Dietary supplements based on borututu: wich is the most suitable choice for bioactive purposes?

Borututu ( Cochlospermum angolensis Welw.) is a widespread tree in Angola used since antiquity by traditional healers for the prevention and treatment of hepatic diseases and for the prophylaxis of malaria [1]. This plant is mostly consumed as infusions but is also available as dietary supplements, such as piiis, capsules, and syrups, among others. In the present study, the aim was to evaluate the proximate composition and energetic contribution of borututu as weii as its composition in hydrophilic (sugars and organic acids) and lipophilic (fatty acids and tocopherols) compounds, given the fact that this plant is directly introduced in some dietary supplements. Furthermore, the bioactivity (antioxidant, hepatoprotective and antimicrobial activities) of three different formulations of borututu (infusion, pills, and syrup) was assessed and compared, and since plant beneficial properties are often ascribed to phenolic compounds [2], the phenolic profile of the formulations was also analysed. Carbohydrates (88 g/100 g) and fat (2.5 g/100 g) were the major and tl1e minor components of the studied borututu dry barks, respectively, with an energetic contribution of 384 kcal/100 g. Fructose was the most abundant sugar (1.3 g/100 g), foilowed by sucrose, trehalose and glucose (1.1, 0.98 and 0.79 g/100 g, respectively). Oxalic (0.70 g/100 g), malic (0.63 g/100 g) and citric (0.57 g/100 g) acids were present in higher amounts but shikimic and fumaric acids were also detected. Among the fatty acids found in borututu, a prevalence of saturated fatty acids (SF A; 48.2%) was observed, whereas polyunsaturated (PUFA) and monounsaturated (MUFA) fatty acids were detected in relative percentages of 30.9% and 20.8%, respectively. P-tocopherol was the most abundant of the four isoforms found in the sample, foiiowed by o-, a- and y-tocopherol, present in concentrations of 597,43, 3.7 and 2.0 g/100 g, respectively. Borututu infusion revealed the highest antioxidant activity, with EC50 values ranging from 20 to 600 J.lg/mL and was the only formulation inhibiting the growth of an HepG2 ceii line, with a Gl5o value of 146 J.lg/mL. This formulation.also revealed the best antimicrobial capacity and proved to be able to inhibit the growth of Escherichia coli, E. coli ESBL, Staphylococcus aureus and Pseudomonas aeruginosa, with MIC values of 50, 6.2, 1.6 and 25 mg!mL, respectively. Pills revealed activity against some of the studied bacterial strains and the syrup did not reveal antimicrobial activity at the studied concentration. Eilagic acids, methyl ellagic acids, eucaglobulinlglobulusin B and (epi)gaiiocatechin-0-gallate were the compounds present in all the different formulations. The highest concentration of phenolic compounds was found in the infusion extract. Protocatechuic acid was the most abundant phenolic compound in the infusions, the only preparation where it was detected, whereas ( epi)gaiiocatechin- 0-gallate was the main phenolic in the pills and eucaglobulinlglobulusin in the syrup. In a general way, borututu proved to be a good source of phytochemicals such as phenolic compounds, with the infusions revealing the best bioactive properties.

Ultrasound and microwave assisted extraction of ergosterol from Agaricus bisporus L.: optimization through response surface methodology

There is scientific evidence demonstrating the benefits of mushrooms ingestion due to their richness in bioactive compounds such as mycosterols, in particular ergosterol [I]. Agaricus bisporus L. is the most consumed mushroom worldwide presenting 90% of ergosterol in its sterol fraction [2]. Thus, it is an interesting matrix to obtain ergosterol, a molecule with a high commercial value. According to literature, ergosterol concentration can vary between 3 to 9 mg per g of dried mushroom. Nowadays, traditional methods such as maceration and Soxhlet extraction are being replaced by emerging methodologies such as ultrasound (UAE) and microwave assisted extraction (MAE) in order to decrease the used solvent amount, extraction time and, of course, increasing the extraction yield [2]. In the present work, A. bisporus was extracted varying several parameters relevant to UAE and MAE: UAE: solvent type (hexane and ethanol), ultrasound amplitude (50 - 100 %) and sonication time (5 min-15 min); MAE: solvent was fixed as ethanol, time (0-20 min), temperature (60-210 •c) and solid-liquid ratio (1-20 g!L). Moreover, in order to decrease the process complexity, the pertinence to apply a saponification step was evaluated. Response surface methodology was applied to generate mathematical models which allow maximizing and optimizing the response variables that influence the extraction of ergosterol. Concerning the UAE, ethanol proved to be the best solvent to achieve higher levels of ergosterol (671.5 ± 0.5 mg/100 g dw, at 75% amplitude for 15 min), once hexane was only able to extract 152.2 ± 0.2 mg/100 g dw, in the same conditions. Nevertheless, the hexane extract showed higher purity (11%) when compared with the ethanol counterpart ( 4% ). Furthermore, in the case of the ethanolic extract, the saponification step increased its purity to 21%, while for the hexane extract the purity was similar; in fact, hexane presents higher selectivity for the lipophilic compounds comparatively with ethanol. Regarding the MAE technique, the results showed that the optimal conditions (19 ± 3 min, 133 ± 12 •c and 1.6 ± 0.5 g!L) allowed higher ergosterol extraction levels (556 ± 26 mg/100 g dw). The values obtained with MAE are close to the ones obtained with conventional Soxhlet extraction (676 ± 3 mg/100 g dw) and UAE. Overall, UAE and MAE proved to he efficient technologies to maximize ergosterol extraction yields.

Nitrogen vs argon: what is the most effective atmosphere for shelf-life extension of buckler sorrel leaves?

Buckler sorrel (Rumex induratus Boiss. & Reut.) is an underutilized leafy vegetable with peculiar sensory properties and potential as a gourmet food. In the food industry, different packaging methods have been used for shelf-life extension, but it is important to know how the quality of minimally processed vegetable is affected by these treatments. Recently, nitrogen and argon have been used for food packaging. Nitrogen is low soluble in water and other food constituents and does not support the growth of aerobic microbes. In turn, argon is biochemically active and appears to interfere with enzymatic oxygen receptor sites. In this study, modified atmospheres enriched with nitrogen and argon were evaluated for shelf-life extension of buckler sorrel leaves. Wild samples were gathered in Bragança, Portugal, considering local consumers’ sites and criteria. Healthy and undamaged leaves were selected, rinsed in tap water, and a portion was immediately analyzed (non-stored control). The remaining fresh material was packaged in polyethylene bags under nitrogen- and argon-enriched atmospheres and a conventional control atmosphere (air). All packaged samples were stored at 4 ºC for 12 days and then analyzed. The headspace gas composition was monitored during storage. Different quality attributes were evaluated, including visual (colour), nutritional (macronutrients, individual sugars and fatty acids) and bioactive (hydrophilic and lipophilic molecules and antioxidant properties) parameters. Different statistical tools were used; the one-way analysis of variance (ANO VA) was applied for analyse the differences among treatments and a linear discriminant analysis (LDA ) was used to evaluate the effects on the overall postharvest quality. The argon-enriched atmosphere better prevent the samples yellowing. The proximate composition did not change significantly during storage. Samples in control atmosphere revealed higher protein and ash contents and lower levels of lipids. The non-stored control samples presented the higher amounts of fructose, glucose and trehalose. The storage time increased the palmitic acid levels and decreased the content in α-linolenic and linoleic acids. The γ- e δ-tocopherols were higher after the 12 days of cold storage. Probably, the synthesis of these lipophilic compounds was a plant strategy to fight against the abiotic stress induced by storage. Higher levels of total phenolics and flavonoids and increased reducing power and β-carotene bleaching inhibition capacity were also found in the stored control samples. Once again, this result may be attributed to the intrinsic plant-protection mechanisms. Overall, the argon atmosphere was more suitable for quality preservation and shelf-life extension of buckler sorrel.

Chromatographic techniques to obtain the biomolecules profile of wild Suillus granulates

In recent years the interest in naturally occurring compounds has been increasing worldwide. Indeed, many of the bioactive compounds currently used as medicines have been synthesized based on the structure of natural compounds [1]. In order to obtain bioactive fractions and subsequently isolated compounds derived from natural matrices, several procedures have been carried out. One of these is to separate and assess the concentration of the active compound(s) present in the samples, a step in which the chromatographic techniques stand out [2]. In the present work the mushroom Sui/Ius granulatus (L.) Roussel was chemically characterized by chromatographic techniques coupled to different detectors, in order to evaluate the presence of nutritional and/or bioactive molecules. Some hydrophilic compounds, namely free sugars, were identified by high performance liquid chromatography coupled to a refraction index detector (HPLC-RI), and organic and phenolic acids were assessed by HPLC coupled to a photodiode array detector (HPLC-PDA). Regarding lipophilic compounds, fatty acids weredetermined by gas chromatography with a flame ionization detector (GC-FID) and tocopherols by HPLC-fluorescence detection. Mannitol and trehalose were the main free sugars detected. Different organic acids were also identified (i.e. oxalic, quinic and fumaric acids), as well as phenolic acids (i.e. gallic and p-hydroxybenzoic acids) and the related compound cinnamic acid. Mono- and polyunsaturated fatty acids were the prevailing fatty acids and a-, ~- and ~-tocopherol were the isoforms of vitamin E detected in the samples. Since this species proved to be a source of biologically active compounds, the antioxidant and antimicrobial properties were evaluated. The antioxidant activity was measured through the reducing power, free radical's scavenging activity and lipid peroxidation inhibition of its methanolic extract, and the antimicrobial activity was also tested in Gram positive and Gram negative bacteria and iri different fungi. S. granulatus presented antioxidant properties in all the performed assays, and proved to inhibit the growth of different bacterial and fungal strains. This study is a first step for classifying S. granulatus as a functional food, highlighting the potential of mushrooms as a source of nutraceutical and biologically active compounds.

Non-fermented and fermented jabuticaba (Myrciaria cauliflora Mart.) pomaces as valuable sources of functional ingredients

abuticaba (Myrciaria cauliflora. Mart) is a highly perishable fruit native to Brazil, which is consumed both fresh and industrially processed in the form of juices, jams, wines and distilled liqueurs. This processing generates a large amount of waste by-products, which represent approximately 50% of the fruit weight. The by-products are of interest for obtaining valuable bioactive compounds that could be used as nutraceuticals or functional ingredients. In this study, fermented and non-fermented jabuticaba pomaces were studied regarding their hydrophilic and lipophilic compounds, as well as their antioxidant properties, including: soluble sugars, organic acids and tocopherols (using high performance liquid chromatography coupled to refraction index, diode array and fluorescence detector, respectively); phenolics and anthocyanins, (using liquid chromatography coupled to diode array detection, and mass spectrometry with electrospray ionization); and fatty acids (using gas-liquid chromatography with flame ionization detection). The analytical data demonstrated that jabuticaba pomaces are a rich source of bioactive compounds such as tocopherols, polyunsaturated fatty acids and phenolic compounds (namely hydrolyzable tannins and anthocyanins) with antioxidant potential. Therefore, jabuticaba pomace may have good potential as a functional ingredient in the fabrication of human foods and animal feed.

Screening natural resources for mineralogenic and osteogenic bioactivities using in vitro and in vivo fish systems

Dissertação de Mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2016

Determination of acrylamide levels in selected commercial and traditional foods in Syria

Purpose: To determine acrylamide (AA) levels in different brands of commercial and traditional foodstuffs available in Syria by ultra-performance liquid chromatography-mass spectrometery (UPLCMS). Methods: A total of 63 samples were analyzed. Food samples were defatted by hexane and then extracted with methanol 98 % in a vortex mixer. Thereafter, Carrez I and Carrez II were added to precipitate proteins from the co-extractives and then centrifuged to obtain a clear aqueous extract that was evaporated to dryness. The extract was dissolved in 1 mL of water, eluted through a preconditioned Oasis HLB cartridge and then filtered. The filtrate was analyzed by UPLC-MS/MS to determine AA content. Results: Among the commercial foods tested, the highest acrylamide quantity was found in potato products (396 ± 3.59 – 1844 ± 5.29 μg kg−1) and the lowest in corn products (183 ± 2.64 – 366 ± 4.58 μg kg−1). This was followed by biscuits (57 ± 2.64 – 1433 ± 2.51 μg kg−1), breakfast cereals (121 ± 8.73 – 245 ± 3.60 μg kg−1), bread (119 ± 1.73 – 263 ± 3.60 μg kg−1) and then coffee (113 ± 2.64 - 64 ± 3.05 μg kg−1). Regarding traditional foods, the highest level of AA was found in AL- Mshabak (481 ± 2.08 μg kg−1) and AL-Awamat (421 ± 2.64 μg kg−1) followed by AL-Namora (282 ± 4.35 μg kg−1) and AL-Kenafa (242 ± 2.64 μg kg−1). It was also observed that the lowest amount of AA was in fried bread (230 μg kg−1), AL-Fatayer (192 ± 3.51 μg kg−1) and AL-Baqlawa (172 ± 4.35 μg kg−1) while Eid Aqras (130 ± 4.58 μg kg−1) and AL-Brazeq (167 ± 3.78 μg kg−1) contained the least amount of AA. Conclusion: The results indicate that the highest levels of AA are found in the most commonly consumed foods. There was significant difference (p < 0.05) in AA levels among different food items and within different brands of the same product.

Desenvolvimento de uma nanoformulação autoemulsificante contendo o alcaloide epiisopiloturina para melhorar sua biodisponibilidade plasmática após administração via oral

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Nanociência e Nanobiotecnologia, 2016.

Estudo da homogeneidade e discriminação de madeiras através de espectroscopia de imagens, imagens digitais e ferramentas quimiométricas

Tese (doutorado)–Universidade de Brasília, Instituto de Química, Programa de Pós-Graduação em Química, 2016.

The initial phase of sodium sulfite pulping of softwood : A comparison of different pulping options

Single stage and two-stage sodium sulfite cooking were carried out on either spruce, pine or pure pine heartwood chips to investigate the influence of several process parameters on the initial phase of such a cook down to about 60 % pulp yield. The cooking experiments were carried out in the laboratory with either a lab-prepared or a mill-prepared cooking acid and the temperature and time were varied. The influences of dissolved organic and inorganic components in the cooking liquor on the final pulp composition and on the extent of side reactions were investigated. Kinetic equations were developed and the activation energies for delignification and carbohydrate dissolution were calculated using the Arrhenius equation. A better understanding of the delignification mechanisms during bisulfite and acid sulfite cooking was obtained by analyzing the lignin carbohydrate complexes (LCC) present in the pulp when different cooking conditions were used. It was found that using a mill-prepared cooking acid beneficial effect with respect to side reactions, extractives removal and higher stability in pH during the cook were observed compared to a lab-prepared cooking acid. However, no significant difference in degrees of delignification or carbohydrate degradation was seen.  The cellulose yield was not affected in the initial phase of the cook however; temperature had an influence on the rates of both delignification and hemicellulose removal. It was also found that the  corresponding activation energies increased in the order:  xylan, glucomannan, lignin and cellulose. The cooking temperature could thus be used to control the cook to a given carbohydrate composition in the final pulp. Lignin condensation reactions were observed during acid sulfite cooking, especially at higher temperatures. The LCC studies indicated the existence of covalent bonds between lignin and hemicellulose components with respect to xylan and glucomannan. LCC in native wood showed the presence of phenyl glycosides, ϒ-esters and α-ethers; whereas the α-ethers  were affected during sulfite pulping. The existence of covalent bonds between lignin and wood polysaccharides might be the rate-limiting factor in sulfite pulping.

Utilisation d’arbres de faible vigueur provenant de forêts feuillues tempérées comme matière première pour la fabrication de granules de bois

La présente étude avait pour objectif de contribuer à une meilleure compréhension de la granulation des bois feuillus par l’évaluation de la possibilité technique de transformer des bois feuillus de faible vigueur (l’érable à sucre et le bouleau jaune) en granules conventionnels et granules de haute qualité, un type prometteur de transport énergétique. Trois études individuelles ont été réalisées et présentées dans cette thèse. La première étude visait à déterminer s’il y a des changements de teneur en extractibles, cendres, et lignine du bois entre les classes de vigueur des arbres. Les teneurs plus élevées en extractibles et en lignine dans les arbres peu vigoureux ont suggéré que ces derniers sont plus appropriés par rapport aux arbres vigoureux pour la conversion en biocombustibles solides. La deuxième étude visait à optimiser des procédés de granulation des bois feuillus. L’étude a porté sur l’influence des paramètres du procédé (la température et la force de compression) et des caractéristiques de la matière première (la taille des particules et la teneur en humidité) sur les propriétés physiques et mécaniques des granules de bois. Le procédé de granulation doit être effectué à une température d’environ 100 °C pour minimiser la force de friction dans le granulateur et à une teneur en humidité d’environ 11,2% pour maximiser la masse volumique et la résistance mécanique des granules produites. Cette étude a également confirmé que les arbres de faible qualité sont plus appropriés pour la fabrication de granules de bois que les arbres vigoureux. La troisième étude visait l’élaboration de granules de haute qualité. L’eau chaude à température élevée a été utilisée pour modifier les propriétés de la matière première avant granulation. Les caractéristiques de granulation du matériau traité ont été significativement améliorées. Les granules produites ont montré des propriétés améliorées incluant une plus faible teneur en cendres, une plus haute densité énergétique, une meilleure résistance à l’eau, et une meilleure résistance mécanique. Les résultats obtenus de toutes ces études ont démontré la nécessité de bien connaître les fondements de la granulation des bois feuillus et les solutions pratiques pour l’utilisation d’arbres feuillus de faible qualité, le premier peut être applicable pour le développement de procédés de granulation et le dernier peut contribuer à long terme à la restauration des forêts feuillues dégradées en termes de santé des forêts et de leur valeur.

Molecular probes for monitoring mitochondrial movement and function

This thesis explores two distinct parts of mitochondrial physiology: the role of mitochondria in generation of reactive oxygen species (ROS) and mitochondrial morphology and dynamics within cells. The first area of research is covered in Chapters 1-8. Mitochondrial biofunctionality and ROS production are discussed in Chapter 1, followed by the strategy of targeting bioactive compounds to mitochondria by linking them to lipophilic triphenylphosphonium cations (TPP) (Chapter 2). ROS sensors relevant to the research are reviewed in Chapter 3. Chapter 4 presents design and synthesis of novel probes for superoxide detection in mitochondria (MitoNeo-D), cytosol (Neo-D) and extracellular environment (ExCellNeo-D). The results of biological validation of MitoNeo-D and Neo-D performed in the MRC MBU in Cambridge are presented in Chapter 5. A dicationic hydrogen peroxide sensor that utilizes in situ click chemistry is discussed in Chapter 6. Preliminary work on the synthesis of mitochondria-targeted superoxide generators, which led to the development of mitochondria-targeted analogue of paraquat, MitoPQ, is presented in Chapter 7. A set of bifunctional probes (BCN-Mal, BCN-E-BCN and Mito-iTag) for assessing the redox states of protein thiols is discussed in Chapter 8 along with their biological validation. The second part of the thesis is aimed at the study of mitochondrial morphology and dynamics and is presented in Chapters 9-11. Chapter 9 provides background on the classes of fluorophores relevant to the research, the phenomenon of fluorescence quenching and the principle of photoactivation with examples of photoactivatable fluorophores. Next, the background on mitochondrial morphology and heterogeneity is presented in Chapter 10, followed by the ways of imaging and tracking mitochondria within cells by conventional fluorophores and by photoactivatable fluorophores exploiting super-resolution microscopy. Chapter 11 presents the design and synthesis of four photoactivatable fluorophores for mitochondrial tracking, MitoPhotoRhod110, MitoPhotoNIR, Photo-E+, MitoPhoto-E+, along with results of biological validation of MitoPhotoNIR. The results and discussion concludes with Chapter 12, which is a summary and suggestions for future work, followed by the chemistry experimental procedures (Chapter 13), materials and methods for biological experiments (Chapter 14) and references.

Physicochemical and antioxidant properties of the pequi (Caryocar brasiliense Camb.) almond oil obtained by handmade and cold-pressed processes.

Abstract: The aim of this study is to characterize physical and chemically and determine the antioxidant capacity of pequi almond oils (PAO) extracted by handmade and by cold-pressing. Both oils showed good quality by acid, peroxide and thiobarbituric acid values. The fatty acid (FA) profile showed a significant presence of monounsaturated FA, mainly oleic acid (53.48 to 55.41%); saturated FA, such as palmitic acid (33.30 to 35.89 %); and polyunsaturated FA (PUFA), such as linoleic acid (5.85 to 7.23%). The total phenolic (TP) and carotenoid content ranged in concentration from 87.56 to 392.00 mg GAE/100 g and 36.03 to 262.40 mg/100 g, respectively. The tocopherol and phytosterol results indicated the predominant presence of α-tocopherol (52 to 67%) and stigmasterol (63 to 68 %). The antioxidant capacity of PAO as measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH?) method oscillated from 58.48 mg/mL to 76.46 mg/mL (IC50), from 10.61 to 40.46 µmol TE/g by the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS?+) method, and from 113.93 to 280.85 µmol TE/100 g and 164.49 to 277.86 µmol TE/100 g, by the lipophilic and hydrophilic oxygen radical absorbance capacity (ORAC) methods, respectively. The oils presented a good oxidative and thermal stability by Rancimat method (IP of 7.33 a 15.91 h) and curves thermogravimetric and differential scanning calorimetry (To 337-363 °C and 159-184 °C, respectively). The results confirmed the presence of compounds that conferred antioxidant capacity and oxidative and thermal resistance for PAO made by handmade or cold-pressing, indicating that these oils can potentially be used for food and non-food applications.

Design of biobased additives for tuning the properties of biopolymers

This work mainly arises from the necessity to support the rapid introduction of different biobased polymers that the industrial sector has been facing lately. Indeed, while considerable efforts are being made to find environmentally and economically sustainable materials, less attention is paid to their need to be properly compounded to fulfil increasingly rigorous technical and quality requirements. Therefore, there is a strong demand for the development of a novel generation of compatible additives able to improve the properties of biobased polymers while respecting sustainability. With this in mind, a new class of biobased plasticizers is herein proposed. Five different ketal-diesters were selectively synthesized starting from levulinic acid, a promising renewable chemical platform. These molecules were added to poly(vinyl chloride) as model polymer to test their plasticizing effectiveness. Complete morphological, thermal and viscoelastic characterizations showed a clear correlation between the structural features of the ketal-esters and the properties of the material. In addition, no significant leaching was found in both hydrophilic and lipophilic environments. Importantly, the proposed ketal-diesters performed comparably and, in some cases, even better than commercial plasticizers. The same molecules were then added to bacterial poly(3-hydroxybutyrate), a semicrystalline polyester characterized by poor thermal and mechanical properties. Morphology assessments showed no phase separation and the plasticizing effectiveness was confirmed by thermal and viscoelastic analyses, while leaching tests showed low extraction values. Readily usable fractions with controlled structure and tailored properties were obtained from highly heterogeneous industrial grade Kraft lignin. These fractions were then added to poly(vinyl alcohol). Promising preliminary results in terms of compatibility were achieved, with thermograms showing only one glass transition temperature. Finally, a fully biobased glycerol-trilevulinate was successfully synthesized by means of a mild and solvent-free route. Its plasticizing effectiveness was evaluated on poly(vinyl chloride), showing a significant decrease of the glass transition temperature of the material.

Innovative photoresponsive materials & supramolecular structures

This thesis focuses on two main topics: photoresponsive azobenzene-based polymers and supramolecular systems generated by the self-assembly of lipophilic guanosines. In the first chapters describe innovative photoresponsive devices and materials capable of performing multiple roles in the field of soft robotics and energy conversion. Chapter 2 describes a device obtained by coupling a photoresponsive liquid-crystalline network and a piezoelectric polymer to convert visible light into electricity. Chapter 3 deals with a material that can assume different shapes when triggered by three different stimuli in different environments. Chapter 4 reports a highly performing artificial muscle that contracts when irradiated. The last two chapters report on supramolecular structures generated from functionalized guanosines dissolved in organic solvents. Chapter 6 illustrates the self-assembly into G-quadruplexes of 8- and 5’-functionalized guanosines in the absence of templating ions. Chapter 7 describes the supramolecular structure generated by the assembly of a lipophilic guanosine in the presence of silver cations. Chapter 6 is reproduced from an already published paper, while the other chapters are going to be submitted to different journals in a couple of months.