Antifungal and antimycotoxigenic metabolites from native plants of northwest Argentina: isolation, identification and potential for control of Aspergillus species.

Extracts from aerial parts of Prosopis ruscifolia, Bidens pilosa, Cercidium praecox and Phoradendron liga were assayed against toxigenic Aspergillus species. They were obtained by sequential extraction of the aerial parts with hexane (fHex), dichloromethane (fDCM), ethyl acetate (fEtOAc) and methanol (fMeOH). The fMeOH from P. ruscifolia showed the highest antifungal spectrum (MIC = 750-1500 µg mL-1; MID = 50-200 µg; DI = 1.7-3.0 mm). Indolizidine alkaloids (juliflorine and juliprosine) and tryptamine were identified with strong (MIC = 188 µg mL-1) and moderate antifungal activities (MIC = 750 µg mL-1), respectively, towards A. parasiticus and A. flavus. The fMeOH, the indolizidine alkaloids and tryptamine synergized the fungitoxic effect of potassium sorbate and propiconazole. They completely suppressed the biosynthesis of aflatoxins at concentrations of 47, 94 and 375 µg mL-1, respectively. Our results indicate that fMeOH and its identified alkaloids are promisory additives of commercial antifungals and are antiaflatoxigenic agents at concentrations below of those required for complete suppression of fungal growth.

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.

Antifungal activity of 4-hydroxy-3-(3-methyl-2-butenyl)acetophenone against Candida albicans: evidence for the antifungal mode of action.

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.

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.

Essential oils from Schinus species of northwest Argentina: Composition and antifungal activity.

The composition of the essential oils from leaves (Sal) and fruits of S. areira (Saf), and fruits of S. fasciculatus (Sff) and S. gracilipes (Sgf) were analyzed by GC/MS. The major compounds identified were sabinene (26.0 +/- 0.5%), bicyclogermacrene (14.5 +/- 0.4%), and E-citral (6.7+/- 0.2%) in Sal oil, limonene (27.7 +/- 0.7%), sabinene (16.0+/- 0.5%), beta-phellandrene (14.6 +/- 0.8%) and bicyclogermacrene (8.1 +/- 0.2%) in Saf oil, sabinene (22.7 +/- 0.6%), alpha-phellandrene (18.7 +/- 0.3%), beta-phellandrene (15.7 +/- 0.4%), and bicyclogermacrene (8.1 +/- 0.2%) in Sff oil and beta-pinene (25.4 +/- 0.8%), alpha-pinene (24.7 +/- 0.7%), and sabinene (13.6 +/- 0.4%) in Sgf oil.The antifungal activity of the four oils was evaluated on strains of Fusarium verticillioides and F. graminearum, and the results compared with the effect of epoxyconazole, pyraclostrobin and thyme oil. The Sff oil had the highest antifungal activity among the Schinus oils tested, with MIC100 (F. graminearum) = 6 per thousand and MIC100 (F. verticillioides) = 12 per thousand. A principal component analysis suggests that 9 constituents (alpha-thujene, alpha-terpinene, p-cymene, gamma-terpinene, terpinolene, 1-terpineol, alpha-calacorene, alpha-phellandrene, and terpinen-4-ol) explain the higher antifungal effect of Sff. The MIC100s of Schinus oils were on average 30-60 and 8.5-17 fold lower than those obtained for thyme oil on F. verticillioides and F. graminearum, respectively. In the case of commercial fungicides, their MIC100s were three orders of magnitude lower than those of Schinus oils. The last ones showed an additive interaction when assayed in mixtures with the commecial fungicides and thyme oil. The results suggest that the doses of fungicides required for control of the Fusarium species can be reduced when they are assayed in mixtures with the Schinus oils.

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.

A thermostable α-galactosidase from Lenzites elegans (Spreng.) ex Pat. MB445947: purification and properties.

An α-galactosidase was isolated from a culture filtrate of Lenzites elegans (Spreng.) ex Pat. MB445947 grown on citric pectin as carbon source. It was purified to electrophoretic homogeneity by ammonium sulfate precipitation, gel filtration chromatography and anion-exchange chromatography. The relative molecular mass of the native purified enzyme was 158 kDa determined by gel filtration and it is a homodimer (Mr subunits = 61 kDa). The optimal temperature for enzyme activity was in the range 60-80 °C. This α-galactosidase showed a high thermostability, retaining 94 % of its activity after preincubation at 60 °C for 2 h. The optimal pH for the enzyme was 4.5 and it was stable from pH 3 to 7.5 when the preincubation took place at 60 °C for 2 h. It was active against several α-galactosides such as p-nitrophenyl-α-D-galactopyranoside, α-D-melibiose, raffinose and stachyose. The α-galactosidase is a glycoprotein with 26 % of structural sugars. Galactose was a non-competitive inhibitor with a Ki = 22 mM versus p-nitrophenyl-α-D-galactoside and 12 mM versus α-D-melibiose as substrates. Glucose was a simple competitive inhibitor with a Ki = 10 mM. Cations such as Hg(2+) and p-chloromercuribenzoate were also inhibitors of this activity, suggesting the presence of -SH groups in the active site of the enzyme. On the basis of the sequence of the N-terminus (SPDTIVLDGTNFALN) the studied α-galactosidase would be a member of glycosyl hydrolase family 36 (GH 36). Given the high optimum temperature and heat stability of L. elegans α-galactosidase, this fungus may become a useful source of α-galactosidase production for multiple applications.

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.

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.

Evaluation of the cytotoxicity, genotoxicity, mutagenicity, and antimutagenicity of propolis from Tucuman, Argentina.

This study evaluates the toxic, genotoxic/mutagenic, and antimutagenic effects of propolis extract from Amaicha del Valle, Tucumán, Argentina. The cytotoxicity assays carried out with the lethality test of Artemia salina revealed that the LD50 was around 100 microg/mL. Propolis extracts showed no toxicity to Salmonella typhimurium TA98 and TA100 strains and Allium cepa at concentrations that have antibiotic and antioxidant activities. Otherwise, for the testing doses, neither genotoxicity nor mutagenicity was found in any sample. The propolis extracts were able to inhibit the mutagenesis of isoquinoline (IQ) and 4-nitro o-phenylenediamine (NPD) with ID50 values of 40 and 20 microg/plate, respectively. From this result, the studied propolis may be inferred to contain some chemical compounds capable of inhibiting the mutagenicity of direct-acting and indirect-acting mutagens. A compound isolated from Amaicha del Valle propolis, 2',4'-dihydroxychalcone, showed cytotoxic activity (LC50 values of 0.5 microg/mL) but was not genotoxic or mutagenic. Furthermore, this compound was able to inhibit the mutagenicity of IQ (ID50 values of 1 microg/plate) but was unable to inhibit the mutagenicity of NPD. Our results suggest a potential anticarcinogenic activity of Amaicha del Valle propolis and the chalcone isolated from it.

Antibacterial activity of Zuccagnia punctata Cav. ethanolic extracts.

The present study was conducted to investigate antibacterial activity of Zuccagnia punctata ethanolic extract against 47 strains of antibiotic-resistant Gram-negative bacteria and to identify bioactive compounds. Inhibition of bacterial growth was investigated using agar diffusion, agar macrodilution, broth microdilution and bioautographic methods. Zuccagnia punctata extract was active against all assayed bacteria (Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Morganella morganii, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia) with minimal inhibitory concentration (MIC) values ranging from 25 to 200 microg/mL. Minimal bactericidal concentration (MBC) values were identical or two-fold higher than the corresponding MIC values. Contact bioautography, indicated that Zuccagnia punctata extracts possess one major antibacterial component against Pseudomonas aeruginosa and at least three components against. Klebsiella pneumoniae and Escherichia coli. Activity-guided fractionation of 1he ethanol extract on a silica gel column yielded a compound (2',4'-dihydroxychalcone), which exhibited strong antibacterial activity with MIC values between 0.10 and 1.00 microg/mL for Proteus mirabilis, Enterobacter cloacae, Serratia marcescens, Morganella morganii, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia. These values are lower than imipenem (0.25-16 microg/mL). Zuccagnia punctata might provide promising therapeutic agents against infections with multi-resistant Gram-negative bacteria.