Pharmacokinetic Characteristics Of Protocatechualdehyde In Plasma And Brain Tissue Of Rats With Cerebral Ischemia/Reperfusion Injury

ObjectiveProtocatechualdehyde,a brain-protective component of Gastrodia elata and Salvia miltiorrhiza,can effectively protect against cerebral ischemia/reperfusion（CIRI）injury in rats.The underlying mechanism is related to anti-inflammation,anti-apoptosis,and neuroprotection.Pharmacokinetic studies have shown that protocatechualdehyde is absorbed rapidly following intragastric administration in normal rats and can pass through the blood-brain barrier（BBB）.Moreover,the pathological state of CIRI can affect the disposal of drugs.It is suggested that protocatechualdehyde may play a neuroprotective role in the brain.Under normal circumstances,drugs play a protective role in the brain,in addition to passing through the BBB for brain tissue distribution,also reach an effective concentration and residence time in the brain.Here,high performance liquid chromatography（HPLC）and microdialysis sampling were used to study the pharmacokinetic characteristics of protocatechualdehyde in plasma and brain tissue of CIRI model rats,which should provide a reference for studies on the mechanism underlying its brain-protective effects.Methods1.Establishment of a method for concentration of protocatechualdehyde in ratsA novel HPLC method was established to determine the concentration of protocatechualdehyde in rat plasma,brain tissue,and brain microdialysate.Methodological verification was performed based on specificity,linear relationship,accuracy,intra-and inter-day precision,stability,extraction recovery rate,and matrix effects.2.Plasma pharmacokinetic characteristics of protocatechualdehyde under CIRI pathologyMiddle cerebral artery occlusion/reperfusion（MCAO/R）model rats were established using the suture-occlusion method.The above established HPLC method was then used to determine the plasma concentrations of protocatechualdehyde（intragastric administration:400 mg/kg）at different time points in normal and MCAO/R model rats.Plasma pharmacokinetic parameters were obtained,and a plasma pharmacokinetic time curve was drawn to compare differences between normal and pathological CIRI rats to determine the plasma pharmacokinetic characteristics of protocatechualdehyde under CIRI pathology.3.Pharmacokinetic characteristics of protocatechualdehyde in brain tissue under CIRI pathology3.1 Distribution of protocatechualdehyde in the brainThe established HPLC method and traditional tissue homogenization were used to determine the distribution of protocatechualdehyde（intragastric administration:400 mg/kg）in different regions of the brain in normal and MCAO/R model rats under the equilibrium phase.The distribution characteristics of protocatechualdehyde in brain tissue were also determined.3.2 Brain pharmacokinetic characteristics of protocatechualdehydeThe established HPLC method and microdialysis sampling were used to determine the concentration of protocatechualdehyde（intravenous administration:100 mg/kg）in local brain regions of normal and MCAO/R model rats at different time points to determine the brain tissue pharmacokinetic characteristics of protocatechualdehyde under CIRI pathology.4.Preliminary study on protocatechualdehyde metabolism in ratsBased on the above experiments,a large metabolic peak was observed in the plasma and brain tissue of experimental animals after administration of protocatechualdehyde.This metabolite peak was confirmed quantitatively as protocatechuic acid.Based on HPLC analysis,we detected the concentration of protocatechuic acid in rat plasma and brain tissue at different time points,constructed a drug-time curve,measured the pharmacokinetic parameters,and evaluated the pharmacokinetic characteristics of protocatechualdehyde metabolites in rats.In addition,the pharmacokinetics of the metabolite were used to further clarify the disposition of the prototype drug（i.e.,protocatechualdehyde）in vivo.Results1.Establishment of a method for concentration of protocatechualdehyde in ratsThe specificity,linearity,accuracy,precision,stability,extraction recovery,and matrix effects of the established HPLC analysis method were all in line with the requirements of biological sample analysis and could be used for pharmacokinetics of protocatechualdehyde in rat plasma,brain tissue,and brain microdialysate.2.Plasma pharmacokinetic characteristics of protocatechualdehyde under CIRI pathologyAfter intragastric administration of protocatechualdehyde,in normal rats,the plasma concentration peaked at 11 min,AUC_（0-t）was 10028.52±2119.42μg/Lh,and t _1/2was 5.60h.In MCAO/R model rats,the plasma concentration peaked at 7.50 min,AUC_（0-t）decreased to 6924.17±3627.70μg/Lh,and t_1/2 was 5.31 h.These results showed that under CIRI pathology,absorption speed accelerated,but absorption degree declined.3.Pharmacokinetic characteristics of protocatechualdehyde in brain tissue under CIRI pathology3.1 Distribution characteristics of protocatechualdehyde in the brainAfter intragastric administration of protocatechualdehyde,in the equilibrium phase,the distribution concentration in the brain of normal rats followed:cortex>striatum>hippocampus>cerebellum.The distribution concentration in the brain of MCAO/R model rats followed:cortex>cerebellum>hippocampus>striatum.Thus,compared with that under normal conditions,the distribution of protocatechualdehyde in brain tissue increased significantly in MCAO/R rats.These results showed that the cortical distribution of protocatechualdehyde was highest during the equilibrium phase,and CIRI pathology increased its distribution and concentration in the brain.3.2 Brain pharmacokinetic characteristics of protocatechualdehydeAfter intravenous injection of protocatechualdehyde,in the cortex of normal rats,AUC_（0-t）was 1295.11±216.57 mg/L·min,T_max was 30 min,and t_1/2 was 17.83 min.In the cortex of MCAO/R model rats,AUC_（0-t）was 2498.58±128.30 mg/L·min,T_max was 30 min,and t_1/2was 10.35 min.These results showed that under CIRI,protocatechualdehyde exposured in the brain increased significantly.4.Preliminary study on metabolism of protocatechualdehyde in ratsPlasma pharmacokinetic characteristics of the metabolite protocatechuic acid were determined after intragastric administration of protocatechualdehyde.In normal rats,the plasma concentration peaked at 11 min,AUC_（0-t）was 32435.13±11970.01μg/Lh,and t_1/2was 2.29 h.In MCAO/R model rats,the plasma concentration peaked at 13 min,AUC_（0-t）was 27511.59±15544.33μg/Lh,and t_1/2 extension was 2.47 h.Thus,plasma exposure to protocatechuic acid was higher than that of protocatechualdehyde when it was administered intragastrically.This may be related to the fact that protocatechualdehyde is mainly metabolized to protocatechuic acid for elimination.The distribution characteristics of protocatechuic acid in the brain were as follows:after intragastric administration of protocatechualdehyde,under the equilibrium phase,the concentration of protocatechuic acid in the brain of normal rats followed:cortex>striatum>cerebellum>hippocampus.The concentration distribution in the brain of MCAO/R model rats was:cortex>striatum>hippocampus>cerebellum.Thus,compared with that under normal conditions,the distribution protocatechuic acid in brain tissue increased under CIRI pathology.Our results showed that protocatechuic acid can be detected in the brain after intragastric administration of protocatechualdehyde,with the highest concentrations found in cortical areas,and the pathological state of CIRI can increase its concentration in the brain.ConclusionsUnder CIRI pathology,the absorption rate of protocatechualdehyde was accelerated,but the degree of absorption was decreased.Furthermore,protocatechualdehyde exposured in the brain increased significantly.In addition,a large amount of protocatechuic acid,a protocatechualdehyde metabolite,was detected in the plasma and brain tissue of normal and MCAO/R model rats.It is believed that protocatechualdehyde is mainly metabolized to protocatechuic acid for elimination.