Antimicrobial activity and partial chemical structure of acylpolyamines isolated from the venom of the spider Acanthoscurria natalensis.

Background: Acylpolyamines are one of the main non-peptide compounds present in spider venom and represent a promising alternative in the search for new molecules with antimicrobial action. Methods: The venom of Acanthoscurria natalensis spider was fractionated by reverse-phase liquid chromatography (RP-HPLC) and the antimicrobial activity of the fractions was tested using a liquid growth inhibition assay. The main antimicrobial fraction containing acylpolyamines (ApAn) was submitted to two additional chromatographic steps and analyzed by MALDI-TOF. Fractions of interest were accumulated for ultraviolet (UV) spectroscopy and ESI-MS/MS analysis and for minimum inhibitory concentration (MIC) and hemolytic activity determination. Results: Five acylpolyamines were isolated from the venom with molecular masses between 614 Da and 756 Da, being named ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756. The analysis of UV absorption profile of each ApAn and the fragmentation pattern obtained by ESI-MS/MS suggested the presence of a tyrosyl unit as chromophore and a terminal polyamine chain consistent with structural units PA43 or PA53. ApAn presented MIC between 128 µM and 256 µM against Escherichia coli and Staphylococcus aureus, without causing hemolysis against mouse erythrocytes. Conclusion: The antimicrobial and non-hemolytic properties of the analyzed ApAn may be relevant for their application as possible therapeutic agents and the identification of an unconventional chromophore for spider acylpolyamines suggests an even greater chemical diversity.

Antimicrobial activity and partial chemical structure of acylpolyamines isolated from the venom of the spider Acanthoscurria natalensis

Background: Acylpolyamines are one of the main non-peptide compounds present in spider venom and represent a promising alternative in the search for new molecules with antimicrobial action. Methods: The venom of Acanthoscurria natalensis spider was fractionated by reverse-phase liquid chromatography (RP-HPLC) and the antimicrobial activity of the fractions was tested using a liquid growth inhibition assay. The main antimicrobial fraction containing acylpolyamines (ApAn) was submitted to two additional chromatographic steps and analyzed by MALDI-TOF. Fractions of interest were accumulated for ultraviolet (UV) spectroscopy and ESI-MS/MS analysis and for minimum inhibitory concentration (MIC) and hemolytic activity determination. Results: Five acylpolyamines were isolated from the venom with molecular masses between 614 Da and 756 Da, being named ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756. The analysis of UV absorption profile of each ApAn and the fragmentation pattern obtained by ESI-MS/MS suggested the presence of a tyrosyl unit as chromophore and a terminal polyamine chain consistent with structural units PA43 or PA53. ApAn presented MIC between 128 µM and 256 µM against Escherichia coli and Staphylococcus aureus, without causing hemolysis against mouse erythrocytes. Conclusion: The antimicrobial and non-hemolytic properties of the analyzed ApAn may be relevant for their application as possible therapeutic agents and the identification of an unconventional chromophore for spider acylpolyamines suggests an even greater chemical diversity.(AU)

Antimicrobial activity and partial chemical structure of acylpolyamines isolated from the venom of the spider Acanthoscurria natalensis

Abstract Background: Acylpolyamines are one of the main non-peptide compounds present in spider venom and represent a promising alternative in the search for new molecules with antimicrobial action. Methods: The venom of Acanthoscurria natalensis spider was fractionated by reverse-phase liquid chromatography (RP-HPLC) and the antimicrobial activity of the fractions was tested using a liquid growth inhibition assay. The main antimicrobial fraction containing acylpolyamines (ApAn) was submitted to two additional chromatographic steps and analyzed by MALDI-TOF. Fractions of interest were accumulated for ultraviolet (UV) spectroscopy and ESI-MS/MS analysis and for minimum inhibitory concentration (MIC) and hemolytic activity determination. Results: Five acylpolyamines were isolated from the venom with molecular masses between 614 Da and 756 Da, being named ApAn728, ApAn614a, ApAn614b, ApAn742 and ApAn756. The analysis of UV absorption profile of each ApAn and the fragmentation pattern obtained by ESI-MS/MS suggested the presence of a tyrosyl unit as chromophore and a terminal polyamine chain consistent with structural units PA43 or PA53. ApAn presented MIC between 128 µM and 256 µM against Escherichia coli and Staphylococcus aureus, without causing hemolysis against mouse erythrocytes. Conclusion: The antimicrobial and non-hemolytic properties of the analyzed ApAn may be relevant for their application as possible therapeutic agents and the identification of an unconventional chromophore for spider acylpolyamines suggests an even greater chemical diversity.

Mycotoxins in cereals and cereal-based products: Incidence and probabilistic dietary risk assessment for the Brazilian population.

A probabilistic dietary risk assessment on mycotoxins was conducted using the Monte Carlo Risk Assessment software, with consumption data from the 2008/2009 Brazilian Household Budget Survey for individuals who were at least 10 years old and occurrence data for 646 samples of rice, maize, wheat, and their products, collected in the Federal District and in the state of Rio Grande do Sul, Brazil. Processing factors were estimated and applied to concentration data. Chronic exposure was estimated for fumonisins (free and bound/hidden), deoxynivalenol (DON) (including the acetylated forms) and zearalenone (ZON) (including alfa-zearalenol) and acute exposure was estimated for DON. For the general population, the chronic exposure exceeded the safe exposure levels at the 95P for DON and at the 99P for fumonisins. Additionally, safe level exceedance occurred at the 97.5P for fumonisins and at the 95P for DON for teenagers, as well as at the 99P for fumonisins for women of child-bearing-age. No exceedances were found for chronic exposure to ZON and acute exposure to DON. Maize couscous contributed most of the total fumonisins (91%) and ZON intakes (~40%) and bread to total intake of DON (~30%). Further studies should be conducted with updated Brazilian consumption data, which should include information for individuals aged less than 10 years old.

Determination of multi-mycotoxins in cereals and of total fumonisins in maize products using isotope labeled internal standard and liquid chromatography/tandem mass spectrometry with positive ionization.

Mycotoxins are secondary fungi metabolites present in foods that cause adverse effects in humans and animals. The aims of this work were to develop and validate a multi-mycotoxin method to determine the presence of aflatoxins, citreoviridin, deoxynivalenol, fumonisins ochratoxin A, zearalenone and some metabolites/derivatives in rice, maize-based products and wheat-based products, and a method to determine total fumonisins (free and bound/hidden forms) in maize-based products. The validated multi-mycotoxin method was based on extraction with acidified acetonitrile and LC-ESI+-MS/MS analysis, with LOQs ranging from 0.5 to 121µg/kg, and proved to be suitable for the multi-mycotoxin analysis in wheat, maize and rice products. Bound/hidden fumonisins were determined after extraction of the free forms using the multi-mycotoxin method, followed by a basic hydrolysis of the unextracted bound/hidden and solid-liquid extraction with low temperature purification (SLE-LTP). The hydrolysis efficiency of the bound/hidden extraction procedure was investigated by analyzing a maize reference material and showed recoveries ranging from 75% (HFB2) to 108% (HFB1). The use of isotope internal standards was crucial for mycotoxins quantification in maize meal and wheat flour, while for rice, external calibration and matrix matched curves gave satisfactory results. All 55 samples of wheat-based products analyzed were contaminated with at least one mycotoxin and 16% of 44 rice samples were also contaminated. The most prevalent mycotoxins were DON and ZON in wheat-based products.

Simultaneous analysis of aflatoxins B1, B2, G1, G2, M1 and ochratoxin A in breast milk by high-performance liquid chromatography/fluorescence after liquid-liquid extraction with low temperature purification (LLE-LTP).

The aims of this study were to optimize and validate a methodology for the simultaneous analysis of aflatoxins B1, B2, G1, G2, M1 (AFB1, AFB2, AFG1, AFG2, AFM1) and ochratoxin A (OTA) in breast milk, and to analyze these mycotoxins in samples obtained from human milk banks in the Federal District, Brazil. The optimized analytical method was based on liquid-liquid extraction with low temperature purification (3.25mL of acidified acetonitrile+0.75mL of ethyl acetate), followed by analysis by high-performance liquid chromatography with fluorescence detector (HPLC/FLD) and a photochemical post-column reactor. Limits of quantification (LOQ) ranged from 0.005 to 0.03ng/mL, recoveries from 73 to 99.5%, and relative standard deviations (RSD) from 1.8 to 17.3%. The LLE-LTP extraction method was shown to be simple and cost-effective, since no columns were needed for clean-up. Only 2 of the 224 breast milk samples analyzed were positive for the mycotoxins, both samples containing AFB2 at the LOQ level (0.005ng/mL). The identity of the mycotoxin detected was confirmed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This result indicates that infants who are fed with breast milk from the milk banks are not at risk from aflatoxin and ochratoxin exposure.