Polysaccharides from the infusions of P. tridentatum, F. angustifoliaand M. suaveolens

The use of plant infusions for medicinal purposes is present in secular traditions of all civilizations. In Portugal, in Trás-os-Montes region, the small shrub (Pterospartum tridentatum), the narrow-leafed ash (Fraxinus angustifolia), and the apple mint (Mentha suaveolens) are plants used for such purposes. According to the popular tradition, the infusions of P. Tridentatum, F. Angustifolia, and M. suaveolens protect against diabetes, high blood pressure, high levels of cholesterol, and uric acid [1].These health benefits are associated with the presence of phenolic compounds, essential oils, and polysaccharides, among others. Although phenolic compounds and essential oils have been studied in detail for more than two decades, the structures of the polysaccharides present in plant infusions and their involvement in the health benefits is still incipient. Infusions were prepared in two different ways: a traditional method, consisting in the infusion of the vegetal material with boiling water during five minutes (Method A), and a more intensive method, consisting in the infusion of the vegetal material with boiling water during four hours (Method B). The high molecular weight material was obtained by concentrating and dialyzing the extracts obtained by Methods A and B. The high molecular weight material obtained with the Method B contained more glycosidic material than the obtained with Method A. The high molecular weight material was fractionated using ethanol precipitation yielding fractions especially rich in uronic acids besides considerable amounts of arabinose, galactose, glucose and rhamnose, suggesting the presence of pectic polysaccharides. Detailed monomeric composition and glycosidic linkages of each fraction will be presented

Composição glicosídica e actividade antioxidante de extractos de infusões de folhas de freixo (Fraxinus angustifolia)

Neste trabalho foram preparadas infusões de folhas de freixo secas. Cada infusão foi dividida em duas fracções, tendo uma sido utilizada para a obtenção do material polimérico por diálise (Dial) e a outra submetida a uma extracção em fase sólida C18, sendo a fase aquosa recolhida composta pelo material não retido (C18 H2O) e a fracção de metanol composta pelo material retido e eluído neste solvente (C18 MeOH). A análise da composição glicosídica mostrou que a fracção C18 H2O era rica em glucose e manose; a fracção C18 MeOH era rica em glucose e a fracção Dial era rica em ácidos urónicos, arabinose, galactose e glucose. A fracção C18 H2O era a mais pobre em compostos fenólicos e a que apresentou menor actividade antirradicalar. As fracções C18 MeOH e Dial apresentaram actividades antirradicalares semelhantes entre si, apesar de possuírem quantidades de compostos fenólicos totais distintas. A fracção Dial, constituída por material de peso molecular superior a 12-14 kDa, foi ainda sujeita a uma análise de ligações glicosídicas, através da análise por GC-MS dos respectivos acetatos de alditol parcialmente metilados. O resultado obtido permitiu inferir a presença de resíduos glicosídicos característicos de arabinogalactanas do tipo II, xiloglucanas e xilanas

Isolation and characterization of polysaccharides from Fraxinus angustifolia infusions

In folk medicine there is a great variety of plants that are used for as infusions, such as Camellia sinensis and Matricaria recutita. Despite the reported biological activities and health benefits of these infusions, most of the knowledge achieved so far is based on folk tradition passed over several generations, without a sound scientific basis. In Trás-os-Montes region, the dried leaves of the narrow-leafed ash “freixo” (Fraxinus angustifolia) are used for medicinal purposes, particularly against high levels of cholesterol, blood pressure and uric acid. These health benefits are associated with the presence of biologically active compounds such as phenolic compounds, essential oils, and polysaccharides, among others. Although phenolic compounds and essential oils have been studied in detail for more than two decades, the structures of the polysaccharides present in plant infusions and their involvement in the health benefits is still incipient. Therefore, in this work we present a study regarding the structure of the polysaccharides present in the infusions of “freixo” dried leaves. “Freixo” leaves were collected and dried according to the popular tradition and used for the preparation of the infusions. The polysaccharides were recovered by dialysis, fractionated by ethanol precipitation and anion exchange chromatography, yielding fractions with distinct ethanol solubility and uronic acid content. Sugar and linkage analysis evidenced the presence of pectic polysaccharides, frequently reported as immunostimulatory active. The assessment of the biological activity of these fractions is in progress

Monomeric composition and linkage analysis of the polysaccharides present in the infusions of Fraxinus angustifolia leaves

In the Trás-os-Montes region, the use of infusions of Fraxinus angustifolia dried leaves as a protection against high levels of cholesterol, blood pressure and uric acid is widespread. Pectic polysaccharides isolated from the infusions of some medicinal plants have been reported as biologically active [1,2]. Pectic poly-saccharides have been described as structurally complex polymers, exhibiting different polymeric building blocks: homogalacturonans (HG), rhamnogalacturonans-I (RG-I), rhamnogalacturonans-II (RG-II) and xylogalacturonans (XG) [3]. The backbone of RG-I can be partly substituted with various side chains, such as arabinans, type-I and type-II arabinogalactans (AG-I and AG-II). The aim of this work is to provide a first insight of the nature of the pectic polysaccharides present in the infusions of F. angustifolia dried leaves

Polysaccharide composition of Fraxinus angustifolia Leave infusions

The use of plant infusions with medicinal purposes, such as the ones from members of the Fraxinus genus, is an ancient practice that has been reported by popular medicine [1]. The health benefits are associated with the presence of phenolic compounds and polysaccharides, among others [2]. Although phenolic compounds have been studied in detail for more than two decades, the characterization of the polysaccharides present in plant infusions and their involvement in the health benefits is still incipient. The polysaccharides of F. angustifolia dried leaves were recovered by dialysis (12-14 kDa cut-off). The high molecular weight material comprised approximately 2% of the total soluble material obtained from the infusions. Total sugars in fraction were 27%, with a monomeric composition rich in Ara (15.2 ± 1.9 %), Gal (17.7 ± 0.4 %), Glc (30.1 ± 1.3 %) and uronic acids (18.9 ± 2.9 %). Analysis of the glycosidic linkage composition showed the presence of (1→6)-, (1→3,6)- and (1→3)-Gal with a proportion of 5:3:1, respectively, and also a high percentage of Ara terminally linked, diagnostic of the presence of Type II arabinogalactans [3]. Similar features have been reported in biological active Type II pectic arabinogalactans obtained from aqueous extracts of the roots of C. tinctorium [4]. The EC50 for the antioxidant activity (DPPH radical assay) was 0.34±0.04 mg/mL, showing lower antioxidant activity than that reported for aqueous extracts of hazel leaves (0.16-0.20 mg/mL) [5], but higher than that reported for the table olives ‘‘alcaparras’’ aqueous extracts (0.47± 0.03) [6]. The high molecular weight material was fractionated by ethanol precipitation in four distinct fractions: WI, a cold water insoluble residue (20.3% yield); Et50, material precipitated with 50% ethanol (17.9%); Et75, material precipitated with 75% ethanol (21.1%); and SN, material that remained soluble in 75% ethanol solution (40.7%). Fractions Et50, Et75 and SN were submitted to solid phase extraction (C18 cartridge). The Et50 and Et75 C18 non-retained fractions were rich in Ara (13.5-17.2%), Gal (27.1-35.7%) and uronic acids (25.9-35.2%), suggesting the occurrence of heterogeneous pectic arabinogalactans. NMR experiments are in progress in order to evaluate the detailed structure of the type II pectic arabinogalactans isolated from the infusions of dried leaves of F. angustifolia

Produção e tecnologia de cereais: processo de maltagem da cevada

O processo de maltagem da cevada consiste na germinação e secagem controlada do cereal, tendo como principal objetivo a obtenção de um produto com atividade enzimática, coloração e estabilidade adequadas. É possível obter maltes com características bastante distintas, no que se refere à sua cor e atividade enzimática, o que também possibilita a obtenção de cervejas com diferentes características. Com este trabalho pretende-se descrever o processo de maltagem da cevada, abordando numa fase inicial a estrutura e composição química do grão de cevada. Serão também descritas as principais modificações químicas que ocorrem ao nível dos principais componentes químicos do grão de cevada.info:eu-repo/semantics/publishedVersio

Produção e tecnologia de cereais: notas breves sobre o cultivo de cevada em Portugal

A cevada é o principal cereal usado no fabrico de cerveja. Portugal é um produtor modesto de cevada, pelo que a indústria cervejeira nacional funciona com base em cevada importada. Em Portugal a cevada cultiva-se quase exclusivamente em sequeiro, sendo as produções unitárias de grão muito baixas quando comparadas com as produtividades obtidas em países situados a latitudes mais elevadas. Neste trabalho procuram explicar-se as causas das baixas produtividades obtidas em Portugal. Sobretudo interessa saber se as baixas produtividades são devidas a causas naturais ou se podem ser alteradas com a introdução de melhorias nas técnicas de cultivo. O trabalho está organizado em quatro tópicos principais: origem e situação atual da cultura; aspetos botânicos e morfologia da planta; adaptação ecológica da cevada; e técnica cultural. Discutem-se ainda brevemente alguns critérios a observar na escolha das cultivares e como esta espécie está integrada nas rotações.info:eu-repo/semantics/publishedVersio

Polysaccharides from the infusions of P. tridentatum, F. angustifolia, and M. suaveolens

The use of plant infusions for medicinal purposes is present in secular traditions of all civilizations. In Portugal, in Trás-os-Montes region, the small shrub (Pterospartum tridentatum), the narrow-leafed ash (Fraxinus angustifolia), and the apple mint (Mentha suaveolens) are plants used for such purposes. According to the popular tradition, the infusions of P. Tridentatum, F. Angustifolia, and M. suaveolens protect against diabetes, high blood pressure, high levels of cholesterol, and uric acid [1].These health benefits are associated with the presence of phenolic compounds, essential oils, and polysaccharides, among others. Although phenolic compounds and essential oils have been studied in detail for more than two decades, the structures of the polysaccharides present in plant infusions and their involvement in the health benefits is still incipient. Infusions were prepared in two different ways: a traditional method, consisting in the infusion of the vegetal material with boiling water during five minutes (Method A), and a more intensive method, consisting in the infusion of the vegetal material with boiling water during four hours (Method B). The high molecular weight material was obtained by concentrating and dialyzing the extracts obtained by Methods A and B. The high molecular weight material obtained with the Method B contained more glycosidic material than the obtained with Method A. The high molecular weight material was fractionated using ethanol precipitation yielding fractions especially rich in uronic acids besides considerable amounts of arabinose, galactose, glucose and rhamnose, suggesting the presence of pectic polysaccharides. Detailed monomeric composition and glycosidic linkages of each fraction will be presented

Caracterização da estrutura e atividade biológica dos polissacarídeos presentes em infusões utilizadas na medicina popular

Doutoramento em QuímicaThe use of plants with medicinal purposes is an ancient practice still very common in developing regions, and is rapidly spreading in industrialized countries. This fact is evidenced by the large number of ethnobotanical studies found in the literature referring that these plants are often used as decoctions and infusions. In most studies the reported biological activities are attributed to the presence of phenolic compounds, due to their antioxidant properties, and to polysaccharides, with its anti-tumoral properties. In “Trás-os-Montes” region, some of the most popular infusions used by the popular medicine are prepared with the dried leaves of Fraxinus angustifolia, the dried shoots of Mentha suaveolens, and the dried inflorescences of Pterospartum tridentatum. However, there are no studies about the polysaccharides present in these infusions. Thus, through the structural characterization of the polysaccharides present in the infusions of F. angustifolia, M. suaveolens, and P. tridentatum, the present PhD thesis intends to evaluate the possible relation between polysaccharides and the immunostimulatory activity that these infusions might present. In a preliminary phase, infusions of F. angustifolia were prepared according to the popular tradition, and it was observed that the obtained water soluble material contained approximately 85% of material non-retained in C18 cartridges, with hydrophilic characteristics, with the remaining 15% comprising retained-material with hydrophobic characteristics. It was also shown that the infusions only contained between 2 and 4% of high molecular weight material (HMWM), which comprised approximately 30% of carbohydrate material. Sugar and methylation analysis of the HMWM suggested the presence of pectic polysaccharides, together with type II arabinogalactans, mannans, and xyloglucans. However, the amount of material obtained is to low for the fractionation, and structural analysis of the polysaccharides present. The 4 h decoction, divided in two periods of 2 h, with water renewal, allowed to increase the HMWM yield, relatively to the infusions traditional infusions. It was also observed that the decoction also allowed to increase the HMWM proportion of carbohydrate material, due to an increase in the proportion of uronic acid present, although the neutral sugar residues seemed to be detected in similar proportions. Therefore, in all the experiments subsequently performed, the HMWM used was obtained through the decoction of F. angustifolia dried leaves, M. suaveolens dried shoots, and P. tridentatum dried inflorescences. x After the fractionation, through ethanol precipitation, and anion exchange chromatography, of the polysaccharides from the HMWM obtained by the decoction of the vegetable material of the distinct studied plants, it was observed the presence of high proportions of pectic polysaccharides, containing type I arabinogalactans, together with minor proportions of type II arabinogalactans, mannans, and xyloglucans. The presence of pectic polysaccharides in the extracts from F. angustifolia was also evidenced through endo-polygalacturonase treatment, and ESI-MS and ESI-MS/MS experiments. The detection of linked pentose and uronic acid residues, also seemed to suggest the presence of xylogalacturonan domains in the pectic polysaccharides from F. angustifolia. The extracts from F. angustifolia dried leaves also contained type II arabinogalactans that exhibited a higher structural diversity than those detected in the M. suaveolens, and P. tridentatum extracts, particularly in the substitution degree of the galactan backbone, and in the extension of the (1→5)-Araf side chains. Moreover, for all the plants studied, it was also observed that the type II arabinogalactans, extracted during the 2nd 2h of the extraction process, exhibited a substitution degree of the galactan backbone higher than those extracted during the 1st 2h. The extracts from P. tridentatum dried inflorescences contained higher proportions of mannans, and also of xyloglucans, both presenting a substitution degree higher than those, which were detected in lower proportion in the extracts of F. angustifolia and M. suaveolens. Through ESI-MS and ESI-MS/MS it was possible to evidence that the mannans present in the extracts of P. tridentatum presented acetyl groups on the O-2 of the mannosyl residues. It was also evidenced that the P. tridentatum mannans were more extensively acetylated than the mannans detected in the coffee infusion, LBG, and other non-conventional mannan sources. Moreover, it was detected the presence of oligosaccharides comprising hexose residues linked to non acetylated pentose residues, suggesting the possible presence of arabinose residues in the mannans from P. tridentatum extracts. The immunostimulatory activity of three fractions isolated from the extracts of F. angustifolia, M. suaveolens, and P. tridentatum, was tested and an increase in the NO production by macrophages, without compromising their cellular viability, was observed. The type I, and type II arabinogalactans detected in the extracts from F. angustifolia, and M. suaveolens seem to have contributed for the observed immunostimulatory activity. For the fraction from P. tridentatum, the mannans acetylation, and the presence of type I, and type II arabinogalactans seemed to contribute for the macrophage immunostimulatory activity observed. The possible presence of storage xyloglucans from the inflorescences seeds, also seems to have contributed for the immunostimulatory activity registered when the macrophages were stimulated with higher extract concentrations. The results obtained allow to conclude that the extracts of F. angustifolia dried leaves, M. suaveolens dried shoots, and P. tridentatum dried inflorescences contained high proportions of pectic polysaccharides, exhibiting type I arabinogalactans, together with other polysaccharides, such as type II arabinogalactans, mannans, and xyloglucans. This polysaccharide mixture seems to have contributed to the immunostimulatory activity of fractions isolated from the extracts of the studied plants. Therefore, as the same type of polysaccharides seem to be present in the decoctions and in the infusions, it seems possible that the polysaccharides might contribute for the therapeutic properties frequently associated by the popular tradition to the infusions of these plants.O uso de plantas com fins medicinais é uma prática antiga, ainda bastante frequente em regiões de países em desenvolvimento, e que se tem vindo a disseminar em países mais industrializados. Este facto é suportado pelo elevado número de estudos etnobotânicos, onde se refere que estas plantas são frequentemente usadas na forma de decocções e infusões. Na maioria dos estudos as atividades biológicas são associadas à presença de compostos fenólicos e às suas propriedades antioxidantes, assim como aos polissacarídeos, nomeadamente devido às propriedades antitumorais. Na região de Trás-os-Montes, algumas das infusões mais utilizadas pela medicina popular têm por base as folhas secas do freixo (Fraxinus angustifolia), a parte aérea seca do mondrasto (Mentha suaveolens) e as inflorescências secas da carqueja (Pterospartum tridentatum). No entanto, não existe nenhum estudo sobre os polissacarídeos presentes nas infusões das folhas secas do freixo (Fraxinus angustifolia), da parte aérea do mondrasto (Mentha suaveolens) ou das inflorescências da carqueja (Pterospartum tridentatum). Deste modo, com a presente dissertação pretende-se, através da caracterização estrutural dos polissacarídeos presentes nas infusões de F. angustifolia, M. suaveolens e P. tridentatum, avaliar a possível relação entre os polissacarídeos e a atividade imunoestimuladora que estas infusões possam apresentar. Numa primeira fase, foram preparadas infusões tradicionais de F. angustifolia, tendo-se mostrado que o material solúvel das infusões continha cerca de 85% de material não retido em cartuchos C18 com características hidrofílicas, sendo os restantes 15% compostos por material retido de características hidrofóbicas. Mostrou-se ainda que as infusões obtidas apenas continham cerca de 2 a 4% de material de alto peso molecular (HMWM), que era composto por cerca de 30% de carboidratos. A análise de açúcares e a análise de metilação do HMWM sugeriu a presença de polissacarídeos pécticos, em conjunto com arabinogalactanas do tipo II, mananas e xiloglucanas. No entanto, a quantidade de material obtido é muito baixa para o fracionamento e a análise estrutural dos polissacarídeos presentes. A decocção de 4 h, divididas em 2 períodos de 2 h, com renovação da água, permitiu um aumento no rendimento em HMWM, relativamente às infusões tradicionais. Verificou-se ainda, que a decocção também permitiu aumentar a proporção de carboidratos no HMWM, particularmente devido ao aumento da proporção de ácido urónico presente, embora os resíduos de açúcares neutros se encontrassem em proporção semelhante. Deste modo, em todas as experiências realizadas posteriormente, o HMWM utilizado foi obtido através da decocção das folhas secas de F. angustifolia, da parte aérea seca de M. suaveolens e das inflorescências secas de P. tridentatum. Após fracionamento por precipitação em etanol e cromatografia de troca aniónica dos polissacarídeos presentes no HMWM obtido através da decocção dos materiais vegetais das diferentes plantas estudadas, verificou-se a presença de elevadas proporções de polissacarídeos pécticos, contendo arabinogalactanas do tipo I, em conjunto com menores proporções de arabinogalactanas do tipo II, mananas e xiloglucanas. A presença de polissacarídeos pécticos nos extratos de F. angustifolia foi também evidenciada através do tratamento enzimático com endopoligalacturonase e de experiências de ESI-MS e ESI-MS/MS. A deteção de resíduos de pentose ligados a resíduos de ácido urónico também pareceu indicar a presença de domínios de xilogalacturonana nos polissacarídeos pécticos de F. angustifolia. Os extratos das folhas secas de F. angustifolia mostraram conter arabinogalactanas do tipo II com uma maior diversidade estrutural do que as que foram detetadas nos extratos de M. suaveolens e P. tridentatum, particularmente no que diz respeito à substituição das cadeias de (1→3)-Galp e à extensão das cadeias laterais compostas por resíduos de (1→5)-Araf. Para além disso, verificou-se que, para todas as plantas estudadas, as arabinogalactanas do tipo II presentes nas frações obtidas durante a segunda parte do processo de extração apresentaram uma substituição das cadeias de (1→3)-Galp maior do que as extraídas durante a primeira parte da extração. Os extratos das inflorescências de P. tridentatum continham maiores proporções de mananas e de xiloglucanas, tendo-se detetado estruturas que apresentaram um maior grau de substituição do que as que foram detetadas em menores proporções nos extratos de F. angustifolia e M. suaveolens. Através das técnicas de ESI-MS e ESI-MS/MS, foi possível demonstrar que as mananas presentes nos extratos de P. tridentatum se encontravam acetiladas no O-2 dos resíduos de manose. Verificou-se que as mananas presentes nos extratos de P. tridentatum se encontravam acetiladas em maior extensão do que as mananas detetadas na infusão do café, na goma de alfarroba e em outras fontes não convencionais de mananas. Para além disso, também foram detetados oligossacarídeos contendo resíduos de pentose, ligados a resíduos de hexose, sugerindo a presença de resíduos de arabinose na estrutura das mananas de P. tridentatum. A atividade imunoestimuladora de três frações isoladas a partir dos extratos de F. angustifolia, M. suaveolens e P. tridentatum foi avaliada, tendo-se observado o aumento da produção de NO por parte de macrófagos, sem comprometer a sua viabilidade celular. As arabinogalactanas do tipo I e II parecem contribuir para a atividade imunoestimuladora observada nas frações dos extratos de F. angustifolia e de M. suaveolens No caso da fração obtida a partir dos extratos de P. tridentatum, a acetilação das mananas, bem como a presença de arabinogalactanas do tipo I e II parecem ter contribuído para a atividade imunoestimuladora dos macrófagos observada. A possível presença de xiloglucanas de armazenamento, provenientes das sementes das inflorescências de P. tridentatum, também poderá contribuir para a atividade imunoestimuladora observada quando os macrófagos foram estimulados com maiores concentrações de extrato. Os resultados obtidos permitem concluir que os extratos das folhas secas de F. angustifolia, da parte aérea de M. suaveolens e das inflorescências de P. tridentatum contêm elevadas proporções de polissacarídeos pécticos, contendo arabinogalactanas do tipo I, misturados com outros polissacarídeos, tais como arabinogalactanas do tipo II, mananas e xiloglucanas. Esta mistura de polissacarídeos mostrou contribuir para a atividade imunoestimuladora de frações isoladas a partir dos extratos das plantas estudadas. Deste modo, e como o mesmo tipo de polissacarídeos parece estar presente nas infusões e decoções, é possível que os polissacarídeos possam contribuir para as propriedades terapêuticas frequentemente atribuídas pela tradição popular às infusões destas plantas

Food products in two cosmetic formulations: determination of the stability and toxicity

The cosmetic market is extending to millions of consumers worldwide and its industry is worth tens of billions of US$ [ ]. The competitive environment and the demands made by the consumers pushed the manufactures to develop new types of cosmetic products and to use new ingredients, such as bee products, to produce formulations with higher quality and efficacy. The aim of this work was to develop two cosmetic formulations for topical application that combine bee products and essential oils from medicinal plants, as well it was to extract and analyse the essential oils of an edible wild mushrooms Clitocybe odora (Buli.) P. Kumm, grown in vitro, to understand the potential of it, as a source of bioactive compounds usable in cosmetics. The effect of two different bee products (hydroalcoholic exti-act of propolis and two honeys of Lavandula ssp. with different geographic provenance) and essential oils from two different species (Thymus Zygis L. subsp. Zygis and Mentha pulegium L.) was tested. For that purpose, several preparations were elaborated to witch one essential oil and a bee product were added. For each type of formulation, one preparation with essential oils but without a bee product was also prepared. In relation to the bee products, the Lavandula spp. honey was used in the proportion of l :50 (v/m) and l :20 (v/m); the hydroalcoholic extract of propolis, using ethanol, was add in a concentration of5% (m/v). The concentration of essential oil used was 7. 86x10^g/g (e. o./ emulsion). Additionally was also tested the toxicity of the two essential oils and the Clitocybe odora volatiles, through the LD50 determination, using Artemia Salina L.. To determine the effect of the bee products in the formulations stabilization were made physicochemical tests and stabilization tests. Was not verified alterations m the relative density of the samples, in the pH or in a vibration test made placing the samples 10 seconds in the vortex. When the samples were place during l 5 days in a daylight lamp, was observed, by comparing with samples just with essential oil in the same conditions, that all the samples with propolis extract changed the colour and the odour. The effect of the bee products in the viscosity of the formulations was also tested. When using a lower spindle rotation velocity, the use of the honey from the North of Portugal and of the propolis extract increased the viscosity of the emulsions. Both the two essential oils tested as the in vitro Clitocybe odora volatiles show to have a LD50<0,1 mg/ml.info:eu-repo/semantics/publishedVersio