| The rational use of pesticides is one of the critical measures to decrease the occurrence of pests and diseases and improve yields in traditional Chinese medicine(TCM)cultivation.However,the registration and the residue limit standard of pesticides used in TCM cultivation,and the detection method of pesticides are not perfect yet,which leads to the abuse of pesticides,and seriously affects the medication safety of TCM.Plant growth regulator pesticides(PGRs)play an important role in promoting the growth and yield of TCM.At present,PGRs lack effective monitoring methods and safety evaluation studies because their toxicity and pathogenicity are lower compared with insecticides,fungicides and herbicides,and their residues have become a potential risk factor affecting the safety of TCM.In order to ensure the quality and medication safety of TCM,a non-directional screening and a quantitative analysis method of pesticide residues in ginseng and licorice was established based on a thorough examination of the types of pesticides that may be used in the current ginseng and licorice cultivation in this study,and the pesticide residues in ginseng and licorice samples were analyzed.Then,aiming at PGRs residues in ginseng and licorice,the toxicity and mechanism of the related pesticides were studied.The main research contents are as follows.1.Study on the non-directional screening method of pesticide multi-residues in ginseng and licoriceIn order to achieve the rapid and comprehensive screening of pesticide multi-residues in ginseng and licorice,a mass spectrometric database of 103 pesticides was established by using ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry(UPLC-QTOF-MSE)technique combined with UNIFI data processing platform in this study firstly.And the fragmentation pathways and rules of carbamate,triazole,organophosphorus and amide pesticides under the mass spectrum were analyzed and summarized,which provided a theoretical basis for qualitative analysis of pesticide compounds.Then,a non-directional screening method for pesticide residues in ginseng and licorice was developed using the Qu ECh ERS pre-treatment method combined with the UPLC-QTOF-MSE technique based on the database information,and the method was validated according to the SANTE/12682/2019 guidelines.Method validation results showed that the screening detection limits(SDL)values of 94 pesticides in ginseng and 72 pesticides in licorice were lower or equal to the maximum residue limits(MRLs)values in domestic and abroad,and could meet the requirements of pesticide residue screening in ginseng and licorice.Using the established method,residues of difenoconazole,dimethomorph,azoxystrobin,tebuconazole,gibberellic acid,and the isomers of gibberellic acid(iso-gibberellic acid)of which was not included in the mass spectrometry database were detected in ginseng,and residues of paclobutrazol were detected in licorice.This method can complete the screening of 94 pesticides in ginseng and 72 pesticides in licorice easily and quickly in the case of no reference substance,which expands the detection range of pesticide residues in ginseng and licorice,and provides a rapid,comprehensive,and effective method for the screening of pesticide residues in ginseng and licorice.2.Study on the quantitative method of pesticide multi-residues in ginseng and licoriceIn order to achieve the quantitative analysis of pesticide multi-residues in ginseng and licorice,a method for the determination of pesticide residues in ginseng and licorice was developed by using a modified Qu ECh ERS method combined with ultra-high performance liquid chromatography-triple quadrupole mass spectrometry(UPLC-QQQ-MS)in this study.Firstly,the conditions of the extraction solvent,extraction salt and adsorbent types and amounts in the Qu ECh ERS method were optimized,and the optimal pretreatment conditions were determined,with the extraction agent being 1% acetonitrile acetate,the extraction salt being 4 g anhydrous magnesium sulfate and 1 g sodium acetate,and the adsorbent being magnetic multi-walled carbon nanotubes(Fe3O4-MWCNTs)prepared by the coprecipitation method.And then,the method was validated according to the SANTE/12682/2019 guidelines.The results showed that the method can meet the quantitative analysis of 93 pesticide residues in ginseng and 88 pesticide residues in licorice,and the method has good precision and recovery,wide linear range,and the limits of quantification can meet the requirements of pesticide residue limit in domestic and abroad.Different batches of ginseng and licorice samples were tested using the established method,and as a result,8 pesticide residues were detected in ginseng and 2 pesticide residues in licorice.This method contains most of the pesticide compounds in non-directional screening,which can further quantify the pesticide residues detected by non-directional screening.Additionally,the method provides a basis for the improvement of pesticide residue monitoring and limit standard systems in ginseng and licorice.3.Study on the hydrolysis characteristics of gibberellic acidGibberellic acid is a kind of PGR,and ginseng seeds can be soaked in gibberellic acid solution to improve germination rates.The results of non-directional screening showed that iso-gibberellic acid residues were detected in ginseng,and it was speculated that iso-gibberellic acid may be the hydrolysate of gibberellic acid in the process of the ginseng seeds soaking based on the application methods of gibberellic acid.In order to clarify the source of isogibberellic acid residues in ginseng,this study investigated the hydrolysis characteristics of gibberellic acid in different temperatures(25℃,37℃ and 50℃)and p H conditions(p H 4.14,7.02 and 9.20)with reference to the standards of "Test Guidelines on Environmental Safety Evaluation of Chemical Pesticides".Firstly,UPLC-QTOF-MSE was used to predict the structure of the hydrolysate of gibberellic acid under different conditions.Then,the quantitative analysis method of gibberellic acid hydrolysate was established by using the putative multiple reaction monitoring strategy(PMRM),which is based on the UPLC-QTOF-MSE and UPLCQQQ-MS technology.Finally,the dynamic changes under different conditions and the residues in ginseng of gibberellic acid and its hydrolysates were investigated using the established method.The results showed that gibberellic acid produced six hydrolysates under different hydrolysis conditions,including iso-gibberellic acid,M367,M237,M283-1,M283-2,and M283-3.Among them,iso-gibberellic acid was the main hydrolysate in the p H 7.02 buffer,with maximum relative contents of 31.8%(25°C),42.0%(37°C),and 42.6%(50°C)at the three temperature conditions,respectively.It was found that the ginseng seeds soaking environment met the reaction conditions for the conversion of gibberellic acid to iso-gibberellic acid based on the application methods of gibberellic acid,which confirmed that iso-gibberellic acid was the hydrolysate of gibberellic acid in the process of seed soaking of ginseng.The results of the study provide an important reference for the rational use and regulation of gibberellic acid.4.Study on the toxicity mechanisms of gibberellic acid and its hydrolysate iso-gibberellic acidIn order to evaluate the residual risk of gibberellic acid and iso-gibberellic acid,this study examined the toxic mechanism of gibberellic acid and iso-gibberellic acid through in vivo experiments,network toxicology and in vitro experiments,and compared the magnitude of their toxicity.Firstly,it was determined that the main toxic target organ of gibberellic acid was the heart by the measurement of organ coefficients and the observation of observing the histopathologic morphology of rats.Then,the potential mechanism of cardiac injury caused by gibberellic acid and iso-gibberellic acid was predicted by network toxicology,and the results showed that "cell death in response to oxidative stress" may be the key pathway for cardiotoxicity caused by gibberellic acid and iso-gibberellic acid.Finally,based on the results of network toxicology analysis,the effects of gibberellic acid and iso-gibberellic acid on cellular activity and oxidative stress indicators(ROS,CAT,and SOD)were examined and evaluated using rat cardiomyocytes H9C2 as a cellular model to verify the predicted results of network toxicology and to compare the magnitude of their toxic effects.The assay results showed that the cell activity decreased both with the gibberellic acid and iso-gibberellic acid exposure dose increase,and iso-gibberellic acid had a higher inhibition rate on cell activity.The cardiac toxicity of gibberellic acid and iso-gibberellic acid may be related to oxidative damage,and iso-gibberellic acid had more obvious oxidative damage to cells.This study not only provides a basis for in-depth study of the toxic effects of gibberellic acid and isogibberellic acid,but also provides a reference for the scientific and rational use and safety evaluation of gibberellic acid.5.Study on the toxicity mechanisms of paclobutrazolPaclobutrazol is a widely used PGR with a high detection rate in licorice.Many studies have shown that paclobutrazol has potentially toxic effects on aquatic organisms,but there are few reports on toxicity studies on mammals.In this study,the hepatorenal toxic effects and mechanisms of subacute oral exposure to different doses of paclobutrazol on rats were investigated.Firstly,the hepatorenal toxicity of paclobutrazol was evaluated by histopathological observation and determination of biochemical indexes.Then,the critical targets,metabolites,and metabolic pathways of hepatorenal toxicity induced by paclobutrazol were screened by an integrated strategy combining metabolomics with network toxicology,combined with molecular docking to elucidate the toxicity mechanism of paclobutrazol.The results indicated that paclobutrazol showed obvious toxicity towards the liver and kidney of rats.The metabolomics and network toxicology analysis showed that CYP1A2,CYP2A6,CYP2E1,MAOA,PLA2G2 A,PTGS1 and XDH were critical targets for paclobutrazol hepatorenal toxicity.After the associated analysis of the above targets and related metabolites,it was found that the paclobutrazol hepatorenal toxicity may be related to the pathway disorders of linoleic acid metabolism,arachidonic acid metabolism,tryptophan metabolism,phenylalanine metabolism and purine metabolism.This preliminary study revealed paclobutrazol hepatorenal toxicity and provided a theoretical basis for the rational and safe use of paclobutrazol.In conclusion,this study provides a screening and a quantitative method for pesticide residues in ginseng and licorice by using liquid chromatography-mass spectrometry as the main analysis technology,and the detection of pesticide residues in different batches of ginseng and licorice samples was completed.Based on the detection results of pesticide residues in ginseng and licorice,a preliminary study was conducted on the toxicity and mechanism of gibberellic acid,iso-gibberellic acid and paclobutrazol.The research results not only provide a scientific basis for the formulation and revision of pesticide residue limit standards in ginseng and licorice but also provide data reference and technical support for the quality and safety of ginseng and licorice. |
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