Hydroethanolic Extract of Grape Peel from Vitis labrusca Winemaking Waste: Antinociceptive and Anti-Inflammatory Activities.

Research background: Extracts from grape pomace, including the wine, show many biological effects such as antioxidant and anti-inflammatory activities. Unfortunately, winemakers discard the bagasse, so the waste is not exploited, although it contains bioactive compounds with antioxidant and anti-inflammatory properties. The work aims to analyze the hydroethanolic extract of peels from Vitis labrusca agro-industrial waste and to evaluate its antinociceptive and anti-inflammatory properties. This study is relevant for reusing a residue and adding value to the grape economic chain. Experimental approach: A representative sample of pomace was obtained and the peels were used to produce the extract. The phenolic compounds were determined by mass spectrometry in multiple reaction monitoring mode and Folin-Ciocalteu colorimetric method, using gallic acid as standard. The biological analyses were carried out using mice orally treated with crude extract at doses of 30, 100 and 300 mg/kg. We evaluated mechanical hyperalgesia by the von Frey method, thermal heat hyperalgesia using a hot plate at 55 °C, paw edema using a pachymeter, and neutrophil recruitment by measurement of myeloperoxidase activity. The nephrotoxicity and hepatotoxicity were evaluated by biochemical analyses using blood samples that were collected after the Vitis labrusca administration. Results and conclusions: In all wet winemaking residues peel mass fraction was 75%, and in dry residues 59%. We identified nine anthocyanins (3-O-glucosides: peonidin, delphinidin, petunidin and malvidin; 3-p-coumaroyl-glucosides: cyanidin, peonidin, petunidin and malvidin, and malvidin-3,5-diglucoside), five flavonoids (apigenin-7-glucoside, luteolin-7-glucoside, quercetin-3-galactoside, isorhamnetin-3-glucoside and myricetin-3-rutinoside), and mass fraction of phenolic compounds, expressed as gallic acid equivalents, was 26.62 mg/g. In vivo assays showed that Vitis labrusca extract at mass fractions 100 and 300 mg/kg reduced carrageenan-induced mechanical and thermal hyperalgesia, 50% of the paw edema, and neutrophil recruitment. In addition, there were no indications of nephrotoxicity and hepatotoxicity. Our extract obtained from winemaking residue has analgesic and anti-inflammatory properties, related at least in part to the presence of phenolic compounds, and it is not toxic to renal and hepatic tissues. Novelty and scientific contribution: This bio-product can be used as an alternative to synthetic anti-inflammatory agents with the same pharmacological potential and fewer side effects. We demonstrated that Vitis labrusca winemaking waste can be used for the production of antinociceptive and anti-inflammatory products (nutraceutical, pharmaceutical and cosmetics) without toxicity, contributing to the environmental economy.

Effects of Subcutaneous Ochratoxin-A Exposure on Immune System of Broiler Chicks.

Ochratoxin A (OTA), an immunosuppressive mycotoxin, can increase the risk of many infectious diseases and contribute to economic losses to the poultry industry. The immunosuppressive effect has mainly been investigated through oral exposure; however, birds may also be contaminated through skin absorption. The present study investigated the influence of OTA exposure on the defense system of broiler chicks through the subcutaneous route and including low doses. Groups of broiler chicks (Cobb), 05 days old, were exposed to subcutaneous inoculation of OTA at concentrations of 0.1; 0.5; 0.9; 1.3; and 1.7 mg OTA/kg body weight. The size of the lymphoid organs, circulating immune cells, and total IgY and IgA levels were evaluated 21 days post inoculation. Subcutaneous OTA exposure decreased the weight of the thymus, spleen, and bursa of Fabricius, and leukocytopenia (p < 0.05) was detected in chicks of the OTA treated groups. In a dose-dependent way, decreased levels of circulating lymphocytes and heterophils (p < 0.05), and increased levels of monocytes (p < 0.05) were detected. Decreased IgY and IgA serum concentrations were noted in the OTA treated groups (p < 0.05). In conclusion, subcutaneous OTA exposure induces immunosuppression even at low levels.

Kaurenoic acid extracted from Sphagneticola trilobata reduces acetaminophen-induced hepatotoxicity through inhibition of oxidative stress and pro-inflammatory cytokine production in mice.

Acetaminophen (paracetamol) is a widely used analgesic and antipyretic drug that is safe at therapeutic doses. However, acetaminophen overdose can be fatal. Currently, the only treatment available is the N-acetyl cysteine. The diterpene kaurenoic acid (ent-kaur-16-en-19-oic acid, KA) is the major constituent of Sphagneticola trilobata (L.) Pruski. KA presents anti-inflammatory, anti-nociceptive and antioxidant properties. In this study, we evaluated the efficacy of KA in a model of acetaminophen-induced hepatotoxicity. KA increased, in a dose-dependent manner, the survival rate after acetaminophen overdose. KA reduced acetaminophen-induced hepatic necrosis and ALT and AST levels. KA decreased acetaminophen-induced neutrophil and macrophage recruitment, oxidative stress and the production of IL-33, TNF-α and IL-1ß, alongside with normalisation of IL-10 levels in the liver. Therefore, KA showed preclinical efficacy in acetaminophen-induced hepatotoxicity and lethality.

Low Doses of Ochratoxin-A Decrease IgY and IgA Production in Broiler Chicks.

The mycotoxin, ochratoxin-A (OTA), produced by some fungi, and is a natural contaminant of many foods and animal feeds worldwide. Due to its toxic effects, the recommended maximum daily intake of OTA for poultry feeds is 0.1 mg OTA/kg (ECR2006/575/EC); this dose does not induce changes in hepatic/renal parameters, but decreases thymus size and serum globulin concentrations. Accordingly, in this study, we assessed quantitatively the total circulating IgY and IgA serum levels, in chicks consuming a 0.1 mg OTA/kg diet (limit) and higher doses (0.3⁻1.1 mg OTA/kg diet) for 14 or 21 days. We also evaluated other immunological parameters (thymus, bursa of Fabricius, and spleen weights and leukocyte profiles) at day 21. Decreased IgY serum levels were observed in all OTA-treated groups (p < 0.05). In the low-dose group, IgA levels were decreased on day 21, but not on day 14. The size of the thymus and the bursa of Fabricius was decreased in all OTA-treated groups (p < 0.05), whereas reduced spleen size and altered leukocyte profiles were detected only in the high-dose group (p < 0.05). We concluded that chronic exposure to OTA, even at the recommended highest dose, affected IgY and IgA production in chicks.

Exposure Assessment of Infants to Aflatoxin M1 through Consumption of Breast Milk and Infant Powdered Milk in Brazil.

Aflatoxin M1 (AFM1) is an important biomarker that can be used to evaluate aflatoxin exposure in both humans and animals. The aim of this study was to evaluate the exposure degree of infants to AFM1 through consumption of breast milk and infant powdered milk in Brazil. For this purpose, the estimated daily intake (EDI) for infants was calculated based on the AFM1 levels analyzed in 94 breast milk (BM) samples collected in Southern Brazil, and 16 infant powdered milk (IPM) samples commonly commercialized in Brazil. AFM1 was detected in 5.3% (n = 5) and 43.8% (n = 7) of BM and IPM samples, with mean levels of 0.003 ng/g and 0.011 ng/g, respectively. All the IPM samples showed AFM1 levels lower than those established by the Brazilian guidelines (5 ng/g), and in most of the samples (81.25%) levels were below the maximum limit tolerated by the European Commission (0.025 ng/g). The EDI of AFM1 for infants aged zero to 12 months old showed values from 0.018 to 0.069 ng/kg body weight/day for BM, and 0.078 to 0.306 ng/kg body weight/day for IPM. Hazard index (HI) values for BM and IPM were less than one, except for IPM intended for infants up to one month. In conclusion, the exposure of infants to AFM1 was low, but continuous monitoring of mycotoxin levels is essential to minimize infant health risk.

Effect of fungicide on Fusarium verticillioides mycelial morphology and fumonisin B1 production.

The effect of fludioxonil + metalaxyl-M on the mycelial morphology, sporulation and fumonisin B 1 production by Fusarium verticillioides 103 F was evaluated. Scanning electron microscopy analysis showed that the fungicide caused inhibition of hyphal growth and defects on hyphae morphology such as cell wall disruption, withered hyphae, and excessive septation. In addition, extracellular material around the hyphae was rarely observed in the presence of fludioxonil + metalaxyl-M. While promoting the reduction of mycelial growth, the fungicide increased sporulation of F. verticillioides compared to the control, and the highest production occurred on the 14 (th) day in the treatments and on the 10 (th) day in the control cultures. Fumonisin B 1 production in the culture media containing the fungicide (treatment) was detected from the 7 (th) day incubation, whereas in cultures without fungicide (control) it was detected on the 10 (th) day. The highest fumonisin B 1 production occurred on the 14 (th) day, both for the control and for the treatment. Fludioxonil + metalaxyl - M can interfere in F. verticillioides mycelial morphology and sporulation and increase fumonisin B 1 levels. These data indicate the importance of understanding the effects of fungicide to minimize the occurrence of toxigenic fungi and fumonisins.

Effect of fungicide on Fusarium verticillioides mycelial morphology and fumonisin B1 production

The effect of fludioxonil + metalaxyl-M on the mycelial morphology, sporulation and fumonisin B1 production by Fusarium verticillioides 103 F was evaluated. Scanning electron microscopy analysis showed that the fungicide caused inhibition of hyphal growth and defects on hyphae morphology such as cell wall disruption, withered hyphae, and excessive septation. In addition, extracellular material around the hyphae was rarely observed in the presence of fludioxonil + metalaxyl-M. While promoting the reduction of mycelial growth, the fungicide increased sporulation of F. verticillioides compared to the control, and the highest production occurred on the 14th day in the treatments and on the 10th day in the control cultures. Fumonisin B1 production in the culture media containing the fungicide (treatment) was detected from the 7th day incubation, whereas in cultures without fungicide (control) it was detected on the 10th day. The highest fumonisin B1 production occurred on the 14th day, both for the control and for the treatment. Fludioxonil + metalaxyl - M can interfere in F. verticillioides mycelial morphology and sporulation and increase fumonisin B1 levels. These data indicate the importance of understanding the effects of fungicide to minimize the occurrence of toxigenic fungi and fumonisins.

Inibidores de amilase em híbridos de milho como fator de resistência a Sitophilus zeamais ( Coleoptera: Curculionidae ) / Amylase inhibitors in corn hybrids as a resistance factor to Sitophilus zeamais ( Coleoptera: Curculionidae )

Sitophilus zeamais Motschulsky é uma das principais pragas do milho armazenado, sendo controlado, principalmente, com inseticidas. Porém, devido às conseqüências indesejáveis desses químicos sobre o meio ambiente e a saúde humana, outras táticas de controle têm sido investigadas, especialmente o desenvolvimento de plantas resistentes. A presença de inibidores de amilase nos grãos de milho pode interferir na digestão do inseto. Esta pesquisa foi desenvolvida com o objetivo de avaliar a resistência de híbridos de milho ao ataque de S. zeamais, correlacionada com a presença de inibidores de amilase e com a composição nutricional dos grãos. Adultos do gorgulho-do-milho foram expostos a amostras de grãos de diferentes híbridos a 27 ± 1°C e 75 ± 5 por cento de UR. Avaliou-se o ciclo biológico, peso dos adultos, índice de suscetibilidade e consumo dos grãos. Os resultados foram correlacionados com a composição dos grãos (teores de umidade, cinzas, lipídios, proteínas e carboidratos) e com a atividade de inibidores de amilase. A atividade inibidora de amilase foi determinada incubando-se o inibidor com 0,2 unidades de amilase durante 30 min. a 25°C, medindo-se posteriormente a atividade amilásica residual pelo método iodométrico. Dos parâmetros nutricionais avaliados, somente o conteúdo de lipídios correlacionou-se significativamente com o índice de suscetibilidade (r = 0,46). Os inibidores de amilase obtidos pela extração com etanol ou tampão correlacionaram-se negativa e significativamente com o índice de suscetibilidade (r = -0,58), sugerindo que esses inibidores contribuem para a resistência de híbridos de milho ao ataque de S. zeamais.

Trends of fumonisin contamination and animal intoxication through monitoring 1991 to 1997 corn crop in the State of Paraná, Brazil.

Eleven feed samples associated with six animal (horse and poultry) intoxication outbreaks (1991) in the state of Paraná, Brazil, were evaluated for fungal and fumonisin contamination. In order to estimate the trend of livestock intoxication, fumonisin contamination was monitored in corn produced both at the commercial level (1991, 1995 crop), and in an experimental field at a local Agronomy Institute (1997 crop). The total mould count in the feed samples ranged from 2.9 x 10(3) to 1.9 x 10(7) CFU/g, with Fusarium verticillioides as the predominant species, at a high count of 2.4 x 10(4)-6.5 x 10(5) CFU/g. Fumonisins (FB1 + FB2) were detected in all corn-based feed samples at levels ranging from 2.89 to 14.54 microg/g. All 27 Northern corn samples (1991 crop) were contaminated with fumonisins at levels ranging from 2.32 to 16.64 microg/g. Twenty-six (96.3%) out of 27 corn samples from the Central-Southern region (1995 crop) were positive for fumonisins (FB1+FB2), with the range of 0.07-3.66 microg/g, while all 37 Northern samples (1995 crop) were contaminated with fumonisins ranging from 0.57 to 9.97 microg/g. Twenty-one out of 37 corn samples from the Northern region (1997 crop) were positive for fumonisins, but at low level (range of 0.05-2.67 microg/g). The results showed a decreasing trend in fumonisin contamination over the years. Nowadays animal intoxication outbreaks rarely occur in this State, as both animal producers and feed industries have become conscious about monitoring of corn and other raw materials at the quality control level.

Characterization of a Hydrophobic Amylase Inhibitor from Corn (Zea mays) Seeds with Activity Against Amylase from Fusarium verticillioides.

ABSTRACT A hydrophobic 19.7-kDa amylase inhibitor (AI) was purified from corn kernels by 95% ethanol extraction and anionic exchange chromatography. The AI has an isoelectric point of 3.6 and was very stable at different pH values and high temperatures, maintaining 47.6% activity after heating to 94 degrees C for 60 min. Amino acid analysis indicated high valine, leucine, glycine, alanine, and glutamic acid/glutamine content, and especially high valine content (41.2 mol%). This inhibitor is not a glycoprotein. It required 30-min preincubation to maximize complex enzyme-inhibitor formation when the amylase from Fusarium verticillioides was tested. The optimal pH of interaction was 6.5. It showed broad-spectrum activity including the following amylases: human saliva, porcine pancreas, F. verticillioides, as well as those from some insects of agricultural importance (Acanthoscelides obtectus, Zabrotes subfasciatus, Sitophilus zeamais, and Prostephanus truncatus). This novel hydrophobic protein not only inhibited the amylase from F. verticillioides but also decreased the conidia germination. Thus, this protein represents an approach to decrease the production of fumonisin in corn, either by using it as a molecular marker to detect fungal resistance or through genetic engineering.

New Amylase Inhibitor Present in Corn Seeds Active In Vitro Against Amylase from Fusarium verticillioides.

A screening for specific amylase inhibitor levels against amylase from Fusarium verticillioides (Fusarium moniliforme), the most relevant mycotoxigenic fungus in corn, was conducted on 37 corn hybrids. The amylase inhibitor levels in these hybrids ranged from 5.5 to 16.0 amylase inhibitor units per gram of corn (AIU/g) in the MASTER and AG5011 hybrids, respectively. The hybrid with the maximum content of inhibitor was used as the source of this new protein. The inhibitor was partially purified using fractional precipitation, gel filtration on Sephadex G-75 column, high performance liquid chromatography (HPLC) Superose HR 10/30 column, and HPLC anion exchange chromatography, obtaining a 20.7-fold purification. Electrophoresis after denaturing and heating under reductive conditions showed an apparent 23.8 kDa molecular mass and an acidic isoelectric point of 5.4, which differs from previous molecular masses reported for other inhibitors present in corn seeds (14 and 22 kDa). This inhibitor showed activity against amylases from human saliva and pancreas, from the fungi F. verticillioides and Aspergillus flavus, and from the insects Acanthoscelides obtectus, Zabrotes subfasciatus, Tribolium castaneum, and Sitotroga cerealella. The mycoflora found in the corn grain indicated Fusarium sp. as the most prevalent fungi (81.1% of the samples), with a count ranging from 1.5 × 102 to 2.4 × 106 CFU/g of corn. The presence of fumonisins was detected in 21 out of the 37 hybrids studied, ranging from 0.05 to 2.67 µg of FB per gram of corn. No correlation could be established between this amylase inhibitor level in the corn seeds and the presence of Fusarium sp. or with the fumonisin content under the experimental conditions of the test.

Anti-Fusarium moniliforme activity and Fumonisin biodegradation by Corn and Silage microflora

A aplicaçäo de microroganismos visando controle de fungos micotoxigêncos ou detoxificaçäo em armazenagem consiste em uma área promissora, já que reduz contaminaçäo de ecossistema por resíduos agrotóxicos. Visando controle biológico, microorganismos isolados de milho e silagem foram analisados perante efeito anti-F, moniliforme (linhagem 113F) em associaçäo com a detoxificaçäo de fumonisinas. Após análise de 150 isolados, selecionou-se quatro bacilos Gram-positivos e uma levedura com melhor atividade inibitória. O halo de inibiçäo variou de 50 a 72.5 mm usando culturas íntegras e 25 a 52.5 mm, para extrato bruto de cultivo. Os isolados S9, S10, S69 (bacilos esporulados) e SE3071 (levedura) degradaram 43 por cento, 48 por cento, 83 por cento r 57 por cento de FB1 respectivamente, em relaçäo à concentraçäo inicial. O pH aumentou gradativamente com o tempo de incubaçäo

Screening and isolation of anti-Fusarium moniliforme compounds producing microorganisms from soil and corn

Fusarium moniliforme, fitopatógeno cosmopolita de milho, é responsável pela produçäo de micotoxina recentemente descoberta, denominada fumonisima. Com a finalidade de avaliar a inibiçäo de F. moniliforme, 34 antagonistas isolados de 60 amostras de solo e de 20 amostras de milho foram testados contra F. moniliforme 113F, produtor de fumonisina. O extrato bruto foi preparado com a cultura de microrganismos selecionados em caldo infuso de cérebro e coraçäo (BHI) e concentrados, adicionando-se etanol na proporçäo 1:1. A presença de organismos inibidores de F.moniliforme ocorreu em 29 amostras de solo, obtendo-se 36 microrganismos antagonistas. Referente ao milho, 15 amostras apresentaram microrganismos inibidores, permitindo o isolamento de 15 antagonistas. A caracterizaçäo destes 51 isolados demonstrou que 5 consistiram de leveduras, 3 cocos Gram-positivos , 3 cocos Gram-negativos e 40 bacilos Gram positivos, destacando-se a predominância do último grupo. Todos os 51 isolados

Aflatoxicose em cunicultura de Londrina, PR / Aflatoxicosis in rabit farm in Londrina, Paraná, Brazil

Resumo: No período de dezembro de 1986 a abril de 1987,a ocorrência de aflatoxicose casou a morte de 560 coelhos na regiäo de Londrina - PR. PAra a diagnose do problema foram analisadoas 6 amostras de raçäo coletadas em diferentes propriedades. A presença de aflatoxina B1, foi detectada em uma amostra de triguilho na concentraçäo de 33 ugKg, duas amostras de raçäo granulada na concentraçäo de 110 e 44 ugKg, e a contagem total de bolores foi de 8,2 x 10(elevado a +6); 2,8 x 10(elevado a +6) e 8,0 x 10(elevado a +3) UFC/G, respectivamente. Na amostra de triguilho predominou Aspergillus flavuis com 6,7 x 10(elevado a +6) UFC/G, enquanto que nas amostras de raçäo granulada, indica que o tratamento térmico pode ter eliminado o fungo, mas näo inativou a toxina (au)