Análise botânica, química e biológica comparativa entre flores das espécies sambucus nigra L. e sambucus australis cham. & Schltdt. e avaliação preliminar da estabilidade

Flores das espécies Sambucus nigra, de origem européia, e Sambucus australis, nativa da América do Sul (Caprifoliaceae), denominadas sabugueiro e sabugueirodo- brasil, respectivamente, são utilizadas popularmente sob forma de infusão, como antiinflamatórias, laxativas e para condições febris resultantes de afecções do trato respiratório. Estudos prévios para S. nigra indicam compostos fenólicos como principais constituintes químicos, sendo estes relacionados às principais atividades biológicas avaliadas. Objetivando comparar esta espécie com Sambucus australis, foram realizadas análises botânicas macro e microscópicas das flores, identificando as principais diferenças entre as espécies, tais como o número de lóculos no ovário e presença de idioblastos cristalíferos em algumas estruturas, e observando os possíveis contaminantes (pedicelos). Também foram determinados os parâmetros farmacopéicos: cinzas totais e perda por dessecação. Após a análise química, foi escolhido o flavonóide rutina como marcador das espécies, para realizar análises quantitativas nas 31 amostras adquiridas e / ou coletadas, utilizando método de CLAE previamente validado. Soluções hidroetanólicas apresentaram maior capacidade de extração do produto alvo. Os limites mínimos de rutina observados para ambas as espécies foram de aproximadamente 0,65%. Também foram quantificados, por método espectrofotométrico, os flavonóides totais expressos em quercetina, sendo 0,93% e 1,46% os teores mínimos determinados para S.nigra e S. australis, respectivamente. O estudo da estabilidade acelerada (50°C ± 90% U.R.), avaliando a degradação dos constituintes químicos presentes permitiu sugerir a cinética de degradação de segunda ordem para da rutina nas duas espécies. Comparações de atividades biológicas das espécies foram realizadas pelos ensaios das atividades antiinflamatória (inibição do edema em pata de rato induzido por carragenina) e antioxidante (DPPH). Os resultados para o primeiro ensaio demonstraram ação equivalente em ambas as espécies para extratos hidroetanólicos a 80% (86% de inibição) e aquosos (81%), com atividade semelhante ao padrão indometacina (~83%); para a atividade antioxidante os extratos hidroetanólicos a 80% foram mais ativos (CE50 = 16 μg/ml) que os aquosos (CE50 = 27 μg/ml) em S. australis, e ambos extratos, superiores ao 28 extrato padronizado Gingko biloba (CE50 = 40 μg/ml) e aos extratos de S. nigra (CE50= 50 μg/ml – hidroetanólico e CE50= 32 μg/ml – aquoso).

Nematicidal activity of Solanum nigrum and S. sisymbriifolium extracts against the root-lesion nematode Pratylenchus goodeyi and its effects on infection and gene expression

The control of Pratylenchus goodeyi a common nematode parasite of banana crop in Madeira Island can benefit from searching for natural nematicides through plants extracts. With this aim we submitted Solanum nigrum and S. sisymbriifolium dried plants to a sequential extraction in the solvent sequence of dichloromethane, acetone, ethanol and water, and to na aqueous extraction of the fresh and dried plants. Analyses with the extracts at several concentrations were used to assess mobility and mortality on P. goodeyi. Results showed that the water extract and aqueous extracts from both plants at a concentration of 10 mg/mL affected nematode mobility and caused mortality but the acetone extract from S. nigrum was the most efficient, causing 100% mortality whereas dichloromethane had no effect on P. goodeyi. Determination of the lipophilic and phenolic compounds present in the two most effective Solanum extracts (acetone and water) and in dichloromethane extract revealed that some of these compounds had nematicidal activity. S. nigrum acetone extract (10 mg/mL) was used to find out the nematicidal potential following the effect at gene expression level and nematode behaviour. Genes coding for calreticulin and beta-1,4- endoglucanase related to parasitism and translocon-associated protein putatively connected to stress were obtained and its relative expression assessed in nematodes exposed to the extract. Results revealed that expression of Pg-CRT decreased showing to influence the infection, Pg-ENG remained steady and Pg-TRAPδ was induced over time exposure. Biological assays showed that P. goodeyi mobility and ability to infect the banana roots were affected as a decrease in the number of nematodes that reached the roots was obtained with the increased exposure time to the extract being implicated in the infection success. The information obtained from this thesis showed that S. nigrum has potential to be used for the development of a new control strategy against plant-parasitic nematodes.

HPLC-DAD methodology for the quantification of organic acids, furans and polyphenols by direct injection of wine samples

This article proposes a simple and sensitive HPLC method with photo-diode array detection for the analysis of organic acids, monomeric polyphenols and furanic compounds in wine samples by direct injection. The chromatographic separation of 8 organic acids, 2 furans and 22 phenolic compounds was carried out with a buffered solution (pH 2.70) and acetonitrile as mobile phases and a difunctionally bonded C18 stationary phase, Atlantis dC18 (250 4.6 mm, 5mm) column. The elution was performed in 12 min for the organic acids and in 60 min for the phenolic compounds, including phenolic acids, stilbenes and flavonoids. Target compounds were detected at 210 nm (organic acids, flavan-3-ols and benzoic acids), 254 nm (ellagic acid), 280 nm (furans and cinnamic acid), 315 nm (hydroxycinnamic acids and trans-resveratrol) and 360 nm (flavonoids). The RSD for the repeatability test (n55) of peak area and retention times were below 3.1 and 0.3%, respectively, for phenolics and below 1.0 and 0.2% for organic acids. The RSDs expressing the reproducibility of the method were higher than for the repeatability results but all below 9.0%. Method accuracy was evaluated by the recovery results, with averaged values between 80 and 104% for polyphenols and 97–105% for organic acids. The calibration curves, obtained by triplicate injection of standard solutions, showed good linearity with regression coefficients higher than 0.9982 for polyphenols and 0.9997 for organic acids. The LOD was in the range of 0.07–0.49 mg/L for polyphenols (cinnamic and gallic acids, respectively) and 0.001–0.046 g/L for organic acids (oxalic and lactic acids, respectively). The method was successfully used to measure and assess the polyphenolic fingerprint and organic acids profile of red, white, rose ´ and fortified wines.

Quantification of polyphenols with potential antioxidant properties in wines using reverse phase HPLC

A RP-HPLC method with photodiode array detection (DAD) was developed to separate, identify and quantify simultaneously the most representative phenolic compounds present in Madeira and Canary Islands wines. The optimized chromatographic method was carefully validated in terms of linearity, precision, accuracy and sensitivity. A high repeatability and a good stability of phenolics retention times (a3%) were obtained, as well as relative peak area. Also high recoveries were achieved, over 80.3%. Polyphenols calibration curves showed a good linearity (r2 A0.994) within test ranges. Detection limits ranged between 0.03 and 11.5 lg/mL for the different polyphenols. A good repeatability was obtained, with intra-day variations less than 7.9%. The described method was successfully applied to quantify several polyphenols in 26 samples of different kinds of wine (red, ros and white wines) from Madeira and Canary Islands. Gallic acid was by far the most predominant acid. It represents more than 65% of all phenolics, followed by p-coumaric and caffeic acids. The major flavonoid found in Madeira wines was trans-resveratrol. In some wines, (–)-epicatechin was also found in highest amount. Canary wines were shown to be rich in gallic, caffeic and p-coumaric acids and quercetin.

Effects of Purified Saccharomyces cerevisiae (1→3)-β-Glucan on Venous Ulcer Healing

Water-insoluble glucan was isolated from the baker’s yeast Saccharomyces cerevisiae. The yeast cells were treated with alkali and the residue then with acid. Chemical and NMR (1D and 2D) analyses showed that a linear (1→3)-β-glucan was purified that was not contaminated with other carbohydrates, proteins or phenolic compounds. The effects of the glucan on wound healing were assessed in human venous ulcers by histopathological analysis after 30 days of topical treatment. (1→3)-β-glucan enhanced ulcer healing and increased epithelial hyperplasia, as well as increased inflammatory cells, angiogenesis and fibroblast proliferation. In one patient who had an ulcer that would not heal for over 15 years, glucan treatment caused a 67.8% decrease in the area of the ulcer. This is the first study to investigate the effects of (1→3)-β-glucan on venous ulcer healing in humans; our findings suggest that this glucan is a potential natural biological response modifier in wound healing

Effects of Purified Saccharomyces cerevisiae (1→3)-β-Glucan on Venous Ulcer Healing

Water-insoluble glucan was isolated from the baker’s yeast Saccharomyces cerevisiae. The yeast cells were treated with alkali and the residue then with acid. Chemical and NMR (1D and 2D) analyses showed that a linear (1→3)-β-glucan was purified that was not contaminated with other carbohydrates, proteins or phenolic compounds. The effects of the glucan on wound healing were assessed in human venous ulcers by histopathological analysis after 30 days of topical treatment. (1→3)-β-glucan enhanced ulcer healing and increased epithelial hyperplasia, as well as increased inflammatory cells, angiogenesis and fibroblast proliferation. In one patient who had an ulcer that would not heal for over 15 years, glucan treatment caused a 67.8% decrease in the area of the ulcer. This is the first study to investigate the effects of (1→3)-β-glucan on venous ulcer healing in humans; our findings suggest that this glucan is a potential natural biological response modifier in wound healing

Atividades Biológicas De Xilana De Sabugo De Milho

The corn cob is an agricultural by-product still little used, this in part due to the low knowledge of the biotechnological potential of their molecules. Xylan from corn cobs (XSM) is a polysaccharide present in greater quantity in the structure of plant and its biotechnology potential is little known. This study aimed to the extraction, chemical characterization and evaluation of biological activities of xylan from corn cobs. To this end, corncobs were cleaned, cut, dried and crushed, resulting in flour. This was subjected to a methodology that combines the use of alkaline conditions with waves of ultrasound. After methanol precipitation, centrifugation and drying was obtained a yield of 40% (g/g flour). Chemical analysis indicated a high percentage of polysaccharides in the sample (60%) and low contamination by protein (0.4%) and phenolic compounds (> 0.01%). Analysis of monosaccharide composition indicated the presence of xylose:glucose:arabinose:galactose:mannose:glucuronic acid in a molar ratio 50:20:15:10:2.5:2.5. The presence of xylan in the sample was confirmed by nuclear magnetic resonance (¹H and ¹³C) and infrared spectroscopy (IR). Tests were conducted to evaluate the antioxidant potential of XSM. This showed a total antioxidant capacity of 48.45 EAA/g sample. However, did not show scavenging activity of superoxide and hydroxyl radical and also reducing power. But, showing a high capacity chelating iron ions with 70% with about 2 mg/mL. The ability to XSM to influence cell proliferation in culture was also evaluated. This polymer did not influence the proliferation of normal fibroblast cells (3T3), however, decreased the rate of proliferation of tumor cells (HeLa) in a dose-dependent, reaching an inhibition of about 50% with a concentration around 2 mg/mL. Analyzing proteins related to cell death, by immunoblotting, XSM increases the amount of Bax, Bcl-2 decrease, increase cytochrome c and AIF, and reduce pro-caspase-3, indicating the induction of cell death induced apoptosis dependent and independent of caspase. XSM did not show anticoagulant activity in the PT test. However, the test of activated partial thromboplastin time (aPTT), XSM increased clotting time at about 5 times with 600 μg of sample compared with the negative control. The presence of sulfate on the XSM was discarded by agarose gel electrophoresis and IR. After carboxyl-reduction of XSM the anticoagulant activity decreased dramatically. The data of this study demonstrate that XSM has potential as antioxidant, antiproliferative and anticoagulant compound. Future studies to characterize these activities of XSM will help to increase knowledge about this molecule extracted from corn and allow their use in functional foods, pharmaceuticals and chemical industries.

Efeito de glucanas do fungo Caripia montagnei em modelo de inflamação intestinal induzida por tnbs em ratos Wistar e em células de carcinoma de cólon humano HT-29

Compounds derived from fungi has been the subject of many studies in order to broaden the knowledge of their bioactive potential. Polysaccharides from Caripia montagnei have been described to possess anti-inflammatory and antioxidant properties. In this study, glucans extracted from Caripia montagnei mushroom were chemically characterized and their effects evaluated at different doses and intervals of treatment. It was also described their action on colonic injury in the model of colitis induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS), and its action on cells of the human colon carcinoma (HT-29). Compounds extracted of C. montagnei contain high level of carbohydrates (96%), low content of phenolic compounds (1.5%) and low contamination with proteins (2.5%). The (FT-IR) and (NMR) analysis showed that polysaccharides from this species of mushroom are composed of α- and β-glucans. The colonic damage was evaluated by macroscopic, histological, biochemical and immunologic analyses. The results showed a reduction of colonic lesions in all groups treated with the glucans of Caripia montagnei (GCM). GCM significantly reduced the levels of IL-6 (50 and 75 mg/kg, p < 0.05), a major inflammatory cytokine. Biochemical analyses showed that such glucans acted on reducing levels of alkaline phosphatase (75 mg/kg, p < 0.01), nitric oxide (p < 0.001), and myeloperoxidase (p < 0.001). These results were confirmed microscopically by the reduction of cellular infiltration. The increase of catalase activity suggest a protective effect of GCM on colonic tissue, confirming their anti-inflammatory potential. GCM displayed cytostatic activity against HT-29 cells, causing accumulation of cells in G1 phase, blocking the cycle cell progression. Those glucans also showed ability to modulate the adhesion of HT-29 cells to Matrigel® and reduced the oxidative stress. The antiproliferative activity against HT-29 cells displayed by GCM (p <0.001) can be attributed to its cytostatic activity and induction of apoptosis by GCM

Propriedades antioxidante, anti-hemostástica e antiproliferativa de galactanas sulfatadas da alga vermelha hypnea musciformis (wulfen) j. V. Lamouroux

Marine algae are one of the major sources of biologic compounds. In extracellular matrix of these organisms there are sulfated polysaccharides that functions as structural components and provides protection against dehydration. The fraction 1.0 (F1.0) rich in sulfated galactans obtained from red seaweed Hypnea musciformis was physicochemical characterized and evaluated for pharmacologic activity through antioxidant activity, cytotoxic action on erythrocytes, anticoagulant, stimulatory action under antithrombotic heparan sulfate synthesis and their effects on cell proliferation and cycle cell progression. The main components of F1.0 were carbohydrates (49.70 ± 0.10%) and sulfate (44.59 ± 0.015%), presenting phenolic compounds (4.79 ± 0.016%) and low protein contamination (0.92 ± 0.001%). Fraction 1.0 showed polidisperse profile and signs in infrared analysis in 1262, 1074 and 930, 900 and 850 attributed to sulfate esters S=O bond, presence of a 3,6- anidrogalactose C-O bond, non-sulfated β-D-galactose and a C-O-SO4 bond in galactose C4, respectively. The fraction rich in sulfated galactans exhibited strong antioxidant action under lipid peroxidation assay with IC50 of 0.003 mg/mL. Besides the inhibition of hemolysis induced by H2O2 in erythrocytes treated with F1.0, this fraction did not promote significant cytotoxity under erythrocytes membranes. F1.0 exhibited low anticoagulant activity causing moderate direct inhibition of enzimatic activity of thrombin. This fraction promoted stimulation around of 4.6 times on this synthesis of heparan sulfate (HS) by rabbit aortic endothelial cells (RAEC) in culture when was compared with non treated cells. The fraction of this algae displayed antiproliferative action under RAEC cells causing incresing on cell number on S fase, blocking the cycle cell progression. Thus F1.0 presented cytostatic and no cytotoxic action under this cell lineage. These results suggest that F1.0 from H. musciformis have antioxidant potential which is a great effect for a compound used as food and in food industry which could be an alternative to food industry to prevent quality decay of lipid containing food due to lipid peroxidation. These polysaccharides prevent the lipid peroxidation once the fraction in study exhibited strong inhibitory action of this process. Furthermore that F1.0 present strong antithrombotic action promoting the stimulation of antithrombotic HS synthesis by endothelial cells, being important for thrombosis preventing, by its inhibitory action under reactive oxygen species (ROS) in some in vitro methods, being involved in promotion of hypercoagulability state.

Caracterização estrutural e avaliação das atividades farmacológicas da fucana B extraída da alga Dictyota menstrualis

Seaweeds are a major source of biologically active compounds . In the extracellular matrix of these organisms are sulfated polysaccharides that functions as structural components preventing it against dehydration. The fraction 0.9 (FucB) rich in sulfated fucans obtained from brown seaweed Dictyota menstrualis was chemical characterized and evaluated for pharmacological activity by testing anticoagulant activity, stimulatory action on the synthesis of an antithrombotic heparan sulfate, antioxidant activity and its effects in cell proliferation. The main components were FucB carbohydrates (49.80 ± 0.10 %) and sulfate (42.30 ± 0.015 %), with phenolic compounds ( 3.86 ± 0.016 %) and low protein contamination ( 0.58 ± 0.001 % ) . FucB showed polydisperse profile and analysis of signals in the infrared at 1262, 1074 and 930 cm -1 and 840 assigned to S = O bonds sulfate esters , CO bond presence of 3,6- anhydrogalactose , β -D- galactose non- sulfated sulfate and the axial position of fucose C4 , respectively. FucB exhibited moderate anticoagulant activity , the polysaccharides prolonged time (aPTT ) 200 ug ( > 90s ) partial thromboplastin FucB no effect on prothrombin time (PT), which corresponds to the extrinsic pathway of coagulation was observed. This stimulation promoted fraction of about 3.6 times the synthesis of heparan sulfate (HS) by endothelial cells of the rabbit aorta ( RAEC ) in culture compared with cells not treated with FucB . This has also been shown to compete for the binding site with heparin. The rich fraction sulfated fucans exhibited strong antioxidant activity assays on total antioxidant (109.7 and 89.5 % compared with BHT and ascorbic acid standards ) , reducing power ( 71 % compared to ascorbic acid ) and ferric chelation ( 71 , comparing with 5 % ascorbic acid). The fraction of algae showed cytostatic activity on the RAEC cells revealed that the increase of the synthesis of heparan sulfate is not related to proliferation. FucB showed antiproliferative action on cell lines modified as Hela and Hep G2 by MTT assay . These results suggest that FucB Dictyota menstrualis have anticoagulant , antithrombotic , antioxidant potential as well as a possible antitumor action, promoting the stimulation of the synthesis of antithrombotic HS by endothelial cells and is useful in the prevention of thrombosis, also due to its inhibitory action on species reactive oxygen ( ROS ) in some in vitro systems , being involved in promoting a hypercoagulable state

Bioprospecção de extratos metanólicos de macroalgas marinhas do litoral do Rio Grande do Norte: avaliação das atividades anticoagulante, antioxidante e antiproliferativa

Seaweeds are organisms known to exhibit a variety of biomolecules with pharmacological properties. The coast of Rio Grande do Norte has over 100 species of seaweeds, most of them not yet explored for their pharmacological potential. Sugars and phenolic compounds are the most studied of these being assigned a range of biological properties, such as anticoagulant , antiinflammatory, antitumor and antioxidant activities. In this work, we obtained methanolic extracts from thirteen seaweeds of the coast of Rio Grande do Norte (Dictyota cervicornis; Dictiopterys delicatula; Dictyota menstruallis; D. mertensis; Sargassum filipendula; Spatoglossum schröederi; Acanthophora specifera; Botryocladia occidentalis; Caulerpa cupresoides; C. racemosa; C. prolifera; C. sertularioides e Codium isthmocladum). They were evaluated as anticoagulant and antioxidant drugs, as well as antiproliferative drugs against the tumor cell line HeLa. None of the methanolic extracts showed anticoagulant activity, but when they were evaluated as antioxidant drugs all of extracts showed antioxidant activity in all tests performed (total antioxidant capacity, sequestration of superoxide and hydroxyl radicals, ferric chelation and reductase activity), especially the algae D. mentrualis, D. cilliolata and C. prolifera, who had the greatest potential to donate electrons.In addition, the ability of iron ions chelation appears as the main antioxidant mechanism of the methanolic extracts of these seaweeds mainly for the extract of the C. racemosa seaweed, which reached almost 100% activity. In the MTT assay, all extracts showed inhibitory activity at different levels againts HeLa cells. Moreover, D. cilliolata (MEDC) and D. menstrualis (MEDM) extracts showed specific activity to this cell line, not inhibiting the viability of 3T3 normal cell line, so they were chosen for detailing the antiproliferative mechanism of action. Using flow cytometry, fluorescence microscopy and in vitro assays we demonstrated that MEDC and MEDM induced apoptosis in HeLa cells by activation of caspases 3 and 9 and yet, MEDC induces cell cycle arrest in S phase. Together, these results showed that the methanolic extracts of brown seaweed D. menstrualis and D. cilliolata may contain agents with potential use in combatting cells from human uterine adenocarcinoma. This study also points to the need for more in-depth research on phytochemical and biological context to enable the purification of biologically active products of these extracts

Evaluation of anti-inflammatory activity of derivatives from aerial parts of Baccharis uncinella

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação da atividade antioxidante e antiproliferativa do extrato aquoso de frutas tropicais

The antioxidant activity of aqueous extracts of five edible tropical fruits (Spondias lutea, Hancornia speciosa, Spondias purpurea, Manilkara zapota and Averrhoa carambola) was investigated using different methods. The amount of phenolic compounds was determined by the Folin-Ciocalteu reagent. The M. zapota had Total Antioxidant Capacity (TAC) higher than the other fruits. Extracts showed neither reducing power nor iron chelation (between 0.01 and 2.0 mg/mL). H. speciosa exhibited the highest superoxide scavenging activity (80%, 0.5 mg/mL). However, at high concentrations (8.0 mg/mL) only A. carambola, S. purpurea and S. lutea scavenging 100% of radicals formed. M. zapota and S. purpurea had higher phenolic compound levels and greater OH radical scavenging activity (92 %, 2.0 mg/mL). Antiproliferative activity was assessed with 3T3 fibroblasts and cervical tumor cells (HeLa). The most potent extract was S. purpurea (0.5 mg/mL), which inhibited HeLa cell proliferation by 52%. The most fruits showed antioxidant and antiproliferative properties, characterizing them as functional foods.