Study On The Relationship Of "Dose-Time-Toxicity" Of Evodia Rutaecarpa And Material Basis Of Liver Injury Caused By Evodia Rutaecarpa

In this study,the"dose-time-toxicity" relationship of liver injury caused by Evodia rutaecarpa was studied.In the basic research of hepatotoxic substances,the basis of hepatotoxic substances of Evodia rutaecarpa was explored by consulting a large number of literatures and combining the basis of hepatotoxic substances locked by prediction software,carrying out content determination,cytotoxicity research,verification of liver injury in experimental animals and toxicokinetics research accompanied by toxicity,In order to provide some basis and reference for the quality standard of Evodia rutaecarpa contained in part I of Chinese Pharmacopoeia.The research contents are as follows:(1)Study on"dose time toxicity" relationship of liver injury caused by Evodia rutaecarpaIn this study,the acute toxicity test of Evodia rutaecarpa aqueous extract showed that the median lethal dose(LD50)of mice was 64.227 g/kg(the following were calculated by crude drug dose),and the maximum tolerance dose(MTD)of rats was greater than 88.45 g/kg;The results of single dose acute liver injury test showed that 40,60 and 80 g/kg could cause liver injury in mice;Repeated administration of liver injury test found that mice in the 40 g/kg dose group showed significant liver injury after continuous administration for 7 days,and mice in the 20 and 40 g/kg dose groups showed significant liver injury after continuous administration for 14 days,while rats did not show significant liver toxicity after giving the equivalent dose of Evodia rutaecarpa aqueous extract for 28 days.(2)Basic study on hepatotoxic substances of Evodia rutaecarpaCombined with literature review and toxicity prediction software Derek nexus,the research object of this chapter is determined as the index components of Evodia rutaecarpa,namely evodiamine,evodiamine and limonin,and other main components,namely evodiamine,evodiamine and dehydrogenated evodiamine,a total of 6 compounds;The content of aqueous extract of Evodia rutaecarpa was determined by ultra high performance liquid chromatography high resolution mass spectrometry(UPLC-Q-TOF/MS).The average contents of evodiamine,evodiamine,limonin,evodiamine and dehydrogenated evodiamine were 0.0834%,0.0419%,1.0921%,0.0016%and 0.4193%respectively;The IC50 of Evodia rutaecarpa aqueous extract was 0.34 mg·mL-1 and that of evodiamine was 70.32 μmoL-1 by cell counting(CCK-8),the IC50 of dehydrogenated evodiamine was 135.60 μmoL-1,while the other groups had no significant inhibitory effect on the proliferation of HepG2 cells;Through the acute liver injury test of the main components of Evodia rutaecarpa,it was found that intraperitoneal injection of 200 mg/kg limonin,evodiamine and 100 mg/kg dehydrogenated evodiamine could significantly increase the serum ALT(P<0.05 or P<0.01)and the liver body ratio(P<0.05 or P<0.01),suggesting that intraperitoneal injection of 200 mg/kg limonin,evodiamine and 100 mg/kg dehydrogenated evodiamine could cause acute liver injury in mice.(3)Study on Toxicokinetics based on LC-MS/MSOn the basis of the previous toxicity study,the concomitant toxicokinetic study was carried out.Limonin was selected as the target analyte,and the Evodia rutaecarpa water extract gavage group,limonin gavage group and limonin intraperitoneal injection group were established.The doses were 60 g/kg,200 mg/kg and 200 mg/kg respectively.The results showed that the AUC0-t and Cmax of Limonin in the plasma of mice in Evodia rutaecarpa Decoction gavage group were 91797.2±31524.4 h·ng/mL and 11130.0±3047.8 ng/mL,the AUC0-t and Cmax of Limonin gavage group were 138.5±139.3 h·ng/mL and 32.1±14.5 ng/mL,and the AUC0-t and Cmax of Limonin intraperitoneal injection group were 10234.5±6917.3 h·ng/mL and 1080.0±296.3ng/mL.To sum up,Evodia rutaecarpa water extract induced liver injury in mice has a certain"dose-time-toxicity" effect.With the extension of administration time and the increase of dose,the degree of liver injury in mice becomes worse;In addition,through in vitro and in vivo toxicity verification,it was found that intraperitoneal injection of Limonin,evodiamine and dehydroaevodiamine could cause liver injury in mice;On this basis,the toxicokinetics showed that limonin was absorbed rapidly in the body.At the equivalent dose,the plasma exposure of Limonin was higher,about 600 times,than that of Evodia rutaecarpa aqueous extract by gavage.At the same time,the plasma exposure of Limonin by intraperitoneal inj ection was higher,about 100 times,than that of monomer by gavage.This paper confirms the "dose-time-toxicity" relationship of Evodia rutaecarpa water extract induced hepatotoxicity,preliminarily explains the correlation between six main components in Evodia rutaecarpa and Evodia rutaecarpa water extract induced hepatotoxicity,reveals the correlation between Evodia rutaecarpa water extract induced hepatotoxicity and limonin exposure in vivo,provides data support for the safe use of Evodia rutaecarpa in clinic,and provides a scientific basis for the preparation of more safe Evodia rutaecarpa preparations,It provides reference data for the Pharmacopoeia to formulate the standard of Evodia rutaecarpa,and also provides a new idea for the study of toxic substances of other toxic traditional Chinese medicine.