Tesi sul tema "Phenolic fractions"

Thesis (MSc (Chemistry and Polymer Science))--University of Stellenbosch, 2009.
ENGLISH ABSTRACT: The separation of apple, cocoa and green tea phenolic compounds by comprehensive 2-dimensional liquid chromatography (2-D-LC) has been studied. In the first dimension, phenolic compounds were separated according to polarity by hydrophilic interaction chromatography (HILIC) on a diol stationary phase with a mobile phase containing acetonitrile, methanol, acetic acid and water. Gradient reversed-phase (RP) LC using a C18 column with fluorescence detection was employed in the second dimension to separate compounds according to hydrophobicity. Compounds were identified using negative electrospray ionisation mass spectrometry (ESI-MS) coupled to both HILIC and RP separations. The coupling of HILIC and RP separations proved to be especially beneficial since this provided simultaneous information on both the polarity and hydrophobicity of phenolics. The low degree of correlation (r2 < 0.21) between the two LC modes afforded peak capacities in excess of 3000 for the off-line method. An on-line method was also developed utilizing a short, small particle-packed column to provide fast separation in the second dimension. A 1 mm i.d. column was used in the first dimension for the on-line system to reduce injection volumes onto the second dimension column. A significantly lower practical peak capacity was measured for the on-line system, due largely to the reduction in second dimension peak capacity. On the other hand, analysis could be performed in an automated fashion using the online system reducing the risk of sample alteration and guaranteeing better operation reliability and reproducibility. Especially the off-line comprehensive HILIC × RP-LC method developed demonstrated its utility in the analysis of various groups of phenolic compounds including proanthocyanidins, phenolic acids, flavonols and flavonol conjugates in a variety of natural products.
AFRIKAANSE OPSOMMING: Die skeiding van fenoliese komponente in appel, kakao en groen tee is deur middel van ‘comprehensive’ 2-dimensionele vloeistof chromatografie (2-D-LC) bestudeer. Hidrofiliese interaksie chromatografie (HILIC) is gebruik om die fenoliese komponente in die eerste dimensie te skei op grond van polariteit, deur gebruik te maak van ‘n diol stationêre fase en mobiele fase bestaande uit asetonitriel, metanol, asynsuur en water. ‘n Gradiënt omgekeerde fase (RP) LC analisie op ‘n C18 kolom met fluorosensie deteksie is in die tweede dimensie gebruik om fenole volgens hidrofobisiteit te skei. Negatiewe elektrosproei-ionisasie massa spektometrie (ESIMS) gekoppel aan HILIC en RP skeidings is gebruik vir identifikasie van fenole. Die koppeling van HILIC en RP skeidings veral voordelig deurdat dit gelyktydige informasie verskaf het oor die polariteit sowel as die hidrofobisiteit van die fenoliese komponente. Die lae graad van korrelasie (r2 < 0.21) tussen die twee LC metodes was verantwoordelik vir piek kapasiteite bo 3000 vir die af-lyn metode. ‘n Aanlyn metode was ontwikkel deur gebruik te maak van ‘n kort, klein partikel gepakte kolom om vinnige skeiding in die tweede dimensie te verseker. 1 mm i.d. kolom was gebruik in die eerste dimensie vir die aanlyn sisteem om die inspuit volume op die tweede dimensie kolom te verminder. Aansienlike laer praktiese piek kapasiteit was gemeet vir die aanlyn sisteem, grootliks toegeskryf aan die reduksie in die tweede dimensie piek kapasitiet. Aan die ander kant, analise kan geoutomatiseerd uitgevoer word deur gebruik te maak van die aanlyn sisteem, wat monster alterasie, beter betroubaarheid en reproduseerbaarhied verseker. Veral die ontwikkelde af-lyn ‘comprehensive’ HILIC × RP-LC metode toon demonstreerbare voordele vir die analiese van verskeie groepe fenoliese komponente, insluitende proantosianiede, fenoliese sure, flavonole en gekonjugeerde flavonole in ‘n verskeidenheid natuurlike produkte.

The food industry is facing the significant challenge of developing new ingredients and foods addressing prevention of the major chronic diseases, while optimizing processing technologies in order to achieve a sustainable use of natural resources. Grape pomace, the byproduct of winemaking, is available on a large scale and is comprised of various compounds associated with potential health benefits. In this context, the overall PhD project aimed at developing an integrated strategy for recovery and reuse of value added fractions of winemaking byproducts in the food system. Grape pomace samples, differing for the varieties and winemaking conditions were collected from winemaking processes and evaluated for valuable components, i.e, dietary fibre, total phenolics, soluble and insoluble proanthocyanidins (n-butanol/HCl assay), individual phenolic compouns (UPLC-DAD-MS), tocopherol and tocotrienol contents (HPLC with fluorimetric detection). This led to the design of a comprehensive recovery plan, bringing about the overall value of winemaking byproducts. The aim of targeting a specific health functionality by grape phenolics was tackled in chapter 1, focused on their protective effects on hyperglyacemia induced damage. In the current scenario, prevention of diabetes complications is of utmost importance due to an upsurge in the prevalence of this disease among the world population (with an estimation of 300 million cases that could be registered by 2030). In vivo study has shown that phenolics can prevent the leading cause of hyperglycemia damage, i.e., non-enzymatic protein glycaton, most likely via radical scavenging, metal chelation and carbonyl trapping. However to target this effect, there is a need for phenolic fractions with high concentration and efficiency. An in vitro model system using fructose as glycating agent and bovine serum albumin as target was set up. The protective ability of grape skin phenolics was evaluated with both a novel methodological approach by 2D-SDS/IEF-PAGE and quantification of the formation of advanced glycation end-products by fluorescence measurements. Structural modifications of BSA upon glycation, as evidenced by changes in pI and Mr were found to be inhibited by the grape skin extracts. The antiglycation activity ranking was: quercetin-3-O-glucoside > malvidin-3-O-glucoside > catechin > procyanidin A2 >> aminoguanidine (reference drug). Despite variability in phenolic content (in the range 4.6-53.6 g gallic acid equivalents (GAE)/kg) and composition among grape skin extracts, upon equalization of the phenolic content, similar antiglycation properties were observed. Grape skins (I50 in the range 9.2-20 µg GAE/mL) were more efficient than the commercial nutraceutical products Leucoselect® (I50 38 µg GAE/mL) and Pycnogenol® (I50 34 µg GAE/mL) and hence could provide cost-effective antiglycation agents, to be used either as food ingredients or as nutraceutical preparations. The task of designing new foods by the incorporation of grape skins, which can deliver both dietary fibers and phenolics, was examined in chapter 2. Grape skins have been proposed as ingredients for “solid” or “gel-like” foods, but knowledge on their possible application in complex food fluids that could represent low caloric vehicle for value added ingredients is lacking. Hence, a tomato puree added with grape skins was studied as a model application and a production process was designed to increase potential health benefit of this product with maximum consumers’ liking. To optimize grape skin ingredient incorporation in tomato puree, six formulations (3% dietary fibre and ~ 0.7% proanthocyanidins) varying in the particle size (assessed by a laser granulometer) were considered in parallel. Stabilization was performed by either an intensive autoclave treatment or an optimized microwave treatment achieving six decimal reductions of the target microorganism (Alicylobacillus acidoterrestris). Upon processing, major changes in the fortified purees were both decrease in proanthocyanidin solubility (by ~ 40%, probably due to interaction with tomato protein) and decrease in flavonol glycosides with parallel increase of their aglycones. However, phenolic contents, reducing capacity and antiglycation activity remained ~ 3 times higher in all the fortified purees than in the controls. These chemical parameters were related to particle size distribution, since with decreasing surface-weighed mean diameter, d(3,2), proanthocyanidin solubility increased. The addition of grape skins played a major role on the color (measured by Hunter colorimeter), storage (G’) and loss (G’’) moduli, complex viscosity (studied by means of dynamic oscillatory measurements) and Bostwick consistency of the formulations. A sensory test based on consumers’ preference disclosed that the formulation having the maximum appreciation was that incorporated with the smallest particle sizes. Liking ratings were found to be inversely correlated to the volume-weighted mean diameter, d(4,3) values. The overall results obtained from this methodological approach represent a basis for the optimization of fibrous byproducts incorporation into complex food fluids. Besides use of grape skins as source of antiglycation and fortifying agents, utilization of grape seeds which forms a major part of the pomace is of importance in the recovery strategy. Chapter 3 focused on assessment of supercritical-CO2 (SC-CO2) extraction for the recovery of oil, as a “green technology” alternative to n-hexane extraction and mechanical extraction. Extraction by SC-CO2 can be a sustainable technology, if the process is carried out at optimum operating conditions ensuring high yield and in a sufficient extractor volume. The breakeven point, which makes the process economically sustainable, is also linked to the quality of the oil obtained. Besides oil, the defatted seeds rich in phenolics were also considered as a relevant co-product of this process. Hence, grape seed phenolics were studied as inhibitors of starch digestion enzymes, which is the first relevant step to control blood glucose level. Extraction by SC-CO2 provided the same oil yield as that of n-hexane extraction (10.1-16.6 g oil/100g seeds). 1H-NMR spectra showed that diacylglycerols and oxidized lipids were present only in trace amounts. The levels of tocols were in the range: 355-559 mg/kg higher than those of the oils obtained by n-hexane extraction and similar to those of oils obtained by mechanical extraction. Tocotrienols, which possess high antioxidant and antiproliferative properties were the prominent compounds present. In the defatted grape seeds, levels of phenolics one order of magnitude higher than those of the grape skins were observed (49-277 g GAE/kg). The anthocyanin rich grape seed extracts showed the highest inhibitory effectiveness towards α-glucosidase (I50 47 µg GAE/mL ~ half than that of the drug acarbose). Inhibitory effectiveness towards α-amylase activity was similar among grape varieties, with I50 values comparable to that of acarbose and correlated to proanthocyanidin contents. The overall results could pave the way for recovery and value-addition of winemaking byproducts for food and nutraceutical uses, including grape skins as a source of antiglycation agents or as dietary antioxidant fibre, grape seeds as a source of tocol rich oil, defatted grape seeds as a source of phenolics.

Health benefits related to consumption of whole grains have been attributed in part to phytochemical and micronutrient composition. Understanding the composition, structure and distribution of these components in different cereal grains is of potential importance in aiding the selection of whole grains and their processed fractions for inclusion in the diet, and as ingredients in development of new food products. The aim of this research was to characterise the chemical components in the botanical fractions of yellow corn, barley, wheat. Manual separation, a tedious and laborious technique that yields pure fractions, suitable for compositional analysis, was used to separate whole grains into pericarp, aleurone layer, germ and endosperm fractions. Component identification and quantification of tissue components was accomplished by several techniques. The study also explored the possibility of using spectral characteristics fluorescence intensity values to provide rapid estimates of the concentrations and distribution of ferulic acid (FA), a major phenolic compound in cereal grains. While composition of phenolic acids and carotenoids was similar, the distribution was significantly different (P < 0.05) among cereal types and grain fractions. Phenolic acids were concentrated in pericarp and aleurone fractions, followed by the germ and the endosperm had the lowest levels. Yellow corn exhibited the highest values. Carotenoids, lutein and zeaxanthin were concentrated in the germ and aleurone layer of wheat and barley while in yellow corn it was in the endosperm and aleurone layer. This is the first study to report on carotenoid composition of aleurone fractions. Mineral elements, thiamine and niacin were higher in wheat aleurone than in purple barley and yellow corn aleurone layers. These findings suggest that yellow corn aleurone layers have potential as a functional food ingredient despite the low micronutrient content. A positive, significant correlation (r= 0.421, p < 0.0001) was found between fluorescence intensity values and ferulic acid concentration. Thus, fluorescence intensity profiles are a promising approach for rapid assessment of FA concentration in grain in-situ. This work has provided information that would act as a database for selection of cereal fractions and guide the miller to obtain grain fractions with enriched levels of phytochemicals and micronutrients.
February 2016

Le colza et le tournesol sont les plantes oléagineuses les plus cultivées en Europe en général, et en France en particulier. Certains industriels se concentrent actuellement sur le développement de procédés industriels d'extraction/purification des protéines des tourteaux de ces deux végétaux. Ces procédés génèrent des coproduits qui sont des effluents aqueux salins riches en composés phénoliques tels que l'acide cholorgénique (CGA, pour le tournesol) et la sinapine (SP, pour le colza). La capture de ces composés phénoliques qui peuvent agir comme anti-oxydants naturels et/ou anti-inflammatoires en nutrition – santé constitue donc une voie de valorisation prometteuse. Les principaux objectifs de ce travail étaient les suivants 1) caractériser et identifier les composés phénoliques des sous-produits d'isolat de protéines à partir de la SFM et de la RSM ; 2) choisir les meilleures résines macroporeuses et étudier le mécanisme d'adsorption des composés phénoliques; 3) optimiser les conditions dans la colonne d’adsorption des composés phénoliques; et 4) évaluer les activités biologiques des fractions phénoliques obtenues, notamment les propriétés antioxydantes et anti-inflammatoires. Par différentes méthodes analytiques, nous avons déterminé que les effluents liquides étaient constitués de composés phénoliques, d'acides aminés, d'hydrates de carbone et de sel, qui ont un poids moléculaire faible et qui peuvent facilement passer à travers une membrane UF/DF. Tous les composés phénoliques ont été identifiés par analyse HPLC et HPLC-ESI-MS, en comparaison avec des standards. Le CGA est le principal composé phénolique des effluents tournesol. Le principal composé phénolique des effluents colza est la SP. Contrairement au tournesol, ils contiennent aussi de nombreux autres composés mineurs. L'adsorption/désorption des composés phénoliques de tournesol et de colza a été évaluée à l'aide de différentes résines macroporeuses, notamment XAD4, XAD7, XAD16, XAD1180 et HP20. Nous avons constaté que tous les composés phénoliques s'adsorbaient facilement sur les résines. Les résines XAD7 et XAD16 ont montré les meilleures propriétés d'adsorption/désorption dans les effluents liquides de tournesol et de colza, respectivement. Les résultats ont montré que l'adsorption de tous les composés phénoliques suit un modèle. Les isothermes d’adsorption suivent un modèle de Langmuir. Selon les paramètres thermodynamiques déterminés, le processus d'adsorption, est dans tous les cas physique et est exothermique. La condition optimale pour l'adsorption sur colonne a été déterminée sur les résines sélectionnées par planification expérimentale et optimisation multicritère. Une méthodologie d'optimisation multicritères basée sur des plans d'expériences a montré que les conditions optimales étaient un débit d'adsorption de 15 BV/h à un pH de 2,7 pour le CGA de la SFM. De l’autre côté, un débit d'adsorption de 13,3 BV/h et un pH compris entre 2 et 5 étaient les conditions optimales pour la sinapine du RSM. Des solutions d’éthanol (50% (v/v) pour l'acide chlorogénique, 70% (v/v) pour la sinapine ont été utilisées pour la desorption. Ces approches ont permis de produire avec succès les fractions phénoliques pour des activités biologiques telles que l'antioxydation et l'anti-inflammation. La fraction phénolique a montré une capacité antioxydante plus élevée que la vitamine C dans le test ABTS (IC50/fractions phénoliques < IC50 vitamine C, p < 0,05). Il a en outre été discuté si les fractions phénoliques obtenues dans le cadre de ce projet présentaient également un effet inflammatoire. La fraction tournesol (CGA) a efficacement inhibé la production de TNF-α, qui est un marqueur pro-inflammatoire lorsqu'un échantillon est traité avec du LPS. Cependant, les fractions colza n'étaient pas efficaces contre les médiateurs pro-inflammatoires. Aucune des fractions n’a montré de cytotoxicité
Rapeseed and sunflower are the most cultivated oilseed plants in Europe in general, and in France in particular. Some industrialists are currently focusing on the development of industrial processes for the extraction/purification of proteins from the oil cakes of these two plants. These processes generate co-products which are saline aqueous effluents rich in phenolic compounds such as cholorgenic acid (CGA, for sunflower) and sinapine (SP, for rapeseed). The capture of these phenolic compounds, which can act as natural antioxidants and/or anti-inflammatory agents in nutrition and health, is therefore a promising way of valorization. The main objectives of this work were: 1) to characterize and identify the phenolic compounds of protein isolate by-products from SFM and RSM; 2) to select the best macroporous resins and to study the adsorption mechanism of phenolic compounds; 3) to optimize the conditions in the phenolic compounds adsorption column; and 4) to evaluate the biological activities of the obtained phenolic fractions, especially the antioxidant and anti-inflammatory properties.By different analytical methods, we determined that the liquid effluents consisted of phenolic compounds, amino acids, carbohydrates, and salt, which have a low molecular weight and can easily pass through a UF/DF membrane. All phenolic compounds were identified by HPLC and HPLC-ESI-MS analysis in comparison with standards. CGA is the main phenolic compound in the sunflower effluent. The main phenolic compound of rapeseed effluents is MS. Unlike sunflower, they also contain many other minor compounds. The adsorption/desorption of sunflower and rapeseed phenolic compounds was evaluated using different macroporous resins including XAD4, XAD7, XAD16, XAD1180 and HP20. We found that all phenolic compounds adsorbed readily onto the resins. XAD7 and XAD16 resins showed the best adsorption/desorption properties in sunflower and rapeseed liquid effluents, respectively. The results showed that the adsorption of all phenolic compounds follows a Langmuir model. According to the determined thermodynamic parameters, the adsorption process, is in all cases physical and is exothermic.The optimal condition for column adsorption was determined on the selected resins by experimental planning and multicriteria optimization. A multicriteria optimization methodology based on design of experiments showed the optimal conditions were adsorption flow rate of 15 BV/h at pH 2.7 for CGA from SFM. Meanwhile, adsorption flow rate of 13.3 BV/h and at pH ranging from 2 to 5 were the optimal conditions for sinapine from RSM. Ethanol solutions 50% (v/v) for chlorogenic acid, 70% (v/v) for sinapine were used for desorption.These approaches successfully produced the phenolic fractions for biological activities such as antioxidation and anti-inflammation. Phenolic fraction showed a higher antioxidant capacity than vitamin C in DPPH and ABTS assays (IC50/phenolic fractions < IC50 vitamin C, p < 0.05. In addition, it was discussed whether the phenolic fractions obtained in this project also showed an inflammatory effect. The sunflower fraction (CGA) effectively inhibited the production of TNF-α, which is a pro-inflammatory marker when a sample is treated with LPS. However, the rapeseed fractions were not effective against proinflammatory mediators. None of the fractions showed cytotoxicity

Karakterizacija  ekstrakata  plodnih  tela  devet autohtonih  vrsta  gljiva  pripadnika  razdela Basidiomycota  obuhvatila  je  preliminarno ispitivanje  hemijskog  sastava  i  ispitivanje biološke  aktivnosti.  Primenom  HPLC‐MS/MS  i GC-FID tehnike  detektovan je i određen sadržaj fenolnih jedinjenja i metil estara masnih kiselina. Primenom  tehnike  elektroforeze  na  mikročipu ("LAB‐ON‐A‐CHIP"  kapilarnom  elektroforezom) izvršena je karakterizacija proteinskih frakcija u odabranim  vrstama  gljiva.  Antioksidantna aktivnost  (sposobnost  neutralizacije  slobodnih  radikala,  redukcioni potencijal), sadržaj ukupnih fenola  i  flavonoida  vodenih  i  etanolnih ekstrakata  ispitan  je  primenom spektrofotometrijskih  metoda.  Antimikrobna aktivnost  metanolnog  i  heksanskog  ekstrakta ispitana  je  na  Gram‐pozitivnim  i  Gram–negativnim bakterijskim  sojevima.  Efekat etanolnih i vodenih  ekstrakata  odabranih vrsta gljiva  na  rast ćelijske  linije  MCF7  (humani adenokarcinom dojke pozitivan na estrogenske i progesteronske receptore), ispitan je MTT i SRB testom, dok je efekat frakcija ispitan primenom MTT  testa.  Sumarno,  ukupni  biopotencijal ispitivanih vrsta na osnovu sprovedenih analiza hemijske  karakterizacije  i  bioloških  testova upućuju opravdanost hipoteze da su odabrane vrste potencijalni  izvori nutraceutika. Svakako je potrebno i neophodno nastaviti dalja istraživanja ovih vrsta.
Characterization of extracts of fruiting bodies of nine native species of mushrooms members of section  Basidiomycota  included  preliminary testing  of  chemical  composition  and  biological activity.  Using  HPLC‐MS  /  MS  and  GC‐FID techniques the content of phenolic compounds and fatty acid methyl esters has been detected and determined. By applying the techniques of electrophoresis  on  microchip  ("LAB‐ON‐A‐CHIP" capillary  electrophoresis)  characterization  of protein fractions in selected types of mushrooms has been performed. The antioxidant activity (the ability  of  neutralizing  free  radicals,  reducing potential),  the  contents  of  total  phenols  and flavonoids  of  water  and  ethanol  extracts  has been  tested  using  the  spectrophotometric method.  The  antimicrobial  activity  of  methanol and  hexane  extracts  was  tested  against  Gram‐positive and Gram‐negative bacterial strains. The effect  of  ethanol  and  aqueous  extracts  of selected fungi species on the growth of the cell line  MCF7  (human  breast  adenocarcinoma positive  for  estrogen  and  progesterone receptors),  has  been  examined  using  MTT  and SRB assay, while the effect of the fractions has been tested by using the MTT assay. In summary, the overall biological potential of the examined species  based  on  results  for  chemical  and biological  characterization  tests  (anti‐oxidative, antimicrobial and anti‐proliferative) indicate the justifiability  of  the  hypothesis  that  examined species  of  mushrooms  are  potential  sources  of nutraceuticals.  It  is  certainly  needed  and necessary to continue further research of these species.

Bio-oil produced from biomass by the fast pyrolysis method is promising as a renewable fuel and as sources of industrial chemicals. In this study, lower cost separation methods of organic acids such as acetic and formic acids and pyrolytic lignin fraction present in bio-oil were investigated to provide basic data needed for future industrial production procedures. The calcium oxide method and a quaternary ammonium anion-exchange resin method were studied to separate organic acids as respective salts and the methanol-and-water method was studied to separate the water-insoluble pyrolytic lignin fraction. The calcium oxide and anion-exchange methods were shown to be effective in separation of organic acids, although further improvements would be needed. The pyrolytic lignin separation method was also shown to give lignin fraction that is effective for up to 40% replacement of phenol in the oriented strand board core-layer binder PF resins.

Doutoramento em Química
The Mediterranean species Cynara cardunculus L. is recognized in the traditional medicine, for their hepatoprotective and choleretic effects. Biomass of C. cardunculus L. var. altilis (DC), or cultivated cardoon, may be explored not only for the production of energy and pulp fibers, but also for the extraction of bioactive compounds. The chemical characterization of extractable components, namely terpenic and phenolic compounds, may valorize the cultivated cardoon plantation, due to their antioxidant, antitumoral and antimicrobial activities. In this study, the chemical composition of lipophilic and phenolic fractions of C. cardunculus L. var. altilis (DC), cultivated in the south of Portugal (Baixo Alentejo region) was characterized in detail, intending the integral valorization of its biomass. The biological activity of cultivated cardoon extracts was evaluated in terms of antioxidant, human tumor cell antiproliferative and antibacterial effects. Gas chromatography-mass spectrometry (GC-MS) was used for the chemical analysis of lipophilic compounds. Sixty-five lipophilic compounds were identified, from which 1 sesquiterpene lactone and 4 pentacyclic triterpenes were described, for the first time, as cultivated cardoon components, such as: deacylcynaropicrin, acetates of β- and α-amyrin, lupenyl acetate and ψ-taraxasteryl acetate. Sesquiterpene lactones were the major family of lipophilic components of leaves (≈94.5 g/kg), mostly represented by cynaropicrin (≈87.4 g/kg). Pentacyclic triterpenes were also detected, in considerably high contents, in the remaining parts of cultivated cardoon, especially in the florets (≈27.5 g/kg). Taraxasteryl acetate was the main pentacyclic triterpene (≈8.9 g/kg in florets). High pressure liquid chromatography-mass spectrometry (HPLC-MS) was utilized for the chemical analysis of phenolic compounds. Among the identified 28 phenolic compounds, eriodictyol hexoside was reported for the first time as C. cardunculus L. component, and 6 as cultivated cardoon components, namely 1,4-di-O-caffeoylquinic acid, naringenin 7-O-glucoside, naringenin rutinoside, naringenin, luteolin acetylhexoside and apigenin acetylhexoside. The highest content of the identified phenolic compounds was observed in the florets (≈12.6 g/kg). Stalks outer part contained the highest hydroxycinnamic acids abundance (≈10.3 g/kg), and florets presented the highest flavonoids content (≈10.3 g/kg). The antioxidant activity of phenolic fraction was examined through 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay. Stalks outer part, and receptacles and bracts extracts demonstrated the highest antioxidant effect on DPPH (IC50 of 34.35 μg/mL and 35.25 μg/mL, respectively). (cont.) abstract (cont.) The DPPH scavenging effect was linearly correlated with the total contents of hydroxycinnamic acids (r = -0.990). The in vitro antiproliferative activity of cultivated cardoon lipophilic and phenolic extracts was evaluated on a human tumor cells line of triple-negative breast cancer (MDA-MB-231), one of the most refractory human cancers to conventional therapeutics. After 48 h of exposition, leaves lipophilic extract showed higher inhibitory effect (IC50 = 10.39 μg/mL) than florets lipophilic extract (IC50 = 315.22 μg/mL), upon MDA-MB-231 cellular viability. Pure compound of cynaropicrin, representative of the main compound identified in leaves lipophilic extract, also prevented the cell proliferation of MDA-MB-231 (IC50 = 17.86 μM). MDA-MB-231 cells were much more resistant to the 48 h- treatment with phenolic extracts of stalks outer part (IC50 = 3341.20 μg/mL) and florets (IC50 > 4500 μg/mL), and also with the pure compound of 1,5-di-O-caffeoylquinic acid (IC50 = 1741.69 μM). MDA-MB-231 cells were exposed, for 48 h, to the respective IC50 concentrations of leaves lipophilic extract and pure compound of cynaropicrin, in order to understand their ability in modelling cellular responses, and consequently important potentially signaling pathways for the cellular viability decrease. Leaves lipophilic extract increased the caspase-3 enzymatic activity, contrarily to pure compound of cynaropicrin. Additionally, leaves lipophilic extract and pure compound of cynaropicrin caused G2 cell cycle arrest, possibly by upregulating the p21Waf1/Cip1 and the accumulation of phospho-Tyr15-CDK1 and cyclin B1. The inhibitory effects of leaves lipophilic extract and cynaropicrin pure compound, against the MDA-MB-231 cell proliferation, may also be related to the downregulation of phospho-Ser473-Akt. The antibacterial activity of cultivated cardoon lipophilic and phenolic extracts was assessed, for the first time, on two multidrug-resistant bacteria, such as the Gram-negative Pseudomonas aeruginosa PAO1 and the Gram-positive methicillin-resistant Staphylococcus aureus (MRSA), two of the main bacteria responsible for health care-associated infections. Accordingly, the minimum inhibitory concentrations (MIC) were determined. Lipophilic and phenolic extracts of florets did not have antibacterial activity on P. aeruginosa PAO1 and MRSA (MIC > 2048 μg/mL). Leaves lipophilic extract did not prevent the P. aeruginosa PAO1 growth, but pure compound of cynaropicrin was slightly active (MIC = 2048 μg/mL). Leaves lipophilic extract and pure compound of cynaropicrin blocked MRSA growth (MIC of 1024 and 256 μg/mL, respectively). The scientific knowledge revealed in this thesis, either by the chemical viewpoint, or by the biological viewpoint, contributes for the valorization of C. cardunculus L. var. altilis (DC) biomass. Cultivated cardoon has potential to be exploited as source of bioactive compounds, in conciliation with other valorization pathways, and Portuguese traditional cheeses manufacturing.
A espécie mediterrânica Cynara cardunculus L. é reconhecida na medicina tradicional, pelos seus efeitos hepatoprotetor e colerético. A biomassa de C. cardunculus L. var. altilis (DC), ou cardo cultivado, poderá ser explorada não só para a produção de energia e fibras de pasta de papel, mas também para a extração de compostos bioativos. A caracterização química dos componentes extratáveis, nomeadamente os compostos terpénicos e fenólicos, poderá valorizar a plantação de cardo cultivado, dadas as suas atividades antioxidante, antitumoral e antimicrobiana. Neste estudo, a composição química das frações lipofílica e fenólica de C. cardunculus L. var. altilis (DC), cultivado no sul de Portugal (região do Baixo Alentejo), foi caracterizada em detalhe, com vista à valorização integral da sua biomassa. A atividade biológica dos extratos de cardo cultivado foi avaliada em termos de efeitos antioxidante, antiproliferativa em células humanas tumorais, e antibacteriano. A análise química dos compostos lipofílicos foi realizada por cromatografia em fase gasosa acoplada à espectrometria de massa (GC-MS). Identificaram-se 65 compostos lipofílicos, dos quais 1 lactona sesquiterpénica e 4 triterpenos pentacíclicos foram descritos, pela primeira vez, como componentes do cardo cultivado, tais como: desacilcinaropicrina, acetatos de β- e α-amirina, acetato de lupenilo e acetato de ψ-taraxasterilo. As lactonas sesquiterpénicas foram a principal família de compostos lipofílicos das folhas (≈94,5 g/kg), maioritariamente representadas pela cinaropicrina (≈87,4 g/kg). Os triterpenos pentacíclicos foram também detetados, em teores consideravelmente elevados, nas restantes partes do cardo cultivado, em especial nos floretos (≈27,5 g/kg). O acetato de taraxasterilo foi o triterpeno pentacíclico maioritário (≈8,9 g/kg nos floretos). Para a análise química dos compostos fenólicos foi utilizada a cromatografia líquida de alta resolução acoplada à espectrometria de massa (HPLC-MS). Entre os 28 compostos fenólicos identificados, o erioditiol hexósido foi descrito pela primeira vez como componente de C. cardunculus L., e 6 como componentes do cardo cultivado, nomeadamente ácido 1,4-di-O-cafeoilquínico, naringenina 7-O-glucósido, naringenina rutinósido, naringenina, luteolina acetil-hexósido e apigenina acetil-hexósido. A concentração mais alta de compostos fenólicos identificados foi observada nos floretos (≈12,6 g/kg). A parte externa dos caules continha o maior teor em ácidos hidroxicinâmicos (≈10,3 g/kg), e os floretos apresentaram o maior teor em flavonoides (≈10,3 g/kg). A atividade antioxidante da fração fenólica foi examinada face ao radical livre 2,2-difenil-1-picril-hidrazilo (DPPH). Os extratos da parte externa do caule, e dos recetáculos e brácteas demonstraram o maior efeito antioxidante, face ao DPPH (IC50 de 34,35 μg/mL e 35,25 μg/mL, respetivamente). (cont.) resumo (cont.) A atividade antioxidante face ao DPPH foi correlacionada linearmente com a concentração total de ácidos hidroxicinâmicos (r = -0.990). A atividade antiproliferativa in vitro dos extratos lipofílicos e fenólicos de cardo cultivado foi avaliada numa linha de células tumorais humanas de cancro da mama de fenótipo triplo-negativo (MDA-MB-231), um dos tipos de cancro humano mais refratários às terapêuticas convencionais. Após 48 h de exposição, o efeito inibitório do extrato lipofílico das folhas (IC50 = 10,39 μg/mL) foi superior ao do extrato lipofílico dos floretos (IC50 = 315,22 μg/mL), sobre a viabilidade celular de MDA-MB-231. O composto puro da cinaropicrina, representativo do composto maioritário identificado no extrato lipofílico das folhas, também inibiu a proliferação das células MDA-MB-231 (IC50 = 17,86 μM). As células MDA-MB-231 foram muito mais resistentes, ao tratamento de 48 h, com os extratos fenólicos da parte externa dos caules (IC50 = 3341,20 μg/mL) e dos floretos (IC50 > 4500 μg/mL), e também com o composto puro do ácido 1,5-di-O-cafeoilquínico (IC50 = 1741,69 μM). As células MDA-MB-231 foram expostas, durante 48 h, às respetivas concentrações de IC50 do extrato lipofílico das folhas e do composto puro da cinaropicrina, de modo a perceber a sua capacidade em modelar respostas celulares, e consequentemente potenciais vias de sinalização importantes para o decréscimo da viabilidade celular. O extrato lipofílico das folhas aumentou a atividade enzimática da caspase-3, ao contrário do composto puro da cinaropicrina. Além disso, o extrato lipofílico das folhas e o composto puro da cinaropicrina causaram paragem do ciclo celular na fase G2, possivelmente através do aumento da expressão proteica de p21Waf1/Cip1 e da acumulação das proteínas fosfo-Tyr15-CDK1 e ciclina B1. Os efeitos inibitórios do extrato lipofílico das folhas e do composto puro da cinaropicrina, contra a proliferação das células MDA-MB-231, poderão também estar relacionados com a diminuição da expressão proteica da fosfo-Ser473-Akt. A atividade antibacteriana dos extratos lipofílicos e fenólicos de cardo cultivado foi avaliada, pela primeira vez, sobre duas bactérias multirresistentes, tais como a bactéria Gram-negativa Pseudomonas aeruginosa PAO1 e a bactéria Gram-positiva Staphylococcus aureus resistente à meticilina (MRSA), duas das principais bactérias responsáveis pelas infeções associadas aos cuidados de saúde. Para tal, determinaram-se as concentrações inibitórias mínimas (MIC). Os extratos lipofílicos e fenólicos dos floretos não revelaram atividade antibacteriana contra P. aeruginosa PAO1 e MRSA (MIC > 2048 μg/mL). O extrato lipofílico das folhas não inibiu o crescimento de P. aeruginosa PAO1, mas o composto puro da cinaropicrina foi ligeiramente ativo (CIM = 2048 μg/mL). O extrato lipofílico das folhas e o padrão puro da cinaropicrina bloquearam o crescimento de MRSA (MIC de 1024 e 256 μg/mL, respetivamente). O conhecimento científico revelado nesta tese, quer do ponto de vista químico, quer do ponto de vista biológico, contribui para a valorização da biomassa de C. cardunculus L. var. altilis (DC). O cardo cultivado tem potencial para ser explorado como fonte de compostos bioativos, em conciliação com outras vias de valorização, e a produção de queijos tradicionais portugueses.

Research Doctorate - Doctor of Philosophy (PhD)
With the realisation of the impending impact of climate change, the scientific community has been working towards generating viable renewable energy sources that do not contribute to the greenhouse effect. Bioethanol is one form of renewable energy that could replace the majority of transportation fuels, however, its production efficiency and commercial viability relies on optimisation of agricultural quality and yields, biomass conversion to sugars, and fermentation of these sugars into ethanol. The focus of this dissertation will be the optimisation of agricultural quality and yields of bioenergy crops. Most food crops have been domesticated for thousands of years providing selection pressure to increase harvest index (i.e. a high ratio of fruit/seed to vegetative biomass), among other traits. Bioenergy crops, however, are grown for their vegetative biomass more than fruit or seed yields, thus farmers seek a very different plant. To meet the demands of this growing industry, rapid improvements in agricultural yields of both soluble sugars and the cell wall fraction of bioenergy crops, driven by biomass increases, are required. Improvements through traditional breeding strategies, including screening of mutagenized populations, is limited by the availability of high-throughput methods for assessing phenotypes within large populations. Improvements through genetic modification are limited by a fundamental understanding of the biogenesis of vegetative biomass, the biosynthesis and composition of cell walls, and the accumulation of soluble sugars. Here I present a toolkit for the rapid improvement of bioenergy crops. A holistic high-throughput screening strategy was developed for the bioenergy crop, Sorghum bicolor, which rapidly quantifies biomass, cell wall composition and soluble sugar concentrations of stalks, and it was subsequently applied to a large mutagenesis population. Setaria viridis was established as a model C4 grass for the study of stalk development (the major source of bioenergy crop biomass). A developing internode was identified as an ideal experimental system for the study of the biogenesis of stalk tissue, the biosynthesis of cell walls and the import of soluble sugars for storage. RNA sequencing was performed in this system as a resource for gene discovery in relation to these three processes. To complement this resource for gene discovery a technique for 3D visualisation of proteins and tissue structure, PEA-CLARITY was developed and Raman confocal imaging was applied to S. viridis stems for in situ determination of cell wall composition. The high-throughput screening method will provide a tool to drive rapid improvements in commercial bioenergy crops and allow rapid assessment of phenotypes in a field setting. The use of S. viridis as a transformable model C4 grass, along with genetic resources for gene discovery and cutting edge imaging techniques, will allow rapid improvements in understanding of the processes that underpin bioenergy crop yields and will undoubtedly uncover opportunities for crop improvement through genetic manipulation. This toolkit will be integral for the improvement of agricultural yields from bioenergy crops and, therefore, the commercial viability of the biofuel industry.

碩士
中山醫學大學
營養學研究所
96
Cranberry shows high antioxidation and biological activities, including anticancer, prevention of cardiovascular disease, prevention of urinary tract infection, prevention of gastric ulcer and so on. Cranberry contains abundant phenolics. Thus this study was focused on the storage stability of phenolics in cranberry and its effect on age-related macular degeneration（AMD）. The results showed that the phenolics in cranberry would reduced. The free quercetin showed the best stability. And the flavonoids and proanthocyanidins were the second. The total phenolics showed the worst stability. In the ethyl acetate extract of cranberry, the condensed tannin containing fraction showed the best antioxidant activity. In the cell experiments, the model of damaged ARPE-19 cells irradiated by blue light （450nm）showed that the condensed tannin containing fraction in cranberry could repair the damaged ARPE-19 cells irradiated by blue-light. Moreover, the oligomeric condensed tannin containing fraction showed the best efficiency. In conclusion, this study clearly showed that cranberry could scavenge free radicals and cause good efficiency on repairing ARPE-19 cells. This probably showed that cranberry could prevent the age-related macular degeneration causing by free radicals.

Soil organic matter (OM) is a complex mixture of compounds, mainly derived from plants and microbes at various states of decay. It is part of the global carbon cycle and is important for maintaining soil quality. OM protection is mainly attributed to its association with minerals. However, clay minerals preferentially sorb specific OM structures, and clay sorption sites become saturated as OM concentrations increase. Therefore, it is important to examine how OM structures influence their association with soil minerals, and to characterize other protection mechanisms. Several techniques, which provide complementary information, were combined to investigate OM composition: Biomarker (lignin phenol, cutin-OH acid, and lipid) analysis, using gas chromatography/mass spectrometry; solid-state 13C nuclear magnetic resonance (NMR) spectroscopy; and an emerging method, solution-state 1H NMR spectroscopy. OM composition of sand-, silt-, clay-size, and light fractions of Canadian soils were compared. It was found that microbial-derived and aliphatic structures accumulated in clay-size fractions, and lignin phenols in silt-size fractions may be protected from further oxidation. Therefore, OM protection through association with minerals may be structure-specific. OM in soils amended with maize leaves, stems, and roots from a biodegradation study were also examined. Over time, lignin phenol composition, and oxidation; and aliphatic structure contribution changed less in soils amended with leaves compared to soils amended with stems and roots. Compared to soils amended with leaves and stems, amendment with roots may have promoted the more efficient formation of microbial-derived OM. Therefore, plant chemistry influenced soil OM turnover. Synthetic OM-clay complexes and soil mineral fractions were used to investigate lignin protection from chemical oxidation. Coating with dodecanoic acid protected lignin from chemical oxidation, and overlying vegetation determined the relative resistance of lignin phenols in clay-size fractions from chemical oxidation. Therefore, additional protection from chemical oxidation may be attributed to OM composition and interactions between OM structures sorbed to clay minerals. Overall, these studies suggest that while association with minerals is important, OM turnover is also influenced by vegetation, and protection through association with clay minerals was modified by OM structure composition. As well, OM-OM interaction is a potential mechanism that protects soil OM from degradation.

Dissertação de Mestrado em Química Farmacêutica Industrial apresentada à Faculdade de Farmácia
O colesterol é um composto de natureza lipídica, essencial ao funcionamento de todas as células do organismo humano, podendo ser sintetizada a nível endógeno. Cerca de 30 a 40% é sintetizado pelo organismo, sendo o restante obtido pela alimentação, em particular por ingestão de produtos de origem animal. As doenças cardiovasculares são a principal causa de morte e hospitalização nos países desenvolvidos e tem vindo a aumentar nos países em via de desenvolvimento. São diversos os fatores de risco, como é o caso das dislipidemias, isto é, elevados níveis de lipídios (colesterol, triglicerídeos ou ambos) transportados pelas lipoproteínas no sangue. Um dos principais problemas associados à dislipidemia é a formação de placas de aterosclerose que podem acumular-se e depositar-se nas artérias. Isto leva a que possa ocorrer uma diminuição da quantidade de sangue que circula nas artérias, podendo levar mesmo à total oclusão da artéria, dando origem ao acidente vascular cerebral (AVC) e ao enfarte do miocárdio, entre outras situações graves. O aumento da concentração de colesterol no sangue (hipercolesterolémia) associado ao consumo de dieta rica em lípidos, o consumo de grandes quantidades de álcool, a falta de exercício físico, e ainda, a diabetes e hipertensão têm sido apontados como as principais causas das doenças cardiovasculares. Atualmente encontra-se disponível uma grande variedade de terapêuticas medicamentosas para o controlo da hipercolesterolémia. Porém os efeitos adversos destas terapias provocam muitas vezes uma fraca adesão e mesmo o abandono da terapêutica levando muitas pessoas a recorrer aos produtos naturais.O recurso a estudos in vitro permite avaliar, de forma rápida e pouco onerosa, o potencial de plantas medicinais no controlo do colesterol e, consequentemente, selecionar as mais promissoras para a validação subsequente através de ensaios in vivo e clínicos. Uma das vias de redução do colesterol exógeno é a inibição da formação de micelas no intestino, essencial à absorção do colesterol. Pretendeu-se com este trabalho otimizar um método que permita estudar in vitro a solubilidade micelar do colesterol na presença de extratos de folhas do Cymbopogon citratus (infuso e infuso deslipidificado) e comprovar com esta metodologia a potencial atividade desta planta, descrita como tendo atividade hipocolesterolémica e atividade hipolipemiante. Adicionalmente, avaliou-se a contribuição dos seus compostos fenólicos (ácidos fenólicos, flavonóides e taninos) para essa mesma atividade. Os resultados obtidos permitiram concluir que ambos extratos, assim como a fração de ácidos fenólicos, flavonóides e taninos apresentam capacidade para a destruição de micelas, particularmente as duas últimas classes de fenólicos, para as quais se obtiveram percentagens de destruição micelar superiores a 90% para concentração exíguas de flavonóides e taninos (1 µg/mL e 25 µg/mL respetivamente). Estes resultados sugerem que este é um dos mecanismos possíveis pelo qual se observa o efeito hipocolesterolémico desta planta e que os compostos fenólicos, maioritariamente os flavonóides e taninos contribuem para essa atividade. Em conclusão, foi otimizado um teste rápido para o estudo in vitro da solubilidade micelar do colesterol na presença de extratos e frações fenólicas do Cymbopogon citratus, o qual permite evidenciar um dos mecanismos conducentes à atividade hipolipemiante e hipocolesterolémia. Este trabalho permitiu ainda aquilatar o potencial hipolipemiante do Cymbopogon citratus, assim como inferir que essa atividade, pelo menos em parte, se relaciona com os compostos fenólicos, nomeadamente flavonóides e taninos.
Cholesterol is a compound of a lipid nature, essential to the functioning of all Human bodily cells, which can be synthetized at an endogenous level. Between 30 and 40% are synthetized within the organism, with the remaining acquired through the diet, in particular through animal products. Cardiovascular diseases are the leading cause of death and hospital admission in the developed countries and is currently increasing in the developing countries. There several risk factors, such as dyslipidaemia, that is, the high amount of lipids (cholesterol, triglycerides, etc.) transported by the lipoproteins of the blood. One of the major problems associated with dyslipidaemia is the formation of arteriosclerosis plaques that can deposit and accumulate in the arteries. This can lead to a diminishing amount of blood circulating in the arteries, up to the full blocking of the artery, which leads to strokes, myocardial infarction, among other serious illnesses. The increase in cholesterol concentration of the blood (hypercholesterolemia), associated to the consumption of a diet rich in lipids, the consumption of great amounts of alcohol, the lack of exercise, hypertension and diabetes have been sown to be the primary causes of heart diseases. Currently there is a great variety of available therapies through medication to control hypercholesterolemia. Nonetheless, the adverse effects of those therapies can often cause a weak acceptance and high abandonment of the therapies, with many people searching for natural products for the same effect. The in vitro study allows the assessment, quickly and cheaply, of the potential of medicinal plants in the control of cholesterol and, sequentially, the selection of the most promising for subsequent validation through in vivo and clinical trials. One of the ways to reduce exogenous cholesterol is the inhibition of the intestinal micelle formation, essential to the absorption of cholesterol. With this essay, the intention was to optimize a method to allow the in vitro study of the micelle solubility of cholesterol in the presence of leaves extracts of the plant Cymbopogon citratus (infusion and non-lipid infusion) and prove, with this methodology, the potential activity of this plant, already described as having hypercholesterolemic activity and lipid-lowering activity in literature. Additionally, the contribution of its phenolic compounds (phenolic acids, flavonoids and tannins) for such activity was evaluated. The results obtained allow to conclude that both extracts, as well as the phenolic acid, flavonoids and tannins show a micelle destruction capacity, particularly the two last classes of phenolic compounds to which percentages over 90% of micelle destruction were obtained for similar flavonoids and tannins concentrations (1 µg/mL and 25 µg/mL respectively). These results suggest that this is one of the possible mechanisms by which the cholesterol-lowering and fat-lowering effects are observed for this plant and mostly flavonoids and tannins are the phenolic compounds contributing to this activity. In conclusion, a rapid-test was optimized for the in vitro study of micelle solubility of cholesterol in the presence of extracts and phenolic fractions of Cymbopogon citratus, which allows for a conducting mechanism to the fat-lowering and cholesterol-lowering activities. This essay allowed also to evaluate the fat-lowering potential of Cymbopogon citratus as well as inferring that this activity is, at the least partly, related to the phenolic compounds, namely flavonoids and tannins.