The role of tannins as antiulcer agents: a fluorescence-imaging based study

Abstract Condensed tannins have been used for many years in folk medicine to treat gastric problems. The mechanism of action that explains why tannins improve gastritis symptoms is based on their ability to chelate metals, antioxidant activity, and their complexation power with other molecules. Even though these uses are well-known, the requirements to become an herbal medicine are much more complex. Herein, we analyzed Stryphnodendron rotundifolium Mart., Fabaceae, extract using MALDI for tannin characterization and carried out a fluorescence-imaging study to prove the gastroprotective effects of tannins as coating agents. Through these methods we show that condensed tannins form a gastroprotective layer. Moreover, we revise and discuss other possible mechanisms of action for phenolic-rich plant extracts and their potential in the development of herbal medicines to treat ulcers and gastritis.

Biological activities and phytochemical profile of Passiflora mucronata from the Brazilian restinga

ABSTRACT In general, Passiflora species have been reported for their folk medicinal use as sedative and anti-inflammatory. However, P. caerulea has already been reported to treat pulmonary diseases. Severe pulmonary tuberculosis, generally caused by Mycobacterium tuberculosis strains resistant to multiple drugs, can lead to deleterious inflammation and high mortality, encouraging new approaches in drug discovery. Thus, the aim of this work was to evaluate the Passiflora mucronata Lam., Passifloraceae, potential for tuberculosis treatment. Specifically, related to antimycobacterial activity and anti-inflammatory related effects (based on inhibition of nitric oxide, tumor necrosis factor-alpha production and antioxidant potential), as well as the chemical profile of P. mucronata. High performance liquid chromatography coupled with diode-array ultraviolet and mass spectrometer analyses of crude hydroalcoholic extract and ethyl acetate fraction showed the presence of flavonoids. Ethyl acetate fraction showed to be as antioxidant as Ginkgo biloba standard extract with EC50 of 14.61 ± 1.25 µg/ml. One major flavonoid isolated from ethyl acetate fraction was characterized as isoorientin. The hexane fraction and its main isolated compound, the triterpene β-amyrin, exhibited significant growth inhibitory activity against Mycobacterium bovis BCG (MIC50 1.61 ± 1.43 and 3.93 ± 1.05 µg/ml, respectively). In addition, Passiflora mucronata samples, specially hexane and dichloromethane fractions, as well as pure β-amyrin, showed a dose-related inhibition of lipopolysaccharide (LPS)-induced nitric oxide production. In conclusion, Passiflora mucronata presented relevant biological potential and should be considered for further studies using in vivo pulmonary tuberculosis model.

Use of GC/MS to identify chemical constituents and cytotoxic activity of the leaves of Phoradendron mucronatum and Phoradendron microphyllum (Viscaceae)

ABSTRACT Phoradendron mucronatum and P. microphyllum are plants that found in tropical and subtropical areas, used in traditional medicine and popularly known as mistle-thrush. The aim of this study was to identify the chemical constituents of different leaf extracts from P. mucronatum and P. microphyllum and assess cytotoxic activity against strains from a human tumour cells. Extracts obtained with hexane, dichloromethane, chloroform and ethyl acetate from the leaves were analysed by gas chromatography coupled with mass spectrometry (GC-MS) and the cytotoxicity was assessed by the MTT method (bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)). The tested human tumour cells were NCI-H292 (human pulmonar mucoepidermoid carcinoma), MCF-7 (human breast adenocarcinoma) and HEp-2 (epidermoid carcinoma of the larynx). Analysis by GC/MS of the extracts from leaves of P. microphyllum and P. mucronatum detected 51 different compounds, such as alkaloids, diterpenes, triterpenes, sterols, alcohols, aldehydes, fatty acids and hydrocarbons. In the cytotoxic evaluation, hexane and ethyl acetate extracts from the leaves P. microphyllum inhibited cell growth of NCI-H292 strains (72.97%) and HEp-2 (87.53%), respectively. The extracts of P. mucronatum species showed an inhibitory effect towards NCI-H292 (83.19%/hexane), MCF-7 (88.69%/dichloromethane) and HEp-2 (93.40%/hexane). The extracts showed cytotoxic activity against the tested strains, especially the P. mucronatum, which presented the highest percentages of inhibition of cell growth.

Antimicrobial activity of Aspilia latissima (Asteraceae)

Abstract We evaluated the antimicrobial activity of Aspilia latissima - an abundant plant from the Brazilian Pantanal region - against Candida albicans, Candida parapsilosis, Candida krusei, Candida tropicalis, Pseudomonas aeruginosa, Enterococcus faecalis, Escherichia coli and Staphylococcus aureus. The crude extracts and fractions showed activity in all tested microorganisms. The chloroform fraction of the leaves and roots showed the most antimicrobial activity against S. aureus, with an MIC of 500 μg/mL. This fraction was submitted to bioautographic assays to characterize the activity of the compounds. Two bands from the leaves (L-A and L-B) and three bands from the roots (R-C, R-D and R-E) were bioactive. Within the root-derived bands, the terpene derivatives stigmasterol, kaurenoic acid and kaura-9(11), 16-dien-18-oic acid were identified. Antibiotic activity of A. latissima is reported for the first time.

Amphidinolide P from the Brazilian octocoral Stragulum bicolor

Abstract Dinoflagellates are an important source of unique bioactive secondary metabolites. Symbiotic species, commonly named zooxanthellae, transfer most of their photosynthetically fixed carbon to their host. The mutualistic relationship provides the organic metabolites used for energy production but there are very few reports of the role of the dinoflagellates in the production of secondary metabolites in the symbiotic association. Corals and other related cnidarians are the most well-known animals containing symbiotic dinoflagellates. In the present paper we describe the isolation of amphidinolide P (1) from the octocoral Stragulum bicolor and its prey, the nudibranch Marionia limceana, collected off the coasts of Fortaleza (Ceará, Brazil). The coral extracts also contained 3-O-methyl derivative (2) of amphidinolide P, together with minor compounds still under investigation. Amphidinolides have been so far reported only in laboratory cultures of Amphidinium sp., thus compounds 1 and 2 represents the first identification of these polyketides in invertebrates. The finding proves the possibility to isolate amphidinolides from a natural symbiosis, enabling further biological and biotechnological studies.

Characterization of intestinal absorption of C-glycoside flavonoid vicenin-2 from Lychnophora ericoides leafs in rats by nonlinear mixed effects modeling

AbstractVicenin-2 (apigenin-6,8-di-C-β-d-glucopyranoside) is present in hydroalcoholic extracts of the Brazilian species Lychnophora ericoides Mart., Asteraceae, leaves, and the biological effects of this compound have been demonstrated including anti-inflammatory, antioxidant and anti-tumor effects in rat models. Given the potential of this compound as a pharmacological agent, the aims of this investigation were to evaluate the extent of intestinal absorption of vicenin-2, and to determine the intestinal permeation profile using an in situ single-pass intestinal perfusion technique. A validated HPLC–UV method was applied to measure the amount of unabsorbed vicenin-2 in the gut after an oral administration of 180 mg kg-1 in five rats. A nonlinear mixed effects model was used to determine the absorption pharmacokinetic parameters assuming a first order absorption and active secretion processes for this compound, wherein the active secretion was characterized by a zero-order process. The population pharmacokinetic parameters obtained were 0.274 min-1 for the first-order absorption rate constant, 16.3% min-1 for the zero-order rate constant; the final percentage of the original dose that was absorbed in vivo was 40.2 ± 2.5%. These parameters indicated that vicenin-2 was rapidly absorbed in the small intestine. In contrast to literature information indicating no absorption of vicenin-2 in Caco-2 cells, our results suggested that vicenin-2 can be absorbed in the small intestine of rats. The finding supports further investigation of vicenin-2 as a viable oral phytopharmaceutical agent for digestive diseases.

First serine protease inhibitor isolated from Rhinella schneideri poison

Background Toad secretions are a source of molecules with potential biotechnological application on a wide spectrum of diseases. Toads from theRhinella family have two kinds of poisonous glands, namely granular and mucous glands. Rhinella schneideritoads produce granular secretions that comprise a great number of molecules, including serine proteases inhibitors. Serine proteases, such as trypsin, chymotrypsin and elastase, are enzymes that have a serine amino acid into its catalytic site and can be found in a large number of vertebrate species and pathogenic microorganisms. Therefore, the present work aims to purify a serine protease inhibitor from Rhinella schneiderigranular secretions.Findings This study presents the protocol used to purify a serine protease inhibitor from the Rhinella schneideri poison. The granular secretion was submitted to dialysis in order to separate the low molecular weight compounds, which were submitted to a reversed phase-fast protein liquid chromatography fractionation step in a C2C18 column. The major fractions were tested over trypsin, chymotrypsin and elastase through colorimetric assay. The inhibition tests were performed with the enzyme in absence (positive control) and presence of fractions, denatured enzyme (negative control) and the respective chromogenic substrate. Rs20 was the compound with the major inhibitory activity over chymotrypsin, inducing a delay in the formation of the chromogenic enzymatic product. The structure characterization of Rs20 was performed by high resolution electronspray ionization-mass spectrometry (HRESI-MS) and gas chromatography coupled with mass spectrometry (GC-MS). HRESI showed an intense signal suggesting the presence of bufadienolide with less than 10 ppm error. In addition, it was observed a low intense signal at m/z399 that could be lithocholic acid, a biosynthetic precursor of bufadienolide. Finally, GC-MS analysis applying NIST library identification reinforced this hypothesis.Conclusions The current study have isolated and partially characterized the function and structure of the first bufadienolide with inhibitory action over chymotrypsin.

First serine protease inhibitor isolated from Rhinella schneideri poison

Background Toad secretions are a source of molecules with potential biotechnological application on a wide spectrum of diseases. Toads from theRhinella family have two kinds of poisonous glands, namely granular and mucous glands. Rhinella schneideritoads produce granular secretions that comprise a great number of molecules, including serine proteases inhibitors. Serine proteases, such as trypsin, chymotrypsin and elastase, are enzymes that have a serine amino acid into its catalytic site and can be found in a large number of vertebrate species and pathogenic microorganisms. Therefore, the present work aims to purify a serine protease inhibitor from Rhinella schneiderigranular secretions.Findings This study presents the protocol used to purify a serine protease inhibitor from the Rhinella schneideri poison. The granular secretion was submitted to dialysis in order to separate the low molecular weight compounds, which were submitted to a reversed phase-fast protein liquid chromatography fractionation step in a C2C18 column. The major fractions were tested over trypsin, chymotrypsin and elastase through colorimetric assay. The inhibition tests were performed with the enzyme in absence (positive control) and presence of fractions, denatured enzyme (negative control) and the respective chromogenic substrate. Rs20 was the compound with the major inhibitory activity over chymotrypsin, inducing a delay in the formation of the chromogenic enzymatic product. The structure characterization of Rs20 was performed by high resolution electronspray ionization-mass spectrometry (HRESI-MS) and gas chromatography coupled with mass spectrometry (GC-MS). HRESI showed an intense signal suggesting the presence of bufadienolide with less than 10 ppm error. In addition, it was observed a low intense signal at m/z399 that could be lithocholic acid, a biosynthetic precursor of bufadienolide. Finally, GC-MS analysis applying NIST library identification reinforced this hypothesis.Conclusions The current study have isolated and partially characterized the function and structure of the first bufadienolide with inhibitory action over chymotrypsin.(AU)

Biomimetic oxidation studies of monensin A catalyzed by metalloporphyrins: identification of hydroxyl derivative product by electrospray tandem mass spectrometry

Monensin A is an important commercially available natural product isolated from Streptomyces cinnamonensins that shows antibiotic and anti-parasitic activities. This molecule has a significant influence in the antibiotic market, but until now there are no studies on putative metabolite formations. Bioorganic catalysts applying metalloporphyrins and mono-oxygen donors are able to mimic the cytochrome P450 reactions. This model has been employed for natural product metabolism studies affording several new putative metabolites and in vivo experiments confirming the relevance of this procedure. In this work we evaluated the potential of 10,15,20-tetrakis (pentafluorophenyl) porphyrin metal(III) chloride [Fe(TFPP)Cl] catalyst models to afford a putative monensin A metabolite. Oxidation agents such as meta-chloroperoxy benzoic acid, iodosylbenzene, hydrogen peroxide 30 wt.% and tert-butyl hydroperoxide 70 wt.%, were used to investigate different reaction conditions, in addition to the analysis of the influence of the solvent. The quantification of total monensin A conversion and the structure of the new hydroxylated putative metabolite were proposed based on electrospray ionization tandem mass spectrometry analysis. The porphyrin tested, afforded moderate conversions of monensin A in all reaction conditions and the selectivity was found to be dependent on the oxidation/medium employed.

Antiproliferative activity of Eremanthus crotonoides extracts and centratherin demonstrated in brain tumor cell lines

The genus Eremanthus is recognized by the predominance of sesquiterpene lactones from the furanoheliangolide type, a class of substances extensively tested against cancer cell lines. Thus, the species E. crotonoides (DC.) Sch. Bip., Asteraceae, obtained on "restinga" vegetation was evaluated against U251 and U87-MG glioma cell lines using the MTT colorimetric assay. Dichloromethane fraction was cytotoxic to both glioblastoma multiforme cell lines. We then conducted UPLC-PDA-ESI-MS/MS analysis of the dichloromethane fraction, which allowed the identification of the sesquiterpene lactones centratherin and goyazensolide. The isolation of centratherin was performed using chromatographic techniques and the identification of this substance was confirmed according to NMR data. Cytotoxic activity of centratherin alone was also evaluated against both U251 and U87-MG cells, which showed IC50 values comparable with those obtained for the commercial anticancer drug doxorubicin. All the tested samples showed cytotoxic activity against glioblastoma multiforme cells which suggests that E. crotonoides extracts may be important sources of antiproliferative substances and that the centratherin may serve as prototype for developing new antiglioblastoma drugs.

Antioxidant activity and chemical composition of the non polar fraction of Gracilaria domingensis (Kützing) Sonder ex Dickie and Gracilaria birdiae (Plastino & Oliveira)

Gracilaria domingensis (Kützing) Sonder ex Dickie and Gracilaria birdiae (Plastino & Oliveira) (Gracilariales, Rhodophyta) are seaweeds that occur on the Brazilian coast. Based on their economic and pharmaceutical importance, we investigated the antioxidant activity of the methanolic, ethyl acetate and hexane extracts of both species. The hexane extracts display a high antioxidant activity and comparative analyses indicated G. birdiae as the most active species. Chemical investigation of these fractions showed several carotenoids and fatty acids, as well as cholesterol and sitosterol derivatives. HPLC-DAD analysis of G. birdiae showed violaxanthin (0.04 μg.mg-1 of dry material), antheraxanthin (5.31 μg.mg-1), aloxanthin (0.09 μg.mg-1), zeaxanthin (0.45 μg.mg-1) and β-carotene (0.37 μg.mg-1) as the major carotenoids. G. domingensis showed a similar carotenoid profile, however, with much lower concentration than G. birdiae. Gas chromatography coupled to mass spectrometry was used to determine other nonpolar compounds of these seaweeds. The main compounds detected in both studied species were the fatty acids 16:0; 18:1 Δ9; 20:3 Δ6,9,12, 20:4 Δ5,8,11,14. We found no specificity of compounds in either species. However, G. birdiae, presented higher contents of carotenoids and arachidonic acid than G. domingensis.

Contribution for the phytochemical studies of Ageratum fastigiatum

Organic extracts from leaves plus branches plus inflorescences of Ageratum fastigiatum (Gardner) R. M. King & H. Rob., Asteraceae, were fractionated through classic chromatography. The steroids stigmasterol, chondrillasterol and campesterol were isolated from hexane extract. The triterpenes lupeol, taraxasterol, α-amyrin, β-amyrin, pseudotaraxasterol, lupeol acetate and α-amyrin acetate were isolated from ethyl acetate extract. Steroids and triterpenes were identified by GC-MS. The coumarin ayapin was isolated from ethanol extract and identified by NMR. Essential oils of the fresh leaves and fresh inflorescences were obtained by hydrodistillation and analyzed for GC-MS. The main components in both essential oils were α-pinene, limonene and germacrene D.

Antimicrobial activity of Aegiphila sellowiana Cham., Lamiaceae, against oral pathogens / Atividade antimicrobiana de Aegiphila sellowiana Cham., Lamiaceae, contra patógenos orais

The antimicrobial activity of Aegiphila sellowiana Cham., Lamiaceae, against oral pathogens is reported. The Minimal Inhibitory Concentrations (MICs) for inhibiting the microorganisms growth were determined using the broth microdilution method from the CLSI M7-A7 protocol. Chlorhexidine was used as the positive control. The ethanol crude extract of the aerial parts of A. sellowiana exhibited activity against the microorganisms tested in this work; however, the activity decreased after partition with n-hexane, dichloromethane, and ethyl acetate. Among the tested fractions, the n-hexane fraction was found to be the most effective against the evaluated oral pathogens. GC-MS analysis of this latter fraction revealed that fatty acids esters, steroids, and aliphatic sesquiterpene hydrocarbons are its major constituents. These compounds may be responsible for the activity of the n-hexane fraction, but other chemical constituents of the dichloromethane, ethyl acetate, and hydroalcoholic fraction may potentialize their activities in the crude extract.

A atividade antimicrobiana de Aegiphila sellowiana Cham., Lamiaceae, contra patógenos da cavidade bucal foi avaliada empregando-se o método de microdiluição em caldo, segundo o protocolo CLSI M7-A7, utilizando-se clorexidina como controle positivo. O extrato etanólico bruto das partes aéreas de A. sellowiana, obtido por maceração, exibiu atividade contra os microrganismos testados neste trabalho, entretanto, os valores de CIM (concentração inibitória mínima) aumentaram após a partição com n-hexano, diclorometano e acetato de etila. Entre as frações testadas, a fração n-hexânica foi a mais efetiva contra os patógenos bucais avaliados, com valores de CIM entre 140 e 350 µg/mL-1. Dados obtidos por CG-EM revelaram que ésteres de ácidos graxos, esteróides e hidrocarbonetos sesquiterpênicos alifáticos são os constituintes majoritários desta fração. Embora esses compostos possam ser os responsáveis pela atividade da fração n-hexânica, outros constituintes químicos presentes nas frações em diclorometano, acetato de etila e hidroalcoólica podem potencializar suas atividades no extrato bruto.

Variabilidade sazonal do teor de saponinas de Baccharis trimera (Less. ) DC (Carqueja) e isolamento de flavona / Seasonal variability in the content of saponins from Baccharis trimera (Less. ) DC (Carqueja) and isolation of flavone

O objetivo deste trabalho foi avaliar a sazonalidade sobre o teor de saponinas e o isolamento e elucidação estrutural de constituinte químico (flavona) de Baccharis trimera (Less.) DC (Carqueja). O cultivo de indivíduos estaminados de B. trimera foi realizado no Campo Experimental da Universidade de Ribeirão Preto - Unaerp, em Ribeirão Preto, no período de janeiro a dezembro de 1998 e foi conduzido em três blocos, sendo que cada um deles era constituído de 48 mudas. As amostras foram colhidas após seis meses do plantio (durante o inverno), três meses após a primeira colheita (durante a primavera) e três meses após a segunda colheita (durante o verão). Após processo de secagem e moagem, as drogas foram submetidas ao ensaio para obtenção do índice de espuma. Os resultados mostraram que não há variabilidade neste índice em função da época de colheita. Parte da droga colhida no verão sofreu análise química para isolamento e elucidação estrutural de componente químico. O isolamento a partir do extrato metanólico de partes aéreas de B. trimera foi realizado através de métodos cromatográficos (CC e CLAE) e a identificação estrutural da substância isolada, através de métodos espectrométricos (UV, RMN ¹H e 13C). Foi identificada a flavona denominada 5,6-diidroxi-7,3,4-trimetoxiflavona.

The aim of this study was to evaluate the seasonal effect on the foam index and the isolation and structural identification of a chemical constituent (flavone) of Baccharis trimera. The culture staminate samples of B. trimera were realized in the experimental cultivation area of the Universidade de Ribeirão Preto - Unaerp, in Ribeirão Preto, state of São Paulo, Brazil from January to December/1998 and were conduced in three blocks, which received 48 seedlings. The samples were harvested six months after installation (during the winter), three months after the first harvest (during the spring) and three months after the second harvest (during the summer). After the drying process and comminution, an assay to obtain the foam index was performed with the plant material. The results didn't show any alteration, independently of harvest times. Part of the dried plant that had been harvested during the summer was extracted to isolate and identify structurally the chemical compound. The isolation from the methanol extracts of the aerial parts of B. trimera has been performed through chromatographic methods (CC and HPLC) and the identification through spectrometric methods (UV, ¹H and 13C NMR). A flavone was identified as 5,6-dihidroxy-7,3,4-trimetoxiflavone.

Differences in secondary metabolites from leaf extracts of Mikania glomerata Sprengel obtained by micropropagation and cuttings / Diferenças nos metabólitos secundários acumulados nos extratos das folhas de Mikania glomerata Sprengel obtidos por micropropagação e estaquia

GC analysis of the dichloromethane extracts obtained from cultivated specimens of Mikania glomerata Sprengel possibilited to verify that cuttings technique led to production of kaurenoic acid and coumarin while the same results have not been observed by propagation process using in vitro techniques.

A análise realizada por CG dos extratos diclorometânicos das folhas de Mikania glomerata Sprengel mostrou que a planta propagada por estaca produziu cumarina e ácido caurenóico, enquanto que o material micropropagado acumulou apenas cumarina.

Esteróides e cumarina em calos de Mikania glomerata Sprengel / Steroides and coumarin from callus of Mikania Glomerata Sprengel

O estudo fitoquímico do extrato diclorometânico dos calos obtidos a partir de segmentos foliares de Mikania glomerata Sprengel resultou no isolamento dos esteróides campesterol, estigmasterol e ß-sitosterol e da cumarina, não tendo sido detectado nenhum diterpeno do tipo caurano