatty acids, and the consumption of this fat is associated with a number of secondary diseases. We evaluate the effects of VCO supplementation on biochemical, inflammatory, and oxidative stress parameters in rats fed with high-fat diet (HFD). After feeding with HFD for 12 weeks, the animals were supplemented with VCO for 30 days. HFD+VCO group increased in diet intake, weight gain, low-density lipoprotein cholesterol level, and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. These findings were accompanied by increased in hepatic lipid profile and fat deposition in the liver. Adipocyte hypertrophy was observed in the HFD+VCO group, which was associated with elevated expression of tumor necrosis factor alpha (TNF-α) in adipose tissue. These results revealed that VCO associated with HFD induced important metabolic alterations, adipose inflammation, and hepatic lipid accumulation in rats.

Bioactive compounds and protective effect of red and black rice brans extracts in human neuron-like cells (SH-SY5Y).

Rice bran is obtained from the rice polishing process, and this by-product contains many bioactive compounds. In this study, the composition of phenolic compounds from red and black rice brans was determined by HPLC-DAD-MS. Additionally, the neuroprotective ability of these brans in SH-SY5Y cells insulted with hydrogen peroxide (H2O2) was evaluated. The phenolic constituents of rice bran were separated into hydrophilic and pellet fractions. The major phenolic compound in both samples was ferulic acid. Cyanidin 3-glucoside was the main anthocyanin in black rice bran. The hydrophilic and pellet fractions showed a protective effect (38-94%) on SH-SY5Y cells insulted by H2O2 in DCFH-DA assay. No extract showed cytotoxicity in the SRB assay. These results suggest a neuroprotective effect of red and black rice brans extracts due to their high antioxidant capacity, along with the absence of cytotoxicity. Thus, they may potentially be used as sources of bioactive compounds.

Effect of temperature and nitrogen concentration on biomass composition of Heterochlorella luteoviridis

Abstract The interest in microalga as a food supplement has grown due their high contents of carotenoids, polyunsaturated fatty acids and proteins. This study evaluated the effect of different temperatures (22, 27 or 32 °C) and sodium nitrate concentrations (12, 24, 36, 48 or 60 mg L-1 of N-NO3) in culture medium on Heterochlorella luteoviridis biomass production and composition. The highest biomass concentration (3.35 g L-1) was observed at the highest N-NO3 concentration. The N-NO3 concentration positively affected protein, carbohydrate and carotenoids contents of biomass. On the other hand, cells cultured at the lowest N-NO3 concentration showed a slight increment in lipid content. The major carotenoid was lutein (30.7 ± 1.4% of total carotenoids), and the polyunsaturated fatty acids were 37 ± 2% of total fatty acids. Low temperature improved the biosynthesis of ω3 type fatty acids by lowering the ω6:ω3 ratio. Overall, our results indicate H. luteoviridis can yield high biomass concentration under autotrophic growth, resulting in a biomass rich in carotenoids, mainly lutein, and ω3 polyunsaturated fatty acids.

Chemical composition of microalgae Heterochlorella luteoviridis and Dunaliella tertiolecta with emphasis on carotenoids.

BACKGROUND: Microalgae have been used as food supplements owing to their high protein, polyunsaturated fatty acid and carotenoid contents. As different carotenoids have distinct properties and the carotenoid composition of microalgae has been poorly explored in the literature, this study determined the complete carotenoid composition of two microalgae species, Heterochlorella luteoviridis and Dunaliella tertiolecta, using high-performance liquid chromatography coupled with diode array detection and tandem mass spectrometry (HPLC-DAD/MS2 ). Additionally, the proximate composition and major minerals were evaluated. RESULTS: The carotenoid composition of the two microalgae was similar, with 13 carotenoids being found in H. luteoviridis and 12 in D. tertiolecta. The major carotenoids were all-trans-lutein (1.18 mg g-1 in H. luteoviridis and 1.59 mg g-1 in D. tertiolecta), all-trans-violaxanthin (0.52 mg g-1 in H. luteoviridis and 0.45 mg g-1 in D. tertiolecta) and all-trans-ß-carotene (0.50 mg g-1 in H. luteoviridis and 0.62 mg g-1 in D. tertiolecta). All-trans-lutein was the predominant carotenoid in both microalgae, representing around 40% (mass fraction) of the total carotenoids. The lutein content found in these microalgae was significantly higher (2-40 times) than that in other important food sources of lutein (e.g. parsley, carrot, red pepper and broccoli). CONCLUSION: The microalgae H. luteoviridis and D. tertiolecta are excellent sources of lutein that could be commercially exploited by the food and pharmaceutical industries. Moreover, it was confirmed that both microalgae are good sources of protein, lipids and calcium. © 2016 Society of Chemical Industry.

Phenolic compounds and carotenoids from four fruits native from the Brazilian Atlantic Forest.

Fruits from the Atlantic Forest have received increasing interest because they contain high levels of bioactive compounds with notable functional properties. The composition of carotenoids and phenolic compounds from fruits found in the Atlantic Forest (jussara, uvaia, araça, and grumixama) was determined by high-performance liquid chromatography coupled to diode array and mass spectrometry detectors. Uvaia showed the highest levels of carotenoids (1306.6 µg/100 g fresh matter (f.m.)). Gallic acid was the major phenolic compound in araça (12.2 mg GAE/100 g f.m.) and uvaia (27.5 mg GAE/100 g f.m.). In grumixama, eight quercetin derivatives were found; the main carotenoids included all-trans-ß-cryptoxanthin (286.7 µg/100 g f.m.) and all-trans-lutein (55.5 µg/100 g f.m.). Uvaia and grumixama contain high amounts of carotenoids, while jussara showed greater levels of phenolic compounds (415 mg GAE/100 g f.m.), particularly anthocyanins (cyanidin 3-rutinoside: 179.60 mg/100 g f.m.; cyanidin 3-glucoside: 47.93 mg/100 g f.m.).

Carotenoids and phenolic compounds from Solanum sessiliflorum, an unexploited Amazonian fruit, and their scavenging capacities against reactive oxygen and nitrogen species.

The composition of carotenoids and phenolic compounds from mana-cubiu (Solanum sessiliflorum), a fruit native to Amazonia, was determined by high-performance liquid chromatography coupled to diode array and mass spectrometry detectors (HPLC-DAD-MS(n)). The antioxidant capacities of the hydrophilic and carotenoid extracts against some reactive oxygen (ROO(•), H(2)O(2), HOCl, and HO(•)) and nitrogen (ONOO(-)) species were also determined. Seventeen carotenoids and three phenolic compounds were found in mana-cubiu. The major carotenoids were (all-E)-ß-carotene (7.15 µg/g of dry weight) and (all-E)-lutein (2.41 µg/g of dry weight). The 5-caffeoylquinic acid (1351 µg/g of dry weight) was the major phenolic compound, representing more than 78% (w/w) of the total phenolic compounds. Moreover, two dihydrocaffeoyl spermidines were found in the hydrophilic extract. Both mana-cubiu extracts were able to scavenge all the tested reactive species. The carotenoid extract was shown to be a potent scavenger of peroxyl radical, while the hydrophilic extract was a potent hydrogen peroxide and hypochlorous acid scavenger.

Scavenging capacity of marine carotenoids against reactive oxygen and nitrogen species in a membrane-mimicking system.

Carotenoid intake has been associated with the decrease of the incidence of some chronic diseases by minimizing the in vivo oxidative damages induced by reactive oxygen (ROS) and nitrogen species (RNS). The carotenoids are well-known singlet oxygen quenchers; however, their capacity to scavenge other reactive species, such as peroxyl radical (ROO•, hydroxyl radical (HO•), hypochlorous acid (HOCl) and anion peroxynitrite (ONOO⁻), still needs to be more extensively studied, especially using membrane-mimicking systems, such as liposomes. Moreover, the identification of carotenoids possessing high antioxidant capacity can lead to new alternatives of drugs or nutritional supplements for prophylaxis or therapy of pathological conditions related to oxidative damages, such as cardiovascular diseases. The capacity to scavenge ROO•, HO•, HOCl and ONOO⁻ of seven carotenoids found in marine organisms was determined in liposomes based on the fluorescence loss of a fluorescent lipid (C11-BODIPY58¹/59¹) due to its oxidation by these reactive species. The carotenoid-bearing hydroxyl groups were generally more potent ROS scavengers than the carotenes, whilst ß-carotene was the most efficient ONOO⁻ scavenger. The role of astaxanthin as an antioxidant should be highlighted, since it was a more potent scavenger of ROO•, HOCl and ONOO⁻ than α-tocopherol.

Development of a novel micro-assay for evaluation of peroxyl radical scavenger capacity: application to carotenoids and structure-activity relationship.

A micro-assay was developed and validated, using a microplate reader in 96-well format, C(11)-BODIPY(581/591) as fluorescent probe and AIBN as ROO() generator. The structure-activity relationship was established for 15 carotenoid standards, indicating that the opening of the ß-ionone ring and the increase of chromophore extension in the carotenoid structure were the major factors leading to the increase of ROO() scavenging capacity. The values for ROO() scavenging capacity were calculated using α-tocopherol as reference compound. Among the studied carotenoids, all-trans-lycopene was the most efficient ROO() scavenger (8.67±0.74) followed by all-trans-astaxanthin (6.50±0.62). All the carotenoids showed to be more effective ROO() scavengers than α-tocopherol and some hydrophilic compounds. Finally, the method was successfully applied to assay the ROO() scavenging capacity of carotenoid extracts from two Amazonian fruits, peach palm (7.83±0.21) and mamey (6.90±0.44).