Development of a reusable polymeric fluorescence sensor based on acryloyl ß-cyclodextrin for the determination of aflatoxin B1 in grain products.

AFB1 is a harmful substance that can be found in agricultural products and can seriously affect human health, even in trace amounts. Therefore, monitoring AFB1 levels to ensure food safety and protect public health is crucial. New, highly reliable, selective, and rapid detection methods are needed to achieve this goal. Our work involves the development of a polymeric membrane sensor using radical polymerization that can accurately detect AFB1. Various spectroscopic techniques (Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM)) were used to obtain information about the structural and morphological properties of the prepared sensor. The sensor displayed fluorescence selectively responsive to AFB1 at the excitation wavelength of 376 nm and emission wavelength of 423 nm. The polymeric fluorescence sensor showed good sensitivity and a wide linear range from 9.61 × 10-10 and 9.61 × 10-9 mol/L for AFB1quantification. The limit of detection (LOD) is as low as 3.84 × 10-10 mol/L for AFB1. Other mycotoxins, such as aflatoxin B2 and aflatoxin G1, did not interfere with the sensor's high selectivity towards AFB1. To test the sensor's effectiveness in detecting AFB1 in real samples, three different grain samples - peanuts, hazelnut butter, and peanuts with a sauce known to contain AFB1 - were utilized. The results were satisfactory and demonstrated that the sensor can be successfully employed in real samples, with an error range of 0.43 % to 12.10 %.

A precursor of Aflatoxin B1, Versicolorin A, impairs the mitochondrial function of human intestinal Caco-2 cells.

Versicolorin A (VERA) is a mycotoxin produced by Aspergillus section Flavi species that is frequently detected in foodstuffs, particularly in corn. VERA is a precursor of aflatoxin B1 (AFB1), which is currently considered to be the most hazardous mycotoxin. While AFB1 has been shown to impair oxidative phosphorylation (OXPHOS), the impact of VERA on mitochondrial function has not been extensively documented until now. The aim of the present study was to investigate the effect of VERA on mitochondrial function in intestinal Caco-2 cells. To this end, OXPHOS was assessed by measuring the oxygen consumption rate using the Seahorse™ real-time analyzer. In contrast to AFB1, a low concentration of VERA (5 µM) was a strong uncoupler of OXPHOS and inhibited respiratory complexes I and III within a few minutes of exposure. After 24 h of exposure, VERA reduced the transcription of all mitochondrial genes encoding proteins involved in the electron transfer chain as well as decreasing the rate of OXPHOS. This effect was associated with the simultaneous down expression of two genes encoding proteins involved in the initiation phase of mitochondrial DNA transcription: POLRMT and TFB2M. Moreover, VERA induced down expression of genes coding for upstream key glycolytic enzymes, hexokinase and phosphofructokinase. These effects led to a reduced rate of ATP production associated with a cytotoxic effect. Given the significant implications of mitochondrial dysfunction for human health, it is crucial to consider the potential involvement of VERA in mitochondrial diseases.

Silver amplified immunosensor via effective fluorogenic Ag+-imidazole aggregation for detection of AFB1.

BACKGROUND: Cereals are susceptible to aflatoxin contamination during storage and transportation, which is highly carcinogenic and teratogenic, and seriously threaten human health. The accurate and rapid detection of total aflatoxin (including aflatoxin B1, B2, G1, and G2) is of great importance for food safety. Conventional fluorescence immunoassays have the advantage of being sensitive and fast; however, these methods can be affected by strong background and matrix interference. Therefore, the development of ultrasensitive, cost-effective, and interference rejection sensors for detecting aflatoxins in moldy grains is vital for food safety and human health. RESULTS: In this paper, a broad-spectrum aflatoxin monoclonal antibody was prepared by using hybridoma cell fusion technology. An aggregation-induced emission (AIE) based immunosensor via silver amplification coupled with a fluorogenic Ag+ probe was established for AFB1 analysis. Silver nanoparticles are decomposed into numerous Ag+ by H2O2, and then Ag+ further specifically binds with imidazole-modified AIE molecules, improving the sensitivity and anti-interference ability of the method. The IC50 and IC15 of AIE-based immunosensor for AFB1 were 0.019 and 0.0014 µg/L, respectively, 2.3-fold and 5.8-fold higher than those of icELISA. The AIE-based immunosensor was also used to analyze AFB1 from actual cereal samples, with spiked recoveries ranging from 72.91 to 115.92 %. In addition, the method was used to detect total aflatoxins in moldy grains. SIGNIFICANCE: Based on the advantages of broad-spectrum aflatoxin monoclonal antibody, high-efficiency metal signal amplification, and functional AIE molecule, a sensitive, accurate, cost-effective, and time-saving method was developed for the analysis of total aflatoxins in cereals. Moreover, the proposed signal amplification strategy shows great potential for analyzing other trace-level small molecular pollutants.

Aflatoxin B1-induced hepatotoxicity through mitochondrial dysfunction, oxidative stress, and inflammation as central pathological mechanisms: A review of experimental evidence.

Aflatoxin B1 (AFB1) is a class of mycotoxin known to contaminate agricultural products, animal feed and animal food products, subsequently causing detrimental effects on human and animal health. AFB1 is the most common and potent aflatoxin found in food and contributes significantly to liver injury as well as the development of hepatocellular carcinoma. Although the liver is a primary target organ for AFB1 toxicity and biotransformation, underlying mechanisms implicated in liver injuries induced by these mycotoxins remain to be fully elucidated for therapeutic purposes. This review aims to dissect the complexities of the pathophysiological and molecular mechanisms implicated in hepatotoxicity induced by AFB1, including mitochondrial dysfunction, oxidative stress and hepatic inflammation. Mechanistically, AFB1 disrupt mitochondrial bioenergetics and membrane potential, promotes mitochondrial cholesterol trafficking and induces mitophagy. Moreover, mitochondrial dysfunction may lead to hepatic oxidative stress as a consequence of uncontrolled production of reactive oxygen species and defects in the antioxidant defense system. Retrieved experimental evidence also showed that AFB1 may lead to hepatic inflammation through gut microbiota dysbiosis, the release of DAMPs and cytokines, and immune cell recruitment. Overall, these mechanisms could be utilized as potential targets to extrapolate treatment for liver injury caused by AFB1.

Construction of carbon-doped iron-based nanozyme for efficient adsorption and degradation to synergistic removal of aflatoxin B1.

The multifunctional composites Fe3O4/GO/NH2-MIL-53(Fe) with excellent adsorption-degradation performance was prepared for the removal of Aflatoxin B1 (AFB1). The adsorption function of Fe3O4/GO/NH2-MIL-53(Fe) was based on the large specific surface area and abundant adsorption sites. The degradation function of Fe3O4/GO/NH2-MIL-53(Fe) was based on the activation of H2O2 by the catalytic active center formed by the coordination of metal ions and oxygen-containing groups in the system, resulting in hydroxyl radicals (·OH), superoxide anion radicals (O2-) and singlet oxygen (1O2). The adsorption of nanozyme accelerated the degradation reaction process, and the adsorption site was further exposed as the degradation process progressed. The synergistic effect realized the efficient removal of AFB1. Construction of Fe3O4/GO/NH2-MIL-53(Fe) as the carbon-doped iron-based nanozyme provided novel approaches of the removal for risks control of AFB1. Accompanied by the AFB1 adsorption, the advanced oxidation of nanozyme to the AFB1 degradation provided a promising way for the synergistic removal of AFB1.

Assessment of the Nutritional Benefits and Aflatoxin B1 Adsorption Properties of Blackberry Seed Cold-Pressed Oil By-Product.

This study explores the potential valorization of blackberry seed oil cake (BBSOC), a by-product of cold-pressed blackberry seed oil (Rubus fruticosus L.), as a nutritionally valuable material with aflatoxin B1 (AFB1) adsorption properties. The chemical and mineral composition, polyphenols, and antioxidant activity of BBSOC flour were assessed. BBSOC was found to be a significant source of fiber (62.09% dry weight) and essential minerals such as Fe (123.48 mg/kg), Mg (1281.40 mg/kg), K (3087.61 mg/kg), and Ca (1568.41 mg/kg). The high polyphenol content, especially ellagic acid, highlighted its biologically active potential. Moreover, BBSOC demonstrated effective biosorption of AFB1 under in vitro conditions at 37 °C, with adsorption efficiencies of 85.36% and 87.01% at pH 3 and 7, respectively. Characterization techniques including SEM, FTIR analysis, Boehm titration, and pH zero charge determination confirmed its AFB1 adsorbing properties. This valorization process reintroduces a secondary product into the food chain, supporting the circular economy and zero-waste concepts. Thus, BBSOC is nutritionally rich and effective in AFB1 biosorption, presenting potential applications as a food or feed additive.

Biological Mechanisms of Aflatoxin B1-Induced Bile Metabolism Abnormalities in Ducklings.

This study investigated the effects and biological mechanisms of aflatoxin B1 (AFB1) on the health and bile metabolism of ducklings. Forty-eight 1-day-old ducklings were randomly assigned to two groups, with six replicates per group. The control group was fed a basic diet, while the AFB1 group received a diet containing 90 µg/kg of AFB1. The experiment lasted for 2 weeks. The results showed that 90 µg/kg AFB1 caused abnormal bile metabolism; damaged liver cell nuclei and mitochondria; and significantly decreased body weight, average daily weight gain, and levels of albumin, total protein, cholesterol, total superoxide dismutase, glutathione peroxidase, and glutathione. It also significantly increased feed conversion efficiency, along with alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bile acids, and malondialdehyde levels. In the liver, the expression levels of CYP7A1, SCD, and other genes were significantly upregulated, while BSEP, FASN, HMGCR, CAT, and other genes were significantly downregulated. In conclusion, AFB1 causes abnormal bile metabolism and impairs the overall health and liver function of ducklings. Its mechanism of action may involve changes in gene expression related to bile acid metabolism, lipid metabolism, oxidative damage, and cancer pathways.

A comparative study of aptasensor vs. immunosensor for ultrasensitive detection of aflatoxin B1 using Ag-pSi SERS substrate.

Numerous SERS based platforms have been designed to address the emerging need for detecting fungal metabolite contamination in foodstuffs, and specifically the Group 1 carcinogen aflatoxin B1. Herein, 4-aminothiophenol modified silver-coated porous silicon was used as the SERS substrate. Two ratiometric responses were individually assessed upon direct target capture using specific aptamers or antibodies. Under optimized physical features, elevated enhancement factor, wide dynamic range, low detection limits and pronounced recycling capabilities were achieved (7.39 × 107, 0.2-200 ppb, 0.0085 and 0.0110 ppb, 7 and 1 regeneration cycles without impairing the performances for aptasensor and immunosensor, respectively). The accuracy and anti-interference responses in several intricate matrices (maize, peanut, wheat, oats and rice) were compared to a routine HPLC method with equivalent recoveries. Overall, the comparative assessment revealed preferable features of reusability, durability and accuracy of the aptasensor over the immunosensor. Furthermore, the results demonstrate the substantial potential of the proposed SERS substrate for diverse on-site analytical applications using simple and portable monitoring instrumentation.

Ultrasensitive aflatoxin B1 detection based on vertical organic electrochemical transistor.

Herein, we presented an ultrasensitive Aflatoxin B1 (AFB1) detection platform based on vertical organic electrochemical transistor (vOECT) first time. Chitosan-graphene nanosheets nanocomposites and AFB1 antibodies were modified on commercial electrodes as immunosensors, in series with gate electrodes of vOECT, operated at enhancement mode with ultrahigh transconductance gm 94 mS to amplify current signals. When AFB1 is added, the impedance of the immunosensors increased due to antigen-antibody immune binding, resulting in a potential decrease in reaction cell. Then, the potential decrease leads to an effective gate voltage VGeff increase, contributing to a significant drain-source current IDS decrease as a consequence of ultrahigh gm of vOECT. As a result, the presented vOECT platform exhibited an ultrahigh sensitivity of ∼1 mA/dec, and an ultralow detection limit of 0.01 fg/mL (S/N = 3), superior to all previous reported values. Furthermore, the platform exhibited satisfactory stability and specificity, and was applied to detect AFB1 in corn samples.

Vitamin U alleviates AFB1-induced hepatotoxicity in pregnant and lactating mice by regulating the Nrf2/Hmox1 pathway.

This study investigated the protective effect of Vitamin U on liver injury induced by aflatoxin B1 (AFB1) in maternal mice. 25 pregnant ICR mice were randomly divided into five groups: the AFB1 group (AF, 0.3 mg AFB1/kg b.w.), the Vitamin U group (U, 50 mg Vitamin U/kg b.w.), the AFB1 + Vitamin U group (AU, 50 mg Vitamin U /kg b.w. + 0.3 mg AFB1/kg b.w.), the control group (DMSO), and the MOCK group (distilled water). They were administered substances by gavage every day for 28 days. Results indicated that exposure to AFB1 increased the liver index and caused histological disruptions. Elevated serum levels of ALT and ALP were observed, along with a significant increase in liver MDA content and a decrease in GSH-Px and T-SOD levels. Moreover, the Keap1 and Hmox1 gene was downregulated with statistical significance, while the IL1ß and TNFα gene were significantly upregulated. Vitamin U was demonstrated by the organized structure of liver cells in tissue slices, effectively reducing liver cell necrosis. This intervention was associated with a significant decrease in serum ALT and ALP activities, as well as a significant decrease in liver MDA content. Additionally, there were significant increases in liver T-SOD and GSH-Px levels, along with upregulation of mRNA and protein expression of Nfr2, Hmox1 and Keap1, and downregulation of mRNA expression of the IL1ß gene. In summary, Vitamin U mitigated oxidative stress-induced liver injury by modulating the Nrf2/Hmox1 signaling pathway and inflammatory factors affected by AFB1.

AFB1-responsive mesoporous silica nanoparticles for AFB1 quantification based on aptamer-regulated release of SERS reporter.

In this study, we propose a novel surface-enhanced Raman scattering (SERS) method for quantifying aflatoxin B1 (AFB1). This method relies on the target-triggered release of a SERS reporter from aptamer-sealed aminated mesoporous silica nanoparticles (MSNs). These MSNs were synthesized to accommodate 4-mercaptophenylboronic acid (4-MPBA) within their well-defined micropores, which were subsequently sealed with AFB1 aptamers. Upon specific binding of AFB1 to its aptamer, the conformational change in the aptamer is regulated by the presence of the target. Consequently, a positive linear relationship between the AFB1 concentration and the 4-MPBA SERS signal was observed. Under optimal conditions, the method exhibited a good linear relationship over the range of 0.1 to 5 ng/mL AFB1, with a limit of detection (LOD) of 0.03 ng/mL. This strategy was validated using wheat samples, yielding results comparable to high performance liquid chromatography-fluorescence detector (P > 0.05), confirming its reliability for detecting AFB1 in complex food matrices.

Enhancing resolution in DNA staining dye-based label-free visual fluorescence aptasensor: Strategy for eliminating non-specific binding-induced signal interference.

By optimizing the quenching capabilities of diverse two-dimensional (2D) nanomaterials such as graphene oxide (GO), Ti3C2 MXene, and MoS2, we have pioneered a label-free fluorescence aptasensor with near-zero background signal, enabling highly sensitive detection of aflatoxin B1 (AFB1). This aptasensor was equipped with a newly synthesized dicationic fluorophore, VLM, which exhibited binding-induced turn-on fluorescence properties. Among the evaluated 2D nanosheets, MoS2 nanosheets were found to exhibit exceptional quenching efficiency for the background emission of the cDNA/VLM complex (cDNA was the complementary DNA of the aptamer), further enhancing the overall performance of our aptasensor. Upon exposure to AFB1, the aptamers underwent conformational switching and target binding, leading to the formation of aptamer/AFB1 complex. Additionally, the aptamers bound complementarily to cDNA, creating aptamer-cDNA duplexes that interacted with VLM, resulting in a robust fluorescence signal. Despite the presence of a weakly fluorescent cDNA/VLM background, this fluorescence could be effectively quenched by the addition of MoS2 nanosheets. Consequently, the label-free fluorescence aptasensor exhibited excellent linearity with AFB1 concentration within 2-3000 ng mL-1, achieving a limit of detection (LOD) of 0.006 ng mL-1. Remarkably, the visual fluorescence captured by a smartphone camera can be processed using extracted grayscale values, consistently revealing a linear relationship with the AFB1 concentration within 2-3000 ng mL-1, with a LOD of 0.197 ng mL-1. This aptasensor demonstrated exceptional sensitivity and a remarkably rapid sample-to-answer detection time of 74 min, showcasing its immense potential as a straightforward, sensitive, and visually intuitive method for rapid AFB1 detection with enhanced resolution.

A smartphone-integrated deep learning strategy-assisted rapid detection system for monitoring dual-modal immunochromatographic assay.

This study focuses on the integration of a custom-built and optimally trained YOLO v5 model into a smartphone app developed with Java language. A dual-modal immunochromatographic rapid detection system based on a deep learning strategy for smartphones was developed for grade determination and predicting the concentration of aflatoxin B1 (AFB1). Innovative distance-type quantum dot microsphere fluorescent immunochromatographic chips enable semi-quantitative analysis by naked eye, and conventional colloidal gold nanoparticle colorimetric strips were also prepared. The compact and versatile hardware device making it easily integrable into smartphones of varying dimensions. Moreover, the wireless charging functionality of smartphones was to tackle power supply challenges. After optimized training, the accuracy, mAP@0.5, precision, and recall metrics of the YOLO v5 model all soared to 98 %. For the dual-modal immunochromatographic chips, the R2 values for the standard curve fits were as high as 0.993, with a broad linear range of 0.05-40 ng/mL and a standard deviation lower than 0.03 at each concentration. Finally, this system determined the grade of the AFB1 concentration with an accuracy of up to 98 % and it exhibited an ultra-sensitive quantitative detection capability with a limit of detection as low as 2.2 pg/mL, showcasing the reliability of the deep learning strategy for practical applications in smartphones. This robust technological foundation paves the way for potentially community-based, family-oriented, and personalized applications.

Homogeneous fluorescence immunoassay based on AuNPs (AgNPs) quenching multicolor QDs@hydrogel beads for the simultaneous ultra-sensitive determination of aflatoxin B1 and capsaicinoids in food.

A capsaicinoids (CPCs) broad spectrum monoclonal antibody with same recognition ability to capsaicin (CPC), dihydrocapsaicin (DCPC), nordihydrocapsaicin (NDCPC), and N-vanillylnonanamide (NV) is prepared. Chitosan (CS) hydrogel is used as the carrier of multicolor quantum dots (QDs) to prepare fluorescence hydrogel beads, CPCs and aflatoxin B1 (AFB1) antibody are coupled with fluorescence hydrogel beads to prepare signal probes. Using AuNPs (or AgNPs) as fluorescence quenching agent to prepare quenching probes followed forming a fluorescence quenching test system. Based on optimal group of signal and quenching probes, a novel, simple, convenient, and ultra-sensitive homogeneous fluorescence immunoassay for the simultaneous detection of CPCs and AFB1 is constructed. The limit of detection (LOD) of assay for AFB1 and CPC is 0.00064 µg L-1 and 0.00049 µg L-1, respectively. This method can realize the simultaneous rapid detection of AFB1 and CPCs in food, which provides a new strategy for the identification of kitchen waste oil.

Mycotoxin Detection through Colorimetric Immunoprobing with Gold Nanoparticle Antibody Conjugates.

Driven by their exceptional optical characteristics, robust chemical stability, and facile bioconjugation, gold nanoparticles (AuNPs) have emerged as a preferred material for detection and biosensing applications in scientific research. This study involves the development of a simple, rapid, and cost-effective colorimetric immuno-sensing probe to detect aflatoxin B1 and zearalenone using AuNP antibody (AuNP-mAb) conjugates. Anti-toxin antibodies were attached to the AuNPs by using the physical adsorption method. The colorimetric immunosensor developed operates on the principle that the optical properties of the AuNP are very sensitive to aggregation, which can be induced by a critical high salt concentration. Although the presence of antibodies on the AuNP surface inhibits the aggregation, these antibodies bind to the toxin with higher affinity, which leads to exposure of the surface of AuNPs and aggregation in a salt environment. The aggregation triggers a noticeable but variable alteration in color from red to purple and blueish gray, as a result of a red shift in the surface plasmon resonance band of the AuNPs. The extent of the shift is dependent on the toxin exposure dose and can be quantified using a calibration curve through UV-Visible-NIR spectroscopy. The limit of detection using this assay was determined to be as low as 0.15 ng/mL for both zearalenone and aflatoxin B1. The specificity of the prepared immunoprobe was analyzed for a particular mycotoxin in the presence of other mycotoxins. The developed immunoprobe was evaluated for real-world applicability using artificially spiked samples. This colorimetric immunoprobe based on localized surface plasmon resonance (LSPR) has a reduced detection limit compared to other immunoassays, a rapid readout, low cost, and facile fabrication.

Cloning of Three Aflatoxin B1 Oxidases of the Dipeptidyl Peptidase III Family and Evaluation of Their Potential for Practical Applications as Decontamination Enzymes.

Aflatoxin B1 (AFB1) produced by some Aspergillus species belongs to the most dangerous contaminants of animal feeds. Development of safe and cost efficient decontamination methods saving feed quality and nutritional value are of paramount importance. The use of recombinant AFB1-detoxifying microbial enzymes represents a promising biotechnological approach meeting the aforementioned requirements. In this study, three AFB1-degrading oxidases (AFOs) from edible basidiomycetes Cantharellus cibarius, Lentinula edodes and Pleurotus eryngii as well as AFO from Armillaria tabescens were expressed in E. coli Rosetta (DE3) and purified by immobilized metal-chelate chromatography. The stabilizing effect of the addition of glycerol and ß-mercaptoethanol during protein extraction is shown. The catalytic constants of the recombinant AFOs (rAFOs) and other characteristics, which might be important for their practical application (and optimal temperature and pH, thermolability, regulation of the activity by metal ions and chelating agents, storage stability) were investigated. Among the obtained enzymes, rAFO from P. eryngii (Pe-AFO), which was characterized by the highest specific activity, thermostability and pH stability (especially at acidic pH range), the lowest Km, and relative resistance to the inhibition by phytate, showed the best AFB1-degrading efficacy. However, Pe-AFO and all other rAFOs significantly decreased the target activity during heating above 45 °C, storage frozen or lyophilization.

Preparation of Soybean Dreg-Based Biochar@TiO2 Composites and the Photocatalytic Degradation of Aflatoxin B1 Exposed to Simulated Sunlight Irradiation.

Aflatoxin B1 (AFB1) is a highly toxic carcinogen severely harmful to humans and animals. This study fabricated SDB-6-K-9@TiO2 composites via the hydrothermal synthesis method to reduce AFB1. The structural characterization results of the photocatalytic composites showed that TiO2 was successfully loaded onto SDB-6-K-9. The different photocatalytic degradation conditions, photocatalyst kinetics, recycling performance, and photocatalytic degradation mechanism were investigated. Photocatalysis with 6 mg of 4%SDB-6-K-9@TiO2 in a 100 µg/mL AFB1 solution presented a reduction of over 95%, exhibiting excellent performance, high stability, and reusability even after five cycles of photocatalytic experiments. Active species trapping experiments confirmed that holes (h+) played the most critical role. After structural analysis and identification of the photocatalytic degradation products, the photodegradation path and photocatalytic oxidation mechanism of 4%SDB-6-K-9@TiO2 were postulated. The results show a new way to improve TiO2's photocatalytic performance, providing a certain theoretical basis for the effective AFB1 reduction.

More than a mutagenic Aflatoxin B1 precursor: The multiple cellular targets of Versicolorin A revealed by global gene expression analysis.

Versicolorin A (VerA), a precursor of the potent carcinogen Aflatoxin B1 (AFB1), is an emerging mycotoxin. Recent research has highlighted the mutagenic and genotoxic properties of VerA, yet several facets of its pronounced toxicity remain unexplored. In the present study, we investigated early (6 h) transcriptomic changes induced by VerA in differentiated intestinal cells in non-cytotoxic conditions (1 and 3 µM) and compared its effects to those of AFB1 at 1 µM. Our findings indicated that VerA led to substantial alterations in global gene expression profiles, while AFB1 did not exhibit the same effects. As expected, both toxins caused alterations in gene expression associated with well-known aspects of their toxicity, including mutagenicity, genotoxicity, oxidative stress, and apoptosis. However, we also observed novel features of VerA toxicity, including the ability to cause mitochondrial dysfunction and to trigger a type-1 interferon response, at least partially mediated by cGAS-STING. VerA also induced changes in the expression of genes involved in the regulation of cell shape and adhesion, transcription/translation as well as genes associated with tumor biology. Our results provide new evidence of the high toxicity of VerA and underscore the importance of further assessing the risks associated with its presence in food.

Aflatoxin B1 contamination reduces the saponins content and anti-osteoporosis efficacy of the traditional medicine Radix Dipsaci.

ETHNOPHARMACOLOGICAL RELEVANCE: The Radix Dipsaci, a traditional Chinese medicine with a history spanning over 2000 years in China, is widely recognized for its hepatorenal tonic properties, musculoskeletal fortifying effects, fracture healing capabilities, and its frequent application in the treatment of osteoporosis. Like many traditional Chinese herbal medicines, preparations from Radix Dipsaci are at risk of contamination by harmful mycotoxins such as aflatoxin B1. AIMS OF THE STUDY: This study aims to evaluate the impact of aflatoxin B1 contamination on Radix Dipsaci in terms of changes in quality, efficacy of anti-osteoporosis and hepatorenal toxicity. MATERIALS AND METHODS: The contamination rates and levels of major mycotoxins were determined in 45 batches of Radix Dipsaci samples using UPLC-MS/MS analysis. The total saponin content and the levels of akebia saponin D in Radix Dipsaci and its decoctions were evaluated through high-performance liquid chromatography (HPLC) analysis. Differences in secondary metabolites between samples without any mycotoxin contamination (N-RD) and those contaminated solely by aflatoxin B1 (AFB1-RD) were compared using metabolomics sequencing and analysis. The anti-osteoporotic efficacy of Radix Dipsaci contaminated with aflatoxin B1 was assessed in a murine model of retinoic acid-induced osteoporosis by quantifying bone mineral content and bone mineral density using dual-energy X-ray absorptiometry. Additionally, the hepatorenal toxicity of Radix Dipsaci contaminated with aflatoxin B1 was evaluated using hematoxylin-eosin staining and enzyme-linked immunosorbent assay (ELISA). RESULTS: The results indicated that aflatoxin B1 (AFB1) was the most frequently detected mycotoxin, found in 37.7% of the Radix Dipsaci samples. AFB1 contamination significantly altered the secondary metabolites of Radix Dipsaci. Specifically, there was a notable decrease in the levels of total saponins and akebia saponin D in the AFB1-contaminated samples, which exhibited a negative correlation with the levels of AFB1 contamination. However, the administration of a water decoction from AFB1-contaminated Radix Dipsaci did not result in significant improvements in bone mineral density, bone mineral salt content, the trabecular number, trabecular area, proportion of trabecular bone volume/tissue volume and trabecular separation in an osteoporosis mouse model. Additionally, we observed that approximately 16.04% of AFB1 could migrate from the raw herbs into the decoction, leading to hepatocyte and kidney cell damage, as well as increased levels of the oxidative stress molecule malondialdehyde and pro-inflammatory cytokines in the liver and kidney tissues of the osteoporosis model mice. CONCLUSION: In summary, Radix Dipsaci is highly susceptible to mycotoxin contamination, particularly aflatoxin B1. The contamination of Radix Dipsaci with AFB1 not only impacts their saponin content and anti-osteoporosis effect but also induces hepatotoxicity and nephrotoxicity.

Data-Independent Acquisition Proteome Technology for Analysis of Antifungal and Anti-aflatoxigenic Properties of Eugenol to Aspergillus flavus.

Several toxicogenic Aspergilli, such as Aspergillus flavus and A. parasiticus, could biosynthesize aflatoxin B1 (AFB1) and other mycotoxins. Chemical fungicides are commonly used to control fungal contamination, but chemical residues may pose significant risks to human health and environmental stability. Consequently, natural antifungal and aflatoxin-inhibiting agents could be sustainable alternatives. Eugenol has been used as an inhibitor of aflatoxins (AFs), which is a common essential oil. Nevertheless, the definite mechanism by which eugenol exerts its inhibitory effect on Aspergillus remains unclear. This research demonstrates that eugenol significantly suppressed fungi growth and AF production as the dose increased (40.9 to 100%). With the proteomics approach, the inhibition pathway of eugenol was investigated. The production of proteins involved in cell wall integrity was notably reduced under eugenol treatment, indicating that eugenol destroys the cell wall integrity of A. flavus. Furthermore, exposure to eugenol downregulated several fungal developmental regulators and subsequently inhibited A. flavus development. Energy metabolism in A. flavus is closely related to its secondary metabolism. Under eugenol treatment, the synthesis of proteins relevant to the pentose phosphate pathway was significantly enhanced, leading to a decrease in the availability of acetyl-CoA, a precursor for AF biosynthesis. Simultaneously, the valine, leucine, and isoleucine biosynthesis pathways were enhanced, further reducing the content of acetyl-CoA. This might be the primary factor in the inhibition of AF biosynthesis by eugenol. Ribosome biogenesis was the most dysregulated pathway based on KEGG data, indicating that eugenol disturbed ribosome biogenesis and affected its normal function in A. flavus. In conclusion, eugenol inhibits the cellular integrity, energy metabolism, and protein synthesis and then suppresses A. flavus development and AF biosynthesis, which provides a clearer grasp of the inhibitory mechanism meaningful for A. flavus and AF contamination control.