Marta A. Vattuone

Comparison of the free radical-scavenging activity of propolis from several regions of Argentina

Propolis is extensively used in Argentine folk medicine. Alcoholic extracts of propolis from different regions of Argentina were prepared. The extracts were analysed for the determination of total flavonoid content (from 13.3 to 42.6 mg/g of propolis) by using the aluminum nitrate method, UV spectrophotometry and thin layer chromatography. All of them contained high total flavonoid content. It was also observed that all samples of ethanolic extracts of propolis showed free radical-scavenging activity in terms of scavenging of the radical DPPH but the highest activities were found for samples from Tucumán and Santiago del Estero. In all cases with 20 microg/ml of soluble principles, the percentage of DPPH degradation was different (Banda Oeste: 67.5%; Verónica: 45%; Forres: 35%; Saenz Peña: 20% and Juan José Castelli: 55%). These results may justify their use as a source of natural antioxidants.

Screening antifungal activities of selected medicinal plants

Plants synthesise a vast array of secondary metabolites that are gaining importance for their biotechnological applications. The antifungal activity of the ethanolic extracts of ten Argentinean plants used in native medicine is reported. Antifungal assays included radial growth inhibition, disk and well diffusion assays and growth inhibition by broth dilution tests. The chosen test fungi were yeasts, microfungi and wood-rot causing Basidiomycetes. Extracts of Larrea divaricata, Zuccagnia punctata and Larrea cuneifolia displayed remarkable activity in the assays against the majority of the test fungi. In addition to the former plants, Prosopanche americana also inhibited yeast growth.

Antioxidant activity of Argentine propolis extracts

Propolis is used in Argentine folk medicine. We have examined its possible protective action against oxidative modification of lipid in unfractionated serum. The kinetics of copper-induced oxidation was continuously monitored by measuring the formation of conjugated dienes, as the increase in the absorbance at 234 nm. According to the kinetics of oxidation, the propolis were classified in three different groups. Group I (CE, CO, BO, MO, BE) inhibited lipid oxidation during the initiation and propagation phases even at low concentrations. Group II (SP, CA, AM) increased the lag-phase for conjugated diene formation. All propolis in groups I and II diminished the maximal rate of diene production and the maximal amount of dienes produced. Group III (PA, RA, FE, VR, TV) had no effect on the lipid oxidation. The extent of lipoprotein oxidation was measured by the thiobarbituric acid reactive substance assay. Generation of malondialdehyde-like substances was inhibited and delayed by the presence of propolis extracts from group I and II. Our results justify the use of propolis (groups I and II) as a source of natural antioxidants.

Screening of antibacterial activity of Amaicha del Valle (Tucumán, Argentina) propolis

Propolis is extensively used in Argentine folk medicine. Alcoholic extracts of propolis from four localities of Amaicha del Valle (El Paraiso, La Banda Este, La Banda Oeste and El Molino), Province of Tucumán and from Cerrillos, Province of Santiago del Estero, Argentina were prepared. All showed antibacterial activity against Gram positive bacteria, the propolis from La Banda Este being the most active (MIC = 7.8 microg/ml) against Streptococcus piogenes, an antibiotic resistant bacterium. Thin layer chromatographic (TLC) separation profiles of propolis from Amaicha del Valle region were similar but differ from the alcoholic extract of the propolis from Cerrillos, another phytogeographical region of Argentina (provincia chaqueña). Bioautographic assays of the TLC profiles showed that several separated compounds of the Amaicha del Valle propolis have antibacterial activity. The difference in composition between Amaicha del Valle and Cerrillos propolis coincides with a different phytogeographical formation.

A molecular based strategy for rapid diagnosis of toxigenic Fusarium species associated to cereal grains from Argentina.

Fusarium species are worldwide causal agents of ear rot in cereals. Their toxigenic potential is a health risk for both humans and animals. In Argentina, most identification of these fungi has been based on morphological and cross-fertility criteria which are time consuming and require considerable expertise in Fusarium taxonomy and physiology. DNA based approaches have been reported as rapid, sensitive and specific alternatives to identify the main fumonisin and trichothecene-producing Fusarium species. In this work, we used PCR assays and the partial sequence of TEF1-alpha gene (Translation Elongation Factor-1 alpha) to identify the fumonisin and trichothecene-producing species in Fusarium isolates from diverse regions of Argentina. The relative efficiency and reliability of those methods to improve mycotoxin risk prediction in this country were also assessed. Species-specific PCR assays were targeted toward multicopy IGS (Intergenic Spacer of rDNA units) and on the toxin biosynthetic genes FUM1 (fumonisins) and TRI13 and TRI7 genes (trichothecenes). PCR assays based on FUM1 gene and IGS sequences allowed detection and discrimination of the fumonisin producers Fusarium proliferatum and Fusarium verticillioides. Molecular identification of nonfumonisin producers from Gibberella fujikuroi species complex was possible after determination of TEF1-alplha gene sequences, which indicated the presence of Fusarium subglutinans, Fusarium andiyazi and Fusarium thapsinum. TEF-1 alpha gene sequences also allowed discrimination of the different species of the Fusarium graminearum complex (F. graminearum sensu lato) as F. graminearum sensu stricto, Fusarium meridionale and Fusarium boothii. The last two species belonged to NIV chemotype and were detected for the first time in the subtropical region of Argentina while F. graminearum sensu stricto was DON producer only, which was also confirmed by specific PCR assays based on TRI137/TRI7 genes. Our results indicated that the PCR assays evaluated in this work are reliable diagnostic tools to detect the main toxigenic Fusarium species associated to cereal grains in Argentina. An extensive epidemiological survey based on the approach presented in this work is currently in progress to know the mycotoxigenic hazard of Fusarium species in cereal grains from the subtropical region of Argentina.

Antibacterial activity of plant extracts from northwestern Argentina

Aims:  To determine the antibacterial and cytotoxic activities of aqueous and ethanolic extracts of northwestern Argentinian plants used in folk medicine. To compare the mentioned activities with those of five commercial antibiotics. To identify the compounds responsible for the antibacterial activity.

Species diversity and toxigenic potential of Fusarium graminearum complex isolates from maize fields in northwest Argentina.

Members of the Fusarium graminearum species complex (Fg complex) are the causal agents of ear rot in maize and Fusarium head blight of wheat and other small grain cereals. The potential of these pathogens to contaminate cereals with trichothecene mycotoxins is a health risk for both humans and animals. A survey of ear rot isolates from maize collected in northwest Argentina recovered 66 isolates belonging to the Fg complex. A multilocus genotyping (MLGT) assay for determination of Fg complex species and trichothecene chemotypes was used to identify 56 of these isolates as F. meridionale and 10 isolates as F. boothii. F. meridionale was fixed for the nivalenol (NIV) chemotype, and all of the F. boothii isolates had the 15-acetyldeoxynivalenol (15ADON) chemotype. The results of genetic diversity analysis based on nine variable number tandem repeat (VNTR) loci supported the hypothesis of genetic isolation between F. meridionale and F. boothii, and provided little evidence of geographic substructure among populations of the dominant pathogen species, F. meridionale. This is the first study to indicate that F. meridionale and F. boothii may play a substantial role in the infection and trichothecene contamination of maize in Argentina. In addition, dominance of the NIV chemotype among Fg complex isolates from Argentina is unprecedented, and of significant concern to food safety and animal production.

Propolis from the northwest of Argentina as a source of antifungal principles

Aims:  To determine the antimycotic and cytotoxic activities of partially purified propolis extract on yeasts, xylophagous and phytopathogenic fungi.

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.

Trichothecene genotypes and chemotypes in Fusarium graminearum complex strains isolated from maize fields of northwest Argentina.

Members of the Fusarium graminearum species complex (Fg complex) cause Gibberella ear rot in maize from northwest Argentina. The potential of these pathogens to contaminate maize grains with type B trichothecenes is a health risk for both humans and animals. We evaluated the reliability of multiplex PCR assays based on TRI3 and TRI12 genes, and single PCR assays based on TRI7 and TRI13 genes to infer trichothecene chemotypes of 112 strains of Fg complex collected from northwest Argentina, checking trichothecene production by chemical analysis. Single and multiplex PCR assays indicated that strains belonging to F. meridionale (87/112) had a NIV genotype. The remainder strains (25/112), which belonged to F. boothii, had a DON genotype (based on single PCR assays) or 15ADON genotype (based on multiplex PCR assays). No strains tested were incorrectly diagnosed with a DON/NIV genotype. Chemical analysis indicated that the F. meridionale strains were NIV producers only (44/87), major NIV producers with unexpected high DON/NIV ratios (36/87), or unexpected major DON producers with minor NIV production (7/87). Strains with atypical DON/NIV production seem to be new phenotypes under a putative NIV genotype, since PCR assays do not provide evidences of a new trichothecene genotype. DON production and absence of its acetylated forms were shown for strains of F. boothii. The inconsistencies between genetic and chemical data highlight the risk of inferring the trichothecenes potentially contaminating food and feedstuffs based only on PCR assays. This study confirms for the first time that strains of Fg complex from maize of northwest Argentina are DON and NIV producers. In addition, dominance of NIV producers in the Fg complex population isolated from maize is unprecedented in Argentina, and of significant concern to food safety and animal production.