Based On The Caenorhabditis Elegans Model, The Toxic Effects And Mechanisms Of Different Processed Products Of Peganum Harmala Were Discussed

Objective:To establish a high performance liquid chromatography(HPLC)method for the simultaneous determination of harmaline and hamine in ethanol extracts from different processed products of seeds of Peganum harmala L.,and to analyze the changes of chemical constituents before and after processing by UPLC-LTQ-Orbitrap-MS.The changes of chemical composition of different processed products were expounded,which provided reference for the study of processing mechanism and scientific basis for perfecting the quality standard of seeds of Peganum harmala L..The toxic effects of different processed products of seeds of Peganum harmala L.were evaluated by using Caenorhabditis elegans(C.elegans)as a model.The chemical composition difference of different processed products and the toxicity to C.elegans were compared,and the correlation between the chemical composition difference of different processed products and the difference of efficacy toxicity was analyzed,and the toxicity mechanism of seeds of Peganum harmala L.was discussed.It provides a scientific basis for the interpretation of the mechanism of the concoction and detoxification of seeds of Peganum harmala L..Using C.elegans as the model organism,the toxicity evaluation model of seeds of Peganum harmala L.was established,which laid a foundation for the application of C.elegans in the toxicity evaluation of traditional Chinese medicine.Methods:1.Analysis of the content of harmaline and hamine in the ethanol extracts of different processed products of seeds of Peganum harmala L.The ethanol extracts from different processed products of seeds of Peganum harmala L.were prepared,and the content of harmaline and hamine was determined by high performance liquid chromatography.2.Rapid identification of chemical constituents in different processed products of seeds of Peganum harmala L.by UPLC-LTQ-Orbitrap-MSOn-line separation and analysis of chemical constituents in different processed products of seeds of Peganum harmala L.by ultra performance liquid chromatography-linear ion trap/electrostatic orbitrap combined high resolution mass spectrometry (UPLC-LTQ-Orbitrap-MS)technique,based on high resolution.The quasi-molecular ion provided by the mass spectrometer and the accurate mass-to-charge ratio of the characteristic fragment ion,combined with the reference substance and related literature data,identify the chemical constituents in different processed products of seeds of Peganum harmala L..3.DPPH method for evaluating the in vitro antioxidant activity of different processed products of seeds of Peganum harmala L.The antioxidant activity of different processed products of seeds of Peganum harmala L.was evaluated by 1,1-diphenyl-2-trinitrophenylhydrazine(DPPH)free radical scavenging capacity assay.The anti-oxidation ability of different processed products was compared by scavenging DPPH radical semi-inhibitory concentration(IC50).4.Toxicity evaluation of different processed products of seeds of Peganum harmala L.on C.elegansAfter exposed to ethanol extracts of different mass concentrations of processed products,C.elegans’lethality,maximum lifespan and median lethal time,individual development,spawning number and locomotion behavior exposure of were observed by stereoscopic microscope.5.Evaluation of the efficacy of different processed products of seeds of Peganum harmala L.on C.elegansThe lifespan of C.elegans exposed to ethanol extracts of different concentrations of processed products was counted under heat stress and oxidative stress conditions.6.Study on the mechanism of the toxic effect of different processed products of seeds of Peganum harmala L.on C.elegansAfter exposure to ethanol extracts from different concentrations of processed,the changes of acetylcholinesterase activity in C.elegans were analyzed by ultraviolet spectrophotometer,Acridine orange staining was used to observe the apoptosis of C.elegans cells under fluorescence microscope.Real-time fluorescence quantitative PCR(qRT-PCR)was used to study the expression difference of genes related to C.elegans.daf-2,tub-1(lifespan-related genes)and ctl-2,sod-3(oxidative stress-related genes)were compared.The difference of expression of hsp-16.1,hsp-16.2(heat shock protein-related gene)and cyp-35A1(P450 family-related gene)between the drug treated group and the control group.Results:1.Analysis of the content of harmaline and hamine in the ethanol extracts of different processed products of seeds of Peganum harmala L.A method for determination of the content of harmaline and hamine by high performance liquid chromatography in different extracts of seeds of Peganum harmala L.was established.The method has high precision,good stability,good repeatability and sample recovery.Meet the requirements of relevant standards.The content of harmaline in the ethanol extracts of seeds of Peganum harmala L.,raw products,plain fried products,wine products,vinegar products was 96.76,88.47,89.81,91.61 mg·g-1,respectively,and the content of harmine was 98.05.93.01,94.84,93.09 mg·g-1.After processing,compared with the raw products,the content of harmaline and hamine in the ethanol extracts of the fried products,wine products and vinegar products decreased.2.Rapid identification of chemical constituents in different processed products of seeds of Peganum harmala L.by UPLC-LTQ-Orbitrap-MSThrough the analysis of mass spectrometry data of different processed products in positive ion mode,20 compounds were inferred and identified in raw products and 21 compounds were inferred and identified in plain fried products,a total of 21 compounds were inferred and identified from the wine products and 21 compounds were identified from the vinegar products.3.DPPH method for evaluating the in vitro antioxidant activity of different processed products of seeds of Peganum harmala L.The antioxidant activity of different processed products of seeds of Peganum harmala L.:Vc>plain fried products>wine products>raw products>vinegar products.4.Toxicity evaluation of different processed products of seeds of Peganum harmala L.on C.elegansCompared with the blank group,the lethality rate of C.elegans increased significantly,the median lethal time and the maximum lifespan,the individual development,the spawning number were significantly decreased in the groups of ethanol extract of different processed products and different concentrations of ethanol extract of seeds of Peganum harmala L.,and the death rate of C.elegans was significantly increased.The head swing frequency,body bending frequency,and Omega/U swing times are significantly reduced.5.Evaluation of the efficacy of different processed products of seeds of Peganum harmala L.on C.elegansDifferent processed products of seeds of Peganum harmala L.can significantly improve the heat resistance and antioxidant capacity of C.elegans.6.Effects of different processed products of seeds of Peganum harmala L.on gene expression of C.elegansCompared with the blank group,the AChE activity of C.elegans was significantly decreased and the degree of cell apoptosis was increased in different ethanol extracts from processed products of seeds of Peganum harmala L..The expression of cyp-35A,ctl-2,daf-2,hsp-16.l,hsp-16.2gene was significantly increased and the expression of sod-3,tub-1 gene was significantly decreased after exposure to seeds of Peganum harmala L..Conclusion:In this experiment,the method for determination of the content of harmaline and hamine in the ethanol extracts of different processed products of seeds of Peganum harmala L.was established.UPLC-LTQ-Orbitrap-MS technique was used to quickly infer the identification of different processed products of seeds of Peganum harmala L..The chemical composition of the product provides scientific data for clarifying the pharmacodynamic basis of the seeds of Peganum harmala L..A model for evaluating the toxicity and pharmacodynamics of different processed products of seeds of Peganum harmala L.was established.The toxic effect of different processed products of seeds of Peganum harmala L.on C.elegans was reduced,indicating that the processing was indeed attenuated.This study demonstrates that C.elegans is suitable for the evaluation of the toxic effect of seeds of Peganum harmala L.,and provides a suitable research method and theoretical basis for further understanding and evaluation of the toxic components of seeds of Peganum harmala L.and the mechanism of processing attenuated,and provides new ideas and methods for the biological evaluation of the traditional Chinese medicine.