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Polysaccharides are well known for their immunostimulatory properties, which are closely related to structural fèatures, such as molecular weight, branching, acetylation degree and acetylation pattem, and presence of speciflc structures [1, 2]. TTie electrospray ionization-mass spectrometry (ESI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) have shown to be powerfül techniques that can provide detailed information regarding the structural features of polysaccharides and help to clarify its relation to some ofthe observed immunostimulatory properties [3-6]. TTie present work desenhes the use of ESI-MS and ESI-MS/MS in the confimation and elucidation of structural features of immunostimulatory polysaccharides present in lhe aqueous extracts of Fraxinus angustifolia dned leaws and Ptemapaitum tridentatum dried inflorescences. The polysaccharides present in the aqueous extracts of F. angustifolia dried leaves and P. fridentafum dried inflorescences were isolated and fractionated using dialysis, ethanol precipitation, and anionexchange chromatography. Sugar and linkage analysis suggested that the aqueous extracts of F. angustifolia were mostly composed of pectic polysaccharides, while those from P. tridentatum contained a mixture of pectic polysacchahdes, galactomannans, and also xyloglucans. Selected fractions isolated from lhe aqueous extracts of F. angustifolia and P. tridentatum evidenced an in vitro macrophage immunostimulatory activity, expressed through the production of nitric oxide (NO). Enzymatic hydrolysis of the isolated polysaccharides, fbllowed by size exclusion chromatography of the resulting oligosaccharides (OS), and ESI-MS and ESI-MS/MS characterization allowed to confirm the presence of pectic polysaccharides, possibly with xylogalaturonan domains, in the aqueous extracts of F. angustifolia. For the aqueous extracts of P. fridentatum it was possible to detect the presence of galactomannans acetylated at 0-2. Moreover, it was possible to identify acetylated OS that presented an acetylation pattem with much more acetyl groups than those observed for galactomannans from coffee infusions [3]. The saponification ofthe fraction isolated from the aqueous extracts of P. tridentatum dried inflorescences, which contained the acetylated galactomannans, caused a decrease in the NO macrophage production, confirming lhe galactomannans acetylation as an important structural feature for lhe expression of the in vitm macrophage immunostimulatory activity previously registered. info:eu-repo/semantics/publishedVersion

The decoctions of prickled broom (Pterospartum tridentatum (L.) Willk.) dried inflorescences have claimed beneficial properties for various health disorders1'2'3. These can be assigned to several constituents, among them, the polysacharides. In order to unveil the contribution of the polysacharides and their structural features to the modulation of the innate immune activity, the hot water extracts from P. tridentatum dried inflorescences were prepared and fractionated by ethanol precipitation and anion exchange chromatography. A fraction that mainly contained pectic polysaccharides and acetylated galactomannans was isolated. info:eu-repo/semantics/publishedVersion

The decoctions of prickled broom (Pterospartum trídentatum (L.) Willk.) dried inflorescences have claimed beneficial properties for various health disorders [1, 2]. These can be assigned to several constituents, among them, the polysacharides. In order to unveil the contribution of the polysacharides and their structural features to the modulation of the innate immune activity, the hot water extracts from P. tridentatum dried inflorescences were prepared and fractionated by ethanol precipitation and anion exchange chromatography. A fraction that mainly contained pectic polysaccharides and acetylated galactomannans was isolated. This fraction evidenced in vitro immunostimulatory activity without compromising cellular viability, as evidenced by the increase registered in the nitric oxide (NO) production by macrophages. This activity decreased 60-75% after saponification, confirming that acetylation is an important structural feature for this biological property. In addition, the treatment of pectic polysaccharides with endo-polygalacturonase showed that type-1 and type-11 arabinogalactans, as well as low molecular weight galacturonans and xyloglucans, may also contribute to macrophage NO production. Thus, the polysaccharides present in the dried inflorescences of P. trídentatum may contribute to the health beneficial properties frequently attributed to the decoctions of this plant. FCT, Centro de Investigação de Montanha (CIMO), QOPNA, CNC, Prof. Dra Ana Mana Carvalho info:eu-repo/semantics/publishedVersion

Xanthones (XH) are a class of heterocyclic compounds widely distributed in nature that hold numerous noteworthy biological and antioxidant activities. Therefore, it is of utmost importance to achieve relevant detailed structural information to understand and assist prediction of their biological properties. The potential relationship between radical-mediated xanthone chemistry in the gas phase and their promising antioxidant activities has not been previously explored. Thanks are due to Fundação para a Ciência e Tecnologia (FCT, Portugal), European Union, QREN, FEDER and COMPETE, for funding the QOPNA research unit (project PEst-C/QUI/UI0062/2013; FCOMP-01-0124-FEDER-037296), and the Portuguese National Mass Spectrometry Network, RNEM (REDE/1504/REM/2005). E. M. P. Silva is also grateful to FCT (ref SFRH/BPD/66961/2009) for a Post-Doc grant. info:eu-repo/semantics/publishedVersion

The hot water extracts from Pterospartum tridentatum dried inflorescences are used in the northeastern portuguese region of Tras-os-Montes due to its therapeutic properties. The therapeutic properties attributed to hot water extracts from plants are often related with polysaccharide macrophage immunostimulatory activity. This work intends to identify the structural features of polysaccharides from Pferospartum tridentatum hot water extracts and to evaluate their putative macrophage immunostimulatory activity. which may contribute to their therapeutic properties. info:eu-repo/semantics/publishedVersion

Pterospartum tridentatum Willk. (prickled broom) is a common autochthonous plant in Portugal. Leaves and stems are normally used in cooking, to flavour rice, roast meat or hunting animals. Leaves are also used as a condiment in fresh salads and, despite of its traditional use, no toxicological evaluation has been performed. P. tridentatum leaves aqueous extract ESI-MS spectrum revealed the presence of several luteolin and isorhamnetin derived phenolic compounds, which can be associated to the health benefits claimed for this plant species. Still, P. tridentatum leaves extract (up to 100 µg plant extract.mg-1 protein) stimulated state 4 and FCCP-stimulated liver mitochondria respiratory rates and inhibited the state 3 respiratory rate. Respiratory control ratio was decreased, indicating a dysfunction in respiratory activity induced by P. tridentatum leaves extract and, in good agreement with the previous results cytotoxicity evaluation by MTT assay (50 and 125 µg plant extract.10-6 cells) showed a decrease on HepG2 cell viability. Overall, the present study suggests that the consumption of P. tridentatum leaves in high amounts or continuously should be regarded as potencially noxious.

The dried leaves of Fraxinus angustifolia and the dried shoots of Mentha suaveolens are used by the population of the Tras-os-Montes, which is a region located in the northeast of Portugal. to prepare hot water extracts due to their therapeutic properties. Recently, the contribution of potysaccharides for the therapeutic properties often attributed to various plants hot water extracts has been highlighted. This work will provide a structural characterization of the polysaccharides present in these hot water extracts and will also evaluate their possible macrophage immunostimutatory activity, which might contribute for the therapeutic properties reported for these hot water extracts. info:eu-repo/semantics/publishedVersion

Pterospartum tridentatum Willk. (prickled broom) is an autochthonous and common plant in Portugal. Leaves and stems are normally used in cooking, to flavour rice, roast meat or hunting animals. Leaves are also used as a condiment in fresh salads and, despite of its traditional use, no toxicological evaluation has been performed. P. tridentatum leaves aqueous extract ESI-MS spectrum revealed the presence of several luteolin and isorhamnetin derived phenolic compounds, which can be associated to the health benefits claimed for this plant species. Still, P. tridentatum leaves extract (up to 100 µg plant extract.mg-1 protein) stimulated state 4 and FCCP-stimulated liver mitochondria respiratory rates and inhibited the state 3 respiratory rate. Respiratory control ratio was decreased, indicating a dysfunction in respiratory activity induced by P. tridentatum leaves extract and, in good agreement with the previous results, cytotoxicity evaluation by MTT assay (50 and 125 µg plant extract.10-6 cells) showed a decrease on HepG2 cell viability. Overall, the present study suggests that the consumption of P. tridentatum leaves in high amounts or continuously should be regarded as noxious.

Pterospartum tridentatum Willk. is an autochthonous and common plant in Portugal, locally known as ‘carqueija’. Leaves and stems are normally used in cooking, to flavour rice, roast meat or hunting animals and leaves are also used as condiment in fresh salads. Despite its traditional use, no evaluation in mitochondrial bioenergetics has been performed. P. tridentatum leaf extract phenolic composition was elucidated by electrospray ionization-mass spectrometry (ESI-MS). M. aquatica extract effects were evaluated over mitochondrial membrane potential by using a Clarktype oxygen electrode in the presence of succinate as respiratory substrate. Cytotoxicological effects of plant extracts were evaluated on HepG2 cells throughout MTT assay. The ESI-MS spectrum of P. tridentatum leaves aqueous extract revealed the presence of several luteolin and isorhamnetin derived phenolic compounds, which can be associated to the health benefits claimed for this plant species. Yet, P. tridentatum leaves extract (up to 100 lg plant extract mg protein _1) stimulated state 4 and FCCP-stimulated liver mitochondria respiratory rates and inhibited the state 3 respiratory rate. Hence, respiratory control ratios were diminished, indicating a phosphorylative efficiency decrease, due to inner mitochondrial membrane permeability induced by P. tridentatum leaves extract. Nevertheless, P/O ratios were not affected in the range of concentrations tested and cytotoxicity evaluation using MTT assay (50 and 125 ug plant extract) showed no significant decrease over HepG2 cell viability. This dual behaviour reflects the P. tridentatum hormetic effects over mitochondrial activity. Conclusion: Overall, the present study suggests that consumption of P. tridentatum leaves should be regarded as safe and present benefits concerning the modern lifestyles.

Pterospartum tridentatum Willk. is an autochthonous and common plant in Portugal, locally known as ‘carqueija’. Leaves and stems are normally used in cooking, to flavour rice, roast meat or hunting animals and leaves are also used as condiment in fresh salads. Despite its traditional use, no evaluation in mitochondrial bioenergetics has been performed. P. tridentatum leaf extract phenolic composition was elucidated by electrospray ionization-mass spectrometry (ESI-MS). M. aquatica extract effects were evaluated over mitochondrial membrane potential by using a Clarktype oxygen electrode in the presence of succinate as respiratory substrate. Cytotoxicological effects of plant extracts were evaluated on HepG2 cells throughout MTT assay. The ESI-MS spectrum of P. tridentatum leaves aqueous extract revealed the presence of several luteolin and isorhamnetin derived phenolic compounds, which can be associated to the health benefits claimed for this plant species. Yet, P. tridentatum leaves extract (up to 100 lg plant extract mg protein _1) stimulated state 4 and FCCP-stimulated liver mitochondria respiratory rates and inhibited the state 3 respiratory rate. Hence, respiratory control ratios were diminished, indicating a phosphorylative efficiency decrease, due to inner mitochondrial membrane permeability induced by P. tridentatum leaves extract. Nevertheless, P/O ratios were not affected in the range of concentrations tested and cytotoxicity evaluation using MTT assay (50 and 125 ug plant extract) showed no significant decrease over HepG2 cell viability. This dual behaviour reflects the P. tridentatum hormetic effects over mitochondrial activity. Conclusion: Overall, the present study suggests that consumption of P. tridentatum leaves should be regarded as safe and present benefits concerning the modern lifestyles.

Mentha aquatica L., commonly known as water mint, is a perennial herb that grows in Europe temperate regions. This plant is used in traditional medicine for the treatment of external inflammation, rheumatism, colds, respiratory problems and difficult menstruation [1]. It has been described as a good source of phenolic compounds with high scavenger activity [2]. In this study, a purified ethanolic extract of M. aquatica was prepared and its specific phenolic composition was determined. The extract of the aerial parts of M. aquatica was prepared according the procedure described by Pereira et al [3] and analyzed by high performance liquid chromatography with diode array detection with quantification of main phenolic compounds, using the external standard method. In order to determine the exact structure of phenolic compounds, the HPLC eluted fractions were manually collected and further analyzed by tandem electrospray mass spectrometry. The purified ethanolic extract of M. aquatica was mainly rich in flavanones comprises eriodictyol, hesperitin and naringenin glycosides. Moreover, the purified ethanolic extract of M. aquatica contained moderate amounts of rosmarinic acid, a phenolic acid very common in Mentha species and in Lamiaceae family.

Leonurus cardiaca L., also known as motherwort, is a plant native from central Europe that it is spread in different temperate countries around the world. The plant has been described to exert several beneficial properties and it is presently used in infusions, decoctions, syrups and tinctures, as well as an ingredient of pharmaceutical formulations. Yet, the plant phenolics, which are frequently associated to the claimed health benefits of Leonurus cardiaca L., are not fully clarified. This topic is focused in the present work. For that, an extract of the aerial parts of the plant was obtained with aqueous ethanolic solution (80%) and it was further purified onto SPE C18-E cartridges. The resulting purified phenolic extract was analyzed by HPLC-DAD and ESI-MSn techniques, for phenolic identification and quantification. Accordingly, phenolic compounds in Leonurus cardiaca L. purified ethanolic extract accounted for 15 mg/g of dry plant (500 ± 49 mg/g of extract). Phenylethanoid glycosides were the most prevalent phenolics with lavandulifolioside and verbascoside representing 50% and 27% of its total quantified phenolic compounds, respectively. Additionally, the present study allowed the identification and quantification of leucoseptoside A and leonoside B for the first time in Leonurus cardiaca L., which together, accounted for 11% of the quantified phenolics. Besides phenylethanoid glycosides, the Leonurus cardiaca L. ethanolic extract also contained flavonoids (10%) and caffeic acid derivatives, albeit the latter were only present in vestigial amounts. With the exception of luteolin-7-O-rutinoside, all the remaining flavonoids were glycosidic quercetin derivatives, which include the isoquercitrin, rutin, rutin-O-glucoside and quercetin-3-O-sophoroside. Possible association of the major phenolics herein identified with specific health benefits of the plant are under investigation.

Lamium album L., a plant commonly known as white dead nettle, has been used in folk medicine in the treatment of several ailments. Some of their benefitial properties are closely related to its phenolic content, however the exact polyphenolic composition is far from being understood. In this context, the present work aims to determine the phenolic composition of an ethanolic extract of L. album using high performance liquid chromatography with diode array detection, electrospray mass spectrometry and nuclear magnetic resonance techniques. The phenolic extract of L. album was mainly composed by the phenylethanoid verbascoside and its isomer isoverbascoside. Still, it also contained significant amounts of isoscutellarein derivatives, which were herein detected for the first time in the genus Lamium. As isoscutellarein derivatives appear as major components of Lamium Album L., it is feasible that they can be correlated to the beneficial human health properties of this plant.

Cytisus multiflorus(white spanish broom) is a plant used in folk medicine in the Iberian Peninsula, where it is claimed to have various health benefits. However, the usage is totally based on the available ethnopharmacological information, as no scientific data regarding its biological effects has been delivered. In this sense, is the aim of this work to contribute to the scientific knowledge of the antioxidant and anti-inflammatory properties of C. multiflorus. The extract was rich in phenolic compounds mainly chrysin-7-O-glycopyranoside and a dihydroxyflavone isomer of chrysin which accounted for 49.4±7.3 mg/g and 21.8±3.8 mg/g, respectively. The low EC50 values, determined by DPPH and reducing power assays, indicated that the C. multiflorus purified ethanolic extract has a high antioxidant capacity. Moreover, the extract did not cause cytotoxicity against RAW 264.7 macrophages for high concentrations and the treatment of this cell line with purified extract induced a significant decrease in the levels of NO. Overall, the gathered data suggests that C. multiflorus is in fact a good antioxidant and anti-inflammatory plant, as believed by the folk knowledge.

Pterospartum tridentatum Willk. (prickled broom) is an autochthonous and common plant in Portugal. Leaves and stems are normally used in cooking, to flavour rice, roast meat or hunting animals. Leaves are also used as a condiment in fresh salads and, despite of its traditional use, no toxicological evaluation has been performed. P. tridentatum leaves aqueous extract ESI-MS spectrum revealed the presence of several luteolin and isorhamnetin derived phenolic compounds, which can be associated to the health benefits claimed for this plant species. Still, P. tridentatum leaves extract (up to 100 µg plant extract.mg-1 protein) stimulated state 4 and FCCP-stimulated liver mitochondria respiratory rates and inhibited the state 3 respiratory rate. Respiratory control ratio was diminished, indicating a decrease in phosphorylative efficiency due to inner mitochondrial membrane induced by P. tridentatum leaves extract. Nevertheless, previous results, cytotoxicity evaluation by MTT assay (50 and 125 µg plant extract) showed no significant decrease on HepG2 cell viability. Overall, the present study suggests that the consumption of P. tridentatum leaves should be regarded as safe.

Cytisus multiflorus is a well known Mediterranean plant presenting diuretic, anti-inflammatory, anti-hypertension and antidiabetic properties [1]. The high phenolic content of C. multiflorus is frequently associated to its health beneficial properties, however its phenolic composition remains unclear. In this work, the phenolic profile of an extract of C. multiflorus flowers was elucidated. After extraction with an aqueous ethanolic solution (80%), the enrich phenolic extract was analyzed by high performance liquid chromatography with diode array detection, electrospray mass spectrometry [2] and nuclear magnetic resonance techniques. The extract of C. multiflorus was mainly composed with chrysin-7-O-β-D-glycopyranoside (MW 416) that accounted for 15.9±2.3 mg/g of dried plant. It also contained considerable amounts of rutin (4.5±0.7 mg/g), a dihydroxyflavone isomer of chrysin (7.0±1.3 mg/g), 2′′-O-pentosyl-6-C-hexosyl-luteolin (3.3±0.5 mg/g), 2′′-O-pentosyl-8-C-hexosyl-luteolin (3.5 ± 0.3 mg/g) and 6′′-O-(3-hydroxy-3-methylglutaroyl)-2′′-O-pentosyl-C-hexosyl-apigenin (3.6±0.7 mg/g), which are not commonly found in the Fabaceae family. Other unusual phenolic compounds found in minor amounts in the ethanolic extract of C. multiflorus flowers comprised the flavones 2′′-O-pentosyl-6-C-hexosyl-apigenin, 2′′-O-pentosyl-8-C-hexosyl-apigenin and 6′′-O-(3-hydroxy-3-methylglutaroyl)-2′′-O-pentosyl-C-hexosyl-luteolin. The present work emerges as an important contribution for the phenolic characterization of Cytisus genus and of Fabaceae family.

The present study investigates the phenolic composition and the antioxidant capacity of an ethanolic extract from flowers of C. multiflorus. As determined by HPLC-DAD, ESI-MSn and NMR combined analysis, this extract was mainly composed of chrysin-7-O-β-D-glycopyranoside, and also contained considerable amounts of rutin, a dihydroxyflavone isomer of chrysin and glycosidic derivatives of luteolin and apigenin. The C. multiflorus ethanolic extract exhibited high DPPH scavenging activity and reducing power with EC50 of 13.4 ± 0.6 and 95.7 ± 4.6 µg/mL, respectively. The exposure of human hepatoblastoma HepG2 cells to the non-toxic concentrations of the extract (50 or 200 µg/mL) resulted in a decreased rate of ROS production in a concentration dependent manner. Moreover, the mixture of standards that simulated the phenolic composition of the plant afforded a protection of about 50% in intracellular ROS production. Hence, the gathered results suggest that C. multiflorus polyphenols are closely associated to its antioxidant properties.

The main aim of this work is to determine the phenolic composition of a Lamium album L. purified extract, in order to contribute for the chemical characterization of this plant. Moreover, it also intends to emphasize its antioxidant properties. As established by HPLC-DAD and ESI-MSn combined analysis, verbascoside was the major phenolic constituent of Lamium album L. purified extract, as previously described by other authors [1]. Still, we could also detect, for the first time in Lamium genus, significant amounts of isobervascoside and of isoscutellarein derivatives. As confirmed by NMR analysis, the major isoscutellarein derivative in Lamium album L. purified extract was the isoscutellarein-7-O-(6-O-acetyl-β-allosyl)(1→2)-β-glucoside, but isoscutellarein-7-O-allosyl(1→2)glucoside and of O-methyl derivatives of these two compounds were also present in considerable amounts. Aditionally, the extract showed good antioxidant activities in in vitro experiments. Overall, the present study contributes for the chemical and biological characterization of L. album L.

The present study aims to evaluate the in vitro antioxidant potential of Lavandula dentata and Mentha aquatica plant extracts. For that, ethanolic extracts of the two plants were prepared and their phenolic composition was determined through combined methods of HPLC-DAD and ESI-MS. Moreover, the antioxidant activity of the plant extracts was estimated by: i) evaluation of DPPH scavenging potential and ii) monitoring the protective effects against the generation of reactive oxygen species (ROS) induced by potassium dichromate in human hepatoblastoma HepG2 cells. M. aquatica ethanolic extract was much enriched in phenolic compounds, in comparison with that of L. dentata. Both extracts contained rosmarinic acid in similar concentrations but M. aquatica also contained significant amounts of other phenolics, including rutinoside derivatives of eriodictyol and luteolin. The plant extracts showed high radical scavenging activity against DPPH radical and significantly diminished intracellular ROS production under oxidative stress conditions. The latter protection was mostly evidenced in the L. dentata extract and was also observed for the rosmarinic acid used as reference. Attending that rosmarinic acid is a major phenolic component of L. dentata and M. aquatica ethanolic extracts, the present results suggest that this phenolic compound can be involved in the antioxidant properties of both plants.

Introduction: Anthemis nobilis L. (roman chamomille) is an autochthonous plant, very common in North America and in Iberian Peninsula territory. The yellow flower buttons of this plant are widely used in traditional medicine and thus, they are commonly found in herbal stores. Anthemis nobilis L. is used as a potential therapeutic agent for all body illnesses, in particular for fever, inflammatory processes, skin infections and as a tranquilizer (1). In accordance, the main goal of this work was the evaluation of A. nobilis therapeutic potential, with focus on its antioxidant and antimicrobial activities. Methodology: Aqueous extracts of A. nobilis button flowers were prepared by maceration with water at room temperature during 2 hours or by decoction. Both extracts were analysed by electrospray mass spectrometry in the negative mode and their phenolic content was determined by the Folin–Ciocalteu method (2). Antioxidant activity was evaluated through ABTS radical-scavenging activity (3),TBARS method (4) and red blood cells haemolysis (5). Potential antibacterial action of the two extracts was evaluated as the antibiogram potential, analyzing Staphyllococcus aureus and Escherichia coli growth. Results: The aqueous extracts (crude and decoccion) of A. nobilis button flowers showed high antioxidant activity, as evaluated by ABTS, TBARS and haemolysis of red blood cells assays. Moreover, this activity was higher for the decoction extract, in good agreement with its greater phenolic content. As revealed by the mass spectrometry analysis, the potent antioxidant ability of aqueous A. nobilis extracts can result from the presence of quinic acid and caffeic acid derivatives. In opposition, the two aqueous extracts of A. nobilis button flowers showed no inhibitory effect on the growth of S. aureus and E. coli (common Gram+ and Gram- bacteria), thus indicating that this plant is not is not suitable for the treatment of health problems associated to those microorganisms.

O tomilho limão é uma espécie do género Thymus cultivada na região mediterrânica e utilizada, desde há várias décadas, no tratamento de diversas afecções respiratórias e como agente antiséptico e antifúngico [1]. Este estudo pretende contribuir para um melhor conhecimento da espécie Thymus citriodorus, através da identificação e quantificação dos seus principais compostos fenólicos. O perfil fenólico do extracto etanólico de Thymus citriodorus foi obtido por análise de cromatografia líquida de alta resolução (HPLC/UV) e a estrutura dos compostos foi confirmada por análise de espectrometria de massa com ionização por electrospray (ESI-MSn) em modo negativo, de cada uma das fracções eluídas. A análise de HPLC/UV permitiu obter o cromatograma representado na Fig. 1, onde se indicam as principais fracções (B, D e E), e outras de intensidade moderada (A, C e F). A análise de ESI-MSn das fracções B e D revelou a existência de derivados glicosídicos das flavonas luteolina (MW 448 Da) e apigenina (MW 446 Da), respectivamente. Este último apresentou a fragmentação (m/z 445→269→225,183) na análise por ESI-MSn, pelo que foi identificado como apigenina-7-O-glucuronato. O ácido rosmarínico (MW 540 Da) foi identificado na fracção E, uma vez que o seu tempo de retenção, espectro UV-Vis e fragmentação eram coincidentes com o composto padrão. Ainda, de acordo com os seus perfis de fragmentação, as fracções A, C e F foram identificadas como eriodictiol-O-glucósido, crisoeriol-O-glucósido, e derivado do ácido cafeico, respectivamente. A quantificação destes compostos indica que apresentam as quantidades de 3.7±0.5, 5.8±0.5 e 2.3±0.9 μg/mg extracto, embora as flavonas luteolina-7-O-glucósido (12±2 μg/mg extracto), apigenina-7-O-glucuronato (9±2 μg/mg extracto) e o ácido rosmarínico (10.4±0.6 μg/mg extracto) apareçam como os compostos maioritários na planta.

A enorme capacidade poluente das águas ruças produzidas em sistemas de extracção de azeite de três fases deve-se à sua elevada acidez e à grande quantidade de sais de potássio, magnésio, fósforo, e de moléculas orgâncias, especialmente polifenóis. açúcares, taninos, polialcoóis, pectinas, lípidos e compostos aromáticos tóxicos [1]. Por outro lado, alguns destes compostos, nomeadamente os compostos fenólicos, possuem actividades biológicas importantes e são por isso do interesse da indústria farmacêutica e alimentar [2]. A quantidade de compostos fenólicos nas águas ruças depende da maturação das azeitonas, da zona de cultivo, do tempo de armazeanamento e dos procedimentos de extracção [3]. O Hidroxitirosol é um dos mais abundantes e i que levanta mais interesse devido às suas propriedades farmacológicas e antioxidantes, podendo ser encontrado nos efluentes na forma livre, mas também na forma ligada como oleuropeína, verbascosídeo ou glicosídeo [4]. Neste trabalho pretendeu-se determinar a composição em compostos fenólicos de amostras de efluentes líquidos provenientes de diferentes lagares de azeite do Norte de Portugal (Amarante, Mirandela, Castelo Rodrigo e Frieira). Para tal, as águas ruças foram extraídas com acetato de etilo, o extracto foi fraccionado por HPLC e as fracções foram recolhidas e analisadas por espectrometria de massa (ESI-MS e ESI-MSn). Este procedimento permitiu verificar que o composto maioritário em todas as amostras era o hidroxitirosol, embora em concentração variável. As amostras continham ainda outros compostos fenólicos comuns, tais como o ácido clorogénico, o ácido cafeico, o ácido p-cumárico, o tirosol e secoiridoides como o ácido elenólico, o oleosideo, o verbascosídeo, a oleuropeína, o ligostrosídeo e alguns falvonóides como a luteolina, a quercetina e o glucosideo da luteolina.

Olive mill wastewaters (OMW) have high contents of phenolic compounds, which possess a wide range of biological activities. Therefore, OMW are regarded as a potent source of biophenols for food and pharmaceutical industries. Important biophenols occurring in OMW include tyrosol, oleuropein and hydroxytyrosol, with the latter being commercially exploited as a novel ingredient with a broad use in dietary supplements, functional foods and natural cosmetics [1 ,2]. Nevertheless, the phenolic profile of OMW is extremely complex and many compounds remain unknown. The elucidation of new phenolic compounds in this matrix is an important task to encourage the search of more bioactive compounds. Indeed, hydroxytyrosyl acyclodihydroelenolate and p-coumaroyl-6' - secologanoside were recently identified in OMW and proved to have greater antioxidant scavenging activity than hydroxytyrosol and oleuropein [3]. The present study evaluates the recovery of some biophenols from three Portuguese OMW. For that an ethyl acetate liquid-liquid extraction procedure, which has been previously optimized for hydroxytyrosol extraction [4], has been used. On the other hand, this work also contributes to the knowledge of OMW phenolic composition, through the elucidation of major phenolic constituents in purified methanol (PME) or aqueous acetone extracts (PAAE).

Pterospartum tridentatum Willk. (prickled broom) is an autochthonous plant, common in Portuguese territory. The yellow flowers are widely used in traditional medicine, as a potential cure for all body illnesses, mainly for throat irritation treatment or for diabetes, hypertension and hypercholesterolemia therapy. Despite its wide traditional use, no toxicological assessment of this plant has been performed, as far as we know. The major goal of this work was the P. tridentatum water extracts safety assessment. Methodology: Chemical characterization of MS analysis of P. tridentatum flower extract was performed by ESI-MS spectroscopy; phenols and quercetins content were also determined. Antioxidant activity was evaluated through -carotene bleaching test and ABTS radical-scavenging activity. Oxygen consumption of isolated mitochondria was determined polarographically with a Clark oxygen electrode and mitochondrial respiratory rates evaluated accordingly to Chance and Williams (1956) and mitochondrial swelling was evaluated the apparent absorbance changes at 520 nm. Results and discussion: A high antioxidant activity of P. tridentatum flower water extract was assessed in good agreement with its ESI-MS spectrum that revealed the presence of several flavonoids, as luteolin-O-(O-acetyl)-glucuronide, luteolin-Oglucuronide or isorhamnetin-O-hexoside. Mitocondrial respiratory rates (state 4, state 3 and FCCP-stimulated respiration) and respiratory indexes (respiratory control and P/O ratios) showed no consistent decrease of respiratory and phosphorylative efficiencies for the concentrations tested (up to 500 μg.mL-1). In conclusion, for the concentration range commonly used P. tridentatum flowers usage can be regarded as harmless and trustworthy and due to its great antioxidant properties it is useful to counteract diabetes mellitus associated diseases.

Cytisus Desf. (Fabaceae) is a diversified genus enclosing approximately sixty species, which are particularly found around the Mediterranean Sea. Many plants of this genus exhibit bioactive properties such as diuretic, hypnotic, anxiolytic, antiparasitic, antidiabetic and antioxidant [1] and, in particular the latter, has been closely associated to the high content in flavonoids [2]. The present work aims to contribute to the knowledge of Cytisus Desf. chemical composition, through the identification of new flavonoids in that genus. The compounds in focus were detected in ethanolic extracts of Cytisus multiflorus flowers by means of HPLC-DAD, ESI-MS and MSn analyses. These included the two isomers 2′′-O-pentosyl-6-C-hexosyl-luteolin and 2′′-O-pentosyl-8-C-hexosyl-luteolin (MW 580 Da), the two isomers 2′′-O-pentosyl-6-C-hexosyl-apigenin and 2′′-O-pentosyl-8-C-hexosyl-apigenin (MW 564 Da), the 6′′-O-(3-hydroxy-3-methylglutaroyl)-2′′-O-pentosyl-C-hexosyl-luteolin (MW 724 Da) and the 6′′-O-(3-hydroxy-3-methylglutaroyl)-2′′-O-pentosyl-C-hexosyl-apigenin (MW 708 Da). Attending that half of the these compounds were herein described for first time in Fabaceae, overall, the present work is a valuable contribution for the phenolic elucidation of Cytisus genus as well as the Fabaceae family.

Propolis it is a natural resinous substance that honeybees (Apis melifera, L.) collect from buds and exudates of plants and transformed in the presence of bee enzymes. This substance plays an important role in the hive as a construction and defence material due to its chemical and biological properties.[1] Propolis composition is extremely complex and dependent on its vegetal source, geographical location and climatic conditions. Typically this natural product includes resin (50%), beeswax (30%), essential oils (10%), pollen (5%) and other organic compounds (5%).[2] Due to the abundance in phenolic compounds (flavonoids, phenolic acids and their esters) and to its biological and pharmacological properties this group of substances became the target of many research studies in propolis. This work outlines an extensive qualitative and quantitative characterization of the phenolic composition of Azorean islands propolis. For that, eleven samples of propolis, from S. Miguel and Terceira island, were extracted and characterized according to the previous work.[3] The HPLC results show a similar chromatographic profile for all samples, despite their origin, with 37 compounds identified by ESI-MS/MSn analysis and confirmed by HPLC-DAD. Although the samples revealed a similar phenolic composition, significant differences in the concentrations of the different compounds were found, probably due to the differences in flora distribution around the beehive, and therefore resin availability for bees.

Thymus species are well known as medicinal plants because of their biological and pharmacological properties, which include anti-asthmatic, anti-septic, antimicrobial and antioxidant. It is believed that part of these beneficial effects are due to the volatile constituents of Thymus, and thus, their essential oil composition has been the focus of many investigations. In contrast, there is only a limited number of data on the composition of other bioactive phytochemicals of Thymus and their potential biological effects. The present study aims to elucidate the phenolic composition of an ethanolic extract of Thymus citriodorus, as well as to determine its antioxidant capacity. The ethanolic extract was obtained by solubilisation of the defatted-dried plant with aqueous ethanol (80%) for twenty minutes, in a total number of five extractions. The total phenolic compounds in the extract accounted for 139±14 mg/g, as expressed as gallic acid equivalents. Further analysis of the ethanolic extract by high performance liquid chromatography (HPLC) and electrospray mass spectrometry in the negative mode allowed to conclude that its main phenolic components were rosmarinic acid (14.0±0.8 μg/mg extract), luteolin-7-O-glucoside (11±2 μg/mg extract), an apigenin derivative (9±2 μg/mg extract), eriodictyol-O-glucoside (5.5±0.7 μg/mg extract) and naringenin-O-glucoside (1.6±0.1 μg/mg extract). Moreover, the ethanolic extract of Thymus citriodorus exhibited a high antioxidative capacity, with EC50 values of 0.32±0.05 mg/ml for the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging potential and EC50 values of 0.8±0.2 mg/ml for the reducing power. Overall, these results suggest that Thymus citriodorus can be a good source of natural antioxidants.

Portugal geographical situation and therefore their floral diversity points towards the existence of typical propolis from temperate zones, where bud exudates of poplar trees (Populus species) are the main source of this honeybee product With the exception of Madeira and Azores Islands, where the floral diversity could present significant differ· ences, it would be expected to find a product rich in flavonoid aglycones (flavones and flavanones), phenolic acids and their esters, characteristic of European propolis, In order to clarify this issue, several propolis samples were collected along the country, including islands, and their colour, ashes, content of wax, phenols and flavonoids, as also their phenolic profile, were determined

The present work aims to determine the phenolic composition of Thymus citriodorus and to estimate its antioxidant capacity. An ethanolic extract was prepared from the plant and its total phenolic and flavone contents were determined. The phenolic extract was fractionated by reversed-phase HPLC and the major phenolic compounds of each fraction were identified by ESI-MS and MSn analysis, in order to establish the specific phenolic profile of the plant. The total phenolic compounds in the ethanolic extract of Thymus citriodorus accounted for 138,75±13,56 mg/g, and its content of flavones was estimated as 27,30±2,15 mg/g. Moreover, the interpretation of the fragmentation pathways under ESI-MS/MS of the collected HPLC fractions allowed concluding that the major phenolic compounds in the plant included some glycoside derivatives of luteonin, naringenin and eriodictyol. Also, the phenolic extract showed a considerable antioxidant activity, as evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical scavenging potential and the reducing potential, suggesting that Thymus citriodorus can be an important source of natural antioxidants.

Olive mill wastewaters (OMW) have been attracting the attention of food and pharmaceutical industries, as it is a potential source of biophenols with a wide range of biological activities. This effluent produced in the olive oil industry contains approximately half of the total phenolic compounds of the olive fruit and it is currently exploited for industrial extraction of hydroxytyrosol [1]. Still, the phenolic composition of OMW is extremely complex, and many compounds are yet unidentified. In this context, the identification of unknown phenolic compounds can encourage the search of new bioactive compounds in OMW and contribute to further valorize this waste. In the present work, six new phenolic compounds were identified in Portuguese olive mill wastewaters. The phenolic extracts of OMW were obtained by methanol and were fractionated on Sep Pack C18 cartridges, by elution of the cartridges with 50% (v/v) methanol. The analysis of the purified fractions by electrospray mass spectrometry in the negative mode showed high relative abundant [M-H]− ions at m/z 539 and m/z 523, corresponding respectively to oleuropein and ligstroside isomers that contain the glucose unit linked to its hydroxytyrosol moiety. Glucoside derivatives of these compounds were also found in the two OMW samples. In particular, the fragmentation pathway of the [M-H]− ion at m/z 863 demonstrated the presence of a diglucoside derivative of the oleuropein isomer, while those of the ions at m/z 685 and m/z 847 were consistent with mono- and a di-glucosides of the ligstroside isomer, respectively. Moreover, the structure of an elenoic derivative of the ion at m/z 685 was also elucidated. Studies regarding the abundance and the iological activities of these compounds are now required to determine their possible industrial exploitation. [1] Agallias A., Magiatis P., Skaltsounis A.-P., Mikros E., Tsarbopoulos A., Gikas E., Spanos I., Manios T. (2007). J. Agric. Food Chem. 55: 2671-2676.

This research outlines an extensive characterization of the phenolic composition of a propolis sample from the northeast of Porto gal. For that, ao ethanolic extract was prepared, fractionated by HPLC and the identification of the phenolic compounds was done by electro spray mass spectrometry in the negative mode. This technical approach allowed the identification of 37 phenolic compounds in the Portoguese propolis sample, including seven that were described for the first time. Two of these new compounds had [M-Hr ions at mlz 403, aod the others had [M-Hr ions at mlz 433, mlz 461, mlz 417, mlz 475, and mlz 565. In general, the molecular weight of these compounds was higher thao the common phenolic compounds of propolis aod their fragmentation pattern suggested that they belong to the flavonoid family probably lioked with a phenylpropanoic acid moiety in the position C5 (mlz 403, 433, 461, 475) and C3 (mlz 403, 417), corresponding to pinocembrio and pinobaoksin derivatives. The ion at mlz 565 seems to be a p-counlaric ester derivative dimer.

The specific interest in naturally occurring antioxidants has lead to an exponential investigation of many traditional medicinal plants, as they are potential candidates for finding new therapeutic and supplementary health products. Cytisus multiflorus, also known as White Spanish Broom, is a leguminous shrub native from Iberian Peninsula that is u sed in folk medicine mainly due to its diuretic and anti-inflammatory properties. Still, despite its common usage for centuries, this plant has been far less studied than other species of the same genus. This study aimed to determine the phenolic composition and to evaluate the antioxidant capacity of Cytisus multiflorus. For that, an ethanolic extract was prepared and its total phenolics and flavone contents were determined. The antioxidant activity was accessed by measuring the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) scavenging potential. Also, the extract was fractionated by reversed-phase HPLC and the major phenolic compounds of each fraction were identified by ESI-MS and MSn analysis, in order to establish the specific phenolic profile of the plant. The total amount of phenolic compounds and of flavones accounted for 140 mg/g and 47 mg/g of the ethanolic extract from Cytisus multiflorus, respectively. Also, it exhibited a high antioxidative capacity, with an EC50 value of 29,7 μg/ml. Moreover, the interpretation of the fragmentation pathways under ESI-MS/MS of the collected HPLC fractions allowed concluding that the main phenolic compounds in the plant included chrysin and some glycoside derivatives of luteonine and quercetin. Thus, the overall results suggest that the phenolic extract of the Cytisus multiflorus can be claimed as a good source of natural antioxidants.

The chemical composition of propolis is highly variable, depending strongly on thc plant sources available to the bees at different locations, the geographic and climatic characteristics of these locations, I n the temperate zone, propol is originates mostly Ii'om bud exudates of poplar trees (Populus spp .. section Aigciros), For this reason, European propolis contains the typical 'poplar bud' phenolics: flavonoid aglicones (flavones and flavanones), phenolic acids and their esters.