Phytochemical and biological study of phenolic components from Geoffroea decorticans stem bark / Estudio fitoquímico y biológico de los componentes fenólicos de la corteza de Geoffroea decorticans

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.

Antifungal activity and cytotoxicity of extracts and triterpenoid saponins obtained from the aerial parts of Anagallis arvensis L.

ETHNOPHARMACOLOGICAL RELEVANCE: Anagallis arvensis L. (Primulaceae) is used in argentinean northwestern traditional medicine to treat fungal infections. We are reporting the isolation and identification of compounds with antifungal activity against human pathogenic yeast Candida albicans, and toxicity evaluation. AIM OF THE STUDY: to study the antifungal activity of extracts and purified compounds obtained form A. arvensis aerial parts, alone and in combinations with fluconazole (FLU), and to study the toxicity of the active compounds. MATERIALS AND METHODS: Disk diffusion assays were used to perform an activity-guided isolation of antifungal compounds from the aerial parts of A. arvensis. Broth dilution checkerboard and viable cell count assays were employed to determine the effects of samples and combinations of FLU + samples against Candida albicans. The chemical structures of active compounds were elucidated by spectroscopic analysis. Genotoxic and haemolytic effects of the isolated compounds were determined. RESULTS: Four triterpenoid saponins (1-4) were identified. Anagallisin C (AnC), exerted the highest inhibitory activity among the assayed compounds against C. albicans reference strain (ATCC 10231), with MIC-0 =1µg/mL. The Fractional Inhibitory Concentration Index (FICI=0.129) indicated a synergistic effect between AnC (0.125µg/mL) and FLU (0.031µg/mL) against C. albicans ATCC 10231. AnC inhibited C. albicans 12-99 FLU resistant strain (MIC-0 =1µg/mL), and the FICI=0.188 indicated a synergistic effect between AnC (0.125µg/mL) and fluconazole (16µg/mL). The combination AnC+ FLU exerted fungicidal activity against both C. albicans strains. AnC exerted inhibitory activity against C. albicans ATCC 10231 sessile cells (MIC50=0.5µg/mL and MIC80=1µg/mL) and against C. albicans 12-99 sessile cells (MIC50=0.75µg/mL and MIC80=1.25µg/mL). AnC exerted haemolytic effect against human red blood cells at 15µg/mL and did not exerted genotoxic effect on Bacillus subtilis rec strains. CONCLUSIONS: The antifungal activity and lack of genotoxic effects of AnC give support to the traditional use of A. arvensis as antifungal and makes AnC a compound of interest to expand the available antifungal drugs.

Isolation, identification and usefulness of antifungal compounds from Zuccagnia punctata for control of toxigenic ear rot pathogens.

Infusion, tincture and decoction of leaves of Zuccagnia punctata Cav. were assayed on growth of Fusarium verticillioides, F. graminearum sensu stricto, F. boothii, F. meridionale, F. subglutinans and F. thapsinum. The tincture showed the lowest IC50 on mycelial growth. A diethyl ether fraction of the tincture showed the highest antifungal activity in microdilution assays on F. verticillioides and F. graminearum. The antifungal constituents were separated by silica gel chromatography and identified as 2',4'-dihydroxychalcone, 2',4'-dihydroxy-3'-methoxychalcone and 7-hydroxy-3',4'-dimethoxyflavone. These chalcones had the lowest MIC and MFC values on F. verticillioides and F. graminearum sensu stricto. 2',4'-Dihydroxychalcone was mildly toxic and the remaining identified compounds were non-toxic in the brine shrimp assay. 2',4'-Dihydroxychalcone in mixtures with commercial food preservatives showed additive effects on F. graminearum sensu stricto and synergistic ones on F. verticillioides. 2',4'-Dihydroxy-3'-methoxychalcone showed synergistic effects in mixtures. Our results suggest that addition of chalcones to food preservatives allows reduction in the doses of the preservatives required for control of Fusarium species.

Antibacterial activities of Ligaria cuneifolia and Jodina rhombifolia leaf extracts against phytopathogenic and clinical bacteria.

Six plant extracts prepared from Ligaria cuneifolia and Jodina rhombifolia were screened for their potential antimicrobial activities against phytopathogens and clinically standard reference bacterial strains. Bioautography and broth microdilution methods were used to study samples antibacterial activities against 7 bacterial strains. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of samples were attained. An antibacterial activity guided isolation and identification of active compounds was carried out for L. cuneifolia methanolic extract (LCME). Both methanolic and aqueous extracts from L. cuneifolia showed inhibitory activities against phytopathogenic bacteria, with MICs ranging from 2.5 to 156 µg mL(-1) for LCME and 5 mg mL(-1) for the aqueous extract. None of the three J. rhombifolia extracts showed significant antibacterial activities against phytopathogenic strains (MIC > 5 mg mL(-1)), except for the aqueous extracts against Pseudomonas syringae (MIC = 312 µg mL(-1)). Only LCME showed bactericidal activities against phytopathogenic strains (MBCs = 78 µg mL(-1)). The LCME exhibited significant inhibitory activity against reference clinical strains: Escherichia coli (MIC = 156 µg mL(-1)) and Staphylococcus aureus (MIC = 78 µg mL(-1), MBC = 312 µg mL(-1)). LCME active compounds were identified as flavonol mono and diglycosides, and gallic acid. The antibacterial activity of purified compounds was also evaluated. A synergistic effect against S. aureus was found between gallic acid and a quercetin glycoside. Hence, anti-phytopathogenic bacteria potential compounds isolated from L. cuneifolia could be used as an effective source against bacterial diseases in plants.

Anti-inflammatory properties of phenolic lactones isolated from Caesalpinia paraguariensis stem bark.

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.

Free radical scavenging activities and inhibition of inflammatory enzymes of phenolics isolated from Tripodanthus acutifolius.

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.

Purification and characterization of an exo-polygalacturonase from Pycnoporus sanguineus.

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.

Antimycotic activity of 5'-prenylisoflavanones of the plant Geoffroea decorticans, against Aspergillus species.

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.