It is evident that herbal medicines continue to be the mainstay of healthcare systems and source of livelihoods of many local communities in South Africa and other developing countries. As a result, there is an overwhelming dependence on medicinal products harvested from natural populations. This dependence has led to local extinction of some important medicinal plants that include Warburgia salutaris and Cassine transvaalensis in South Africa. Cultivation has great potential to relieve the pressure on natural populations. However, some traditional practitioners and scientists believe that cultivation may weaken medicinal properties and that increased secondary metabolites may form only under stress conditions, respectively. This is certainly true in some cases especially where infections with pathogens, browsing by herbivores or competition takes place in nature. It is however not clear how true this is with environmental stresses. The overall aim of this study was to evaluate to what degree different environmental conditions influenced antimicrobial and antioxidant activities of plants cultivated outside their natural environment. In order to address the aim of the study, exploratory and in-depth studies were undertaken. The exploratory study comprised long-lived Combretum collinum Fresen. (Combretacea), Terminalia sericea Burch. ex DC. (Combretaceae) and Sclerocarya birrea (A. Rich.) Hochst. (Anacardiaceae). Short-lived herbaceous Tulbaghia violacea Harv. (Alliaceae) and Hypoxis hemerocallidea Fish, C.A.Mey,&Avé- Lall, (Hypoxidaceae), were included as part of the exploratory study. The in depth studies were further undertaken, also with short-lived herbaceous Leonotis dysophylla Benth. (Lamiaceae), Bulbine frutescens (L.) Willd. (Asphodelaceae) and T. violacea. Acetone leaf extracts of all plants were studied for antimicrobial activity against bacteria (Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis) and fungi (Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus). Extracts were also studied for antioxidant activity against Trolox and L-ascorbic acid standard oxidants using 2,2’-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) and 2,2- diphenyl-2-picryl-hydrazyl (DPPH) free radicals, respectively. The exploratory study tested the effect of different rates of annual rainfall (≥870 mm/year, 651 mm/year and 484 mm/year) on the antibacterial activity of C. collinum, T. sericea and S. birrea growing in nature. The minimum inhibitory concentration (MIC) of acetone extracts of air-dried leaves was determined by using microplate serial dilution technique. Thin layer chromatography (TLC) and bioautography determined chemical constituents and antibacterial activity of extracts, respectively. The majority of extracts had low MIC values, which indicated good antibacterial activity against test bacteria (MIC of 240 μg/ml - 60 μg/ml). Leaf extracts of C. collinum and S. birrea against S. aureus (range of 390 – 100 μg/ml), E. coli (310 -70 μg/ml) and P. aeruginosa (520 - 70 μg/ml) had antibacterial activity increased significantly with low rate of annual rainfall. However, extracts of T. sericea against P. aeruginosa (240 - 100 μg/ml) and E. faecalis (150 - 820 μg/ml) had antibacterial activity significantly increased and decreased, respectively. Extracts of C. collinum and S. birrea against E. faecalis as well as T. sericea against S. aureus and E. coli did not show any clear correlation between activity and different rates of annual rainfall. Inconsistent results suggest that other factors in nature such as genetic variability, age difference, pathogens, herbivores or allelopathy (competition) might have influenced the antibacterial activity of extracts. The results indicate that the antimicrobial activity of plants growing in nature may be highly variable. In order to eliminate possible effect of those factors common in nature, another exploratory study was undertaken using clone T. violacea and H. hemerocallidea of similar age (Chapter 3). Plants were grown under controlled conditions that included irrigation with 1000 ml of distilled water in intervals of 3, 14 and 21 days outside natural environment. Dry mass of all plants was reduced significantly (P≤0.05) with watering interval of 21 days, which indicated the effect of water stress. Air-dried leaves of all plants were finely ground and extracted with acetone. Extracts had good antibacterial activity as attested by low MIC values (< 1 mg/ml) across watering intervals. Differences in the antibacterial activity of the extracts against test bacterial between water treatments were not statistically significant (P≤0.05). Furthermore, there was no clear correlation between the activity of extracts and water treatments in terms of the MIC and total activity values or chemical constituents. The results in general suggest that cultivation under optimal watering intervals may not necessarily weaken the biological activity of extracts. To complement the above findings, in depth studies were also undertaken with clone L. dysophylla, T. violaceae and B. frutescens of similar age growing under controlled conditions outside natural environment. The studies determined the influence of a wide range of water (50 ml – 500 ml) and temperature (15°C and 30°C) treatments on antibacterial, antifungal and antioxidant of extracts. With the exception of a crassulacean acid metabolism (CAM) plant, B. frutescens, transpiration, dry mass and leaf areas of the other two plants were reduced significantly (P 0.05) under high temperature of 30°C and lowest water supply of 50 ml. Acetone leaf extracts had some biological activity. Differences in the majority of antibacterial and antifungal activities of extracts between water and temperature treatments were not statistically significant. With the exception of the influence of temperature, the majority of the antioxidant activity of extracts was almost similar between water treatments. However, the significant reduction of the antioxidant activity of all extracts under high temperature of 30°C was indicative of great sensitivity to high temperatures. The overall findings suggest that the biological activity of plants is more likely to vary widely in nature than under controlled conditions outside the natural environment. This is an indication that natural environment cannot always guarantee high and stable biological activity. As a result, beliefs by some traditional practitioners and scientists that cultivation weakens medicinal properties and good secondary metabolites form only under stress, respectively, cannot be widely substantiated. Therefore, the study encourages cultivation of medicinal plants. It has potential to optimise yield of biomass production, and ensure uniform and quality biological activity as well as reduce misidentification. / Thesis (PhD)--University of Pretoria, 2012. / Paraclinical Sciences / unrestricted

Phenolic compounds in African green leafy vegetables (GLVs) may have a significant impact on human health. However, there is little information on the phenolic composition, antioxidant properties, as well as biological and cellular protective effects of these vegetables. The effects of boiling and extraction solvent on these compounds and on their antioxidant properties are also unknown. Phenolic content, antioxidant activity and cellular protective effects of four African GLVs in comparison with spinach, an exotic GLV, was determined. African GLVs had appreciable levels of total phenolics and antioxidant activity and in higher quantities compared to spinach. Boiling decreased the antioxidant content and activity of these vegetables and 75% acetone was more effective in extracting antioxidants from the GLVs compared to water. GLVs with high levels of phenolics also contained higher levels of antioxidant activity, suggesting that phenolics are likely to have contributed to radical scavenging activity of these vegetable extracts, even though the degree of scavenging varied in each extract of the vegetable species. The flavonoid compositions of raw and boiled African GLVs and spinach were determined using high-performance liquid chromatography. Epicatechin and rutin were the most dominant flavonoids found in both water and 75% acetone extracts. Among water extracts, pumpkin contained higher concentrations of detected flavonoids, while among the acetone extracts, cowpea exhibited higher concentrations. The effect of boiling was dependent on the type of vegetable and the specific flavonoids. There were no major differences observed between the type of flavonoids detected in extracts of African GLVs and those in spinach. However, similar to the results of total phenolics and antioxidant activity, the 75% acetone extracts of African GLVs also exhibited higher amounts of flavonoids than spinach. The protective effects of GLVs against oxidative haemolysis were dependent on the type of vegetable species. Boiling had variable effects depending on the species. The highest level of protection of erythrocytes against oxidative damage was offered by amaranth extracts, while extracts of raw jute mallow contributed to the damage of erythrocytes. The highest antioxidant protection activity against oxidative damage in plasmid DNA was offered by extracts of jute mallow and lowest by spinach.<p. For the cell viability assays, GLVs were evaluated to determine their cytotoxicity levels and functional role in oxidative damage. The results of the long-term cell viability (i.e. MTT, NR and CV) assays indicated no cytotoxicity, while the short-term cell viability (i.e. DCF) assay indicated that all extracts of raw GLVs were significantly (p < 0.05) cytotoxic to SC-1 fibroblast and human adenocarcinoma colon cancer (Caco-2) cells than extracts of cooked samples, and the levels of toxicity in the extracts of spinach was higher than in African GLVs. These results indicate that there was an initial cytotoxic effect as extracts of raw GLVs were added to the cells. However, after about 72 h, the cells recovered from the initial shock and started proliferating as usual. In the presence of peroxyl radicals, extracts of African GLVs exhibited higher protective effects against oxidative damage in both types of cell cultures than extracts of spinach. These results indicate that these protective effects could be attributed to the presence of phenolics and antioxidant properties of these extracts. Although boiling reduced the antioxidant content and activity of African GLVs, the levels remained higher than in spinach. Boiling also decreased the cytotoxicity and cell damage caused by extracts of raw GLVs samples. African GLVs are consumed after boiling, and therefore the observed cytotoxicities might not be experienced in practical terms. African GLVs have therefore a potential to reduce the risk and development of diseases associated with oxidative stress in communities that consume these vegetables. / Thesis (PhD)--University of Pretoria, 2012. / Centre for Nutrition / PhD / Unrestricted

L’hépatite est une inflammation du foie pouvant être causée par divers agents. Elle reste un problème de santé publique majeur dans le monde, compte tenu du coût de plus en plus élevé des médicaments. Une meilleure compréhension des mécanismes d’action des plantes médicinales apparaît intéressante pour développer des traitements alternatifs. L’objectif de cette étude était donc d’évaluer les propriétés hépatoprotectrice, puis d’identifier les mécanismes qui pourraient soutendre l’action de Neoboutonia velutina (NV), une plante médicinale camerounaise.Les extraits aqueux (NVH) et éthanolique (NVE) de NV ont été préparés, se référant à la préparation traditionnelle. Les analyses phytochimique et toxicologique ont été effectuées et le potentiel antioxydant évalué in vitro et in vivo. De même, l’effet antiinflammatoire des deux extraits a été évalué sur les cellules et chez la souris. L’hépatite aiguë a été induite par le tétrachlorure de carbone (CCl4) ou la Concanavalin A (ConA), chez la souris qui recevait ou non différentes doses d’extrait par gavage. De plus, l’extrait aqueux a été fractionné pour l’identification des composés bioactifs.NVE contiendrait ainsi des stérols et polyterpènes. Cet extrait a présenté une capacité anti-radicalaire meilleure que celle de NVH. Mais, il s’est avéré plus toxique que NVH; ce qui a limité nos investigations sur cet extrait. En revanche, NVH contiendrait des saponines et glycosides et a révélé une très faible toxicité. De plus, un remarquable effet protecteur de NVH a été noté contre les dommages causés par le CCl4. Cet effet protecteur s’est traduit par une diminution dose-dépendante et significative des transaminases sériques et une importante diminution des lésions hépatiques. Ceci, associé à la capacité antiinflammatoire in vitro et in vivo. En outre, un composé anti-radicalaire a été isolé de NVH. Par ailleurs, dans le modèle ConA, NVH n’a présenté qu’un faible effet protecteur. Ce qui suggère une efficacité sélective de cet extrait.En conclusion, nous avons démontré dans notre étude, un effet hépatoprotecteur de NV à travers l’extrait aqueux qui a présenté un effet hépatoprotecteur modèle dépendant. Cet effet, semble être médié au moins, par la capacité anti-radicalaire de la plante. Nos résultats présentent ainsi les premiers arguments en faveur de l’utilisation traditionnelle de NV contre les hépatites. Des études plus poussées permettraient de mieux comprendre les mécanismes d’action de cette plante et d’exploiter au mieux son potentiel thérapeutique, sans risque de toxicité. Ainsi, bien que présentant une toxicité, l’extrait éthanolique qui mime la préparation traditionnelle, a révélé un potentiel thérapeutique qui pourrait être très intéressant à très faibles doses. / Hepatitis is a liver inflammation caused by different agents. It remains a public health problem worldwide since current treatment methods are increasingly expensive. Medicinal plants are known as an important source of new molecules. A better knowledge of these natural resources appears interesting to develop alternative treatments. The aim of this study was then to evaluate the hepatoprotective effect of Neoboutonia velutina (NV), a Cameroonian medicinal plant, and decipher underlying mechanisms.NV aqueous (NVH) and ethanol (NVE) extracts have been prepared referring to the traditional use. Phytochemical and toxicological analyses were performed in vitro and in vivo. Similarly, extracts antioxidant and antiinflammatory potential was assessed on cells (or not) and in mice. Acute hepatitis was induced with carbon tetrachloride (CCl4) or Concanavalin A (ConA), in mice receiving or not different extracts doses by gavage. NVH fractionation was done to identify active compounds.NVE was containing sterols and polyterpens. Though it displayed a high radical scavenging capacity compared to NVH, it appeared more toxic. Thus, for assays, priority was given to NVH, containing saponins and glycosides. NVH showed a radical scavenging capacity with a very low toxicity. It remarkably protected mice from CCl4-induced liver injuries. As shown by significant dose dependent transaminases serum level decrease and liver injury important limitation. These, associated with NVH anti-inflammatory capacity. Furthermore, NVH fractionation led to a radical scavenging compound isolation. Otherwise, in ConA model NVH displayed weak effects. These findings suggested a selective NVH efficacy. In summary, we showed that NVH presents a model dependent hepatoprotective effect that may be mediated at least, through its radical scavenging property. Our findings are in line with Neoboutonia velutina traditional use and provide the first scientific arguments in favor of the traditional use of NV against hepatitis. Additional studies are needed to better understand NV mechanisms of action and then ensured its safe use. NVE mimics the traditional preparation. Even though that extract appeared toxic, it exhibited a therapeutic potential that could be interesting at very low doses.

Phenolics in finger millet (FM) grain, including tannins, may impact significantly on its antimicrobial properties, functionality and health-promoting potential. Unfortunately, the location of tannins in the grain is unknown and there is limited information on the influence of variety on grain phenolic composition and antioxidant activity (AA). The effect of phenolics in FM grain on its malt fungal load and on the functional quality of its food products, including baked goods, is barely known. Twenty two FM grain types of varied visual kernel colour were analysed to determine the influence of grain type on phenolic composition, AA, and tannin localisation in the grain. Condensed tannins, anthocyanins and flavan-4-ols were detected. Light coloured grain types had no tannins and had much lower total phenolics (TP) relative to the pigmented types, and types that stained black with the Bleach test had much higher tannin content and much higher AA. The grains that stained black with the Bleach test and had high tannin content (0.60 to 2.08 mg catechin eqivalents/100 mg, db) had a dark coloured testa layer, indicating that the tannins were located in that layer. The results indicate that occurrence of tannins in FM is a varietal property and the tannins are predominantly responsible for the AA of the grain. Germinative energy (GE), enzymic activity, and total fungal count [TFC], and infection levels of 12 FM grain types of varied phenolic content were measured to determine the impact of phenolics in FM grain on its malt quality. The malt quality of high-phenol FM types was much higher than that of the low-phenol types, with respect to enzymic activity. TFC was negatively correlated with grain total phenolics (TP) and amount of phenolic type (APT) and there were some negative correlations between fungal species infection levels and TP and APT (p<0.05). GE and enzymic activity were positively correlated with TP and APT (p<0.05) and negatively correlated with TFC (p<0.01). The data indicate that phenolics in FM grain impact positively on its malt quality by contributing to its antifungal activity. Cookies in which wheat cake flour was substituted with 15, 35 and 55% (w/w) of either a non-tannin or a high-tannin FM flour were analysed to assess the impact of FM phenolics on cookie quality and AA (health-promoting potential). FM-substituted cookies, particularly those with high levels of the high-tannin FM, were inferior to cake flour cookies (control), with respect to spread, texture and integrity and their dark colour decreased their acceptance by a consumer panel. However, the acceptability of cookies containing up to 35% of either FM type was similar to that of control cookies. Cookies containing the high-tannin FM had antioxidant activities that were similar to or higher than the antioxidant activities of several plant products on the market. Thus, potentially health-promoting cookies can be made by substituting up to approximately 35% wheat with a high-tannin FM. The study indicates that high-phenol FM grain types have good malt quality, which is partly due to the antifungal activity of their phenolics. Although FM phenolics, particularly tannins, seem to affect cookie quality negatively, they contribute significantly to their health-promoting potential. / Thesis (PhD)--University of Pretoria, 2009. / Food Science / unrestricted

Amongst cereals, sorghum is one of the major sources of phenolic compounds. South African cultivars have not been profiled for their phenolic content and antioxidant activity to highlight their potential benefits. Thus, South African sorghum cultivars representing different sorghum types were evaluated for total phenolic content, condensed tannin content and antioxidant activity and the effect of cultivar on their antioxidant activity. The presence of phenolic antioxidants in the different sorghum cultivars, created an opportunity to develop a sorghum product as a vector of the antioxidants. Cookies were a product of choice due to their shelf stability and high nutrient density. Sorghum cookies were produced from 70%, 90% and 100% extraction rate flours. The effects of flour extraction rates and cultivar on the total phenolic content, condensed tannin content and antioxidant activity of the cookies were determined. Consumer sensory evaluation was used to evaluate sorghum cookie acceptability against a wheat flour cookie. Total phenolic content of the cultivars, determined by the Folin-Ciocalteu method was 0.20 to 1.42 g catechin equivalents (CE)/100 g. The total phenolic content was 3 to 7 times higher in condensed tannin cultivars than in tannin-free cultivars. Using the modified Vanillin-HCl method, condensed tannins were only measurable in the condensed tannin cultivars. They ranged between 5.16 and 8.39 g CE/100 g. Subsequently, the antioxidant activity of the condensed tannin cultivars measured by the ABTS radical scavenging assay was up to 4 times higher than in the tannin-free cultivars. The high phenolic content and antioxidant activity of condensed tannin cultivars was attributed to the contribution of condensed tannins. Therefore, condensed tannin cultivars are a major source of antioxidants compared to tannin-free cultivars. For each sorghum cultivar, cookies of 100% extraction rate flours had 2 to 3 times higher total phenolics compared to those of 70% extraction rate flours, while antioxidant activity was 2 to 10 times higher. Cookies of the condensed tannin sorghum had 2 to 5 times more phenolics compared to those of condensed tannin-free sorghum. Antioxidant activity was 145 to 227 ìMol Trolox equivalents (TE)/g in cookies of condensed tannin sorghum compared to 10 to 102 ìMol TE/g in those of condensed tannin-free sorghum. Processing sorghum flours to cookies seemed to reduce phenolic and antioxidant activity, but considering the flour component in the formula, cookie antioxidant activity was slightly higher than that of flours. The texture of all sorghum cookies was less acceptable compared to that of wheat cookies. The consumers showed a slight overall liking of the condensed tannin-free sorghum and wheat flour cookies. The cookies from condensed tannin flours were neither liked nor disliked. Since generally wheat flour is used for making cookies, the similarities in the overall liking of the condensed tannin-free sorghum cookies and the wheat flour cookies indicate strong potential of sorghum flour for cookie making. Therefore, sorghum cookies have a potential as a functional ready-to-eat snack, with target consumers such as school children in feeding schemes to improve their health and nutrition status. / Dissertation (MSc)--University of Pretoria, 2009. / Food Science / unrestricted

Lipid peroxidation has been implicated in the onset and progression of many degenerative diseases, including cardiovascular disease, Alzheimer’s disease and cancer. Accordingly, for more than 50 years, considerable effort has been devoted to the design of synthetic compounds or the discovery of natural products that can slow lipid peroxidation. Despite the enormous investments made to date, no clear antioxidant strategies have emerged for the treatment and/or prevention of degenerative disease. We argue that this is because of a lack of fundamental understanding of the chemical reactivity of these compounds in relevant contexts. Herein, we describe studies of our optimized synthetic radical-trapping antioxidant (RTA) – the tetrahydronaphthyridinols (THNs). We first present the synthesis of a series of THN analogs of α-tocopherol (Nature’s premier lipid-soluble radical-trapping antioxidant) with varying sidechain substitution and then demonstrate how systematic changes in the lipophilicity of these potent antioxidants impact their peroxyl radical-trapping activities in lipid bilayers and mammalian cell culture. Their regenerability by water-soluble reductants in lipid bilayers, binding to human tocopherol transport protein (hTTP), and cytotoxicity were also evaluated to provide insight on whether this type of antioxidant can be potentially pushed toward animal studies. We also describe analogous studies of natural products such as the garlic-derived thiosulfinate allicin and the grape-derived polyphenol resveratrol. These compounds have attracted significant attention in the past 20 years due to their purported health benefits, which are often ascribed to their purported radical-trapping activities. To date, systematic studies on their radical-trapping activities in solution, lipid bilayers and mammalian cells have been lacking. We have determined that allicin and petivericin, while effective RTAs in solution, are not so in lipid bilayers. Moreover, the compounds are not antioxidants in cell culture, but instead kill the cells. Similarly, resveratrol and its dimers pallidol and quadrangularin A, are found to be inefficient RTAs in lipid bilayers. Our studies to date rather suggest that they autoxidize readily to produce hydrogen peroxide, which may induce expression of phase 2 antioxidant enzymes, affording cytoprotection. Our insights underscore the need for systematic studies of antioxidant activity in multiple contexts.

Le caoutchouc naturel (CN), un produit dérivé du latex d'Hevea brasiliensis, est connu pour ses propriétés mécaniques supérieures pour certaines à celles de ses concurrents synthétiques. Néanmoins, le haut degré d'insaturation du poly(cis-1,4-isoprene) le rend susceptible à la thermo-oxydation. Heureusement, le CN est doté de composés non-isoprènes dont certains ont des propriétés antioxydantes. Les lipides sont les plus importants non-isoprènes retenus dans le caoutchouc et contiennent des molécules à activité antioxydante en particulier les tocotriènols. Il est connu que durant la maturation de coagula de latex, la composition chimique et les propriétés du caoutchouc obtenu sont altérées, mais les mécanismes complexes de cette altération ne sont pas encore complètement élucidés. Dans cette étude, l'évolution de certaines molécules antioxydantes natives pendant la maturation a été suivie en relation avec certaines propriétés physiques du caoutchouc. Deux expérimentations de maturation ont été mises en œuvre. La première mettait en jeu des conditions non contrôlées de maturation suivies d'un procédé de confection du caoutchouc basé sur celui des feuilles fumées (RSS) ou non (USS). La seconde a été conduite dans un dispositif expérimental dédié permettant le contrôle des facteurs de l'environnement tels que l'humidité relative, la température et la concentration en oxygène. Le procédé de confection du caoutchouc était dans ce cas basé sur celui des caoutchoucs spécifiés techniquement (TSR). L'évolution des échantillons pendant la maturation a été étudiée à différentes échelles : propriétés en masse (P0, P30 et PRI), mésostructure (% gel, Mw and Mn) et composition biochimique (lipides). En parallèle, l'activité antioxydante in vitro des extraits lipidiques correspondants a été mesurée en utilisant une méthode DPPH optimisée. La quantité et la qualité des lipides extraits évoluent pendant la maturation, en particulier en aérobiose. La quantité totale de lipides décroit, avec, en début de maturation, une libération d'acides gras dont la quantité diminue ensuite, avec une disparation des espèces insaturées en premier. La quantité de γ-tocotrienol libres extraits change peu au cours de la maturation alors que sa forme estérifiée montre un enrichissement en acides gras saturés. L'activité antioxydante de l'extrait lipidique mesurée in vitro est corrélée avec la concentration de γ-tocotrienol libre mais pas avec les valeurs de P30 et PRI qui estiment la résistance du caoutchouc à la thermo-oxydation. Cette absence de corrélation pourrait être due à la différence des conditions de mesure in-vitro de celles existantes au sein du matériau caoutchouc. La localisation des antioxydants dans le caoutchouc et en particulier leur possibilité physique d'interagir avec les doubles liaisons du poly(cis-1,4-isoprene) ou avec des espèces oxydantes reste à étudier afin de comprendre ce qui régit la chute de P30 au cours de la maturation. Des lipides non extractibles ou des molécules non-isoprènes plus polaires (protéines, polyphénols, …) pourraient également influencer la résistance du caoutchouc à la thermo-oxydation. / Natural rubber (NR), a derived product from H. brasiliensis latex, is known for its high mechanical properties that are, for some, superior to those of its synthetic counterparts. However, the high degree of unsaturation of poly(cis-1,4-isoprene) makes it susceptible to thermo-oxidation. Fortunately, NR is endowed with non-isoprene components of which some have antioxidant properties. Especially, lipids, the main non-isoprene component retained in NR, have been reported to contain antioxidant substances, especially tocotrienols. It is well known that during the maturation of latex coagula, both NR physical properties and chemical composition are altered, but the complex mechanisms of this alteration are still to be elucidated. In the present work, the evolution of some native antioxidant molecules during maturation was followed in relation with some physical properties. Two experimental conditions of maturation were chosen. The first experiment involved uncontrolled conditions based on traditional unsmoked (USS) or ribbed smoked sheet (RSS) processing, while the second was performed in a dedicated maturation device with full control of environmental factors (relative humidity, temperature and oxygen content) followed by a processing based on that of Technically Specified Rubber (TSR). The evolution of samples during maturation was studied at different scales: bulk properties (P0, P30 and PRI), mesostructure (% gel content, Mw and Mn) and biochemical composition (lipids components). In parallel, in vitro antioxidant activity of NR lipid extracts was also investigated using an optimized DPPH method. Lipid quantity and quality evolved during maturation, especially under aerobic conditions. The total amount of lipid extract decreased, with a release of free fatty acids at early stage of maturation followed by a later decrease, unsaturated fatty acids being the first to disappear. The amount of extractable free γ-tocotrienol did not change much during maturation, while its esterified form was enriched in saturated fatty acids. The antioxidant activity measured in vitro correlated well with free γ-tocotrienol concentration but not with the resistance of rubber to thermo-oxidation assessed by P30 or PRI. Indeed, the in vitro conditions of measurement were far from those occurring inside rubber material. The localization of antioxidants in rubber and especially their physical possibility to interact with the double bonds of poly(cis-1,4-isoprene) or with oxidant species should be further investigated to understand what drives the drop of P30 along maturation time. Non extractable lipids or more polar non-isoprene molecular species (proteins, polyphenols, etc…) could also influence the resistance to thermo-oxidation.

Reações de oxidação lipídica são responsáveis pelo desenvolvimento de sabores e odores desagradáveis tornando os alimentos impróprios para consumo, sendo necessário o uso de antioxidantes. O ácido ascórbico (AA) é um antioxidante muito eficaz, que exibe função vitamínica, no entanto é relativamente instável. Com o objetivo de aumentar a estabilidade do AA e, consequentemente, facilitar sua aplicação em produtos alimentícios, os métodos de encapsulação por coacervação complexa e por dispositivos microfluídicos foram testados. Primeiramente foi apresentada a Revisão Bibliográfica no Capítulo 1, e em seguida, a encapsulação por coacervação complexa, como será visto no Capítulo 2. Neste caso, nove tratamentos foram obtidos utilizando-se gelatina e goma arábica como materiais de parede e analisados com relação à morfologia, por microscopia ótica e eletrônica de varredura, umidade, atividade de água, higroscopicidade, solubilidade, potencial zeta, espectroscopia no infravermelho por transformada de Fourier (Ftir), tamanho e distribuição de tamanho de partículas, cor instrumental, eficiência de encapsulação e estabilidade do material encapsulado. No capítulo 3 serão apresentados resultados obtidos na encapsulação do AA pelo método de dispositivos microfluídicos. Cinco tratamentos foram obtidos, sendo analisados com relação à morfologia, por microscopia ótica, eletrônica de varredura e confocal, eficiência de encapsulação, tamanho e distribuição de tamanho de partícula e estabilidade do material encapsulado. A obtenção das microcápsulas de AA pelos dois métodos citados foi viável uma vez que apresentaram altos valores de eficiência de encapsulação e ótima atuação em relação à proteção do AA durante estocagem. Comparando-se os dois métodos, as cápsulas obtidas por dispositivos microfluídicos conferiram melhor estabilidade ao ácido ascórbico, no entanto amostras obtidas por coacervação complexa foram aplicadas em salsicha devido a maior quantidade de AA presente em sua constituição. O efeito da aplicação das microcápsulas nas salsichas foi avaliado durante 40 dias de armazenamento refrigerado como será visto no Capítulo 4. Cinco tratamentos foram elaborados e analisados de acordo com a estabilidade da emulsão cárnea durante o processamento, umidade, atividade de água, alteração do pH, determinação da cor instrumental, perfil de textura instrumental, estabilidade oxidativa pelo método de substâncias reativas ao ácido tiobarbitúrico (TBARS) e aceitação sensorial. A aplicação das microcápsulas de AA em salsicha foi possível sem comprometer a qualidade do produto final. Todos os dados obtidos foram analisados estatisticamente por análise de variância ANOVA e teste de Tukey, ao nível de 5% de significância com auxílio do programa SAS. Os experimentos relacionados à encapsulação por coacervação complexa e aplicação das microcápsulas em salsicha foram realizados no Laboratório de Produtos Funcionais, nas dependências do Departamento de Engenharia de Alimentos da FZEA/USP. Os experimentos relacionados à utilização de dispositivos microfluídicos foram realizados nos laboratórios do professor David A. Weitz, da Escola de Engenharia e Ciências Aplicadas de Harvard, da Universidade de Harvard, Cambridge, Estados Unidos. / Lipid oxidation reactions are responsible for the development of unpleasant tastes and odors making food unfit for consumption. For this reason, the use of antioxidant is necessary. Ascorbic acid (AA) is a very effective antioxidant with vitamin function, however it is relatively unstable. With the aim of increasing the stability of AA and thus improve its application in food products, the methods of encapsulation by complex coacervation and microfluidic devices were tested. First of all the Literature Review is presented in Chapter 1. The encapsulation by complex coacervation can be seen in Chapter 2. For this methodology, nine treatments were obtained using gelatin and gum Arabic as encapsulant agent and analyzed regarding to morphology by optical and scanning electron microscopy, moisture, water activity, hygroscopicity, solubility, Zeta Potential, Fourier transform infrared Spectroscopy (FTIR), particle size and particle size distribution, instrumental color, encapsulation efficiency and stability of the encapsulated material. The results obtained for AA encapsulation by microfluidic device will be presented in Chapter 3. Five treatments were obtained and analyzed regarding to morphology by optical, scanning electron and confocal microscopy, encapsulation efficiency, particle size and particle size distribution and stability of the encapsulated material. The production of AA microcapsules by the two methods mentioned was feasible once that showed high levels of encapsulation efficiency and optimal performance regarding to the protection of AA during storage. Comparing the two methods, the microcapsules obtained by microfluidic device conferred better stability to AA, however samples obtained by complex coacervation were applied in sausage due to the greater amount of AA in its constitution. The effect of the application of microcapsules in sausages was evaluated during 40 days at refrigerated storage as it will be seen in Chapter 4. Five treatments were prepared and analyzed according to the stability of the meat emulsion during processing, moisture, water activity, pH changes, determination of instrumental color, instrumental texture profile, oxidative stability by the method of thiobarbituric acid reactive substances (TBARS) and sensory acceptance. The application of AA microcapsules in sausage was possible without compromising the quality of the final product. All data were statistically analyzed by ANOVA and Tukey test, at 5% of significance with the use of SAS software. The experiments related to encapsulation by complex coacervation and application of microcapsules in sausage were carried out at Laboratory of Functional Products, at Department of Food Engineering, FZEA / USP. The experiments related to the use of microfluidic devices were performed in the laboratories of Professor David A. Weitz, at School of Engineering and Applied Sciences of Harvard, at Harvard University, Cambridge, USA.

Diversos estudos têm demonstrado o envolvimento benéfico da utilização do aminoácido glutamina em meios de cultura, favorecendo a organogênese dos tecidos vegetais cultivados. Sabe-se que as fontes de nitrogênio podem influenciar na produção endógena de fitormônios, entretanto o papel exato da glutamina ainda não está bem estabelecido. Em Ananas comosus (L.) Merr., a adição de glutamina ao meio de cultura exerceu efeito promotor sobre a taxa de organogênese e o vigor do crescimento das gemas caulinares a partir de bases foliares. Além da glutamina, discute-se se o estresse resultante da explantação também poderia estar envolvido com a indução do processo organogenético, acarretando na produção de espécies reativas de oxigênio e na alteração do estado redox endógeno. Esse estresse para ser benéfico, entretanto, deveria estar restrito a certo limite. O presente trabalho visou compreender o efeito favorável da glutamina na organogênese adventícia em bases foliares de abacaxizeiro cultivadas in vitro. O envolvimento da glutamina com uma possível diminuição do estresse oxidativo durante o período de indução da organogênese também foi abordado. Para tanto, buscou-se correlacionar a influência do suprimento de glutamina no meio de cultura com os teores endógenos de peróxido de hidrogênio, glutationa e ascorbato. O estado redox da glutationa e do ascorbato durante o período de indução da organogênese adventícia também foi analisado. Além disso, foram analisadas as atividades de duas enzimas antioxidantes nesses explantes foliares, a superóxido dismutase e a catalase. Tentativamente, a glutationa foi adicionada ao meio de cultura, contendo ou não glutamina, visando conhecer o efeito desse antioxidante no processo organogenético. Os resultados mostraram que a glutationa substituiu, mas não intensificou, o efeito benéfico da glutamina sobre a taxa de organogênese das bases foliares de abacaxizeiro. Esse antioxidante não substituiu o efeito positivo do aminoácido no ganho de massas fresca e seca dos eixos caulinares formados, no entanto atuou favoravelmente na formação de um maior número de gemas adventícias por explante inoculado. Ao que parece, o cultivo in vitro das bases foliares gerou um estresse oxidativo nesses tecidos logo no início do período de cultivo, a julgar pela alta concentração de H2O2 detectada nas primeiras 24 horas. Entretanto, essa possível condição estressante foi controlada ao longo do período de cultivo, retornando a uma homeostase do tecido e conferindo condição para que as células se reprogramassem para seguir a uma rota de organogênese caulinar. A glutamina pareceu favorecer a manutenção de um estado redox reduzido tanto de ASC quanto de GSH durante o período em que houve o possível estresse oxidativo. Os resultados das atividades das enzimas antioxidantes sugeriram que a CAT pode ter sido responsável pela regulação do conteúdo endógeno de H2O2, já que a SOD não apresentou alterações expressivas ao longo do período de indução da organogênese tanto em SIM quanto em SIMGln. Em conjunto, os resultados sugerem que o estresse oxidativo causado pelo cultivo in vitro pode ter gerado uma sinalização importante para que a organogênese se inicie, sendo que a glutamina exerceria um papel de manter o estado redox dos tecidos foliares reduzido no momento da maior concentração de H2O2 endógeno. / A positive influence of glutamine on organogenesis of in vitro cultured plant tissues has been demonstrated by several studies. It is well known that the endogenous synthesis of phytohormones can be influenced by nitrogen sources, although it is not completely established in which way glutamine acts in this process. The addition of this amino acid to the culture medium has enhanced the organogenesis rate and resulted in a better vigor of the shoots that were originated from the leaf bases of Ananas comosus (L.) Merr. cultured in in vitro conditions. It is also suggested that the tissue excision may result in a stressful condition by increasing the production of reactive oxygen species and changing the endogenous redox state, which might be involved in the induction of organogenic process. However, this stress should be beneficial only if restricted. The aim of this work was to comprehend the positive influence of glutamine on the in vitro adventitious organogenesis of pineapple leaf bases. It was also attempted to determine whether the glutamine would be involved on a possible oxidative stress decrease during the organogenesis induction. In order to answer these questions, we tried to correlate the presence of glutamine in the culture medium and the endogenous hydrogen peroxide, glutathione, ascorbate levels. The redox state of these antioxidants is also analyzed during the induction of adventitious shoot organogenesis. Moreover, two antioxidants enzymes activities are quantified in the leaf explants: catalase and superoxide dismutase. The glutathione influence on the process was also investigated, considering the glutamine presence or not. It was done in order to establish the effect of this antioxidant in the organogenic process. The results showed that glutathione could replace, but not enhance, the positive effect of glutamine on the organogenesis rate of pineapple leaf bases. This antioxidant did not substitute the positive effect presented by the glutamine on the acquisition of fresh and dry masses by the new shoots. On the other hand, glutathione enhanced the number of adventitious buds per explant. Apparently, the excision of the leaf bases and its subsequent cultivation in the induction culture medium resulted in the tissue oxidative stress early in the first 24 hours of incubation. This could be inferred by the high H2O2 concentrations detected during this period. However, this possible stressful condition was controlled during the culture period, leading to the return of the homeostasis of the tissue and allowing the cells to become determined to shoot organogenesis. During the probable period of oxidative stress, glutamine seemed to maintain the reduced redox state on both ASC and GSH. The results of the antioxidant enzymes activities suggested that CAT may have been responsible for the regulation of the endogenous H2O2 levels, while SOD did not showed significant changes during the induction of organogenesis of leaf bases cultivated either in SIM or SIMGln. Taken together, the results obtained in this work suggest that the oxidative stress caused by the excision of the leaf tissues and its in vitro cultivation may be an important signal to the induction of the leaf organogenesis. Furthermore, the glutamine may have a role in the maintenance of the reduced redox state when higher levels of endogenous H2O2 are present in the tissues.

A espécie Campomanesia phaea (O. Berg.) Landrum é popularmente conhecida como \"cambuci\". É uma das diversas espécies brasileiras de Myrtaceae com fruto comestível e ocorre nos estados de Minas Gerais e São Paulo, principalmente na Serra do Mar. Os frutos possuem intenso aroma agradável e são importantes fontes de compostos fenólicos. Apesar do excelente sabor e aroma do cambuci, a alta adstringência dos frutos, devido ao elevado conteúdo de taninos, reduz a sua aceitabilidade e aplicabilidade na indústria alimentícia. Uma maneira eficaz para remoção da adstringência é submeter o fruto a um processo de destanização, tal como atualmente utilizado para o caqui. O objetivo deste trabalho foi avaliar a variabilidade dos frutos provenientes de uma mesma região e de diferentes regiões, caracterizar frutos em diferentes estádios de maturação, avaliar a eficiência de tratamentos de destanização e determinar o efeito da destanização sobre o potencial funcional, através da determinação do teor e perfil de compostos fenólicos, capacidade antioxidante in vitro e inibição das enzimas do metabolismo de carboidratos. Os tratamentos testados foram: câmaras de etileno, câmara de etanol, ambiente anóxico, etanol no cálice e imersão em etanol. Os resultados mostram que frutos de uma mesma localidade apresentam variações no tamanho e na acidez. Frutos de diferentes regiões apresentaram variação significativa nos teores de fenólicos totais, minerais e na capacidade antioxidante. Com a maturação não foi observado grande variação no teor de sólidos solúveis totais e nem na acidez, porém o conteúdo de fenólicos decresce do estádio mais jovem para o mais maduro. Os processos de destanização com vapor e imersão em etanol foram os mais eficientes na redução do conteúdo de taninos, no entanto esses processos reduziram a capacidade antioxidante e a capacidade de inibição das enzimas &#945;-amilase e &#945;-glicosidase dos frutos. / The specie Campomanesia phaea (O. Berg.) Landrum is popularly known as \"cambuci\". It is one of several Brazilian species of Myrtaceae with edible fruit and occurs in the states of Minas Gerais and São Paulo, especially in the mountains of the sea. The fruits have an intense aroma and are important sources of phenolic compounds. Despite the excellent flavor and aroma of cambuci, high astringency of the fruit, due to the high tannin content, reduces its acceptability and applicability in the food industry. An effective way to astringency removal is to submit the fruit to a deastringency process, as currently used for the persimmon. The aim of this study was to evaluate the variability of the fruits from the same region and from different regions, characterize fruits in different maturation stages, evaluate the efficiency of treatments to astringency removal, and determine the effect of astringency removal about the functional potential, by determining the content and profile of phenolic compounds, antioxidant capacity, and in vitro inhibition of enzymes of carbohydrate metabolism. Treatments tested for removing the astringency were: ethylene chamber, ethanol chamber, anoxic environment, ethanol in the cup, and immersion in ethanol. The results showed that fruit in the same locality exhibit variations in size and acidity. Fruits from different regions showed significant variation in levels of total phenolics, minerals and antioxidant capacity. During maturation it was not observed a wide variation in the content of total soluble solids and acidity, but the phenolic content decreased from the younger stage to the more mature stage. The processes of detannization with ethanol steam and dipped in ethanol were the most effective in reducing the tannin content, however these processes led to reduced antioxidant capacity and capacity of inhibition of the enzymes &#945;-amylase and &#945;-glucosidase in fruits.