Quantitation And Hepatotoxicity Of Multiple Components In Euodiae Fructus Formula Granules

Euodia rutaecarpa,the dried nearly ripe fruits of Euodia rutaecarpa?Juss.?Benth.,Euodia rutaecarpa?Juss.?Benth.var.officinalis?Dode?Huang or Euodia rutaecarpa?Juss.?Benth.var.bodinieri?Dode?Huang,has been widely used in clinic.The Euodiae fructus formula granules are made from Euodia rutaecarpa pieces conforming to the pharmacopoeia regulation and manufactured by modern technology of extract,concentration,separation,spray drying,granulation and packaging.Compared with traditional decoction,formula granules are safer,healthier,and more convenient for carrying and taking without decoction.In the study,a HPLC-MS method was developed for simultaneous quantification of ten components in Euodiae Fructus formula granules and a MTT method was developed for the hepatotoxicities study of four alkaloids and limonin to define the hepatotoxicity material basis of Fructus Euodiae.They can provide previous experimental basis and reference for the quality control and safety evaluation of Euodiae Fructus formula granules.Part one Simultaneous quantification of ten components in Euodiae fructus formula granules by LC-MSObjective:To establish a highly sensitive method for determining the evodiamine,rutaecarpin,dehydroevodiamine,evocarpine,rutin,hesperidin,hyperoside,limonin,caffeic acid and chlorogenic acid in Euodiae fructus formula granules.Methods:The chromatographic separation was performed on a Diamonsil C₁₈ column?150×4.6 mm,5?m?.The column temperature was kept at 40?and the injection volume was 10?L.The mobile phase consisted of A?0.12%HAc-1 mmol/L NH₄Ac solution?and B?acetonitrile?was used with gradient elution as follow at the flow rate of 0.8 mL/min:0⁶ min,85%A⁷2%A;6¹0 min,72%A³2%A;10¹1 min,32%A¹0%A;11²2 min,10%A.The total run time was 22 min.Then returned to the initial proportion?85:15,V/V?quickly and lasted for 10 minutes to equilibrate the column.The detection was accomplished by multiple reaction monitoring?MRM?in positive and negative mode at the same time.The ionspray voltage was set to5500 and-4500 V respectively and the turbo spray temperature was 650?.Nitrogen was used as the nebulizer gas?60 psi?and heater gas?65 psi?.The curtain gas was kept at 25 psi.Results:There were linear relationships between the peak area and drug concentrations.The correlation coefficients were all higher than 0.9954.The The RSDs of intra-day and inter-day precisions were lower than 2.48%and2.90%.All the analytes were stable within 24 h.The average recovery was in the range of 95.7%¹04.6%.Conclusions:The method is simple,highly sensitive and specific.It is an ideal method for the determination of simultaneous quantification of ten components in Euodiae Fructus formula granules.And it can provide scientific basis for the quality control and evaluation of Euodiae Fructus formula granules.Part two The hepatotoxicity material basis study of Fructus EuodiaeObjective:To test the toxic effects of evodiamine,rutaecarpin,dehydroevodiamine,evocarpine and limonin on HepG2 cells and to investigate the potential hepatotoxicity components in Fructus Euodiae.Methods:Human hepatocellular carcinoma HepG2 cells were incubated with different concentrations of five monomers?evodiamine,rutaecarpin,dehydroevodiamine,evocarpine and limonin?separately for 24 hours and the effects of them on HepG2 cells proliferation were analyzed by MTT method.Results:The MTT assay showed that 5²00?mol/L evodiamine,5²00?mol/L rutaecarpin,50²00?mol/L dehydroevodiamine,25²00?mol/L evocarpine and 10²00?mol/L limonin inhibited human hepatocellular carcinoma HepG2 cells viability obviously?P<0.01 or P<0.05?,and the effects were correlated to the doses of drugs.Conclusions:Five monomers in Fructus Euodiae?evodiamine,rutaecarpin,dehydroevodiamine,evocarpine and limonin?show different degrees of hepatotoxicity.