Studies On Chemical Constituents And Processing Methods For Detoxification Of Emilia Sonchifolia

Emilia sonchifolia(L.)DC is an annual herb of the genus Emilia in the family Asteraceae,which is cool and bitter in nature,and the whole herb is used as a medicine to clear heat and detoxify the body,reduce inflammation and diuresis,dissipate blood stasis and swelling,and is mainly used in the treatment of gynecological inflammation,enteritis,dysentery and other diseases,and is one of the raw materials of proprietary Chinese medicines such as Hua hong tablets,Hua hong granules,and Yang er xing pi granules.However,Emilia sonchifolia(L.)DC contains pyrrolizidine alkaloids(PAs),a class of toxic components widely distributed in flowering plants,can protect the plants themselves from external insects and pathogens,and have hepatotoxic,cytotoxic,genotoxic,pulmonary toxic and carcinogenic effects.In order to ensure the safety of the medicine of Emilia sonchifolia(L.)DC,based on the previous research results of the group,the herbs of Emilia sonchifolia(L.)DC were concocted in order to reduce the amount of toxic PAs in Emilia sonchifolia(L.)DC,and risk assessment of the herbs before and after concoction were performed.In addition,the hepatotoxicity and anti-inflammatory activity were verified by in vitro cellular assay,and the chemical components were identified.The main research findings were as follows:1 Research on the chemical compositions of Emilia sonchifolia(L.)DCIn this study,using various chromatographic separation techniques 18 compounds were isolated and purified from 80%ethanolic extracts of the Emilia sonchifolia(L.)DC whole herb,and their structures were identified based on physicochemical properties and spectral data.As follows:dibutyl phthalate(1),Ursolic acid(2),Ethyl-4-hydroxyphenylacetate(3),p-hydroxyacetophenone(4),aurantiamide acetate(5),p-hydroxystyreneol(6),isovanillic acid(7),p-hydroxyphenyl acetic acid(8),1,3,5-trimethoxybenzene(9),Norharmane(10),adenosine(11),6-hydroxymethyl-3-pyridinol(12),Otosenine(13),desacetyldoronine(14),acetylsenkirkine(15),senkirkine(16),Floridanine(17),Onetine(18),of which,except for compounds 5,8,13,16,all were isolated for the first time from the Emilia sonchifolia(L.)DC plant.2 Research on the processing method of Emilia sonchifolia(L.)DC medicinal materials and preliminary risk assessment.The preliminary research of our group found that the content of PAs in the herbs of Emilia sonchifolia(L.)DC seriously exceeded the standard,especially the content of Senkirkine(SK)was higher,accounting for more than 95%of the total PAs.In this study,the content of SK before and after concoction was used as an indicator to study the preparation of dried whole herbs of Emilia sonchifolia(L.)DC,and by adding different excipients to prepare the herbs,a method that can effectively reduce the content of SK was explored.Determination of SK content in different processed products by UPLC-MS/MS.The results showed that after soaking the herbs in 5%NaHCO3 solution for at least 6 hours,the SK content in the concoctions obtained by drying the herbs at 70℃ could be reduced by more than 97%,and this preparation method was applied to 6 batches of the herbs from different origins,and the results showed that the SK content in all 6 batches of the concoctions of the herbs was reduced by more than 97%.The results of the risk assessment showed that this processing method can effectively reduce the risk level of Emilia sonchifolia(L.)DC,reduce the toxicity of the herbs,and provide ideas and methods for the safe and rational clinical use of Emilia sonchifolia(L.)DC.3 In vitro evaluation of the toxicity of Emilia sonchifolia(L.)DC before and after preparation on human normal liver LO2 cellsAfter entering the body,PAs are mainly metabolized in the liver by hepatic cytochrome P450 enzymes to produce metabolic pyrrole adducts leading to hepatotoxicity,genotoxicity and even tumor.In this study,human normal hepatic LO2 cells rich in P450 enzymes were selected as a model for in vitro toxicity assessment,and the extracts of the herbs from the herb before and after concoction were applied to LO2 cells for 24 h,48 h and 72 h.The results showed that the cell survival rate of the herbs from the herb after concoction was significantly higher than that of the herbs before concoction,and the cell survival rate of the pre-concoction group showed a decreasing trend with the prolongation of drug action,while the cell survival rate of the concoction group remained in a certain stable state.This indicates that the NaHCO3 concoction of Emilia sonchifolia(L.)DC has a toxicity-reducing effect.4 In vitro evaluation of the anti-inflammatory activity of RAW264.7 cells induced by lipopolysaccharide(LPS)before and after preparation of Emilia sonchifolia(L.)DCThe in vitro anti-inflammatory activity screening of the herbal extracts of Emilia sonchifolia(L.)DC before and after processing showed that the alcoholic extract of Emilia sonchifolia(L.)DC after concoction showed different degrees of inhibition of LPSinduced NO production in RAW264.7 cells,indicating that Emilia sonchifolia(L.)DC after concoction with baking soda still had some anti-inflammatory activity.It was verified that this processing method can effectively reduce the risk of hepatotoxicity caused by PAs in Emilia sonchifolia(L.)DC herbal while retaining certain anti-inflammatory activity,providing a feasible method for the safe and rational clinical use of Emilia sonchifolia(L.)DC medicinal materials.5 ConclusionIn this study,four PAs-like compounds,which have never been isolated from the plant,and two known PAs-like alkaloids were obtained from the study of the chemical composition of Emilia sonchifolia(L.)DC,and the presence of these alkaloids confirmed the hepatotoxicity of the bitter red herb.The hepatotoxicity and anti-inflammatory activity of the herb were studied in vitro and the results showed that the PAs content in the herb was significantly reduced after the herb was concocted with 5%NaHCO3,which greatly reduced the risk of hepatotoxicity and retained the anti-inflammatory activity of the original herb.6 A literature review of relevant aspects of processing methods for toxic Chinese medicinal herbs was conducted,providing comprehensive references for the further research.