RESUMO
The stem bark of Geoffroea decorticans (Gill.ex Hook. etArn.) Burk. was used medicinally to cure several skin affections; however, phytochemical and biological antecedents were not found. Analyses of purified methanolic extract from G. decorticans bark (PFGB), realized by silylation derivatization for GC/MS, C18-CC and HPLC followed by two-dimensional TLC and UV-Vis spectroscopy, allowed to characterize nine phenolic compounds, among these, two methoxy flavonoids. Antibacterial assays of PFGB showed the highest activity (MICs = 125 µg/mL) against Staphylococcus aureus (25923) and Enterococcus faecalis (29212) ATCC strains. Moreover, PFGB showed the highest intracellular antioxidant activity at low concentration (5 µg/mL), evaluated by using the fluorescent DA-H2DCF probe on lymphocyte culture; cytotoxic effects on lymphocytes activated or not by LPS were not observed, through Trypan Blue Exclusion and MTT colorimetric assays. The results obtained from the ethnomedicinal approach of this work contribute to the scientific validation of the vulnerary medicinal use of G. decorticans.
La corteza de Geoffroea decorticans (Gill.ex Hook. EtArn.) Burk. se utiliza con fines medicinales para curar diferentes afecciones de la piel; sin embargo, no encontramos antecedentes fitoquímicos y biológicos que validen las propiedades medicinales atribuidas. Analizamos el extracto metanólico purificado de corteza de G. decorticans (PFGB), por CG-EM de la muestra derivatizada por sililación, C18-CC y HPLC seguido de CCF bidimensional, y espectroscopia UV-Vis; estos métodos nos permitieron caracterizar nueve compuestos fenólicos, entre estos, dos metoxi-flavonoides. Los ensayos antibacterianos de PFGB mostraron mayor actividad (CIMs = 125 µg/mL) contra las cepas ATCC de Staphylococcus aureus (25923) y Enterococcus faecalis (29212). Además, PFGB evidenció la mayor actividad antioxidante intracelular a baja concentración(5 µg/mL), evaluada en cultivo de linfocitos, mediante el uso de sonda fluorescente DA-H2DCF; no se observaron efectos citotóxicos sobre linfocitos activados o no por LPS, a través de ensayos colorimétricos con MTT y test de exclusión con azul Tripán. Los resultados obtenidos del abordaje etnomedicinal de este trabajo, contribuyen con la validación científica del uso medicinal vulnerario de G. decorticans.
Assuntos
Extratos Vegetais/química , Fabaceae/química , Antibacterianos/química , Fenóis/análise , Espectrofotometria/métodos , Staphylococcus aureus/efeitos dos fármacos , Extratos Vegetais/farmacologia , Testes de Sensibilidade Microbiana , Cromatografia Líquida/métodos , Enterococcus faecalis/efeitos dos fármacos , Casca de Planta , Metanol , Medicina Tradicional , Antibacterianos/farmacologiaRESUMO
The main secondary metabolite of Senecio nutans is 4-hydroxy-3-(3-methyl-2-butenyl)acetophenone (4HMBA). The antifungal activity of this compound and three derivatives was assessed using Candida albicans. 4HMBA exhibited the highest antifungal activity among the assayed compounds. The Fractional Inhibitory Concentration (FIC = 0.133) indicated a synergistic fungicidal effect of 4HMBA (5 mg L(-1)) and fluconazole (FLU) (0.5 mg L(-1)) against the C. albicans reference strain (ATCC 10231). Microscopy showed that 4HMBA inhibits filamentation and reduces cell wall thickness. Our findings suggest that 4HMBA is an interesting compound to diminish resistance to commercial fungistatic drugs such as fluconazole.
Assuntos
Acetofenonas/farmacologia , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Acetofenonas/química , Antifúngicos/química , Candida albicans/ultraestrutura , Testes de Sensibilidade a Antimicrobianos por Disco-Difusão , Relação Dose-Resposta a Droga , Viabilidade Microbiana/efeitos dos fármacosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Caesalpinia paraguariensis (D. Parodi) Burkart stem bark infusion (CPBI) is traditionally used in Argentina because their "vulnerary" properties. AIM OF THE STUDY: CPBI was studied throughout bio-guided purification procedures conducted by in vitro biological assays in order to isolate the main bioactive compounds. MATERIAL AND METHODS: Anti-inflammatory activity was assessed by enzyme inhibition assays of Hyaluronidase (Hyal) and inducible Nitric Oxide Synthase (iNOS). The antioxidant properties were evaluated by DPPH free radical scavenging assay, lipid peroxidation inhibition assay on erythrocyte membranes, and a cell-based assay that included the fluorescent probe (DCFH-DA) for indicating reactive oxygen species (ROS) generation. Bioactive compounds were purified by chromatographic methods and their structures elucidated using spectroscopic methods (ESI-MS and 1D/2D-(1)H/(13)C-NMR). RESULTS: Four main bioactive compounds were isolated from CPBI: ellagic acid (1), 3-O-methylellagic acid (2), 3,3'-di-O-methylellagic acid (3) and 3,3'-di-O-methylellagic-4-ß-D-xylopyranoside (4). These were bioactive at concentrations in which are present in CPBI, being compounds 2 and 3 the best enzyme inhibitors of Hyal and iNOS, reaching the 90% inhibitory concentration (IC90) values ranging from 2.8 to 16.4 µM, that are better than that of the positive controls, aspirin (IC90: no reached) and aminoguanidine (IC90: 20.2 µM) respectively. Compounds 2 and 3 were also better scavengers for lipoperoxides than butylated hydroxytoluene (BHT), reaching the 90% effective concentration (EC90) at 1.2-4.5 µg/ml, and for DPPH radical (2.5-7.3 µg/ml); moreover compounds were able to exert its scavenging action on intracellular ROS. Structural features relevant to the biological activities are discussed. CONCLUSIONS: This work provides scientific validity to the popular usage of CPBI.
Assuntos
Anti-Inflamatórios/farmacologia , Caesalpinia , Lactonas/farmacologia , Fenóis/farmacologia , Preparações de Plantas/farmacologia , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Antioxidantes/farmacologia , Bioensaio , Compostos de Bifenilo/química , Caesalpinia/química , Fracionamento Químico , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Membrana Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Hialuronoglucosaminidase/antagonistas & inibidores , Hialuronoglucosaminidase/metabolismo , Lactonas/química , Lactonas/isolamento & purificação , Peroxidação de Lipídeos/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Estrutura Molecular , Óxido Nítrico Sintase Tipo II/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/metabolismo , Fenóis/química , Fenóis/isolamento & purificação , Fitoterapia , Picratos/química , Casca de Planta , Preparações de Plantas/química , Preparações de Plantas/isolamento & purificação , Caules de Planta , Plantas Medicinais , Espécies Reativas de Oxigênio/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Leaf extracts from Tripodanthus acutifolius (Ruiz and Pavón) Van Tieghem have long been used in Argentinean traditional medicine as anti-inflammatory, however, there is no scientific evidence which supports this use in the literature. AIM OF THE STUDY: The present study was conducted to evaluate the ability of five phenolic compounds purified from infusion prepared from Tripodanthus acutifolius leaves to inhibit key enzymes in inflammatory processes. As anti-inflammatory compounds frequently possess free radical scavenging activities, purified substances were comparatively evaluated to asses their free radical scavenging properties. Genotoxic effects were also evaluated. MATERIALS AND METHODS: Compounds were evaluated on their ability to inhibit hyaluronidase and cyclooxygenase-2 (COX-2) activities to assess their anti-inflammatory capacities. Free radical scavenging activity was assessed by: 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH), superoxide anion assay and the inhibition on lipid peroxidation. Genotoxicity was evaluated by Bacillus subtilis rec assay. RESULTS: Fractionation of Tripodanthus acutifolius infusion yielded a novel phenylbutanoid derivative (tripodantoside) and four known flavonoid glycosides (rutin, nicotiflorin, hyperoside and isoquercitrin). Flavonoids produced higher inhibition on hyluronidase activity (IC(50) approximately 1.7 mM) than tripodantoside (IC(50)=27.90 mM). A similar COX-2 inhibition activity was exerted by tripodantoside and monoglycosilated flavonoids (IC(50) approximately 50 microM). Compounds were strong radical scavengers, with effective concentration 50 (EC(50)) values for DPPH in the range of 2.7-6.3 microg/mL, and for superoxide anion in the range of 3.9-8.7 microg/mL. All compounds scavenged peroxyl radicals in the lipid peroxidation assay. The substances showed no genotoxic effects. CONCLUSIONS: The anti-inflammatory effects, free radical scavenging activities and lack of genotoxicity of purified compounds may support the folk use of infusion from Tripodanthus acutifolius leaves as anti-inflammatory.
Assuntos
Anti-Inflamatórios/farmacologia , Inibidores de Ciclo-Oxigenase 2/farmacologia , Sequestradores de Radicais Livres/farmacologia , Loranthaceae , Fenóis/farmacologia , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/toxicidade , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/genética , Compostos de Bifenilo/química , Bovinos , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/química , Inibidores de Ciclo-Oxigenase 2/isolamento & purificação , Inibidores de Ciclo-Oxigenase 2/toxicidade , Dano ao DNA , Relação Dose-Resposta a Droga , Flavonoides/farmacologia , Sequestradores de Radicais Livres/química , Sequestradores de Radicais Livres/isolamento & purificação , Sequestradores de Radicais Livres/toxicidade , Humanos , Hialuronoglucosaminidase/antagonistas & inibidores , Hialuronoglucosaminidase/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Fenóis/química , Fenóis/isolamento & purificação , Fenóis/toxicidade , Picratos/química , Folhas de Planta , Quercetina/análogos & derivados , Quercetina/farmacologia , Proteínas Recombinantes/metabolismo , Rutina/farmacologiaRESUMO
The present work describes the purification and characterization of a novel extracellular polygalacturonase, PGase I, produced by Pycnoporus sanguineus when grown on citrus fruit pectin. This substrate gave enhanced enzyme production as compared to sucrose and lactose. PGase I is an exocellular enzyme releasing galacturonic acid as its principal hydrolysis product as determined by TLC and orcinol-sulphuric acid staining. Its capacity to hydrolyze digalacturonate identified PGase I as an exo-polygalacturonase. SDS-PAGE showed that PGase I is an N-glycosidated monomer. The enzyme has a molecular mass of 42kDa, optimum pH 4.8 and stability between pH 3.8 and 8.0. A temperature optimum was observed at 50-60 degrees C, with some enzyme activity retained up to 80 degrees C. Its activation energy was 5.352calmol(-1). PGase I showed a higher affinity towards PGA than citric pectin (Km=0.55+/-0.02 and 0.72+/-0.02mgml(-1), respectively). Consequently, PGase I is an exo-PGase, EC 3.2.1.82.
Assuntos
Glicosídeo Hidrolases/metabolismo , Poligalacturonase/metabolismo , Pycnoporus/enzimologia , Citrus/microbiologia , Meios de Cultura , Eletroforese em Gel de Poliacrilamida , Fermentação/genética , Frutas/microbiologia , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/isolamento & purificação , Ácidos Hexurônicos/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Pectinas/genética , Pectinas/isolamento & purificação , Pectinas/metabolismo , Poligalacturonase/genética , Poligalacturonase/isolamento & purificação , Análise de Sequência de DNA , Especificidade por Substrato , TemperaturaRESUMO
The antifungal activity of the ethanolic extract (EE), (3R)-5,7,2',3'-tetrahydroxy-4'-methoxy-5'-prenylisoflavanone (1) and (3R)-7-2'-3'-trihydroxy-4'-methoxy-5'-prenylisoflavanone (2) isolated from Geoffroea decorticans was evaluated against four different species of Aspergillus. Their effect was compared with that displayed by synthetic products. The antifungal activity was assayed by bioautography, hyphal radial growth, hyphal extent and microdilution in liquid medium. The percentage of hyphal radial growth inhibition produced by EE varied between 18.4+/-0.1 and 39.6+/-0.2 for Aspergillus nomius VSC23 and Aspergillus nomius 13137, respectively; and the same value for 1 and 2 were between 31.2+/-0.1-60.8+/-1.5 and 28.9+/-0.7-57.2+/-0.6 for Aspergillus flavus (IEV 018) and Aspergillus nomius 13137, respectively. The values of MIC/MFC determined for EE, 1 and 2 were compared with the actions of ascorbic and sorbic acids, and clotrimazole. The sequence of antifungal potency was clotrimazole>1>2>ascorbic acid>sorbic acid>EE. Consequently, EE as well as the purified substances from Geoffroea decorticans would be used as biopesticides against Aspergillus species. The cytotoxicity was evaluated.