Antioxidant profile of Stevia rebaudiana Bertoni cultivated in Portugal and submitted to different conservation conditions

The demand for natural sweeteners has been gaining more and more importance due to the great controversy associated with the use of some synthetic sweeteners as cyclamates, aspartame and acesulfame-K. The steviol glycosides (E 960) are a group of natural sweeteners of generalized use; these compounds are obtained from Stevia rebaudiana Bertoni, a sweet plant native from South America (Carocho et al., 2015). However, Stevia rebaudiana Bertoni may have other uses to be exploited, in particular due to its antioxidant capacity. This plant is already produced in Portugal but it is important to evaluate if the plant chemical composition is maintained regardless of culture conditions. Therefore, in this study, stevia samples were cultivated in Braganca (northeastern of Portugal) in a field trial with defined culture conditions. After harvesting, the plants were submitted to two different treatments: kept fresh by freezing (-20°C) and oven-dried (30°C). The antioxidant profile of the samples was studied through evaluation of free radicals scavenging activity, reducing power, phenolic compounds (HPLC-DAD-ESI/MS), tocopherols (HPLC-fluorescence) and free sugars (HPLC-RI). Significant differences were observed: while oven-dried samples showed the highest antioxidant activity and phenolic compounds concentration (mainly 5-O-caffeoylquinic acid and 3,5-O-dicaffeoylquinic acid), the frozen fresh samples had the highest values of total tocopherols and total sugars. These results confirm that the plants grown in Bragança have excellent bioactive secondary metabolites responsible for the observed antioxidant capacity.

Effets préventifs et thérapeutiques des polyphénols dans un modèle in vitro et in vivo de maladie inflammatoire de l’intestin : caractérisation des polyphénols de la pelure de pomme et de la canneberge par spectrométrie de masse

La muqueuse intestinale est exposée à des agents oxydants provenant de l’ingestion d’aliments modifiés, de cellules immuno-inflammatoires et de la flore intestinale. Une diète élevée en fruits et légumes peut diminuer le stress oxydant (SOx) ainsi que l’inflammation via plusieurs mécanismes. Ces effets bénéfiques peuvent être attribuables à leur contenu élevé en polyphénols. La première étude de mon doctorat consistait à tester l’hypothèse que les polyphénols extraits de pelures de pomme (DAPP) pouvaient diminuer le stress oxydant et l'inflammation impliqués dans les maladies inflammatoires de l'intestin (MII). Nous avons caractérisé les polyphénols des DAPP par spectrométrie de masse (LC-MS) et examiné leur potentiel antioxydant et anti-inflammatoire au niveau des cellules intestinales. L’identification des structures chimiques des polyphénols a été effectuée par LC-MS. Le SOx a été induit par l’ajout du complexe fer/ascorbate (Fe/Asc, 200 µM/2 mM) et l’inflammation par la lipopolysaccharide (LPS, 200µg/mL) à des cellules intestinales Caco-2/15 pré-incubées avec les DAPP (250 µg/mL). L’effet du SOx est déterminé par le dosage du malondialdéhyde (MDA), de la composition des acides gras polyinsaturés et de l’activité des enzymes antioxydantes endogènes (SOD et GPx). L’impact des DAPP sur l’inflammation a été testé par l’analyse de l’expression des marqueurs inflammatoires: cyclooxygénase-2 (COX-2), le facteur de nécrose tumorale alpha (TNF-a et l’interleukine-6 (IL-6) et les facteurs de transcription NF-KB, Nrf-2 et PGC1α par immunobuvardage. Nos données ont montré que les flavonols et les flavan-3-ols constituent les composés polyphénoliques majoritaires des DAPP. L’ajout de Fer2+/Asc a provoqué une augmentation de la peroxidation lipidique comparativement aux cellules contrôles, un appauvrissement des acides gras polyinsaturés n-3 et n-6, et une modulation des enzymes antioxydantes, se traduisant par une augmentation de l’activité de la SOD et une diminution de la GPx. En contrepartie, les DAPP ont exhibé leur potentiel à corriger la plupart des perturbations, y compris l’expression protéique anormalement élevée du COX-2 et la production de la prostaglandine E2 (PGE2), ainsi que l’inflammation telle que réflétée par les facteurs NF-κB, TNF-α et IL-6. Par ailleurs, les mécanismes sous-jacents à ces changements bénéfiques des DAPP ont fait intervenir les facteurs de transcription antioxydants (Nrf-2, PGC1α). Vraisemblablement, cette première étude a permis de démontrer la capacité des DAPP à amoindrir le SOx et à réduire l’inflammation, deux processus étroitement impliqués dans les MII. Dans la deuxième étape de mon doctorat, nous avons voulu comparer les résultats de DAPP à ceux des polyphénols dérivant de la canneberge qui est considérée par la communauté scientifique comme le fruit ayant le plus fort potentiel antioxydant. À cette fin, nous avons caractérisé l’effet des composés polyphénoliques de la canneberge (CPC) sur le SOx, la défense antioxydante et l’inflammation au niveau intestinal tout en définissant leur métabolisme intraluminal. Les différents CPC ont été séparés selon leur poids moléculaire par chromatographie et leurs structures chimiques ont été identifiées par LC-MS. Suite à une pré-incubation des cellules Caco-2/15 avec les extraits CPC (250 µg/mL), le Fe/Asc et la LPS ont été administrés comme inducteurs du SOx et de l’inflammation, respectivement. La caractérisation globale des CPC a révélé que les acides phénoliques composaient majoritairement l’extrait de canneberge de petit poids moléculaire (LC) alors que les flavonoïdes et les procyanidines dimériques/trimériques représentaient l’extrait de poids moléculaire moyen (MC) tout en laissant les procyanidines oligo et polymériques à l’extrait de haut poids moléculaire (HC). Les CPC ont permis de restaurer la plupart des perturbations engendrées dans les Caco-2/15 par le Fe/Asc et le LPS. Les CPC exhibaient le potentiel d’abaisser les niveaux de MDA, de corriger la composition des acides gras polyinsaturés n-3 et n-6, d’augmenter l’activité des enzymes antioxydantes (SOD, GPx et CAT) et d’élever l’expression de Nrf2 et PGC1α. En outre, les CPC pouvaient aussi réduire les niveaux élevés des protéines inflammatoires COX-2, TNF-α et IL-6 ainsi que la production des PGE2 par un mécanisme impliquant le NF-κB. Au niveau mitochondrial, les procyanidines oligomériques ont réussi à corriger les dysfonctions reliées à la production d’énergie (ATP), l’apoptose (Bcl-2, Cyt C et AIF) et le statut des facteurs de transcription mitochondriaux (mtTFA, mtTFB1, mtTFB2). Dans le but de bien comprendre les mécanismes d’action des CPC, nous avons défini par LC-MS les composés polyphénoliques qui ont été transportés ou absorbés par l’entérocyte. Nos analyses soulignent le transport (i) des acides cinnamiques et benzoïques (LC); (ii) la quercétine glycosylée et conjuguée et les procyanidines dimériques de type A (MC); et (iii) l’épicatéchine et les procyanidines oligomériques (HC). Les processus de métabolisation (méthylation, glucuronidation et sulfatation) au niveau de l’entérocyte ont probablement permis le transport de ces CPC surtout sous leur forme conjuguée. Les procyanidines oligomériques ayant un degré de polymérisation supérieur à 2 (HC) ont semblé adhérer aux cellules Caco-2/15. L’épicatéchine suivi par les procyanidines dimériques de type A ont été trouvés majoritaires au niveau des mitochondries. Même si nous ignorons encore l’action biologique de chaque composé polyphénolique, nous pouvons suggérer que leurs effets combinatoires exercent des fonctions antioxydantes, anti-inflammatoires et mitochondriales dans le modèle intestinal Caco-2/15. Dans une troisième étape, nous avons procédé à l’évaluation des aspects préventifs et thérapeutique des DAPP tout en sondant les mécanismes sous-jacents dans une étude préclinique. À cette fin, nous avons exploité le modèle de souris avec colite expérimentale provoquée par le Dextran Sulfate de Sodium (DSS). L’induction de l’inflammation intestinale chez la souris C57BL6 a été effectuée par l’administration orale de DSS à 2.5% pendant 10 jours. Des doses physiologiques et supra-physiologiques de DAPP (200 et 400 mg/kg/j, respectivement) ont été administrées par gavage pendant 10 jours pré- et post-DSS. L’inflammation par le DSS a provoqué une perte de poids, un raccourcissement du côlon, le décollement dystrophique de l’épithélium, l’exulcération et les infiltrations de cellules mono et polynucléaires au niveau du côlon. De plus, le DSS a induit une augmentation de la peroxidation lipidique, une régulation à la baisse des enzymes antioxydantes, une expression protéique à la hausse de la myéloperoxidase (MPO), du COX-2 et de la production des PGE2. Par ailleurs, les DAPP ont permis de corriger ou du moins d’alléger la plupart de ces anomalies en situation préventive ou thérapeutique, en plus d’abaisser l’expression protéique de NF-κB et des cytokines inflammatoires (TNF-a et l’IL-6) tout en stimulant les facteurs de transcription antioxydants (Nrf-2, PGC1α). Conséquemment, les polyphénols des DAPP ont exhibé leur puissant pouvoir antioxydant et anti-inflammatoire au niveau intestinal dans un modèle in vivo. Leurs actions sont associées à la régulation des voies de signalisation cellulaire et des changements dans la composition du microbiote. Ces trois projets de recherche permettent d’envisager l’évaluation des effets préventifs et thérapeutiques des DAPP cliniquement chez les patients avec des désordres inflammatoires de l’intestin.

GC-MS Analysis, Antimicrobial and Antioxidant Activities of Extracts of the Aerial Parts of Conyza sumatrensis

Phytochemical analyses as well as antimicrobial and antioxidant activities of the extracts of C. sumatrensis aerial parts were investigated in this study. METHODS: The aerial parts of C. sumatrensis were air dried, weighed and exhaustively extracted with hexane, ethyl acetate and methanol successively. The crude extracts were screened for metabolites. These extracts of the plant were evaluated for antimicrobial and antioxidant activities using agar diffusion and DPPH method respectively. The extracts were also analysed using Gas chromatography – Mass spectrometry, and the chromatogram coupled with mass spectra of the compounds were matched with a standard library. RESULTS: Preliminary phytochemical investigation of crude n-hexane, ethyl acetate and methanol extracts of the aerial parts of Conyza sumatrensis revealed the presence of anthraquinones, flavonoids, terpenoids, phenolics, tannin, glycosides and carbohydrate. All the crude extracts gave a clear zone of inhibition against the growth of the test bacteria ( Staphylococcus aureus , Escherichia coli , Bacillus subtilis , Pseudomona aeruginosa, Salmonella typhi , Klebsiellae pneumonae ) at moderate to high concentrations, as well as test fungi ( Candida albicans , Aspergillus niger , penicillium notatum and Rhizopus stolonifer ) at high concentration. Methanolic extract exhibited significant radical scavenging property with IC50 of 17.08 μg/mL while n-hexane and ethyl acetate extracts showed no significant antioxidant activity. GC-MS of N-hexane extract showed a total number of eleven chemical constituents with α-Farnesene and spathulenol being the most abundance compounds constituting 20.27 and 22.28% of the extract respectively. Ethyl acetate extract revealed thirteen compounds with two most abundant compounds, cis-β-farnesene (16.64 %) and cis-pinane (21.09 %). While methanolic extract affords seventeen compounds with Ephytol being the most abundant compound (19.36 %).

Chemotaxonomic studies on Schwenckia americana LINN.

This study investigated the chemotaxonomic studies on Schwenckia americana Linn., a member of the family Solanaceae predominantly found mostly in low grass fields, Nigeria. The habit is annual herbaceous weed with slender stem characterized with free branching and growing up to 45cm in height.They are used mainly as medicine. The leaves are simple, entire, elliptic to ovate in shape, smooth, variable, petiolate and larger at the lower region of stem and narrowing to smaller almost sessile and oblanceolate towards the apical regions which are alternately arranged and acrescently structured from the top to the base upto 3.7 ± 1.5cm long and 2.4±0.6cm wide. The inflorescence is a panicle of 15 or more flowers occurring at stem terminal.The flowers are whitish tubular structures measuring up to 1.0±0.2cm in length and 0.1cm in diameter. The petals are whitish up to 0.9 ±0.2cm in length and sepals are greenish up to 0.1cm in length. The berry fruit is greenish when unripe and pale yellowish when ripe up to 0.3±0.1cm in diameter. The seeds are blackish and spherical or triangular shaped with rough edges measuring up to 0.1cm in diameter. The epidermal studies reveal anomocytic stomata whereas the trichomes are simple uniseriate forms wth bulge heads. The anatomy of mid-ribs and petioles showed bicollateral vascular systems. There are three vascular traces and the node is unilacunar. The petioles have 2 rib traces at primary growth phase. At secondary growth phase, the mid-rib and petiole revealed vascular arcs and the stem has a ring of open vascular system. The cytological studies showed a diploid chromosome number of 2n = 24 and n = 12 for the haploids. Alkaloids, saponins, tannins, phlobatannins, flavonoids, combined anthraquinones, free anthraquinones and cardiac glycosides are present.

EVALUATION OF THE ANTI-FUNGAL PROPERTIES OF EXTRACTS OF Daniella oliveri

The in vitro anti-fungal activity of leaf and stem bark of Daniella oliveri Rolfe was investigated against selected yeasts and moulds including dermatophytes. Water and methanol were used to extract the powdered leaf and stem bark using cold infusion. Antimicrobial activity was assessed by agar-well diffusion. Phytochemical analysis was carried out using standard procedures. The plant extracts were active against the test organisms at concentrations ranging from 3.125-100 mg/mL. The methanol extracts were more active than the aqueous extracts with the highest inhibition against the yeasts, Candida albicans and Candida krusei (MIC values of 3.125 mg/mL and 6.25 mg/mL respectively). Epidermophyton floccosum and Trichophyton interdigitale were the least inhibited of all the fungal strains. Phytochemical screening revealed the presence of tannins, anthraquinones, flavonoids, cardiac glycosides, alkaloids and saponins. The anti-fungal activity of Daniella oliveri as shown in this study indicates that the plant has the potential of utilisation in the development of chemotherapeutic agents for the treatment of relevant fungal infections.

In vivo antileishmanial activity and chemical profile of polar extract from Selaginella sellowii

The polar hydroethanolic extract from Selaginella sellowii (SSPHE) has been previously proven active on intracellular amastigotes (in vitro test) and now was tested on hamsters infected with Leishmania (Leishmania) amazonensis (in vivo test). SSPHE suppressed a 100% of the parasite load in the infection site and draining lymph nodes at an intralesional dose of 50 mg/kg/day × 5, which was similar to the results observed in hamsters treated with N-methylglucamine antimonate (Sb) (28 mg/Kg/day × 5). When orally administered, SSPHE (50 mg/kg/day × 20) suppressed 99.2% of the parasite load in infected footpads, while Sb suppressed 98.5%. SSPHE also enhanced the release of nitric oxide through the intralesional route in comparison to Sb. The chemical fingerprint of SSPHE by high-performance liquid chromatography with diode-array detection and tandem mass spectrometry showed the presence of biflavonoids and high molecular weight phenylpropanoid glycosides. These compounds may have a synergistic action in vivo. Histopathological study revealed that the intralesional treatment with SSPHE induced an intense inflammatory infiltrate, composed mainly of mononuclear cells. The present findings reinforce the potential of this natural product as a source of future drug candidates for American cutaneous leishmaniasis.

Bioactive compounds of citrus as health promoters

Citrus are a group of fruit species, quite heterogeneous in many aspects, including chemical composition of the fruit. Since ancient times, some citrus fruits were used to prevent and cure human diseases. In the recent decades, it has been demonstrated that fruits can actually help prevent and cure some diseases and above all, they are essential in a balanced diet. Citrus fruits, as one of the groups of fruit species, with greater importance in the world, have been studied for their effects on human health. Some species of citrus were referred as potential antioxidant based therapy for heart disease, cancer and inflammation. Fruit peels and seeds have also high antioxidant activity. The health benefits of citrus fruit have mainly been attributed to the high level of bioactive compounds, such as phenols (e.g., flavanone glycosides, hydroxycinnamic acids), carotenoids and vitamin C. These compounds are present in the fruit pulp and hence in the juice. But some bioactive compounds can be found in parts of the fruit which usually are not used for human food. The content of bioactive compounds depends on the species and cultivar, but also depends on the production system followed in the orchard. Citrus fruits, their derivatives and their by-products (peel, pulp and oil) are reach in different bioactive compounds and its maturity, postharvest and agroindustry processes influence their composition and concentration. The aim of this chapter was to review the main bioactive compounds of the different components of citrus and their relationship to health.

Effect of fertilization and type of pruning on quality of fresh and dehydrated Stevia rebaudiana

Mestrado em Engenharia Alimentar - Instituto Superior de Agronomia - UL

Antipyretic, analgesic and anti-inflammatory activities of methanol extract of root bark of Acacia jacquemontii Benth (Fabaceae) in experimental animals

Purpose: To investigate the ethnomedicinal claims regarding the use of Acacia jacquemontii Benth. (Fabaceae) in fever, pain and inflammation. Methods: The methanol root bark extract (AJRBM) of the plant was used in the studies. Preliminary phytochemical screening of the extract was carried out according to established methods. Analgesic, anti-inflammatory and antipyretic activities were evaluated using acetic acid-induced writhing, carrageennan-induced rat paw edema and Brewer’s yeast-induced pyrexia models, respectively. The extract was administered at doses of 50 and 100 mg/kg. Aspirin (300 mg/kg, p.o.) was used as a reference drug in all models. Normal saline (10 mL/kg p.o.) was used as negative control. Results: Phytochemical screening results indicate the presence of cardioactive glycosides, tannins, flavonoids and saponins. In the acetic acid-induced writhing model, the methanol extract exhibited significant (p < 0.05) analgesic effect with 58.98 % reduction in writhing response at a dose of 100 mg/kg, compared with untreated control group. The extract significantly (p < 0.05) reduced carrageenan-induced edema at doses of 50 and 100 mg/kg to 36.84 and 47.36 %, respectively, after 1 h of extract administration. The extract exhibited predominantly dose-dependent antipyretic effect in Brewer’s yeast-induced pyrexia model. Maximum reduction in body temperature to 37.07 and 38.29 ºC at doses of 50 and 100 mg/kg, respectively, was observed, compared with untreated group (38.90 ºC) after 1 h, but this was not significant (p < 0.05). Conclusion: The plant extract exerts inhibitory effect on peripheral pain stimuli, edema and dosedependent anti-pyrexia, and thus justifies the ethnomedicinal use of Acacia jacquemontii Benth. in the management of pain, fever and inflammation.

Phytopharmacological and ethnomedicinal uses of the Genus Berberis (Berberidaceae): A review

Plants belonging to Berberis are reported in several folklore medicinal pharmacopeias and are used in traditional medicines in Asia and European countries. The plants have been used in the preparation of various traditional and synthetic medicines since pre-historic times for wound healing, fever, eye disease, jaundice, vomiting during pregnancy, rheumatism, kidney and gall balder stones, and several other illnesses. Their healing properties are appear to be due to the presence of secondary metabolites and important alkaloids with different pharmacological activities. Their antibacterial, antifungal, antiviral, anti-diabetic, and anti-tumor activities as well as positive effects on the cardiovascular and body immune systems have been reported. Root extracts of some species of the plant genus contain quinine which acts as a powerful anti-malarial agent. The main chemical constituents of Berberis plants are alkaloids, steroids, glycosides, flavonoids, saponins, terpenoids and reducing sugars. Of these alkaloids, berberine is the most important. The present review focuses on recent advances in phytopharmacological and ethnomedicinal uses of plants belonging to Berberis genus.

Anthrone and oxanthrone C,O-diglycosides from Picramnia teapensis

Two C,O-diglycosylated compounds, the anthrone picramnioside F, and the oxanthrone mayoside C, were isolated from the stem bark of Picramnia teapensis, along with the previously reported anthraquinones, 1-O-beta -D- and 8-O-beta -D-glucopyranosyl emodin. The compounds were separated by recycling-HPLC, and their structures were determined on the basis of spectroscopic analysis. CD measurements were used to establish the absolute configuration of the anthrone and oxanthrone. The antifungal activity of 1-O-beta -D- and 8-O--D-glucopyranosyl emodin against Leucoagaricus gongilophorus was shown to be similar to that of the lignan sesamin. (C) 2000 Elsevier B.V. Ltd. All rights reserved.

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

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

The natural plant stress elicitor cis-jasmone causes cultivar-dependent reduction in growth of the stink bug, Euschistus heros and associated changes in flavonoid concentrations in soybean, Glycine max.

To test the hypothesis that the plant stress related elicitor cis-jasmone (cJ) provides protection in soybean pods against the seed-sucking stink bug pest, Euschistus heros, the growth of E. heros on cJ-treated pods was investigated using three soybean cultivars differing in insect susceptibility, i.e. BRS 134 (susceptible), IAC 100 (resistant) and Dowling (resistant). E. heros showed reduced weight gain when fed cJ-treated Dowling, whereas no effect on weight gain was observed when fed other treated cultivars. Using analysis of variance, a three factor (cultivar x treatment x time) interaction was observed with concentrations of the flavonoid glycosides daidzin and genistin, and their corresponding aglycones, daidzein and genistein. There were increases in genistein and genistin concentrations in cJ-treated Dowling at 144 and 120 h post treatment, respectively. Higher concentrations of malonyldaidzin and malonylgenistin in Dowling, compared to BRS 134 and IAC 100, were observed independently of time, the highest concentrations being observed in cJ-treated seeds. Levels of glycitin and malonylglycitin were higher in BRS 134 and IAC 100 compared to Dowling. Canonical variate analysis indicated daidzein (in the first two canonical variates) and genistein (in the first only) as important discriminatory variables. These results suggest that cJ treatment leads to an increase in the levels of potentially defensive isoflavonoids in immature soybean seeds, but the negative effect upon E. heros performance is cultivar-dependent.