Ochratoxin A (OTA) is ubiquitous in the food and feed fields. It has strong hepatotoxicity and nephrotoxicity, seriously threatening the health of humans and animals. Enzymatic degradation of mycotoxins is considered to be a promising method to control mycotoxin contaminations. In this study, a new ochratoxin A amidohydrolase from Microbulbifer thermotolerans (MiADH) was obtained. After heterologous expression in Escherichia coli and purification, the recombinant protein was studied regarding the hydrolysis activity, hydrolysis products, enzymatic properties, and substrate binding mode. MiADH can degrade OTA into ochratoxin α (OTα) and phenylalanine, demonstrating a detoxifying ability. It demonstrated the best performance at 70 ℃ and pH 8.0, and Cu²⁺ had the strongest inhibitory effect on the activity of MiADH. MiADH with good thermal stability exhibited huge potential for industrial application. Rational design guided by three-dimensional structural models and substrate docking analysis revealed the important amino acids affecting substrate binding and obtained multiple mutants with improved activity. Among these mutants, V324A had the highest activity, which was 4.2-fold that of the wild type. The identification of MiADH enriches the ochratoxin A degradation enzyme library and provides a new candidate enzyme for the biological detoxification of ochratoxin A in the food and feed industry.
Amidohydrolases/chemistry* ; Ochratoxins/metabolism* ; Substrate Specificity ; Escherichia coli/metabolism* ; Recombinant Proteins/metabolism* ; Actinomycetales/genetics*

An indirect competitive enzyme-linked immunosorbent assay( ic-ELISA) was developed for the rapid detection of ochratoxin A( OTA) in nutmeg( Myristicae Semen),ginger( Zingiberis Rhizoma) and turmeric( Curcumae Longae Rhizoma). The matrix matching standard curve was used instead of the standard curve of sample diluent,and the sample extract and sample diluent were optimized. The sensitivity( IC_(50)) of this method for OTA in nutmeg,ginger and turmeric were determined as 0. 146,0. 157 and 0. 153 ng·m L~(-1),respectively and the limits of detection( LODs) were 0. 040,0. 032 and 0. 031 ng·m L~(-1),respectively. The recovery of samples ranged from 75. 99% to 122. 3%,with RSD<10%. Two positive samples for nutmeg and one positive sample for turmeric occurred in 50 samples,and the highest OTA contamination value was 1 167. 8 μg·kg~(-1). The results were further confirmed by LC-MS/MS. It shows that the developed ic-ELISA method is simple,rapid and sensitive,and can be applied for rapid and high-throughput screening of OTA in nutmeg,ginger and turmeric,as well as some other CHMs.
Chromatography, Liquid ; Drug Contamination ; Drugs, Chinese Herbal/analysis* ; Enzyme-Linked Immunosorbent Assay ; High-Throughput Screening Assays ; Ochratoxins/analysis* ; Tandem Mass Spectrometry

A one-step dual flow immunochromatographic assay (DICGA), based on a competitive format, was developed for simultaneous quantification of ochratoxin A (OTA) and zearalenone (ZEN) in corn, wheat, and feed samples. The limit of detection for OTA was 0.32 ng/ml with a detection range of 0.53‒12.16 ng/ml, while for ZEN it was 0.58 ng/ml with a detection range of 1.06‒39.72 ng/ml. The recovery rates in corn, wheat, and feed samples ranged from 77.3% to 106.3% with the coefficient of variation lower than 15%. Naturally contaminated corn, wheat, and feed samples were analyzed using both DICGA and liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the correlation between the two methods was evaluated using a regression analysis. The DICGA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of OTA and ZEN in food safety control.
Animal Feed ; Calibration ; Chromatography, Affinity ; Chromatography, Liquid ; Colloids ; Food Contamination/analysis* ; Food Safety ; Gold ; Immunoassay/methods* ; Inhibitory Concentration 50 ; Limit of Detection ; Metal Nanoparticles ; Ochratoxins/analysis* ; Regression Analysis ; Reproducibility of Results ; Sensitivity and Specificity ; Triticum ; Zea mays ; Zearalenone/analysis*

Ochratoxin A (OTA) is found not only nephrotoxic, teratogenic, neurotoxic, and immunotoxic, but also reprotoxic for human and animals. In the recent decade, more attention has been paid to the impact of OTA on human reproduction and the studies of its underlying mechanisms. Many studies show that OTA affects the function of the reproductive system by acting as an endocrine disrupter and, as a testicular toxin, decreases sperm quality and even induces testis cancer. This review summarizes the toxicological characteristics and toxicokinetic process of OTA as well as recent progress in the studies of various toxic effects of OTA and their underlying mechanisms, hoping to call the attention from more people to the toxicity of OTA to male reproductive health.
Animals ; Endocrine Disruptors ; pharmacokinetics ; toxicity ; Fertility ; drug effects ; Humans ; Male ; Ochratoxins ; pharmacokinetics ; toxicity ; Reproduction ; drug effects ; Spermatozoa ; drug effects ; Testicular Neoplasms ; chemically induced ; Testis ; drug effects

Ochratoxin A (OTA) is a toxic secondary metabolite mainly produced by Aspergillus and Penicillium species, existing in a variety of foodstuffs and Chinese medicines. OTA is difficult to be detected in practice because of the characteristics such as trace amounts, toxicity, existing in complex matrices. In the numerous detection technologies, colloidal gold chromatographic techniques are highly sensitive, specific, cost-effective and user-friendly, and are being used increasingly for OTA screening. Recently, with the development of aptamer technology and its application in chromatographic technique, a newly colloidal gold aptamer chromatographic technique has been developed. This review elaborates the structures and principles of both traditional and newly colloidal gold chromatographic techniques, focuses on newly colloidal gold aptamer chromatographic technique, summarizes and compares their use in rapid detection of OTA. Finally, in order to provide a reference for better research of related work, the development trends of this novel technique are prospected.
Base Sequence ; Chromatography ; methods ; Gold Colloid ; chemistry ; Molecular Sequence Data ; Ochratoxins ; analysis

A new chromene (1) and six known compounds identified as 6-hydroxymellein (2), 6-hydroxy-5-methylmellein (3) nectriapyrone (4), chermesinone A(5), chermesinone B(6), and pomopxanthone A(7), were isolated in our investigation of the cytotoxic constituents from the fermented rice substrate of endophytic fungus Phomopsis sp. HCCB03519. The structures of these com pounds were elucidated through spectroscopic data analysis. All compounds exhibited inhibitory activities against cancer cell lines. Compound 7 showed stronger inhibition against cancer cells than the positive control 5-Fu.
Antineoplastic Agents ; chemistry ; pharmacology ; Ascomycota ; chemistry ; Benzopyrans ; chemistry ; pharmacology ; Cell Line, Tumor ; Fluorouracil ; chemistry ; pharmacology ; Humans ; Isocoumarins ; chemistry ; pharmacology ; Molecular Structure

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

A method was developed for the determination of ochratoxin A (OTA) in human urine by HPLC-FLD after molecularly imprinted polymer solid phase extraction (MIP-SPE) column. After the pH being adjusted to 2.5 with 0.1 mol x L(-1) HC1, sample was cleaned up with MIP-SPE column for ochratoxin A, the analyte was analyzed by high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD), and finally all the positive results were confirmed by LC-MS/MS. Recoveries from urine samples spiked with OTA at levels ranging from 2 to 20 ng x mL(-1) were 90.6%-101.9%, and RSDs were 0.1%-1.6%. Sixty-five volunteers living in Beijing took part in the study, of which 5 were found containing OTA in their urine and the highest value was 0.091 ng x mL(-1). The MIP-SPE column was firstly applied to purify and concentrate OTA in human urine, this method is simple, rapid and reliable and can be used to determine the contents of OTA in human urine.
Chromatography, High Pressure Liquid ; methods ; Female ; Humans ; Male ; Molecular Imprinting ; Ochratoxins ; urine ; Polymers ; Reproducibility of Results ; Sensitivity and Specificity ; Solid Phase Extraction

Two novel isocoumarins, erigeronone C (1) and D (2), were isolated from the ethanol extract of the whole plant of Erigeron breviscapus (Vant.) Hand.-Mazz (Compositae). Their structures were respectively elucidated as 8, 9-dihydroxypyrano [3, 2-c] isochromen-4, 6-dione (1) and 4, 7-dihydroxy-3-(3-hydroxy-4-oxo-4H-pyran-2-yl)-1H-isochromen-1-one (2) on the basis of spectral analyses. Both structures of 1 and 2 possess a gamma-pyrone moiety and that is rare in natural products.
Erigeron ; chemistry ; Isocoumarins ; chemistry ; isolation & purification ; Molecular Structure ; Plants, Medicinal ; chemistry

OBJECTIVETo establish a method for the content determination of 5-methylmellein, 5-hydroxymellein, 5-carboxylmellein and genistein in Wuling capsules simultaneously by HPLC.

METHODFour components were determined by HPLC on a Kromasil KR100-5C18 column (4.6 mm x 250 mm, 5 microm) with acetonitrile and 0. 2% phosphoric acid as mobile phase in a gradient elution. The flow rate was 1.0 mL x m min(-1), and the column temperature was set at 30 degrees C.

RESULT5-hydroxymellein showed a good linear relationship at the range of 7.86-157.2 ng, the average recovery was 101.0% with RSD 1.3%. 5-carboxylmellein showed a good linear relationship at the range of 9.57-191.4 ng, the average recovery was 98.6% with RSD 1.5%. Genistein showed a good linear relationship at the range of 28.80-576.0 ng, the average recovery was 98.6% with RSD 1.8%. 5-methylmellein showed a good linear relationship at the range of 21.46-429.2 ng, the average recovery was 99.2% with RSD 1.8%.

CONCLUSIONThe established method is feasible and the repeatability is good. The method can be used for quality control of Wuling capsules.

Capsules ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Genistein ; analysis ; Ochratoxins ; analysis ; chemistry ; Reproducibility of Results