Study On Material Basis And Mechanism Of Reducing Hepatotoxicity Of Euodiae Fructus By Processing With Licorice

ObjectsTo expound the material basis of detoxification by processing with licorice,the components of three extracts from crude Euodiae Fructus（CEF）and Euodiae Fructus processed by Licorice（LPEF）were studied,and the acute hepatotoxicity of the three extracts before and after processing was compared in combination with multi-component pharmacokinetics and metabolomics research.To verify the scientific connotation of detoxification by processing with licorice,the hepatotoxicity and metabolite profile of different extracts were analyzed before and after proceding.To clarify the detoxification mechamism of EF（euodiae frucuts）by integrating toxicological,pharmacokinetic and metabonomics data.Methods1.CCD-RSM（central composite design and response surface methodology）method was used to optimize the processing technology of Euodiae Fructus.The influencing factors were investigated such as frying temperature,frying time and the proportion of CEF vs licorice juice,and Liquiritin limonin,Evodiamine and Rutaecarpine were picked up as evaluation indexes.2.To systematically explore the changes of chemical components in EF before and after processing,three different extracts:ethanol extract（EE）,water extract（WE）and volatile oil（VO）were studied.High performance liquid chromatography（HPLC）,high performance liquid chromatography-quadrubar time of flight mass spectrometry（HPLC-QTOF-MS）and gas chromatography-mass spectrometry（GC-MS）were used to analyze the three extracts of CEF and LPEF to establish its fingerprints.Thus,the change rule of components before and after processing could be traced.3.Multiple approaches and indexes were used to evaluate the hepatotoxicity of the drugs in the rats with stomach excess-cold syndrome,including general behaviors,biochemical analysis(SOD,MDA,Na⁺,K⁺-ATPase,Ca²⁺-Mg²⁺-ATPase,ALT,AST,TNF-α,IL-1βand IL-6),protein expressions（Bax,Bcl-2 and Caspase 3 proteins）and histopathological examination.The hepatotoxicity of 3 doses of three extracts of CEF and LPEF were systematically investigated,and the differences in hepatotoxicity were analyzed and compared comprehensively between the different extracts,three doses,CEF and LPEF.4.A rapid and efficient UPLC-MS-MS method was established for the simultaneous detection of the four active components（Evodiamine,Rutaecarpine,Evocarpineand Dihydroevocarpine）in rat plasma.This method was also applied to the pharmacokinetic studies after rat oral administration of CEF and LPEF.DAS 3.1 pharmacokinetic analysis software was used to fit the pharmacokinetic parameters of each compound,and the parameters were compared before and after processing to find the material basis of detoxification of Licorice juice processing in vivo.5.Based on the UPLC-LTQ-Orbitrap MS technology,the differences of metabolic profiles in plasma,liver,feces and urine of rats withstomach excess-cold syndrome were analyzed to identify potential biomarkers and related enrichment pathways of hepatic toxicity of EF,and to explore the overall mechanism of detoxification of LPEF through metabolic pathway analysis.Results1.The preparation method of licorice juice was determined as follows:licorice herbs were cut into segments of 1-2cm and soaked,then heated and reflow extraction for three times,concentrated filtrate into extract,and diluted with water into licorice juice with a content of0.06g·m L^-1 before use.The optimal processing parameters were determined as follows:the dosage ratio of CEF to Licorice juice was 1:1（mass/volume ratio）,the soaking time was 6 hours,the frying temperature was 120℃±10℃,and the frying time was 20min.2.The main medicinal ingredients of licorice:glycyrrhizin,glycyrrhizic acid and glycyrrhetinic acid were added into LPEF after processing;The content of organic acid decreased by more than 50%after processing.After processing,the total volatile oil,α-pinene and linalool contents decreased by 42.8%,23.6%and 41.1%respectively.Hence,the decrease of total volatile oil,α-pinene and linalol contents,organic acid content,and the increase of glycyrrhiza in LPEF were the material basis for reducing hepatotoxicity of CEF.3.The stomach excess-cold syndrome animal modelwas successfully established by intragastric administration of cold Zhimu（Anemarrhena asphodeloides Bunge.）,the model could well simulate the gastric excessed cold syndrome in human body,it was suitable for the study of hepatotoxicity of CEF and LPEF.According to the analysis of general behaviors and biochemical indexes,WB analysis of apoptotic proteins expression and histopathological examination,it was found that all the three extracts in the high-dose group caused significant liver damage,the order of hepatotoxicity of these extracts were as follows from big to small:WE,EE and VO.The liver injury of all extracts was significantly alleviated after processing,and there was no significant difference in liver injury between VO group of LPEF and the control group,so processed by licorice could significantly reduce the liver injury caused by CEF.The mechanism of attenuation might be that licorice could reduce the peroxidation stress damage to liver tissue,regulate liver energy metabolism,reduce inflammatory release related indexes and regulate the expression of bcl-2,Bax and Caspase3 proteins.On the other hand,the decrease of hepatotoxicity after processing may be related to the antagonism of licorice.4.A fast and sensitive UPLC-MS-MS method was developed,which could simultaneously quantify four active components（Evodiamine,Rutaecarpine,Evocarpineand Dihydroevocarpine）in rat plasma.This method has the characteristics of faster and more accurate.Four alkaloids could be detected simultaneously within 15 min,methodological indexes such as recovery,precision and stability etc.meet the requirements of verification guidelines for biological analysis method of Chinese Pharmacopoeia 2020 edition,which could be used for the pharmacokinetic study of alkaloids in CEF and LPEF.By comparing the pharmacokinetic parameters in plasma of rats,it was found that the area under the drug time curve and half-life of Evodiamine,Rutaecarpine,Evocarpine and Dihydroevocarpinewere significantly increased after processing.The T_max of Evodiamine,Rutaecarpine and Evocarpine decreased significantly(the T_maxof Dihydroevocarpine did not change),indicating that processing enhanced the pharmacodynamic effects of the four components.5.The metabolite profiles in plasma,liver,feces and urine of rats with stomach excess-cold syndrome model were compared and analyzed.The results showed that:（1）CEF could cause the disorder of metabolites in rats,and 10 metabolites in plasma,15 metabolites in liver,9metabolites in urine and 14 metabolites in feces were screened out.（2）CEF can cause the disorder of metabolic pathways such as alanine,aspartic acid and glutamic acid metabolism,glutathione metabolism,arginine synthesis,glycerol phospholipid metabolism and primary bile acid biosynthesis.This is the mechanism of acute liver injury caused by Evodia officinalis.（3）High dose of LPEF could also cause liver damage but the damage was much slighter than CEF.The mechanism of LPEF to protect liver was mainly regulating alanine,aspartic acid and glutamic acid metabolism,glutathione metabolism,arginine synthesis and glycerophospholipid metabolism,but it had no obvious effect on the disturbance of primary bile acid metabolic pathway.6.CEF will not cause liver damage if it is used for a short time in accordance with the recommended dose of Chinese pharmacopoeia.The liver injury mechanism of high dose of CEF was related to peroxide injury,inflammatory response factor and mitochondrial injury.Some toxic compounds contained in CEF could produce drug-protein adduct,leading to immune liver injury.High dose of LPEF can also cause liver damage to a certain extent,but the damage degree is much lower.The total amount of VO and contents inα-pinene and linalool in the volatile oil,organic acids decreased significantly in combined with the synergistic detoxification of glycyrrhizin and glycyrrhizic acid,which constituted the main material basis for the detoxification of LPEF.The addition of active ingredients in licorice plays an antagonistic role in improving the body’s adaptability to external stimuli and protecting the body,playing a"slow release"role so as to jointly achieve the effect of liver protection and detoxification.Processing could regulate the metabolism of glycerol phospholipid and amino acids,but it had no obvious effect on the primary bile acid generation induced by CEF.ConclusionProcessing with licorice could significantly reduce the liver injury of EF.The detoxification of EF was mainly related to the following facts:the content of volatile oil and its hepatotoxic componentsα-pinene and linalool,organic acids and other hepatotoxic compounds decreased greatly after processing;synergistic effect of licorice components;changes in pharmacokinetic characteristics of toxic substances;and licorice may reduce liver toxicity by regulating the metabolism of glutathione,arginine biosynthesis,alanine,aspartic acid and glutamic acid,glycerol phospholipid.