In vitro activity of extracts and isolated polyphenols from West African medicinal plants against Plasmodium falciparum

The aim of the study was to screen 11 selected traditional medicinal plants from West Africa for their in vitro antiplasmodial activity in order to determine the activity of single and of combination of plant extracts and to examine the activity of isolated pure compounds. Ethanolic and aqueous extracts of the 11 selected plants and pure compounds from Phyllanthus muellerianus and Anogeissus leiocarpus were tested in vitro against Plasmodium falciparum 3D7. Proliferation inhibitory effects were monitored after 48 h. Among the plants and pure compounds investigated in this study, geraniin from P. muellerianus, ellagic, gentisic, and gallic acids from A. leiocarpus, and extracts from A. leiocarpus, P. muellerianus and combination of A. leiocarpus with P. muellerianus affected the proliferation of P. falciparum most potently. Significant inhibitory activity was observed in combination of A. leiocarpus with P. muellerianus (IC50 = 10.8 mu g/ml), in combination of A. leiocarpus with Khaya senegalensis (IC50 = 12.5 mu g/ml), ellagic acid (IC50 = 2.88 mu M), and geraniin (IC50 = 11.74 mu M). In general growth inhibition was concentration-dependent revealing IC50 values ranging between 10.8 and -40.1 mu g/ml and 2.88 and 11.74 mu M for plant extracts and pure substances respectively. Comparison with literature sources of in vivo and in vitro toxicity data revealed that thresholds are up to two times higher than the determined IC50 values. Thus, the present study suggests that geraniin from P. muellerianus; ellagic acid, gallic acid, and gentisic acid from A. leiocarpus; and combination of extracts from A. leiocarpus with either P. muellerianus or K. senegalensis could be a potential option for malaria treatment.

LC-DAD/ESI-MS/MS study of phenolic compounds in ash (Fraxinus excelsior L. and F. americana L.) heartwood. Effect of toasting intensity at cooperage

The phenolic composition of heartwood extracts from Fraxinus excelsior L. and F. americana L., both before and after toasting in cooperage, was studied using LC-DAD/ESI-MS/MS. Low-molecular weight (LMW) phenolic compounds, secoiridoids, phenylethanoid glycosides, dilignols and oligolignols compounds were detected, and 48 were identified, or tentatively characterized, on the basis of their retention time, UV/Vis and MS spectra, and MS fragmentation patterns. Some LMW phenolic compounds like protocatechuic acid and aldehyde, hydroxytyrosol and tyrosol, were unlike to those for oak wood, while ellagic and gallic acid were not found. The toasting of wood resulted in a progressive increase in lignin degradation products with regard to toasting intensity. The levels of some of these compounds in medium-toasted ash woods were much higher than those normally detected in toasted oak, highlighting vanillin levels, thus a more pronounced vanilla character can be expected when using toasted ash wood in the aging wines. Moreover, in seasoned wood, we found a great variety of phenolic compounds which had not been found in oak wood, especially oleuropein, ligstroside and olivil, along with verbascoside and isoverbascoside in F. excelsior, and oleoside in F. americana. Toasting mainly provoked their degradation, thus in medium-toasted wood, only four of them were detected. This resulted in a minor differentiation between toasted ash and oak woods. The absence of tannins in ash wood, which are very important in oak wood, is another peculiar characteristic that should be taken into account when considering its use in cooperage. Copyright (C) 2012 John Wiley & Sons, Ltd.

Effetti del chitosano su composti polifenolici in colture cellulari di vite: aspetti molecolari e metabolici

Polyphenols, including flavonoids and stilbenes, are an essential part of human diet and constitute one of the most abundant and ubiquitous group of plant secondary metabolites. The level of these compounds is inducible by stress or fungal attack, so attempts are being made to identify likely biotic and abiotic elicitors and to better understand the underlying mechanism. Resveratrol (3,5,4’-trihydroxystilbene), which belongs to the stilbene family, is a naturally occurring polyphenol, found in several fruits, vegetables and beverages including red wine. It is one of the most important plant polyphenols with proved benefic activity on animal health. In the last two decades, the potential protective effects of resveratrol against cardiovascular and neurodegenerative diseases, as well as the chemopreventive properties against cancer, have been largely investigated. The most important source of polyphenols and in particular resveratrol for human diet is grape (Vitis vinifera). Since stilbenes and flavonoids play a very important role in plant defence responses and enviromental interactions, and their effects on human health seem promising, the aim of the research of this Thesis was to study at different levels the activation and the regulation of their biosynthetic pathways after chitosan treatment. Moreover, the polyphenol production in grape cells and the optimisation of cultural conditions bioreactor scale-up, were also investigated. Cell suspensions were obtained from cv. Barbera (Vitis vinifera L.) petioles and were treated with a biotic elicitor, chitosan (50 μg/mL, dissolved in acetic acid) to promote phenylpropanoid metabolism. Chitosan is a D-glucosamine polymer from fungi cell wall and therefore mimes fungal pathogen attack. Liquid cultures have been monitored for 15 days, measuring cell number, cell viability, pH and grams of fresh weight. The endogenous and released amounts of 7 stilbenes (trans and cis isomers of resveratrol, piceid and resveratroloside, and piceatannol), gallic acid, 6 hydroxycinnamic acids (trans-cinnamic, p-coumaric, caffeic, ferulic, sinapic and chlorogenic acids), 5 catechines (catechin, epicatechin, epigallocatechin-gallate (EGCG), epigallocatechin and epicatechin-gallate) and other 5 flavonoids (chalcon, naringenin, kaempferol, quercetin and rutin) in cells and cultural medium, were measured by HPLC-DAD analysis and total anthocyanins were quantified by spectrophotometric analysis. Chitosan was effective in stimulating trans-resveratrol endogenous accumulation with a sharp peak at day 4 (exceeding acetic acid and water controls by 36% and 63%, respectively), while it did not influence the production of the cis-isomer. Compared to both water and acetic acid controls, chitosan decreased the release of both trans- and cis-resveratrol respect to controls. No effect was shown on the accumulation of single resveratrol mono-glucoside isomers, but considering their total amount, normalized for the relative water control, it was possible to evidence an increase in both accumulation and release of those compounds, in chitosan-treated cells, throughout the culture period and particularly during the second week. Many of the analysed flavonoids and hydroxycinnamic acids were not present or detectable in trace amounts. Catechin, epicatechin and epigallocatechin-gallate (EGCG) were detectable both inside the cells and in the culture media, but chitosan did not affect their amounts. On the contrary, total anthocyanins have been stimulated by chitosan and their level, from day 4 to 14, was about 2-fold higher than in both controls, confirming macroscopic observations that treated suspensions showed an intense brown-red color, from day 3 onwards. These elicitation results suggest that chitosan selectively up-regulates specific biosynthetic pathways, without modifying the general accumulation pattern of other flavonoids. Proteins have been extracted from cells at day 4 of culture (corresponding to the production peak of trans-resveratrol) and separated by bidimensional electrophoresis. The 73 proteins that showed a consistently changed amount between untreated, chitosan and acetic acid (chitosan solvent) treated cells, have been identified by mass spectrometry. Chitosan induced an increase in stilbene synthase (STS, the resveratrol biosynthetic enzyme), chalcone-flavanone isomerase (CHI, that switches the pathway from chalcones to flavones and anthocyanins), pathogenesis-related proteins 10 (PRs10, a large family of defence proteins), and a decrease in many proteins belonging to primary metabolisms. A train of six distinct spots of STS encoded by the same gene and increased by chitosan, was detected on the 2-D gels, and related to the different phosphorylation degree of STS spots. Northern blot analyses have been performed on RNA extracted from cells treated with chitosan and relative controls, using probes for STS, PAL (phenylalanine ammonia lyase, the first enzyme of the biosynthetic pathway), CHS (chalcone synthase, that shares with STS the same precursors), CHI and PR-10. The up-regulation of PAL, CHS and CHI transcript expression levels correlated with the accumulation of anthocyanins. The strong increase of different molecular weight PR-10 mRNAs, correlated with the 11 PR-10 protein spots identified in proteomic analyses. The sudden decrease in trans-resveratrol endogenous accumulation after day 4 of culture, could be simply explained by the diminished resveratrol biosynthetic activity due to the lower amount of biosynthetic enzymes. This might be indirectly demonstrated by northern blot expression analyses, that showed lower levels of phenylalanine ammonia lyase (PAL) and stilbene synthase (STS) mRNAs starting from day 4. Other possible explanations could be a resveratrol oxidation process and/or the formation of other different mono-, di-glucosides and resveratrol oligomers such as viniferins. Immunolocalisation experiments performed on grape protoplasts and the subsequent analyses by confocal microscope, showed that STS, and therefore the resveratrol synthetic site, is mostly associated to intracellular membranes close to the cytosolic side of plasma membrane and in a smaller amount is localized in the cytosol. STS seemed not to be present inside vacuole and nucleus. There were no differences in the STS intracellular localisation between the different treatments. Since it was shown that stilbenes are largely released in the culture medium and that STS is a soluble protein, a possible interaction of STS with a plasma membrane transporter responsible for the extrusion of stilbenes in the culture medium, might be hypothesized. Proteomic analyses performed on subcellular fractions identified in the microsomial fraction 5 proteins taking part in channel complexes or associated with channels, that significantly changed their amount after chitosan treatment. In soluble and membrane fractions respectively 3 and 4 STS and 6 and 3 PR-10 have been identified. Proteomic results obtained from subcellular fractions substantially confirmed previous result obtained from total cell protein extracts and added more information about protein localisation and co-localisation. The interesting results obtained on Barbera cell cultures with the aim to increase polyphenol (especially stilbenes) production, have encouraged scale up tests in 1 litre bioreactors. The first trial fermentation was performed in parallel with a normal time-course in 20 mL flasks, showing that the scale-up (bigger volume and different conditions) process influenced in a very relevant way stilbenes production. In order to optimise culture parameters such as medium sucrose amount, fermentation length and inoculum cell concentration, few other fermentations were performed. Chitosan treatments were also performed. The modification of each parameter brought relevant variations in stilbenes and catechins levels, so that the production of a certain compound (or class of compounds) could be hypothetically promoted by modulating one or more culture parameters. For example the catechin yield could be improved by increasing sucrose content and the time of fermentation. The best results in stilbene yield were obtained in a 800 mL fermentation inoculated with 10.8 grams of cells and supplemented with chitosan. The culture was fed with MS medium added with 30 g/L sucrose, 25 μg/mL rifampicin and 50 μg/mL of chitosan, and was maintained at 24°C, stirred by marine impeller at 100 rpm and supplied of air at 0.16 L/min rate. Resveratroloside was the stilbene present in the larger amount, 3-5 times more than resveratrol. Because resveratrol glucosides are similarly active and more stable than free resveratrol, their production using a bioreactor could be a great advantage in an hypothetical industrial process. In my bioreactor tests, stilbenes were mainly released in the culture medium (60-80% of the total) and this fact could be another advantage for industrial applications, because it allows recovering the products directly from the culture medium without stopping the fermentation and/or killing the cells. In my best cultural conditions, it was possible to obtain 3.95 mg/L of stilbenes at day 4 (maximum resveratrol accumulation) and 5.13 mg/L at day 14 (maximum resveratroloside production). In conclusion, chitosan effect in inducing Vitis vinifera defense mechanisms can be related to its ability to increase the intracellular content of a large spectrum of antioxidants, and in particular of resveratrol, its derivates and anthocyanins. Its effect can be observed at transcriptional, proteomic (variation of soluble and membrane protein amounts) and metabolic (polyphenols production) level. The chitosan ability to elicit specific plant matabolisms can be useful to produce large quantities of antioxidant compounds from cell culture in bioreactor.

Neuroprotective strategies against neurodegeneration in cellular model systems

In the present study we analyzed new neuroprotective therapeutical strategies in PD (Parkinson’s disease) and AD (Alzheimer’s disease). Current therapeutic strategies for treating PD and AD offer mainly transient symptomatic relief but it is still impossible to block the loss of neuron and then the progression of PD and AD. There is considerable consensus that the increased production and/or aggregation of α- synuclein (α-syn) and β-amyloid peptide (Aβ), plays a central role in the pathogenesis of PD, related synucleinopathies and AD. Therefore, we identified antiamyloidogenic compounds and we tested their effect as neuroprotective drug-like molecules against α-syn and β-amyloid cytotoxicity in PC12. Herein, we show that two nitro-catechol compounds (entacapone and tolcapone) and 5 cathecol-containing compounds (dopamine, pyrogallol, gallic acid, caffeic acid and quercetin) with antioxidant and anti-inflammatory properties, are potent inhibitors of α-syn and β-amyloid oligomerization and fibrillization. Subsequently, we show that the inhibition of α-syn and β-amyloid oligomerization and fibrillization is correlated with the neuroprotection of these compounds against the α-syn and β-amyloid-induced cytotoxicity in PC12. Finally, we focused on the study of the neuroprotective role of microglia and on the possibility that the neuroprotection properties of these cells could be use as therapeutical strategy in PD and AD. Here, we have used an in vitro model to demonstrate neuroprotection of a 48 h-microglial conditioned medium (MCM) towards cerebellar granule neurons (CGNs) challenged with the neurotoxin 6-hydroxydopamine (6-OHDA), which induces a Parkinson-like neurodegeneration, with Aβ42, which induces a Alzheimer-like neurodegeneration, and glutamate, involved in the major neurodegenerative diseases. We show that MCM nearly completely protects CGNs from 6-OHDA neurotoxicity, partially from glutamate excitotoxicity but not from Aβ42 toxin.

Amalgamersatz

Amalgamersatz:Neue Wege zur Herstellung von Dentalkompositen mit geringem Polymerisationsschrumpf auf (Meth-)Acrylat-Basis Aufgrund der ästhetischen und gesundheitlichen Bedenken wird seit Jahrzehnten nach einer Alternative für Amalgam als Zahnfüllmaterial gesucht. Der größte Nachteil von organischen Monomeren liegt in der Volumenkontraktion während der Aushärtung, welche sich negativ auf die Materialeigenschaften auswirkt. Aus diesem Grund war das Hauptziel dieser Arbeit, eine Minimierung des Schrumpfes bei der radikalischen Polymerisation zu erreichen. Dazu wurden verschiedene, zum Teil neue, (Meth-)Acrylate synthetisiert und auf ihre Einsetzbarkeit als Bestandteil von Dentalkompositen geprüft.Um die Volumenkontraktion während der Polymerisation zu minimieren, wurde die Beweglichkeit der polymerisierbaren Gruppe eingeschränkt. Im ersten Teil der Arbeit wurden dazu flüssigkristalline Substanzen eingesetzt. Durch Mischen von flüssigkristallinen Diacrylaten konnte eine Mesophase im gewünschten Temperaturintervall von 25 bis 35 °C erhalten werden. Der Einsatz dieser Flüssigkristalle zeigte einen positiven Einfluss auf den Polymerisationsschrumpf. Zudem wurden neue Monomere synthetisiert, deren Methacrylgruppe in direkter Nachbarschaft zum Mesogen angebunden wurde, um die Stabilität der erhaltenen Polymere zu erhöhen.Im zweiten Teil der Arbeit wurde die Beweglichkeit der polymerisierbaren Gruppe durch eine Fixierung an einem starren Kern reduziert. Als Grundkörper wurden Polyphenole, enzymatisch polymerisierte Phenole und ߖCyclodextrin verwendet. Bei den modifizierten Polyphenolen auf Basis von Gallussäure und 3,5-Dihydroxybenzoesäure konnte eine leichte Reduzierung des Polymerisationsschrumpfes erreicht werden. Mit HRP (Horseradish Peroxidase) katalysierten enzymatisch polymerisierten Phenole konnte dagegen nicht photochemisch vernetzt werden, da diese Oligomere in Lösung gefärbt vorlagen. Zudem zeigten die freien, phenolischen Hydroxygruppen eine sehr geringe Reaktivität. Die besten Ergebnisse wurden mit modifizierten ߖCyclodextrinen als Komponente einer Komposite erreicht. Dabei wurde in einem Fall sogar eine leichte Volumenexpansion während der Polymerisation erzielt.

Sviluppo di un metodo elettrochimico per la determinazione della capacità antiossidante

The aim of the present thesis, carried out at the Analytical Group of the Faculty of Industrial Chemistry in Bologna, is to develop a new electrochemical method for the determination of the Antioxidant Capacity (AOC). The approach is based on the deposition of a non-conducting polymeric film on the working electrode surface and the following exposition to the radicals OH· produced by H2O2 photolysis. The strongly oxidant action of hydroxyl radicals degrades, causing an increase of the Faradic current, relevant to the redox couple [Ru(NH3)6]2+/3+ monitored by cyclic voltammetry(CV); the presence of an antioxidant compound in solution slows down the radical action, thus protecting the polymeric film and blocking the charge transfer. The parameter adopted for the quantification of the AOC, was the induction time, called also lag phase, which is the time when the degradation of the film starts. Five pure compounds, among most commonly antioxidant, were investigated : Trolox®(an analogue water-soluble of vitamin E), (L)-ascorbic acid, gallic acid, pyrogallol and (-)- epicatechin. The AOC of each antioxidant was expressed by TEAC index (Trolox® Equivalent Antioxidant Capacity), calculated from the ratio between the slope of the calibration curve of the target compound and the slope of the calibration curve of Trolox®. The results from the electrochemical method, have been compared with those obtained from some other standardized methods, widely employed. The assays used for the comparison, have been: ORAC, a spectrofluorimetric method based on the decrease of fluorescein emission after the attack of alkylperoxide radicals, ABTS and DPPH that exploit the decoloration of stable nitrogen radicals when they are reduced in presence of an antioxidant compound and, finally, a potentiometric method based on the response of the redox couple [Fe(CN)6]3-/ [Fe(CN)6]4-. From the results obtained from pure compounds, it has been found that ORAC is the methodology showing the best correlation with the developed electrochemical method, maybe since similar radical species are involved. The comparison between the considered assays, was also extended to the analysis of a real sample of fruit juice. In such a case the TEAC value resulting from the electrochemical method is higher than those from standardized assays.

Impacts of groundwater discharge at Myora Springs (North Stradbroke Island, Australia) on the phenolic metabolism of eelgrass, Zostera muelleri, and grazing by the juvenile rabbitfish, Siganus fuscescens

Myora Springs is one of many groundwater discharge sites on North Stradbroke Island (Queensland, Australia). Here spring waters emerge from wetland forests to join Moreton Bay, mixing with seawater over seagrass meadows dominated by eelgrass, Zostera muelleri. We sought to determine how low pH / high CO2 conditions near the spring affect these plants and their interactions with the black rabbitfish (Siganus fuscescens), a co-occurring grazer. In paired-choice feeding trials S. fuscescens preferentially consumed Z. muelleri shoots collected nearest to Myora Springs. Proximity to the spring did not significantly alter the carbon and nitrogen contents of seagrass tissues but did result in the extraordinary loss of soluble phenolics, including Folin-reactive phenolics, condensed tannins, and phenolic acids by ?87%. Conversely, seagrass lignin contents were, in this and related experiments, unaffected or increased, suggesting a shift in secondary metabolism away from the production of soluble, but not insoluble, (poly)phenolics. We suggest that groundwater discharge sites such as Myora Springs, and other sites characterized by low pH, are likely to be popular feeding grounds for seagrass grazers seeking to reduce their exposure to soluble phenolics.

Compostos fenólicos de resíduos agroindustriais: identificação, propriedades biológicas e aplicação em matriz alimentar de base lipídica

A geração de resíduos sólidos pelas atividades agroindustriais tem criado a demanda por um reaproveitamento tecnológico desses materiais. Assim, o objetivo deste trabalho foi avaliar o potencial bioativo e tecnológico de resíduos agroindustriais, como fontes naturais de compostos fenólicos com atividade antioxidante. Foram analisados resíduos agroindustriais vinícolas, de indústrias produtoras de polpas congeladas de frutas (açaí, cajá, cupuaçu e graviola) e provenientes do beneficiamento de café e de laranja. Inicialmente, foi realizado um estudo para a determinação das condições ótimas de extração, empregando planejamento experimental multivariado com delineamento composto central rotacional, cujos resultados foram avaliados empregando a técnica de superfície de resposta. Na sequência, foram feitos a triagem dos resíduos, baseada na atividade antioxidante, e a caracterização fenólica dos extratos hidroalcoólicos obtidos dos resíduos agroindustriais. De acordo com os resultados de atividade antioxidante, engaço de uva da variedade Chenin Blanc (EC) e semente de açaí (SA) foram os resíduos selecionados, os quais seguiram para as etapas de concentração e fracionamento bioguiado de sua(s) molécula(s) bioativa(s), as quais foram posteriormente identificadas por UHPLC-ESI-LTQ-MS. Extratos brutos e concentrados foram avaliados in vitro quanto à capacidade de desativação de espécies reativas de oxigênio (radicais peroxila, ânion superóxido e ácido hipocloroso) e então, aplicados em óleo de soja, emulsão e suspensão de lipossomos, a fim de se avaliar a efetividade desses extratos como antioxidante natural em matrizes lipídicas. Concentrações intermediárias de etanol (40-60%) e alta temperatura (96°C), exceto para semente de açaí (25°C), foram as condições ótimas para a extração de antioxidantes dos resíduos agroindustriais. Epicatequina, ácido gálico, catequina e procianidina B1 foram os compostos de maior ocorrência, quando avaliados pela técnica de HPLC-DAD. O EC apresentou a maior atividade antioxidante global e SA a maior atividade entre os resíduos de polpas de frutas, laranja e café. A concentração dos extratos brutos de EC e SA, pela resina Amberlite XAD®-2, produziu aumento significativo da atividade antioxidante. Além disso, extratos brutos e concentrados apresentaram atividade antiproliferativa e anti-inflamatória. Os extratos concentrados foram fracionados por meio de Sephadex LH-20, a partir da qual foi possível identificar quatro frações de maior bioatividade para o EC e três para o SA. Procianidina B1, catequina, epicatequina e resveratrol foram identificados no extrato concentrado e frações de EC. Dezoito procianidinas poliméricas, catequina, epicatequina foram os principais compostos identificados em SA, por meio de UHPLC-ESI-LTQ-MS. Resveratrol também foi encontrado em SA pela primeira vez. Quando avaliados em óleo de soja, EC e SA demonstraram atividade pro-oxidante. Contudo, elevada atividade antioxidante foi verificada quando essas amostras foram aplicadas em sistemas lipídicos coloidais, pois retardaram o consumo de oxigênio em uma emulsão óleo/água e o período de indução na produção de dienos conjugados em uma suspensão de lipossomos. Portanto, os resíduos agroindustriais EC e SA possuem potencial tecnológico de reaproveitamento industrial podendo ser considerados possíveis matérias-primas para a obtenção de extratos ricos em antioxidantes ou pela extração de antioxidantes naturais de uso pelas indústrias farmacêutica e/ou de alimentos.

Allen's commercial organic analysis; a treatise on the properties, modes of assaying, and proximate analytical examination of the various organic chemicals and products employed in the arts, manufactures, medicine, etc., with concise methods for the detection and estimation of their impurities, adulterations, and products of decomposition ...

Vols. 3-9 edited by W.A. Davis and Samuel S. Sadtler.

Phenolic acids in Australian Melaleuca, Guioa, Lophostemon, Banksia and Helianthus honeys and their potential for floral authentication

Eight phenolic acids and two abscisic acid isomers in Australian honeys from five botanical species (Melaleuca, Guioa, Lophostemon, Banksia and Helianthus) have been analyzed in relation to their botanical origins. Total phenolic acids present in these honeys range from 2.13 mg/100 g sunflower (Helianthus annuus) honey to 12.11 mg/100 g tea tree (Melaleuca quinquenervia) honey, with amounts of individual acids being various. Tea tree honey shows a phenolic profile of gallic, ellagic, chlorogenic and coumaric acids, which is similar to the phenolic profile of an Australian Eucalyptus honey (bloodwood or Eucalyptus intermedia honey). The main difference between tea tree and bloodwood honeys is the contribution of chlorogenic acid to their total phenolic profiles. In Australian crow ash (Guioa semiglauca) honey, a characteristic phenolic profile mainly consisting of gallic acid and abscisic acid could be used as the floral marker. In brush box (Lophostemon conferta) honey, the phenolic profile, comprising mainly gallic acid and ellagic acid, could be used to differentiate this honey not only from the other Australian non-Eucalyptus honeys but also from a Eucalyptus honey (yellow box or Eucalyptus melliodora honey). However, this Eucalyptus honey could not be differentiated from brush box honey based only on their flavonoid profiles. Similarly, the phenolic profile of heath (Banksia ericifolia) honey, comprising mainly gallic acid, an unknown phenolic acid (Phl) and coumaric acid, could also be used to differentiate this honey from tea tree and bloodwood honeys, which have similar flavonoid profiles. Coumaric acid is a principal phenolic acid in Australian sunflower honey and it could thus be used together with gallic acid for the authentication. These results show that the HPLC analysis of phenolic acids and abscisic acids in Australian floral honeys Could assist the differentiation and authentication of the honeys. © 2005 Elsevier Ltd. All rights reserved.

A galloylated cyanogenic glycoside from the Australian endemic rainforest tree Elaeocarpus sericopetalus (Elaeocarpaceae)

A cyanogenic glycoside -6'-O-galloylsambunigrin - has been isolated from the foliage of the Australian tropical rainforest tree species Elaeocarpus sericopetalus F. Muell. (Elaeocarpaceae). This is the first formal characterisation of a cyanogenic constituent in the Elaeocarpaceae family, and only the second in the order Malvales. 6'-O-galloylsambunigrin was identified as the principal glycoside, accounting for 91% of total cyanogen in a leaf methanol extract. Preliminary analyses indicated that the remaining cyanogen content may comprise small quantities of sambunigrin, as well as di- and tri-gallates of sambunigrin. E. sericopetalus was found to have foliar concentrations of cyanogenic glycosides among the highest reported for tree leaves, up to 5.2 mg CN g(-1) dry wt. (c) 2006 Elsevier Ltd. All rights reserved.

Composition of a protein-like fluorophore of dissolved organic matter in coastal wetland and estuarine ecosystems

This study demonstrates the compositional heterogeneity of a protein-like fluorescence emission signal (T-peak; excitation/emission maximum at 280/325 nm) of dissolved organic matter (DOM) samples collected from subtropical river and estuarine environments. Natural water samples were collected from the Florida Coastal Everglades ecosystem. The samples were ultrafiltered and excitation–emission fluorescence matrices were obtained. The T-peak intensity correlated positively with N concentration of the ultrafiltered DOM solution (UDON), although, the low correlation coefficient (r2=0.140, p<0.05) suggested the coexistence of proteins with other classes of compounds in the T-peak. As such, the T-peak was unbundled on size exclusion chromatography. The elution curves showed that the T-peak was composed of two compounds with distinct molecular weights (MW) with nominal MWs of about >5×104 (T1) and ∼7.6×103 (T2) and with varying relative abundance among samples. The T1-peak intensity correlated strongly with [UDON] (r2=0.516, p<0.001), while T2-peak did not, which suggested that the T-peak is composed of a mixture of compounds with different chemical structures and ecological roles, namely proteinaceous materials and presumably phenolic moieties in humic-like substances. Natural source of the latter may include polyphenols leached from senescent plant materials, which are important precursors of humic substances. This idea is supported by the fact that polyphenols, such as gallic acid, an important constituent of hydrolysable tannins, and condensed tannins extracted from red mangrove (Rhizophora mangle) leaves exhibited the fluorescence peak in the close vicinity of the T-peak (260/346 and 275/313 nm, respectively). Based on this study the application of the T-peak as a proxy for [DON] in natural waters may have limitations in coastal zones with significant terrestrial DOM input.

Quintosana e seus derivados conjugados com ácido gálico: avaliação de seu potencial antioxidante e de interferência na formação de cristais de oxalato de cálcio

Chitin is the second most abundant polysaccharide in nature and its derivative chitosan has been widely studied due to its unique chemical and pharmacological properties. However, studies show that when this molecule is used as food, drug, etc. it tends to accumulate in renal tissue and promotes an increase in calcium excretion. Nevertheless, the effect of chitosan on the formation of calcium oxalate (OxCa) crystals has never been evaluated. The formation of kidney stones (urolithiasis) is the disease that most often affects the kidneys and the urinary system. In addition, this is a disease with high prevalence and recurrence. Many molecules with antioxidant activity have been shown to decrease the potential for in vitro OxCa crystals formation. Thus, the aim of this study was to evaluate the effect of low molecular weight chitosan and its derivatives conjugated to gallic acid (AG) as antioxidant and inhibitor of OxCa crystals formation. The physico-chemical analysis confirmed the identity of chitosan. This molecule was subjected to five antioxidant tests and showed an excellent copper chelating activity. However, chitosan did not show other significant antioxidant activity. When chitosan was subjected to in vitro crystal formation tests, it increased the number of OxCa monohydrate crystals, modified the morphology of the crystals, modified the proportions between populations of crystals in solution and increased the zeta potential of these crystals formed. Four molecules of chitosan conjugated with GA were obtained. The physico-chemical analysis confirmed that chitosan and AG were covalently bonded. However, the amount of GA liked to chitosan did not increase even when 10 times more GA was used in experiment. When these derivatives were subjected to antioxidant tests, all chitosan conjugates showed higher antioxidant potential than their precursors. However, they showed different activity between them, which indicating that the position where AG is conjugated is an important factor for chitosan-GA activity. When conjugated chitosans were submitted to in vitro crystal formation tests, a reduction in the crystals number was observed when compared with those formed in the presence of unconjugated chitosan. Chitosan has a strong capacity for inducing OxCa monohydrate crystal formation, as well as modify their morphology and zeta potential. Over all, the process of conjugating AG to chitosan led to an increase in antioxidant potential of this molecule and was also able to decrease its capacity of inducing in vitro crystal formation

Extração, caracterização e avaliação bioativa do extrato de Arrabidaea chica

The use of plants for medicinal purposes is ancient, with widespread application in medicinal drugs. Although plants are promising sources for the discovery of new molecules of pharmacological interest, estimates show that only 17% of them have been studied for their possible use in medicine. Thus, biodiversity of Brazilian flora represents an immense potential for economic use by the pharmaceutical industry. The plant Arrabidaea chica, popularly known as “pariri”, is common in the Amazon region, and it is assigned several medicinal properties. The leaves of this plant are rich in anthocyanins, which are phenolic compounds with high antioxidant power. Antioxidant compounds play a vital role in the prevention of neurological and cardiovascular diseases, cancer and diabetes, among others. Within the anthocyanins found in Arrabidaea chica, stands out Carajurin (6,7-dihydroxy-5,4’- dimethoxy-flavilium), which is the major pigment encountered in this plant. The present work aimed to study on supercritical extraction and conventional extraction (solid-liquid extraction) in leaves of Arrabidaea chica, evaluating the efficiency of the extractive processes, antioxidant activity and quantification of Carajurin contained in the extracts. Supercritical extraction used CO2 as solvent with addition of co-solvent (ethanol/water mixture) and were conducted by the dynamic method in a fixed bed extractor. The trials followed a 24-1 fractional factorial design, the dependent variables were: process yield, concentration of Carajurin and antioxidant activity; and independent variables were: pressure, temperature, concentration of co-solvent (v/v) and concentration of water in the co-solvent mixture (v/v). Yields (mass of dry extract/mass of raw material used) obtained from supercritical extraction ranged from 15.1% to 32%, and the best result was obtained at 250 bar and 40 °C, co-solvent concentration equal to 30% and concentration of water in the co-solvent mixture equal to 50%. Through statistical analysis, it was found that the concentration of co-solvent revealed significant effect on the yield. Yields obtained from conventional extractions were of 8.1% (water) and 5.5% (ethanol). Through HPLC (High-performance liquid chromatography) analysis, Carajurin was quantified in all the extracts and concentration values (Carajurin mass/mass of dry extract) ranged between 1% and 2.21% for supercritical extraction. For conventional extraction, Carajurin was not detected in the aqueous extract, while the ethanol extract showed Carajurin content of 7.04%, and therefore, more selective in Carajurin than the supercritical extraction. Evaluation of antioxidant power (radical 2,2-diphenyl-1-picrylhydrazyl – DPPH – sequestration method) of the supercritical extracts resulted in EC50 values (effective concentration which neutralizes 50% of free radicals) ranged from 38.34 to 86.13 μg/mL, while conventional extraction resulted in EC50 values of 167.34 (water) and 42.58 (ethanol) μg/mL. As for the quantification of total phenolic content (Folin-Ciocalteau analysis) of the supercritical extracts resulted in values ranged from 48.93 and 88.62 mg GAE/g extract (GAE = Gallic Acid Equivalents), while solid-liquid extraction resulted in values of 37.63 (water) and 80.54 (ethanol) mg GAE/g extract. The good antioxidant activity cannot be attributed solely to the presence of Carajurin, but also the existence of other compounds and antioxidants in Arrabidaea chica. By optimizing the experimental design, it was possible to identify the experiment that presented the best result considering the four dependent variables together. This experiment was performed under the following conditions: pressure of 200 bar, temperature of 40 °C, co-solvent concentration equal to 30% and concentration of water in the co-solvent mixture equal to 30%. It is concluded that, within the studied range, it is possible to purchase the optimum result using milder operating conditions, which implies lower costs and greater ease of operation.

Ocean acidification and the loss of phenolic substances in marine plants

Rising atmospheric CO2 often triggers the production of plant phenolics, including many that serve as herbivore deterrents, digestion reducers, antimicrobials, or ultraviolet sunscreens. Such responses are predicted by popular models of plant defense, especially resource availability models which link carbon availability to phenolic biosynthesis. CO2 availability is also increasing in the oceans, where anthropogenic emissions cause ocean acidification, decreasing seawater pH and shifting the carbonate system towards further CO2 enrichment. Such conditions tend to increase seagrass productivity but may also increase rates of grazing on these marine plants. Here we show that high CO2 / low pH conditions of OA decrease, rather than increase, concentrations of phenolic protective substances in seagrasses and eurysaline marine plants. We observed a loss of simple and polymeric phenolics in the seagrass Cymodocea nodosa near a volcanic CO2 vent on the Island of Vulcano, Italy, where pH values decreased from 8.1 to 7.3 and pCO2 concentrations increased ten-fold. We observed similar responses in two estuarine species, Ruppia maritima and Potamogeton perfoliatus, in in situ Free-Ocean-Carbon-Enrichment experiments conducted in tributaries of the Chesapeake Bay, USA. These responses are strikingly different than those exhibited by terrestrial plants. The loss of phenolic substances may explain the higher-than-usual rates of grazing observed near undersea CO2 vents and suggests that ocean acidification may alter coastal carbon fluxes by affecting rates of decomposition, grazing, and disease. Our observations temper recent predictions that seagrasses would necessarily be "winners" in a high CO2 world.