Rapid detection of aflatoxin B_1 in Chinese herbal medicines by indirect and direct competitive enzyme-linked immunosorbent assays: a comparative analysis / 中国中药杂志

The indirect competitive enzyme-linked immunosorbent assay(ic-ELISA) and direct competitive enzyme-linked immunosorbent assay(dc-ELISA) were performed for the rapid detection of aflatoxin B_1(AFB_1) in Astragali Radix, Zingiberis Rhizoma Recens, Jujubae Fructus, and Nelumbinis Semen with self-made antigens and antibodies. Different extraction methods were investigated to reduce the matrix effects of different medicinal parts in Chinese herbal medicines. The sensitivity of dc-ELISA method was improved by optimizing the molar ratio of AFB_1 to horseradish peroxidase(HRP). In this study, the sensitivity(IC_(50)) of ic-ELISA and dc-ELISA was 0.046 and 0.023 ng·mL~(-1), with the limit of detection(LOD) of 0.007 and 0.004 ng·mL~(-1), respectively. The detection time was 3 h and 50 min for ic-ELISA and dc-ELISA, respectively. The recovery rates were within the range of 62.96%-104.4%, with RSDs of less than 10%. Confirmed by LC-MS/MS, three positive samples of Nelumbinis Semen were detected from 53 samples. Two ELISA methods established in this study were accurate, rapid and sensitive, and can be used for rapid screening of AFB_1 in Chinese herbal medicines such as Astragali Radix, Zingiberis Rhizoma Recens, Jujubae Fructus, and Nelumbinis Semen. In addition, the advantages and limitations of the two methods were compared and discussed, which can provide a reference for the testing institutions to choose the proper method.

Investigation of aflatoxins and toxigenic fungi contamination during post-harvest processing of Polygalae Radix / 中草药

Objective: To investigate aflatoxins contamination B1, B2, G1, G2 (AFB1, ATB2, AFG1, AFG2), toxigenic fungi species and potential contamination sources of Polygalae Radix during post-harvest processing, and analyze the main ways of aflatoxins contamination. Methods: Twenty-one Polygalae Radix samples were collected from multiple steps during the post-harvest processing in this study. Aflatoxin levels in these samples were determined by immunoaffinity column and HPLC coupled with post-column photochemical derivatization. Dilution-plate method was applied for the fungi isolation followed by strain identification based on morphological characterization and molecular approaches. Results: Aflatoxins were detected in 15 samples, but none of them exceeded the limit set by Chinese Pharmacopoeia. The fungal counts increased significantly from newly harvested samples to post-sweating, and the counts further increased to the maximum (2 × 108 CFU/g) after xylem-removing, then decreased after drying. In contrast, fungal counts of samples dried directly after harvesting did not change much throughout the processing. There was a significant positive correlation between fungal counts and water activity (Aw). A total of 209 fungal belonged to five genera were identified from the samples, and Penicillium was the predominant genus. Cladosporium and Fusarium were increased after sweating, and then Aspergillus increased after xylem-removing and drying. One A. parasiticus strain was confirmed to be able to produce AFB1, AFB2, AFG1, and AFG2. Conclusion: Aflatoxins contamination happened in both field production and post-harvest processing of Polygalae Radix. Especially, the contamination of Penicillium spp. should be paid more attention.

Aflatoxins and fumonisins in feed from a broiler operation system from São Paulo state, Brazil / Aflatoxinas e fumonisinas em rações de um sistema de produção de frangos de corte no estado de São Paulo, Brasil

ABSTRACT: The aim of the present study was to assess the occurrence of aflatoxins (AFs) and fumonisins (FBs) in feed ingredients (corn and soybean meal) and finishing feed in a broiler operation system, as well was to evaluate their effect on the productivity of 20 batches of broilers produced and the histology status of broilers' liver after slaughter. Corn samples presented the highest frequencies of AFs and FBs, at mean levels of 29.1 and 2,100µg/kg, respectively. Soybean samples presented mean levels of 1.5 and 70µg/kg for AFs and FBs, respectively. Batches of broilers receiving feed containing FB levels higher than 1,000µg/kg had lower weight gain and higher mortality rates, while those fed rations with AFs equal or above the limit of quantification (LOQ) of the analytical method presented higher scores of histological changes in the liver. A dilution effect was observed for AFs and FBs from ingredients, especially corn, to feed during manufacture, whilst not enough to prevent losses in productivity. Results of this trial highlighted the need for strict control of mycotoxins in corn intended for broilers.

RESUMO: O objetivo do presente trabalho foi verificar a ocorrência de aflatoxinas (AFs) e fumonisinas (FBs) em ingredientes (milho e farelo de soja) e na ração de abate sobre a produtividade de uma empresa integradora de frangos de corte, bem como avaliar seus efeitos sobre produtividade de 20 lotes de frangos produzidos pela empresa e a histologia dos fígados dos frangos após o abate. As amostras de milho apresentaram as maiores frequências de AFs e FBs, em concentrações médias de 29,1 e 2.100µg/kg, respectivamente. As amostras de farelo de soja apresentaram níveis médios de 1,5 e 70µg/kg para AFs e FBs, respectivamente. Os lotes de aves que receberam ração contendo níveis de FBs maiores que 1,000µg/kg apresentaram menor ganho de peso e maior percentual de mortalidade, enquanto que as que receberam ração com AFs iguais ou superiores ao limite de quantificação (LQ) do método analítico apresentaram maior grau de alteração histopatológica no fígado. Houve efeito de diluição de AFs e FBs dos ingredientes, especialmente o milho, à ração no processo de fabricação, porém não suficiente para evitar perdas na produtividade. Os resultados do estudo reforçam a necessidade do controle estrito de micotoxinas no milho destinado à alimentação de frangos de corte.

In vitro ability of beer fermentation residue and yeast-based products to bind aflatoxin B1

This study aimed to verify the in vitro ability of beer fermentation residue (BFR) containing Saccharomyces cerevisiae cells and five commercial products that differed in the viability and integrity of S. cerevisiae cells to remove aflatoxin B1 (AFB1) from a citrate-phosphate buffer solution (CPBS). BFR was collected at a microbrewery and prepared by drying and milling. The commercial yeast-based products were as follows: inactive intact yeast cells from beer alcoholic fermentation, inactive intact yeast cells from sugarcane alcoholic fermentation, hydrolyzed yeast cells, yeast cell walls and active yeast cells. Adsorption assays were performed in CPBS spiked with 1.0 μg AFB1/mL at pH 3.0 and 6.0 for a contact time of 60 min at room temperature. Analysis of AFB1 in the samples was performed by high performance liquid chromatography. AFB1 adsorption by the products ranged from 45.5% to 69.4% at pH 3.0 and from 24.0% to 63.8% at pH 6.0. The higher percentages (p < 0.05) of AFB1 binding at both pH values were achieved with products containing hydrolyzed yeast cells or yeast cell walls rather than intact cells. The AFB1 binding percentages of BFR were 55.0 ± 5.0% at pH 3.0 and 49.2 ± 4.5% at pH 6.0, which was not significantly different (p > 0.05) from commercial products containing inactive intact yeast cells. The results of this trial indicate that the yeast-based products tested, especially the BFR, have potential applications in animal feeds as a suitable biological method for reducing the adverse effects of aflatoxins.

Aflatoxicoses: prejuízos causados ao desempenho zootécnico de frangos de corte, boas práticas agrícolas e métodos biológicos de detoxicação / Aflatoxicosis: losses in poultry performance, agricultural good practices and biological methods of detoxification

As aflatoxicoses são micotoxicoses causadas por toxinas produzidas por estirpes de fungos do gênero Aspergillus. Cau- sam prejuízos importantes à produção animal, em especial àquelas que têm nos grãos sua matéria-prima base para preparação de ração, como a avicultura de corte. Atualmente, diversas técnicas, tais como uso de substâncias químicas, métodos físicos, estratégias de colheita, armazenagem e processamento de alimentos, estão sendo estudadas para quese diminua e, se bem aplicadas, evite os efeitos deletérios causados por essas substâncias à saúde animal. O objetivo desta revisão é descrever os principais efeitos das aflatoxinas sobre o desempenho zootécnico na produção de frangos de corte, as alterações nas matérias-primas de ração e os avanços científicos em metodologias de detoxificação biológica e de boas práticas agrícolas.

Aflatoxicosis are mycotoxicosis caused by toxins produced by strains of fungi of the genus Aspergillus. They cause important losses to animal production, especially to those that use grains like base raw-material to prepare feeds, like poultry production. Currently, many techniques are being studied to prevent the deleterious effects caused by these substances to animal health, including the use of chemical substances, physical methods, strategies for harvest, storage and processing food. The objective of this review is to describe the main effects of aflatoxins on poultry performance, the alterations in raw-materials of feeds and scientific advances on methods for biological detoxification and agricultural good practices.

Mecanismos moleculares involucrados en la mutagenicidad inducida por aflatoxina B1 / Molecular mechanism involved in the mutagenicity induced by Aflatoxin B1 / Mecanismos moleculares envolvidos na mutagenicidade induzida pela aflatoxina B1

La aflatoxina B1 (AFB1) es una micotoxina identificada como el más potente hepatocarcinógeno. El metabolito que resulta del proceso de detoxificación de la AFB1 en el hígado tiene la capacidad de reaccionar con el ADN genómico, generando el aducto AFB1-ADN; durante la replicación del ADN este aducto induce la transversión G:C—>T:A. Polimorfismos en los genes que codifican las enzimas encargadas de la activación y detoxificación de la AFB1 y enzimas de reparación del ADN han sido asociados con el riesgo de desarrollar carcinoma hepatocelular (CHC). Adicionalmente, en poblaciones con alta exposición a aflatoxina y alta prevalencia de infección por el virus de la hepatitis B (VHB) se ha demostrado un sinergismo entre estos dos factores de riesgo para el desarrollo de CHC.

The aflatoxin B1 (AFB1) is a mycotoxin that has been identified as the most potent hepatocarcinogen. The metabolite resulting from detoxification process of AFB1 in liver, has the ability to react with the genomic DNA, generating AFB1-DNA adducts; during DNA replication process, this adduct induced the G:C—>T:A transversion. Polymorphism in genes encoding for enzymes involved in the activation and detoxification of AFB1 and DNA repair enzymes has been associated with the risk of hepatocellular carcinoma (HCC) development. Additionally, in populations of high exposure to aflatoxin and high prevalence of hepatitis B virus (HBV) infection, has been demonstrated a synergism between these two risk factors for the development of HCC.

Aflatoxina B1 (AFB1) é uma micotoxina identificado como o hepatocarcinogen mais potente. O metabolito resultante do processo de desintoxicação de AFB1 no fígado, tem a capacidade de reagir com o ADN genómico, gerando AFB1 DNA-aducto; transversão durante a replicação do ADN deste aducto induz G:C—>T:A. Polimorfismos em genes que codificam as enzimas envolvidas na activação e na desintoxicação de AFB1 e enzimas de reparação do ADN têm sido associados com o risco de desenvolvimento de carcinoma hepatocelular (HCC). Além disso, em populações com elevada exposição a aflatoxina e elevada prevalência da infecção com vírus da hepatite B (VHB) tem sido mostrado um sinergismo entre estes dois factores de risco para o desenvolvimento de carcinoma hepatocelular.

Effects of Ginkgo biloba extract on biomarker expressions during aflatoxin B_1-induced hepatocarcinogenesis in Wistar rats / 肿瘤

Objective:To study the effect of Ginkgo biloba extract (EGb761) on metabolism of aflatoxin B_1(AFB_1) in Wistar rats. Methods:Seventy one Wistar rats were divided into three groups at random: group A (AFB_1 group), group B (AFB_1+EGb761 group), and group C (control group). The rats in groups A and B were given AFB_1(intraperitoneal injection, 100-200 μg/ kg body weight, 1-3 times/week). The rats in group B were fed the food containing EGb761 while the rats in groups A and C were given normal food. Blood samples were collected and liver biopsy was performed on the 14th, 28th and 42nd week. All the rats were sacrificed at the 64th week. The incidence of hepatoma was observed. The hepatic phase Ⅰ drug-metabolizing enzyme CYP450 and phase Ⅱ enzyme GST were detected by spectrometry. The serum AFB_1-lysine adduct was determined by high performance liquid chromatography (HPLC). The expression of 8-hydroxydeoxyguanosine(8-OHdG) was measured by immunohistochemistry. Results:The incidence of hepatocellular carcinoma (HCC) in group B was significantly lower than that in group A (26.92% vs 76.00%,P<0.001). No hepatocellular carcinoma developed in group C. EGb761 had no effects on the activities of CYP450 and GST in rat liver tissues. The level of AFB_1-lysine adduct reached the peak (4 356.01 pg/mg albumin) at the 14th week in group A. EGb761 significantly inhibited the formation of AFB_1-lysine adducts in serum by 13.07% at the 14th week (P＝0.033), and 73.63% at the 42nd week (P＝0.002). The expression of 8-OHdG protein in rat liver tissues in group B was significantly lower than that in group A at the 28th, 42nd, and 64th week (P<0.05). Conclusion:The main mechanism underlying the effect of EGb761 in blocking hepatogenesis induced by AFB_1 may not be fully related with its influence on the activity of liver phase Ⅰ and phase Ⅱ metabolizing enzymes. EGb761 inhibites the production of AFB_1-lysine addcuts, decreases the expression of 8-OHdG protein, and finally alleviates the DNA oxidative injury, which may be one of the mechanisms for the effects of EGb761 in inhibiting or delaying hepatogenesis induced by AFB_1.

Determination AFB_1 Content of Part Seed and Fruit Traditional Chinese Medicine by ELISA / 医学研究杂志

Objective Determination AFB1 content of Part Seed and fruit Traditional Chinese Medicine by ELISA.Method ELISA.Results Sample were Pollution different degree with AFB1,the content of the mildews were high during storage.Conclusion It is very necessary to establish standard of the AFB1 content.

Effects of heating processing method on AFB1 content in TCM cut crude drug / 中华中医药杂志

Objective: To compare the in uence of various processing methods on AFB1 content in TCM cut crude drug. Methods: ELISA was used to determine the AFB1 content. Results: Heating processing method can decrease the content of AFB1 in TCM cut crude drug. However, it cannot make AFB1 content meet the scope of security when the AFB1 content in TCM cut crude drug was too high. Various heating processing methods had di erent e ect on the content of AFB1. Conclusion: Heating processing method had certain in uence on the content of AFB1. It was necessary to establish limitation standard of the AFB1 content. The AFB1 content limit in the less oil-bearing fruit TCM cut crude drug shouldn,t be higher than 5?g/kg and in the more oil-bearing seed TCM cut crude drug it shouldn't be higher than 20?g/kg. This was the same limit as the national food standards

Effect of diet on aflatoxin B1-DNA binding and aflatoxin B1-induced glutathione S-transferase placental form positive hepatic foci in the rat

Effects of diets on hepatic aflatoxin B1 (AFB1)- DNA binding and AFB1-induced glutathione S- transferase placental (GST-P) form positive hepatic foci have been examined in young male Fischer rats. Animals were fed either AIN-76A or Purina Chow (PC) diet for 1 wk before AFB1- DNA binding studies in vivo and in vitro. Animals were injected i.p. with AFB1 (1 mg/kg body wt) and 3 days later were given either AIN-76A or PC diet with or without 0.1% phenobarbital (PB) in their drinking water. All animals were sacrificed 10 wks after AFB1 dosing for analysis of AFB1-induced GST-P positive hepatic foci by immunochemistry. Two h after i.p. injection of AFB1, hepatic AFB1-DNA binding in AIN-76A fed rats was twice as much as those in PC fed animals without affecting GSH levels. There was no significant effect of diet on either cytochrome P-450 content, GSH levels or microsomal cytochrome P-450 mediated AFB1-DNA binding to exogenous DNA. There was a 40% increase in cytosolic GSH S-transferase activity with 1-chloro-2,4-dinitrobenzene as a substrate in PC fed animals compared to those given AIN- 76A diet. The number and area of AFB1-induced GST-P positive hepatic foci were twice and fivefold as much in AIN-76A fed compared to those in PC fed rats. The number of AFB1-induced GST-P positive foci was increased 5-10 fold in the presence of PB in both groups. In summary, the present data indicate that feeding of PC diet compared to AIN-76A diet inhibits the initiation phase whereas AIN-76A stimulates the promotion phase of AFB1 hepatocarcinogenesis in rats by inhibiting AFB1-DNA binding and increasing AFB1-induced hepatic foci respectively.

INFLUENCE OF SELENIUM-ENRICHED MALT ON AFLATOXINB_1 INDUCED UNSCHEDULED DNA SYNTHESIS (UDS) OFPERIPHERAL WHITE BLOOD CELLS / 营养学报

The blocking effect of selenium-enriched malt (Se-malt) on AFB1-in-duced UDS was reported. When the rats were fed with food containing lppm Se in form of Se-malt for two weeks, the UDS values of leukocytes treated with 6?10-7M AFB1 in vitro was decreased from 54.8 ?14.0 opm/106 WBC to 35.1?10.0 cpm/106 WBC (p