| Elsholtzia blanda(Benth.)Benth,is a kind of traditional Chinese folk medicine,also called as Si-fang-hao,Si-leng-hao or Bai-xiang-ru in the local.The plant mainly distributed in the southwest of China.In folk,Elsholtzia blanda is available for a variety of disorders such as hepatitis,dysentery,cold,faucitis,tonsillitis,toothache,acute gastroenteritis,acute and chronicity pyelonephritis.Recent studies showed that the extracts of Elsholtzia blanda Benth.(EEB) had the obvious pharmacological activities in the treatments of cardiovascular disorders.EEB could improve the recovery of myocardial function and showed a capacity to resist ischemic damage through lowering blood viscosity in the animal models.Elsholtzia blanda is a medicinal herb,from which,luteolin-7-O-β-D-glucoside,luteolin,luteolin-5-O-β-D-glucoside, 5-hydroxyl-7,8-dimethoxy-flavone and 5-hydroxyl-6,7-dimethoxy -flavone were isolated,and the content of the former component,luteolin-7-O-β-D-glucoside,is much higher than the last three ones.Previous studies have demonstrated the pharmacological activities of EEB,but so far there's no report on the absorption,disposition,metabolism and excretion of luteolin in EEB in vivo.ADME reveal the dispositions of drugs in vivo,and the influence of therapeutic effect and toxicity of drugs after those dispositions.For this reason,the understanding of ADME after oral administration of EEB in rats is very important in definiting the potential biological activity of it in vivo.Aim To establish an RP-HPLC method for determination of luteolin from EEB in biological specime,to qualitatively detect metabolites in rats' urine,bile and feces,and to study the pharmacokinetics of luteolin in rats.Methods 1) Absorption:①An RP-HPLC method was established for determination of free and total luteolin in SD rats' plasma and gastrointestinal tract at 0.17,0.5,1,2,4,6 h after administration of EEB to 24 male SD rats(4 rats per one time spot) to understand the absorption of luteolin and luteolin glycoside in gastrointestinal tract;②Drug blood samples from caudal vein was gotten after oral administration to 12 SD rats(6 female and 6 male) at 0.17,0.33,0.5,0.67,0.83,1,2,3,4,5,6,7,8 h.The plasma samples were determined by RP-HPLC after being deproteinized with trichloroacetic acid and extracted with ethyl acetate. Drug-time curve and pharmacokinetics parameters can be obtaind.2)Distribution:An RP-HPLC method was established for determination of total luteolin in SD rats' plasma and organs at 0.17,0.5,1,2,4,6 h after administration to 24 male SD rats(4 rats per one time spot ).3)Metabolism:Rat urine(4～6 h),feces(0～24 h) and bile(2～4 h) were collected after oral administration.By using UPLC-MS,UPLC-MS/MS and UPLC-PDA,the metabolites of luteolin glycoside,including isomers,were analyzed from bile,urine and feces of rats dosed with EEB and metabolic pathway was proposed.4)Excretion:Rat urine,feces and bile were collected after oral administration to 10 male SD rats in different time portions.After deconjugation withβ-glucuronidase/sulfatase,the levels of luteolin in urine,feces and bile were measured by HPLC.The excretory rate and excretion pathway were obtained.Results 1) Absorption:①Luteolin glycoside was hydrolyzed to aglycone luteolin in intestinal tract,and then luteolin was resorpted.The main form of luteolin existed in gastrointestinal tract after administration of EEB is aglycone;②The concentration-time curve of luteolin after oral administration of EEB was fitted to two compartment open model.Two factors analysis of variance were adopted in the evaluation of gender and time spots for collection of blood.The result suggested that the gender-based difference in blood-drug concentrations had statistical significance.2) Distribution:The content of luteolin in liver and kidney were higher than in cardia and lung. The content of luteolin in kidney,cardia and lung were showed max in 1 h.It was similar to the peak time in blood.However,in liver,the drug was distributed quickly,and showed max at 0.17 h.And because of the sensitivity of detection,luteolin can not be detected in other tissues.3) Metabolism:Metabolites identified include luteolin monoglucuronides,methyl luteolin monoglucuronides,methyl luteolin sulfate,luteolin sulfate,methyl luteolin and luteolin in urine, as well as methyl luteolin monoglucuronides,luteolin monoglucuronide sulfate,luteolin sulfates in bile,and luteolin in feces.And all of those were aglycone luteolin's PhaseⅡconjugates.However,luteolin glycoside and its phaseⅡconjugates can not be detected in urine,bile,feces and plasma.Luteolin glycoside was possibly hydrolyzed to aglycone luteolin in intestinal tract,and then luteolin was resorpted in vivo.The main metabolic pathway of luteolin in rats was phaseⅡmetabolism.4) Excretion:PhaseⅡconjugates of luteolin were excreted through kidney to urine.Besides this,Luteolin goes with the excretion of bile to the intestine,and then most of it egest through faecal excretion.This is the major excretion pathway.The total accumulative excretion of the dose was about 37.6%(11.1%in urine;26.5%in feces) for luteolin.Conclusion The developed method is sensitive,specific,accurate,and is appropriate for determination of luteolin in vivo.After oral administration of EEB to rats,the main constituent, luteolin glycoside was hydrolyzed to aglycone luteolin in intestinal tract,and then luteolin was resorpted quickly.Simultaneously,luteolin was quickly distributed in major tissues.The main metabolic pathway of luteolin in rats was phaseⅡmetabolism,and luteolin were mainly excreted through feces in the form of aglycone.Understanding ADME of EEB in rats has an important significance in guiding clinical use of the drug and in further research.
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