Nematicidal activity of Trichoderma spp. and isolation of an active compound

Trichoderma spp. play an important role in biotic control, and several are efficacious against nematodes. To study the potential of Trichoderma species in controlling nematodes, fungal filtrates of 329 Trichoderma strains were evaluated for their nematicidal activity against Panagrellus redivivus and Caenorhabditis elegans. Fifteen strains exhibited nematicidal activity against P. redivivus, and 14 strains showed activity against C. elegans. The strain YMF1.02647 showed strong nematicidal activity against both nematodes, and the culture broth could cause more than 90% mortality to the tested nematodes within 48 h. A nematicidal compound was isolated from ethyl acetate extract of Trichoderma YMF1.02647 based on bioassay-guided fractionation. The compound was identified as trichodermin according to the spectroscopic data, which could kill more than 95% both P. redivivus and C. elegans in 72 h at 0.4 g l⁻¹.     

Detection of estrogenic activity in Slovenian wastewaters by bioassay

Estrogenic activity has been detected in aquatic ecosystems across the world. However, there is a lack of such data for Slovenian wastewaters and surface waters. The Slovenian monitoring program of effluents discharged into surface waters does not require that emissions of natural and synthetic estrogens into aquatic environments be assessed and controlled. In our study, we assessed the potential estrogenicity of wastewater samples from three wastewater treatment plants using a yeast estrogen screen assay (YES assay). Due to the high inhibition of yeast growth in samples obtained during our first sampling period, it was impossible to detect any estrogenic activity. An additional silica gel clean-up step reduced the toxicity of samples collected during our second sampling period; as a result, we were able to record up to 95% relative estrogenic activity inhibition. Deconjugation of the estrogens did not significantly influence our results. We detected estrogenic activity using a YES assay in almost all influent and effluent samples tested, which suggests that the wastewater treatment plants (WWTPs) examined do not effectively remove (xeno)estrogens from wastewaters. Our results suggest that a YES assay is an appropriate screening method for monitoring estrogenic activity in effluents. However, prediction of the potential impacts of wastewater (xeno)estrogens on aquatic organisms require additional in vitro and in vivo assays.     

Copper stress-induced changes in biomass accumulation,antioxidant activity and flavonoid contents in Belamcanda chinensis calli

Plant Cell, Tissue and Organ Culture (PCTOC) - Here, the effects of copper stress (CS) on biomass accumulation, physiological parameters of oxidative stress and flavonoid production were...     

Ibuprofen, a unique anti-inflammatory compound with antifungal activity against dermatophytes

Ibuprofen showed significant antifungal activity in vitro against dermatophytes at pH 5 (MIC: 5–40 μg ml^-1). In this respect it is comparatively more efficient than two well known and medically used antifungal compounds, benzoic and salicylic acids. This compound with anti-inflammatory activity which is not found in any other conventional antifungal organic acids, may have clinical prospects.     

Antifeedant activity of ethanolic extract from Flourensia oolepis and isolation of pinocembrin as its active principle compound

The ethanolic extract from Flourensia oolepis aerial parts showed strong antifeedant activity against the pest larvae, Epilachna paenulata, with an antifeedant index (AI%) of 99.1% at 100 μg/cm². Based on chromatographic fractionation of the extract, guided by bioassays on E. paenulata, the flavanone pinocembrin (1) was isolated as the most active principle. In a choice assay, compound 1 showed strong antifeedant activity against E. paenulata, Xanthogaleruca luteola and Spodoptera frugiperda with an AI% of 90, 94 and 91% (p < 0.01) respectively, at 50 μg/cm². The dosages necessary for 50% feeding inhibition of the insects (ED₅₀) were 7.98, 6.13 and 8.86 μg/cm², respectively. The feeding inhibitory activity of 1 against E. paenulata was compared with the activity of other structurally related flavonoids like naringenin, which was inactive up to 100 μg/cm², catechin which was nearly 6 times less active than 1, and quercetin which was equally active as 1. The effect of these on the feeding behavior of E. paenulata was also studied.     

The isolation, purification and biological activity of a novel antibacterial compound produced by Pseudomonas stutzeri

A novel compound designated zafrin [4beta-methyl-5, 6, 7, 8 tetrahydro-1 (4beta-H)-phenanthrenone] was isolated from a crude extract of a marine bacterium identified as Pseudomonas stutzeri. Zafrin showed strong antibacterial activity against both Gram-positive and Gram-negative bacteria. The compound was purified and its structure was elucidated by spectroscopic methods including 1H-nuclear magnetic resonance (NMR), 13C-NMR, 1D-NMR and 2D-NMR spectroscopy. It could be demonstrated that a purified solution of zafrin was active against several human pathogens, including Staphylococcus aureus, and Salmonella typhi. By contrast, zafrin did not inhibit the growth of eukaryotic organisms Candida albicans and Schizosaccharomyces pombe. The minimal inhibitory concentration for Gram-positive bacteria ranged from 50 to 75 microg mL(-1) and varied between 75 and 125 microg mL(-1) for Gram-negative bacteria. Zafrin lysed Bacillus subtilis cells grown in an osmotically protected medium, suggesting that it does not act upon the cell wall. Further investigation using B. subtilis indicated that the compound is bactericidal and is likely to target the cell membrane.     

Arundifungin, a novel antifungal compound produced by fungi: biological activity and taxonomy of the producing organisms

Echinocandins, the lipopeptide class of glucan synthase inhibitors, are an alternative to ergosterol-synthesis inhibitors to treat candidiasis and aspergillosis. Their oral absorption, however, is low and they can only be used parenterally. During a natural product screening program for novel types of glucan synthesis inhibitors with improved bioavailability, a fungal extract was found that inhibited the growth of both a wild-type Saccharomyces cerevisiae strain and the null mutant of the FKS1 gene (fks1::HIS). The mutant strain was more sensitive to growth inhibition, suggesting that the fungal extract could contain an inhibitor of glucan synthesis. A novel acidic steroid, named arundifungin, was purified from a fungal extract obtained from a liquid culture of Arthrinium arundinis collected in Costa Rica. Arundifungin caused the same pattern of hallmark morphological alterations in Aspergillus fumigatus hyphae as echinocandins, further supporting the idea that arundifungin belongs to a new class of glucan synthesis inhibitors. Moreover, its antifungal spectrum was comparable to those of echinocandins and papulacandins, preferentially inhibiting the growth of Candida and Aspergillus strains, with very poor activity against Cryptococcus. Arundifungin was also detected in nine other fungal isolates which were ecologically and taxonomically unrelated, as assessed by sequencing of the ITS1 region. Further, it was also found in two more Arthrinium spp from tropical and temperate regions, in five psychrotolerant conspecific isolates collected on Macquarie Island (South Pacific) and belonging to the Leotiales, and in two endophytes collected in central Spain (a sterile fungus belonging to the Leotiales and an undetermined coelomycete).     

First chromatographic isolation of an antifungal thaumatin-like protein from French bean legumes and demonstration of its antifungal activity.

A protein, with a molecular weight of 20 kDa, and an N-terminal sequence analogous to those of thaumatin-like proteins (TLPs) and thaumatins, was first isolated from the legume of the French bean Phaseolus vulgaris cv Kentucky wonder using a simple procedure involving affinity and ion exchange chromatography. The protein was adsorbed on both CM-Sepharose and Affi-gel Blue Gel. It was the first leguminous TLP-like protein demonstrated to exert antifungal activity against Fusarium oxysporum, Pleurotus ostreatus, and Coprinus comatus but not against Rhizoctonia solani.     

Pyrroloquinoline quinone, a method for its isolation and identification by mass spectrometry.

Procedures for the unambiguous detection and for the isolation and mass spectrometric identification of pyrroloquinoline quinone (PQQ) are presented. The procedure involved acid hydrolysis of protein in the presence of phenylhydrazine and successive isolation and identification of the formed adduct using mass spectrometry. In HPLC the phenylhydrazone of PQQ gave many methylated products, of which the predominant compound was the pentamethylated derivative. After reaction of the phenylhydrazone derivative of PQQ (PHPQQ) with ammonia, a product was obtained which did not contain phenylhydrazine and which formed a pentamethylated derivative as the main methylation product. The HPLC profiles of the methylated products of PHPQQ and of its ammonia derivative were very characteristic and could be used for identification in addition to mass spectrometry. However, prolonged treatment of proteins with phenylhydrazine during hydrolysis can result in the formation of a material that resembles PQQ in some aspects of its behaviour. Thus, analysis by MS is essential for unambiguous identification. This analytical procedure was applied to pig plasma benzylamine oxidase, pig aorta lysyl oxidase, pig kidney diamine oxidase and bovine serum albumin with negative results. However, samples of pronase contained variable quantities of non-covalently bound PQQ: this can lead to erroneous identification of PQQ in enzyme after pronase digestion.     

Ascherxanthone B from Aschersonia luteola, a new antifungal compound active against rice blast pathogen Magnaporthe grisea

Aims:  Fungicide resistance now exists in the rice blast fungus, Magnaporthe grisea , necessitating the need for new active agents. Fungi isolated from habitats in Thailand were screened with reference to this problem.
Methods and Results:  A new, reliable in vitro screening system based on a microdilution plate format was set up using a virulent strain of M. grisea THL 16. Culture broth extracts from approximately 800 fungal strains were investigated, one of these, Aschersonia luteola BCC 8774, was found to produce an active fungicidal compound, ascherxanthone B, with an IC₉₀ value of 0·58 μg ml⁻¹ (0·95 μmol l⁻¹). An in vivo study of anti-blast efficacy of ascherxanthone B showed a positive effect in disease reduction.
Conclusions:  Previous report has shown that a species of Aschersonia produces ascherxanthone A. Research on the species, A. luteola BCC 8774, led to the discovery of related novel metabolite, ascherxanthone B with fungicidal properties.
Significance and Impact of the Study:  Current methods of rice blast control seem to fail leading to increase in crop losses. Our discovery of the anti-blast activity shown by ascherxanthone B is the first step in the development of a potentially novel fungicide.     

Wasalexins A and B, new phytoalexins from wasabi: isolation, synthesis, and antifungal activity

The chemical structure determination of two phytoalexins from wasabi (Wasabia japonica, syn. Eutrema wasabi), a plant resistant to virulent isolates of the blackleg fungus [Leptosphaeria maculans (Desm.) Ces. et de Not., asexual stage Phoma lingam (Tode ex Fr.) Desm.], as well as their synthesis and antifungal activity towards isolates of P. lingam and P. wasabiae is reported.     

Quantitative determination of the antifungal compound 6-pentyl-α-pyrone (6PAP) using a simple plate bioassay

An assay relating bioactivity to levels of the antifungal compound 6-pentyl-α-pyrone (6PAP) has been developed. Using Cladosporium strain NZ660US as indicator at a spore concentration of 5×10⁶ spores overlay⁻¹ on malt (adjusted to pH 3·5) agar plates, this assay allows quantitative determination of 6PAP down to a detection limit of 0·5% v/v. This will allow rapid screening of Trichoderma culture extracts to compare the bioactivity of isolates grown under different culture conditions, and can be used in combination with analytical chemical analysis to identify the presence of other bioactive metabolites.     

Combination of HPLC chromatogram and hypoglycemic effect identifies isoflavones as the principal active fraction of Belamcanda chinensis leaf extract in diabetes treatment

In previous study, we demonstrated the hypoglycemic effect of aqueous extract of Belamcanda chinensis leaves in rats. Here, we separated the aqueous extract of B. chinensis leaves and investigated the spectrum-effect relationships between HPLC chromatograms and hypoglycemic activities of different isolates from B. chinensis leaf extract. Sequential solvent extraction with petroleum ether, chloroform, acetic ester and n-butanol provided several isolates showing similar hypoglycemic activities, making it difficult to discriminate the active fractions. Stepwise elution through HP20 macroporous resin by water, 40% and 95% ethanol provided isolates with distinct hypoglycemic activities, representing a simple, rapid and efficient preparative separation method. Combination of HPLC chromatogram and pharmacological effect targeted a hypoglycemic activity-related region in HPLC chromatogram. Each peak in this region was analyzed by UV spectrum scan. Most of them were flavonoids in which tectoridin and swertisin were known flavonoids with anti-diabetic activities. In together, this work provides a general model of combination of HPLC chromatography and pharmacological effect to study the spectrum-effect relationships of aqueous extract from B. chinensis leaves, which can be used to find principle components of B. chinensis on pharmacological activity.     

Bioassay-guided isolation of a vasorelaxant active compound from Kaempferia galanga L.

Bioassay-guided fractionation was performed on a crude dichloromethane extract of Kaempferia galanga L. using chromatography techniques. Screening of the extract for biological activity started with the brine shrimp lethality bioassay, followed by the study of its antihypertensive activity on anaesthetized rats, which involved monitoring of the extract's effect on mean arterial blood pressure. The components of the fractions obtained from the separation procedures were analyzed using gas chromatography (GC). The yield of the CH(2)Cl(2) extract was 0.29% of the crude plant extract. Analysis of the data for brine shrimp lethality test using the Finney computer program showed that this extract exhibited potent bioactivity with an ED(50) value of 7.92+/-0.13 microgml(-1). Intravenous administration of the extract induced a dose-related reduction of basal mean arterial pressure (MAP) (130+/-5 mmHg) in the anaesthetized rat, with maximal effects seen after 5-10 min of injection. The gas chromatogram showed that the common compound in the active fractions obtained from the bioassay-guided fractionation of the CH(2)Cl(2) extract was ethyl cinnamate. This vasorelaxant active compound, ethyl cinnamate, was isolated as a colorless oil. Ethyl p-methoxycinnamic acid was also isolated as white needles but did not exhibit any relaxant effect on the precontracted thoracic rat aorta.