[Preparation of highly sensitive monoclonal antibody against aflatoxin B_1 and its application in rapid detection of contamination in Ziziphi Spinosae Semen].

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.

An integrated strategy for rapid on-site screening and determination of prometryn residues in herbs.

We produced a prometryn-specific monoclonal antibody and propose a strategy for convenient on-site detection of prometryn residues in herbs for the first time. This strategy has perfect applicability in a complex herbal medicine matrix. The strategy combines a semiquantitative immunochromatographic strip assay with a heterologous indirect competitive ELISA. When there was no matrix interference, the ELISA had a half-maximal inhibitory concentration of 2.6 ng·mL-1 and a limit of detection of 0.2 ng·mL-1. The immunochromatographic strip assay can be completed within 5 min with a visual limit of detection of 1 ng·mL-1. Although the sample matrix had different effects on the sensitivity of the antibody, excellent repeatability and accuracy were achieved. The method was successfully applied for the screening and determination of prometryn residue in multiple complex herb samples for the first time, and the results were in good agreement with those obtained by liquid chromatography-tandem mass spectrometry. The proposed strategy is rapid, of high-throughput, and of low cost, and may be a promising choice for on-site detection of prometryn in different kinds of herbs. Graphical abstract.

A comprehensive analysis of 201 pesticides for different herbal species-ready application using gas chromatography-tandem mass spectrometry coupled with QuEChERs.

Developing an analysis of multi-pesticide residues for different herbal species-ready applications is a challenge. In the present work, a comprehensive analysis was proposed for rapid detection of 201 pesticides in various medicinal herbs. Samples were extracted and cleaned up with a high throughput pretreatment approach (modified QuEChERS), and then detected by gas chromatograph coupled to an electron impact ionization triple quadrupole mass spectrometer (GC-EI-MS/MS). The clean-up procedure has been optimized using four types of representative medicinal herbs with different primary or secondary metabolites. Moreover, a mixture of analyte protectants (APs) was to improve the peak shape and intensity of some compounds. The performance of the method was validated according to the European Union SANTE/11813/2017 regulatory guidelines. The limit of quantification (LOQ) was determined to be ≤10 ng mL-1, and the recovery was between 70.0%-120.0%, with ≤20% RSD for the majority of pesticides. Sixty samples belonging to different species of medicinal herbs (such radix, flos, cortex, fructus, and seeds) were analyzed to evaluate the applicability of the optimized method. High frequency of chlorpyrifos was found in Citri reticulatae pericarpium, Crataegi fructus and Cuscutae semen samples.

[QuEchERS pretreatment coupled to gas chromatography and tandem mass spectrometry to fast determination of 34 pesticide residues in Glycyrrhizae Radix et Rhizoma].

This study proposed a quantitative method for 34 pesticides including organochlorine,organophosphorus and pyrethroids in Glycyrrhizae Radix et Rhizoma herbs and medicinal slices,and analyzed the pesticide residues of collected Glycyrrhizae Radix et Rhizoma samples from different regions. With acetonitrile extraction and optimized Qu Ech ERS purification,the 32 batches of Glycyrrhizae Radix et Rhizoma herbs and medicinal slices were analyzed by matrix matching standard curve quantitative analysis under GC-MS/MS multi-response monitoring( MRM) mode. This study investigated the pretreatment of Glycyrrhizae Radix et Rhizoma samples based on the Qu Ech ERS method of Chinese Pharmacopoeia( 2015 edition,4),and the result showed that the recoveries of some pesticide was low and pigment has a strong interference in analysis,which result in worse purification effect. Therefore,this paper further optimized the Qu Ech ERS method and corrected the matrix matching standard curve method,and compensated the qualitative and quantitative effects of matrix effects on the detected target compounds in Glycyrrhizae Radix et Rhizoma. The results showed that 34 kinds of pesticide had good linear( R~2 of 0. 996 4 or higher) within a covering 0. 01-0. 2 mg·kg~(-1) concentration range. The limits of quantitation are less than 0. 01 mg·kg~(-1). This method was further applied to the simultaneous determination of 34 pesticide residues of typical organochlorine,organophosphorus and pyrethroids in 32 batches of Glycyrrhizae Radix et Rhizoma herbs and medicinal slices. Six batches containing beta-endosulfan,thiosulphate,o,p'-DDD and thrta-cypermethrin were detected,but none of them exceeded the limit of pesticide residues stipulated in the Chinese Pharmacopoeia and the EU Pharmacopoeia. This study indicates that the established method is rapid,convenient,accurate,and sensitive,which provides a rapid and efficient method for the simultaneous determination of typical organochlorine,organophosphorus and pyrethroids in Glycyrrhizae Radix et Rhizoma.

6'-Hydroxy Justicidin B Triggers a Critical Imbalance in Ca2+ Homeostasis and Mitochondrion-Dependent Cell Death in Human Leukemia K562 Cells.

Justicia procumbens (J. procumbens) is a traditional Chinese herbal medicine which was used for the treatment of fever, pain, and cancer. A compound 6'-hydroxy justicidin B (HJB) isolated from J. procumbens exhibits promising biological properties. However, the mechanism of action and the in vivo behavior of HJB remain to be elucidated. In this study, we investigated the mechanism of action of HJB on human leukemia K562 cells and its pharmacokinetic properties in rats. The results demonstrated that HJB significantly inhibited the proliferation of K562 cells and promoted apoptosis. Besides, HJB resulted in decreased mitochondrial membrane potential deltaPSIm, increased the level of the calcium homeostasis regulator protein TRPC6 and cytosolic calcium. The activity of caspase-8, caspase-9 and the expression of p53 were significantly increased after treatment with HJB. Additionally, HJB has rapid absorption rate and relative long elimination t1/2, indicating a longer residence time in vivo. The results indicate that HJB inhibited the proliferation of K562 cells and induced apoptosis by affecting the function of mitochondria and calcium homeostasis to activate the p53 signaling pathway. The pharmacokinetic study of HJB suggested it is absorbed well and has moderate metabolism in vivo. These results present HJB as a potential novel alternative to standard human leukemia therapies.