By investigating the contamination status and predicting the exposure risk of mycotoxin in Coicis Semen, we aim to provide guidance for the safety supervision of Chinese medicinal materials and the formulation(revision) of mycotoxin limit standards. The content of 14 mycotoxins in the 100 Coicis Semen samples collected from five major markets of Chinese medicinal materials in China was determined by UPLC-MS/MS. The probability evaluation model based on Monte Carlo simulation method was established after Chi-square test and One-way ANOVA of the sample contamination data. Health risk assessment was performed on the basis of margin of exposure(MOE) and margin of safety(MOS). The results showed that zearalenone(ZEN), aflatoxin B_1(AFB_1), deoxynivalenol(DON), sterigmatocystin(ST), and aflatoxin B_2(AFB_2) in the Coicis Semen samples had the detection rates of 84%, 75%, 36%, 19%, and 18%, and the mean contamination levels of 117.42, 4.78, 61.16, 6.61, and 2.13 μg·kg~(-1), respectively. According to the limit standards in the Chinese Pharmacopoeia(2020 edition), AFB_1, AFs and ZEN exceeded the standards to certain extents, with the over-standard rates of 12.0%, 9.0%, and 6.0%, respectively. The exposure risks of Coicis Semen to AFB_1, AFB2, ST, DON, and ZEN were low, while 86% of the samples were contaminated with two or more toxins, which needs more attention. It is suggested that the research on the combined toxicity of different mycotoxins should be strengthened to accelerate the cumulative exposure assessment of mixed contaminations and the formulation(revision) of toxin limit standards.
Humans ; Mycotoxins/analysis* ; Coix ; Aflatoxin B1/analysis* ; Chromatography, Liquid/methods* ; Food Contamination/analysis* ; Tandem Mass Spectrometry/methods*

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.
Aflatoxin B1/analysis* ; China ; Chromatography, Liquid ; Enzyme-Linked Immunosorbent Assay ; Tandem Mass Spectrometry

A highly sensitive monoclonal antibody against aflatoxin B_1(AFB_1) was prepared and an indirect competition enzyme-linked immunosorbent assay(ic-ELISA) was established based on the antibody which was used for high-throughput and rapid screening of AFB_1 contamination in Chinese herbal medicines to ensure the safety of medication. In this study, the structure of AFB_1 was modified by improved oxime method, and the carrier protein was coupled by EDC-NHS method to obtain the complete antigen of AFB_1, which was more convenient and environmental friendly. The Balb/c female mice were immunized using increasing the immunization dose and various ways of injection, and finally the AFB_1 monoclonal antibody was prepared. The AFB_1 monoclonal antibody belongs to IgG_(2 b) immunoglobulin by identifying its immunological characteristics, and its sensitivity(IC_(50)) can reach 0.15 μg·L~(-1), and the affi-nity is 2.81×10~8 L·mol~(-1). The cross-reaction rates of AFB_2, AFG_1, and AFG_2 were 35.07%, 8.75%, and 1.15%, respectively, and there was almost no cross-reactivity with other mycotoxins. Based on the high sensitivity and specificity of the antibody, an ic-ELISA method was established and applied to the determination of AFB_1 contamination in Ziziphi Spinosae Semen. According to the matrix matching standard curve, the linear concentration range for AFB_1 was 0.05-0.58 μg·L~(-1)(R~2=0.992), the recoveries were 88.00%-119.0%, and the detection limit was 1.69 μg·kg~(-1). The AFB_1 in 33 batches of Ziziphi Spinosae Semen samples was determined by ic-ELISA, and the contamination level was 3.62-206.58 μg·kg~(-1). The linear correlation coefficient between the detection results of ic-ELISA and UHPLC-MS/MS was 0.996, and there were no false positive and false negative cases. It indicates that the established ic-ELISA is accurate and reliable, and could provide a simple and effective technique for fast screening of AFB_1 contamination in Ziziphi Spinosae Semen, and also could be considered as the reference for the detection and monitoring of AFB_1 contamination in other Chinese herbal medicines.
Aflatoxin B1 ; analysis ; Animals ; Antibodies, Monoclonal ; Drug Contamination ; Enzyme-Linked Immunosorbent Assay ; Female ; Mice ; Semen ; chemistry ; Tandem Mass Spectrometry

Objective: This study aimed to explore the protective effect of procyanidin B2 (PCB2) on acute liver injury induced by aflatoxin B (AFB ) in rats. Methods: Forty Sprague Dawley rats were randomly divided into control, AFB , AFB + PCB2, and PCB2 groups. The latter two groups were administrated PCB2 intragastrically (30 mg/kg body weight) for 7 d, whereas the control and AFB groups were given the same dose of double distilled water intragastrically. On the sixth day of treatment, the AFB and AFB + PCB2 groups were intraperitoneally injected with AFB (2 mg/kg). The control and PCB2 groups were intraperitoneally administered the same dose of dimethyl sulfoxide (DMSO). On the eighth day, all rats were euthanized: serum and liver tissue were isolated for further examination. Hepatic histological features were assessed by hematoxylin and eosin-stained sections. Weight, organ coefficient (liver, spleen, and kidney), liver function (serum alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total bilirubin, and direct bilirubin), oxidative index (catalase, glutathione, superoxide dismutase, malondialdehyde, and 8-hydroxy-2'-deoxyguanosine), inflammation factor [hepatic interleukin-6 (IL-6) mRNA expression and serum IL-6], and bcl-2/bax ratio were measured. Results: AFB significantly caused hepatic histopathological damage, abnormal liver function, oxidative stress, inflammation, and bcl-2/bax ratio reduction compared with DMSO-treated controls. Our results indicate that PCB2 treatment can partially reverse the adverse liver conditions induced by AFB . Conclusion Our findings indicate that PCB2 exhibits a protective effect on acute liver injury induced by AFB .
Aflatoxin B1 ; toxicity ; Animals ; Biflavonoids ; administration & dosage ; pharmacology ; Catechin ; administration & dosage ; pharmacology ; Chemical and Drug Induced Liver Injury ; drug therapy ; etiology ; Male ; Poisons ; toxicity ; Proanthocyanidins ; administration & dosage ; pharmacology ; Protective Agents ; administration & dosage ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley

The purpose of this study was to investigate the anti-injury effect and protective mechanism of hydrogen-enriched water in a rat model of acute liver injury induced by aflatoxin B (AFB). Healthy male Sprague-Dawley (SD) rats were randomly divided into control group, model group (AFB group) and hydrogen-enriched water treatment group (AFB+H group). The rat model of acute liver injury induced by AFB was established by single intragastric administration of AFB (2.0 mg/kg), and then the rats were treated with hydrogen-enriched water intragastrically. HE staining was used to observe the pathological changes of liver tissue. Blood samples were taken from vena cava to measure serum liver function indexes. Live tissue was sampled to detect malondialdehyde (MDA) and reduced glutathione (GSH) contents. Western blot was used to detect phosphorylation levels of MAPK signaling pathway proteins (ERK, JNK and p38 MAPK). The results showed that, compared with the AFB group, the AFB+H group exhibited increased body weights, alleviated acute liver injury, decreased activities of serum glutamic-pyruvic transaminase and glutamic oxaloacetic transaminase, as well as total bilirubin level in the serum. Meanwhile, hydrogen-enriched water decreased MDA content and increased GSH content in liver tissue. AFB-increased phosphorylation levels of ERK, JNK and p38 MAPK in liver tissue were down-regulated significantly by hydrogen-enriched water treatment. These results suggest that hydrogen-enriched water can alleviate liver injury induced by AFB, and its mechanism may be related to the reduction of oxidative stress and the inhibition of MAPK signal transduction pathway activation.
Aflatoxin B1 ; Animals ; Chemical and Drug Induced Liver Injury ; pathology ; prevention & control ; Deuterium Oxide ; therapeutic use ; Liver ; pathology ; MAP Kinase Signaling System ; Male ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley

This study aimed to analyze the residues of aflatoxin B_1( AFB_1) in Ziziphi Spinosae Semen from different producing areas and to assess the health risk of aflatoxin B_1 residue based on the obtained data. A total of 72 samples of Ziziphi Spinosae Semen from different areas were detected by IAC-HPLC-FLD. Based on the data of AFB_1 pollution,a probabilistic assessment model with Monte Carlo simulation was developed. Then,the risk assessment of AFB_1 exposure by Ziziphi Spinosae Semen intake was carried out by MOE( margin of exposure). The results showed that 32 out of 72 of samples( 44. 4%) were found to be contaminated with AFB_1,and the average and maximum concentration of AFB_1 in samples was 5. 42 μg·kg~(-1) and 55. 09 μg·kg~(-1),respectively. After health risk assessment,the average and 97. 5%( 90% confidence interval) exposure level of daily exposure of AFB_1 by Ziziphi Spinosae Semen intake were 0. 008 6( 0. 008 1-0. 009 2) and 0. 057 3( 0. 053 2-0. 061 4) μg·kg~(-1)·d~(-1),respectively. The results showed common use of Ziziphi Spinosae Semen had low level of risk associated with AFB_1. However,the high consumption of Ziziphi Spinosae Semen showed a higher risk than common intake,requiring attention. This study laid a foundation for clinical safe prescription of Ziziphi Spinosae Semen.
Aflatoxin B1/analysis* ; Chromatography, High Pressure Liquid ; Drug Contamination ; Plant Preparations/analysis* ; Risk Assessment ; Ziziphus/chemistry*

OBJECTIVETo investigate the effect of docosahexaenoic acid (DHA) on invasiveness of aflatoxin B1 (AFB1)-induced hepatocellular carcinoma cells in vitro.

METHODSHepG2.2.15 cells were exposed to different concentrations of AFB1 and DHA plus AFB1. The cell migration and invasion were assessed using wound-healing and Transwell assay, and flow cytometry was used to analyze the cell cycle changes. The ultrastructural changes of the cells were observed by transmission electron microscopy.

RESULTSCompared with the control group, the cells exposed to2 µmol/L AFB1 showed obviously enhanced migration and invasion with decreased cell ratio in G1/G1 phase and increased cell ratio in G2/M phase but no changes in S phase cells; transmission electron microscopy revealed the presence of multiple nucleoli and significantly increased mitochondria and Golgi apparatus in the exposed cells. Compared with AFB1-exposed cells, the cells treated with DHA and AFB1 showed decreased migration and invasion abilities, and the G1/G1 phase cells increased and G2/M phase cells decreased significantly; ultrastructurally, the cells contained single nucleoli with decreased mitochondria and vacuolization occurred in the cytoplasm.

CONCLUSIONDHA can significantly inhibit AFB1-induced enhancement of cell migration and invasion in hepatocellular carcinoma cells in vitro.

Aflatoxin B1 ; pharmacology ; Carcinoma, Hepatocellular ; pathology ; Cell Cycle ; Cell Movement ; drug effects ; Docosahexaenoic Acids ; pharmacology ; Golgi Apparatus ; Hep G2 Cells ; Humans ; Liver Neoplasms ; pathology ; Mitochondria ; Neoplasm Invasiveness

A simple and cost-effective indirect competitive enzyme-linked immune sorbent assay (ic-ELISA) was developed to rapidly screen the content of aflatoxin B1 (AFB1) in lotus seeds, and the results were confirmed by ultra-fast liquid chromatography-tandem mass spectrometry( UFLC-MS/MS). Matrix-matched calibration expressed a good linearity ranging from 0. 171 to 7. 25 µg · L(-1) for AFB, with R2 > 0.978. The medium inhibitory concentration( IC50 ) for AFB1 was 1.29 µg · L(-1), the recovery for AFB1 was 74.73% to 126.9% with RSD < 5%, and the limit of detection (IC10) was 0.128 µg · L(-1). The developed ic-ELSIA method was applied to rapid analysis of AFB, in 20 lotus seeds samples and the results indicated that the contents of AFB, in samples 1-15 were in the range of 1. 19- 115. 3 µg · kg(-1) and in 40% of the samples exceeded the legal limit(5 µg · kg(-1)), while the contamination rate of AFB, in samples 16-20 was 40%. Pearson correlation coefficient(r) reached 0.997 for AFB1 content in the samples detected by ic-ELSIA and UFLC-MS/MS methods. The results proved that the developed ic-ELISA method is simple, sensitive and reliable, and can be used for rapid and high-throughput screening of AFB1 in lotus seeds
Aflatoxin B1 ; analysis ; Drug Contamination ; Enzyme-Linked Immunosorbent Assay ; methods ; Loteae ; chemistry ; Seeds ; chemistry

Fumonisin B1 (FB1) is a carcinogenic mycotoxin found in commodities such as corn and corn-originated products. An aptamer-based method for detection of FB1 was developed using the fluorescent dye PicoGreen, which can recognize and bind double-stranded DNA. A peak fluorescence of PicoGreen was obtained in 15 min in the presence of FB1 aptamer, which formed a double-stranded hybridizer DNA with its complementary strand. The excitation and emission wavelengths for PicoGreen detection were 480 nm and 520 nm, respectively. The sensitivity of this aptamer/PicoGreen-based method was 0.1 μg/L. This method showed a good linearity for FB1 concentration ranging from 0.1 to 1 μg/L. The entire detection procedure for FB1 could be completed within 40 min. No cross reactions were observed with any other mycotoxins against aflatoxin B1, ochratoxin A, citrinin and zearalenone, demonstrating high specificity towards FB1 aptamer. Agreement between commercial, antibody-based enzyme-linked immunosorbent assay (ELISA) kit and aptamer method was excellent with a kappa value of 0.857. Taken together, this aptamer/PicoGreen-based method is more cost-effective, time-saving and useful than ELISA for detection of FB1.
Aflatoxin B1 ; Enzyme-Linked Immunosorbent Assay ; Fluorescence ; Fluorescent Dyes ; chemistry ; Fumonisins ; analysis ; Mycotoxins ; analysis ; Ochratoxins ; Organic Chemicals ; chemistry ; Staining and Labeling ; Zea mays

Some unique subclasses of Camelidae antibodies are devoid of the light chain, and the antigen binding site is comprised exclusively of the variable domain of the heavy chain (VHH). The recombinant VHHs have a high potential as alternative reagents for the next generation of immunoassay. In particular, they might be very useful for molecular mimicry. The present study demonstrated an alpaca immunized with the F(ab')2 fragment of anti-aflatoxin B1 mAb and developed an important anti-idiotypic (anti-Id) responses. Antigen-specific elution method was used for panning private anti-Id VHHs from the constructed alpaca VHH library. The selected VHHs were expressed, renatured, purified, and then identified by a competitive enzyme-linked immunosorbent assay (ELISA). Our findings indicated that the VHH would be an alternative tool for haptens mimicry studies.
Aflatoxin B1 ; immunology ; Amino Acid Sequence ; Animals ; Antibodies, Anti-Idiotypic ; biosynthesis ; chemistry ; isolation & purification ; Camelids, New World ; immunology ; Immunoglobulin Heavy Chains ; chemistry ; isolation & purification ; Molecular Sequence Data