Caracterización química, propiedades antioxidantes y evaluación de los efectos neuroprotectores de bebidas fermentadas

La fresa es una excelente fuente de compuestos bioactivos, sin embargo, es un fruto muy perecedero. La elaboración de productos fermentados supone una valiosa estrategia para disminuir las pérdidas económicas por excedentes de fruta y aumentar su vida útil. Por otro lado, resulta de gran interés el desarrollo de procesos biotecnológicos que preserven los compuestos bioactivos presentes en el fruto de partida. En esta Tesis Doctoral se ha evaluado la composición antociánica, color y actividad antioxidante de productos fermentados elaborados a partir de puré de fresa, concretamente, fermentados alcohólicos, acéticos y glucónicos, así como la estabilidad de una bebida obtenida a partir de estos productos. Asimismo, se ha estudiado el efecto neuroprotector de estos fermentados frente a la toxicidad del péptido β-amiloide (Aβ) y a la producción de Especies Reactivas de Oxígeno (ROS). Por otro lado, se ha demostrado la bioactividad del ácido protocatéquico (uno de los principales metabolitos de antocianos) frente a la agregación y toxicidad del péptido Aβ y de la proteína α-sinucleína (αS) in vitro. Además, se ha analizado el efecto de otros compuestos bioactivos (melatonina y otros compuestos indólicos) presentes en fresa y otros productos fermentados frente al proceso de fibrilación y neurotoxicidad del péptido Aβ. Los resultados obtenidos han permitido la identificación de nuevos compuestos antociánicos en fermentados alcohólicos y acéticos. Al mismo tiempo, se ha comprobado cómo el proceso que mejor preserva los antocianos es la fermentación glucónica, que se ha seleccionado como base de la bebida elaborada. Además, los fermentados alcohólicos y glucónicos protegen a las células neuronales de la toxicidad del péptido Aβ. Asimismo, el ácido protocatéquico posee un efecto inhibidor de la formación de fibras de Aβ y αS y efectos neuroprotectores demostrado por diversas técnicas in vitro. Por último, el estudio de los compuestos indólicos frente al proceso de agregación y la toxicidad del Aβ ha puesto de manifiesto que la serotonina es un potente inhibidor de la formación de fibras y que la melatonina y los compuestos indólicos relacionados con su síntesis presentan un efecto preventivo frente a la muerte neuronal producida por este péptido.The strawberry is an excellent source of bioactive compounds, however, it is a very perishable fruit. The elaboration of fermented products supposes a valuable strategy to prevent the economic losses due to strawberry surplus and to increase the shelf life. On the other hand, the use of biotechnological processes that preserve the bioactive compounds is a relevant aspect to take into consideration. In this PhD Thesis, anthocyanin composition, colour and antioxidant activity of different fermented products made from strawberry purée by alcoholic, acetic and gluconic fermentations have been evaluated. Additionally, the stability of a beverage obtained from these products has been studied. Moreover, the neuroprotective effect of these products against amyloid-β (Aβ) toxicity and Reactive Oxygen Species (ROS) production has been investigated. In addition, the bioactivity of protocatechuic acid (one of the most important anthocyanin metabolite) against the aggregation and toxicity of Aβ peptide and α-synuclein (αS) protein in vitro has been demonstrated. Furthermore, it has been confirmed that other bioactive compounds (melatonin and indolic related compounds) described in strawberry and in other fermented products present an effect on the inhibition of the Aβ aggregation and the Aβ-induced toxicity. The obtained results have led to the identification of new anthocyanin compounds in alcoholic and acetic fermented products. Moreover, it has been proven that gluconic fermentation is the process, from those studied in this Thesis, that better preserves the anthocyanin profile. Thus, it was the one selected as substrate to elaborate the beverage. Furthermore, alcoholic and gluconic fermented extracts protect neuronal cells against Aβ toxicity. Protocatechuic acid possesses an inhibitory effect of the Aβ and αS fibril formation and neuroprotective properties demonstrated by several in vitro techniques. Finally, serotonin has been proved to be a potent inhibitor of the fibril formation. Melatonin and the related indolic compounds presented a preventive effect against the cell death produced by Aβ

Inhibition of VEGF-Induced VEGFR-2 Activation and HUVEC Migration by Melatonin and Other Bioactive Indolic Compounds

Excessive concentrations of vascular endothelial growth factor (VEGF) trigger angiogenesis, which causes complications such as the destabilization of atherosclerotic plaques and increased growth of tumors. This work focuses on the determination of the inhibitory activity of melatonin and other indolic related compounds on VEGF-induced VEGF receptor-2 (VEGFR-2) activation and an approximation to the molecular mechanism underlying the inhibition. Quantification of phosphorylated VEGFR-2 was measured by ELISA. Migration wound-healing assay was used to determine cell migration of human umbilical vein endothelial cells (HUVECs). This is the first time that melatonin, 3-indolacetic acid, 5-hydroxytryptophol, and serotonin are proved to significantly inhibit VEGF-induced VEGFR-2 activation in human umbilical vein endothelial cells and subsequent angiogenesis. 3-Indolacetic acid showed the highest inhibitory effect (IC50 value of 0.9704 mM), followed by 5-hydroxytryptophol (35% of inhibition at 0.1 mM), melatonin (30% of inhibition at 1 mM), and serotonin (24% of inhibition at 1 mM). An approximation to the molecular mechanism of the inhibition has been proposed, suggesting that indolic compounds might interact with the cell surface components of the endothelial membrane in a way that prevents VEGF from activating the receptor. Additionally, wound-healing assay revealed that exposure of HUVECs to melatonin and 3-indolacetic acid in the presence of VEGF significantly inhibited cell migration by 87% and 99%, respectively, after 24 h. These data demonstrate that melatonin, 3-indolacetic acid, 5-hydroxytryptophol, and serotonin would be good molecules for future exploitation as anti-VEGF signaling agents.España, Ministerio de Ciencia e Innovación AGL2013-47300-C3-2-

Phenolic Compounds of Grapes and Wines: Key Compounds and Implications in Sensory Perception

Phenolic compounds are a wide family of thousands of natural bioactives well-known for their overwhelming demonstrated health benefits. Particularly in wines, polyphenols and quality are closely interconnected. Indeed, these compounds possess a critical role due to their contribution to organoleptic wine quality as color, astringency, and bitterness. The profile or the composition of certain polyphenols has been even proposed as an analytical tool for authenticity certification. In this sense, although important progress has been achieved, the understanding of the relationship between the quality of a particular wine and its phenolic composition remains one of the major challenges in enology research. But why? If there is an adjective to define wine, it is “complex.” This final complexity of a wine begins with the enormous polyphenolic variability that may be present in grapes influenced by ripening, genetic, or environmental factors, among others. Winemaking process (alcoholic and malolactic fermentation) and wine aging with or without wood contact produce endless reactions giving rise to complex transformations (copigmentation, cycloaddition, polymerization, and oxidation) of polyphenols. This chapter gathers the most relevant information about the composition, variations, and transformations of phenolic compounds from grape to wine including their influence on sensory properties

Anthocyanins: Dietary Sources, Bioavailability, Human Metabolic Pathways, and Potential Anti-Neuroinflammatory Activity

The objectives of this chapter are to summarize and discuss (i) the anthocyanins structure and content in foodstuffs and their dietary intake (ii) the anthocyanins bioavailability and human metabolic pathways and (iii) the in vitro and in vivo potent anti-neuroinflammatory effects of anthocyanins and their metabolites. Indeed, anthocyanins are polyphenolic compounds belonging to the group of flavonoids, and are one of the most commonly consumed polyphenols in a normal diet. They are responsible of red, blue and purple color of several fruits and vegetables and their intake has been related with several human health benefits. The anthocyanins structures diversities as well as their content in various fruits, vegetables and cereals is addressed. Moreover, despite the growing evidence for the protective effects of anthocyanins, it is important to highlight that the in vivo bioavailability of these compounds is relatively low in comparison to their more stable metabolites. Indeed, after consumption, these bioactives are subjected to substantial transformations in human body. Phase I and II metabolites generated by intestinal and hepatic enzymatic reactions, and phenolic acids produced by gut microbiota and their metabolized forms, are the most important metabolic anthocyanins forms. For this reason, the study of the biological properties of these circulating metabolites represents a more in vivo realistic situation. Although the anthocyanin bioavailability researches in humans are limited, they will be discussed together with a global metabolic pathway for the main anthocyanins. Moreover, several works have demonstrated that anthocyanins can cross the blood brain barrier, and accumulate in brain endothelial cells, brain parenchymal tissue, striatum, hippocampus, cerebellum and cortex. Consequently, the study of anthocyanins as potent therapeutic agents in neurodegenerative diseases has gained relevance and the principal and the most recent studies are also discussed in the book chapter

Isotopic labelling-based analysis elucidates biosynthesis pathways in Saccharomyces cerevisiae for Melatonin, Serotonin and Hydroxytyrosol formation

Yeasts can synthetise bioactive compounds such as Melatonin (MEL), Serotonin (SER) and Hydroxytyrosol (HT). Deciphering the mechanisms involved in their formation can lead to exploit this fact to increase the bioactive potential of fermented beverages. Quantitative analysis using labelled compounds, 15-N2 l-tryptophan and 13-C tyrosine, allowed tracking the formation of the above-mentioned bioactive compounds during the alcoholic fermentation of synthetic must by two different Saccharomyces cerevisiae strains. Labelled and unlabelled MEL, SER and HT were undoubtedly identified and quantified by High Resolution Mass Spectrometry (HRMS). Our results prove that there are at least two pathways involved in MEL biosynthesis by yeast. One starts with tryptophan as precursor being known for the vertebrates’ pathway. Additionally, MEL is produced from SER which in turn is consistent with the plants’ biosynthesis pathway. Concerning HT, it can be formed both from labelled tyrosine and from intermediates of the Erlich pathway.Ministerio de Economía y Competitividad AGL2016-77505-C3-2-RMinisterio de Ciencia, Innovación y Universidades PID2019-108722RB-C32Junta de Andalucía, Consejería de Economía y Conocimiento P18-RT-309

Polyphenolic characterization of merlot, tannat and syrah skin extracts at different degrees of maturity and anti-inflammatory potential in RAW 264.7 cells

(1) Background: Both sensory quality and healthy attributes of Vitis vinifera grapes used for winemaking are closely related with the polyphenolic composition of their skins. (2) Methods: In this study, the polyphenolic characterization (flavan-3-ols, procyanidins, flavonols, stilbenes, anthocyanins) was investigated by ultra performance liquid chromatography coupled to a triple quadrupole mass spectrometer (UPLC-QqQ-MS). Skins from Vitis vinifera Merlot, Tannat, and Syrah red grape varieties cultivated in the south of France at different stages of ripening in 2018 were used. The anti-inflammatory and the antioxidant potential of the extracts were evaluated by the measure of nitric oxide (NO) and the intracellular reactive oxygen species production (ROS) in lipopolysaccharide (LPS)-stimulated macrophages. (3) Results: 41 polyphenols were quantified in all samples. Generally, the flavan-3-ol and procyanidin content decreased during ripening whereas the anthocyanins and stilbenes increased. In addition, as a novelty of this work, a wide identification and characterization of monomeric and oligomeric stilbenes was assessed by using authentic standards isolated in our laboratory, some of them (parthenocissin A and miyabenol C) reported for the first time in Merlot, Tannat and Syrah cultivars. The before-veraison skin extracts of all studied varieties, exhibited higher NO and ROS productions inhibition (>50%) proving both antioxidant and antiinflammatory properties

New Insights into the Exploitation of Vitis vinifera L. cv. Aglianico Leaf Extracts for Nutraceutical Purposes

The leaves of Vitis vinifera L. have been used for a long time in traditional medicine for the treatment of many ailments. Grape polyphenols, indeed, have been demonstrated to be able to defend against oxidative stress, responsible for various disorders such as cancer, diabetes and neurodegenerative diseases. The effects of different extraction techniques, Soxhlet (SOX), Accelerated Solvent (ASE 40, ASE 50) and Ultrasound Assisted Extraction (UAE) were studied in this work to evaluate their impact on the chemical profile and bioactive potential of Vitis vinifera L. (cv. Aglianico) leaf extracts. The phytochemical profile was investigated by HPLC-DAD and 9 phenolic compounds were identified and quantified in the extract. Moreover, the antioxidant, anticholinesterase and antityrosinase activities were evaluated. In detail, the total polyphenol content and antioxidant activity (2,2-diphenyl-1-picrylhydrazyl, Oxygen Radical Absorbance Capacities and β-Carotene Bleaching assays) were evaluated and compared to assess the Relative Antioxidant Capacity Index (RACI). To test the inhibitory activity of extracts towards cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition assays were performed. SOX and ASE 50 have shown the highest value of RACI, 0.76 and 0.65, respectively. Regarding enzymatic inhibitory activity, ASE 50 (IC50 = 107.16 ± 8.12 μg/mL) and SOX (IC50 = 171.34 ± 12.12 μg/mL) extracts exhibited the highest AChE and BChE inhibitory activity, respectively, while UAE (IC50 = 293.2 ± 25.6 μg/mL, followed by SOX (IC50 = 302.5 ± 38.3 μg/mL) showed the highest tyrosinase inhibition value. Our results demonstrated for the first time that Aglianico leaves are important sources of phenols that could be used to prevent oxidative stress and be potentially helpful in diseases treatable with tyrosinase and cholinesterase inhibitors, like myasthenia gravis or Alzheimer’s.Italian Ministry of the Economic Development F/200099/03/X45—CUP, B31B19000590008 COR, C31G1800021000

J. Agric. Food Chem.

Stilbene metabolites are attracting great interest because many of them exhibit similar or even stronger biological effects than their parent compounds. Furthermore, the metabolized forms are predominant in biological fluids; therefore, their study is highly relevant. After hemisynthesis production, isolation, and structural elucidation, three glucuronide metabolites for oxyresveratrol (ORV) were formed: trans-ORV-4′-O-glucuronide, trans-ORV-3-O-glucuronide, and trans-ORV-2′-O-glucuronide. In addition, two glucuronide metabolites were obtained for gnetol (GN): trans-GN-2′-O-glucuronide and trans-GN-3-O-glucuronide. When the metabolism of ORV and GN is studied in vitro by human and rat hepatic enzymes, four of the five hemisynthesized compounds were identified and quantified. Human enzymes glucuronidated preferably at the C-2′ position, whereas rat enzymes do so at the C-3 position. In view of these kinetic findings, rat enzymes have a stronger metabolic capacity than human enzymes. Finally, ORV, GN, and their glucuronide metabolites (mainly at the C-3 position) decreased nitric oxide, reactive oxygen species, interleukin 1β, and tumor necrosis factor α production in lipopolysaccharide-stimulated macrophages

Anthocyanins in Blueberries Grown in Hot Climate Exert Strong Antioxidant Activity and May Be Effective Against Urinary Tract Bacteria

Anthocyanins are extensively studied for their health-related properties, including antibacterial activity against urinary tract infections (UTI). Among common fruits, blueberries, with their remarkable antioxidant capacity, are one of the richest sources. Anthocyanin-rich extracts were obtained from four varieties: Snowchaser, Star, Stella Blue and Cristina Blue, grown in the hot climate of Southern Spain. Their total anthocyanins contents (TAC) were determined spectrophotometrically, and the anthocyanin profile by ultra high performance liquid chromatography - tandem mass spectrometer (UHPLC-MS/MS). Their antioxidant activity was assessed by oxygen radical absorbance capacity (ORAC) assay, while antibacterial activity against strains isolated from UTI patients was assessed in vitro, helping to select the varieties with the highest bioactive potential. Star showed the highest TAC and antioxidant activity (1663 ± 159 mg of cyanidin-3-O-glucoside (cy-3-O-glu) equivalents/100 g fresh weight (FW), 6345 ± 601 μmol Trolox equivalents (TE)/100 g FW, respectively), followed by Cristina Blue, Stella Blue and Snowchaser. As far as we know, this is the first time that cyanidin-3-rutinoside has been identified in blueberries. The extracts inhibited all the tested strains, MICs ranging from 0.4 mg/mL (for Stella Blue extract against UTI P. aeruginosa) to 9.5 mg/mL (for all extracts against UTI K. pneumoniae ssp. pneumoniae). This is the first study that assessed in vitro the antibacterial activity of blueberries against Klebsiella pneumoniae, Providencia stuartii and Micrococcus spp. strains isolated from UTI.España, University of Sevilla, VI Plan Propio de Investigación y Transferencia (VIPPIT‐2019‐I.5

Evidences of hydroxytyrosol as an anti-inflammatory agent in Parkinson’s disease: insights into the mechanisms of action

Ye