Analysis And Detection Of Pyrrolizidine Alkaloids In Perrin And In Vitro Neurotoxicity Studies

Objectives:Pyrrolizidine alkaloids(PAs)are a class of secondary metabolites produced by flowering plants.PAs are the most widely distributed type of natural toxins.At present,about 3%of flowering plants(about 6000 species)in the world contain PAs.Most PAs have hepatotoxicity,nephrotoxicity,tumorigenicity and mutagenicity.PAs can be transformed into humans and animals through the ingestion of Chinese herbal medicines containing PAs.Research found that the commonly used medicinal plant Eupatorium fortunei Turcz.and its closely related species may contain PAs.However,the types and contents of PAs in E.fortunei have not yet been deeply explored.Therefore,this study intends to establish an efficient,fast and sensitive detection method based on the combined method of liquid chromatography and mass spectrometry with E.fortunei as the representative medicinal material.Characterize the composition of PAs in E.fortunei and its related preparations from different sources,and explore the differences in the distribution of PAs in E.fortunei from different sources.Then carry out risk assessment and safety early warning of E.fortunei and its proprietary Chinese medicine preparations.At present,there are few studies on the neurotoxicity of PAs,so this article will also evaluate the potential in vitro neurotoxicity of PAs and E.fortunei extracts through cell experiments.In addition,this article will also conduct preliminary research on the production processes that may affect the transfer of PAs.Methods and results:1.Establishment of LC-MS/MS detection method for pyrrolizidine alkaloidsIn this study,a combination of liquid chromatography and mass spectrometry was used.A total of 34 PAs reference substances were purchased,and a detection method suitable for PAs in the traditional Chinese medicine E.fortunei and related preparations was established.31 PAs were well separated within 30 minutes.The established method was investigated for linearity,limit of detection,limit of quantification,repeatability,precision and recovery rate,and all methodological investigation results met the requirements.Therefore,this chapter has successfully established an efficient,sensitive,fast and suitable LC-MS/MS detection method for E.fortunei medicinal materials and its preparations.2.Detection and analysis of pyrrolizidine alkaloids in E.fortunei and its preparationsThis chapter collected 30 batches of E.fortunei samples from 12 different provinces in China and 4 traditional chinese medicine preparation containing E.fortunei After all samples are processed,the established LC-MS/MS method is used for detection and analysis.The results showed that PAs were detected in all samples.A total of 8 PAs were detected,among which the most content are Intermedine,Intermedine N-oxide,Lycopsamine and Lycopsamine N-oxide.The total content of PAs in 30 batches of E.fortunei samples ranged from 0.1773 to 61.8068μg/g.The total content of PAs in the 4 kinds of pharmaceutical preparations is between 0.86 and 36.96 μg/g.After risk assessment,it is believed that taking E.fortunei and its related preparations within a short period of time will not cause health risks.However,if long-term use of such drugs may pose a great threat to human health,the use of such drugs still needs to be vigilant.3.In vitro neurotoxicity evaluation of pyrrolizidine alkaloidsNeural progenitor cells(NPCs)were selected as cell models to evaluate the neurotoxicity of 9 PAs in vitro.The MTT method and immunofluorescence staining method were used to explore the proliferation and differentiation toxicity of PAs on NPCs.The results showed that Intermedine(Im)and Intermedine N-oxide(ImNO)showed a significant inhibitory effect on the proliferation of NPCs at a concentration of 30 μmol/L.Lycopsamine N-oxide(LyNO),Senkirkine(Sk)and Senecivernine N-oxide(SvNO)at the concentration of 10～30 μmol/L showed an effect on the proliferation of NPCs significant inhibition.Intermedine N-oxide(ImNO)and Europine(Eu)have a significant inhibitory effect on the differentiation of NPCs into OPCs at a concentration of 10 μmol/L.In addition,this study found that Eupatorium fortunei Turcz.extract also has a certain inhibitory effect on the proliferation and differentiation of NPCs.However,considering the bioavailability and transformation of PAs after entering the body,whether the ingestion of PAs will cause toxicity to the nervous system needs further research.4.The effect of different production processes on pyrrolizidine alkaloidsThis chapter compares the effects of four different extraction processes:methanol extraction,water decoction,water extraction and alcohol precipitation,and steam distillation on the content of PAs in E.fortunei extract.It was found that the steam distillation method can remove most of the PAs.In addition,this chapter also explores the influence of changes in different process parameters(extraction temperature and extraction time)on the content of PAs.The results show that PAs are relatively stable in the extraction temperature range of 60～100℃.The content of PAs in the extract of E.fortunei decoction pieces did not change significantly within 60 min.PAs is difficult to decompose or transform within a certain range of conditions.Conclusions:This subject established an efficient,fast and sensitive LC-MS/MS detection method containing 31 PAs.Through testing of E.fortunei samples and related pharmaceutical preparations,it was found that all samples contained PAs,and the content of PAs detected in most samples was relatively high.In addition,through the in vitro neurotoxicity study of NPCs on PAs,it is found that some PAs have a certain inhibitory effect on the proliferation and differentiation of NPCs.E.fortunei extract containing PAs is also neurotoxic in vitro.At the same time,this study also found that PAs is relatively stable,and it is difficult to decompose and transform under certain temperature and time changes.By comparing different extraction processes,it is found that the steam distillation method can greatly reduce the content of PAs in the extract.This study is helpful to understand the distribution status of PAs in E.fortunei and related preparations.It is possible to formulate relevant limit standards.At the same time,it can also provide reference for the clinical safe use and risk assessment of E.fortunei and related preparations.It provides an experimental basis for reducing the content of pyrrolizidine alkaloids in traditional Chinese medicines and their preparations,and improving the safety of medicines.