| Objective: To observe the effect of the catalpol on permanent focal cerebral ischemia of the functional recovery of rats and study the influence of catalpol on neurological severity scores and the change of brain histomorphology after injected different dosages to model rats by different time point. Moreover, establish the methods of HPLC to measure the catalpol in rats'serum to study the pharmacokinetics of the Catalpol in rats. Methods:1. Research the protection of the catalpol on permanent cerebral ischemia of ratsThe model was established by permanent cerebral artery occlusion (pMACAO) in rats with occlusor thread. After model appraisement, catalpol was selected and divided into low, middle and high dosage groups (1mg/kg, 5mg/kg, 10mg/kg). Meanwhile compared citicoline group (0.5mg/kg), solvent group injected the same volume of normal saline, and model groups that didn't use the catalpol. The number of groups is six. Moreover, the sham-operated group was established. In this experiment, there were 7 groups and 9 rats per group, after established model operation 6 hours, 6 rats in each group injected in abdominal cavity every day and kept the successive medication 7 days. The neurological severities were scored on the 1st, 4th and the 7th day, the evaluation includes Bederson neurological function score, muscular energy evaluation and beam-walking test. Then selected 3 rats from eac group.After anesthesia and citromint heart filling, around 2mm thick brain parts peri-optic chiasma were selected as HE staining and Nissl's staining samples from which we can do pathohistological observation. Select the other 3 rats from each group, carried anesthesia after established model operation 6 hours. Injecte in the rats'abdominal cavity every day and keep the successive medication 3 days and then got the parts of the rats'brain as the TTC staining samples at the third day, from that we can measure the infracted volume.2. The research of catalpol in pharmacokinetics in ratsThe research of chromatographiccondition and methodological specificity.The column: Hypersil C-18 (250mm×4.0mm,5um); pre column: C-18 (10mm×4.6mm, 5um); mobile phase: water-acetonitrile (99.5:0.5, V/V); flow rate: 1.0ml/min; sample size: 20ul; column temperature:40℃; detect wave length: 210nm. Use this condition to measure standard curve, absolute recovery, method recovery, and the degree of precision.The method of pharmacokinetics: divided 15 healthy rats into low, middle and high dosage groups randomly, 5 for each group. Catalpol was injected by vena caudalis 3.5mg/kg, 7.0mg/kg and 14.0mg/kg follow the 3 different dosage groups. After injection 1 min, 5 min, 15 min, 30 min, 45 min, 90 min, 120 min, 240 min, 420 min and 600 min, got the blood from the ossa orbitalis'venous plexus of the rats, the measure the concentration of drug in blood. Then display the data from the experiment, used pharmacokinetics software 3P87 to analysis these data then got the pharmacokinetic parameters of catalpol in rats.Results:1. The protection of the catalpol on permanent cerebral ischemia of rats.The effect on neurological action in rats: at the 7th day during the administration, from the Bederson neurological function score, it was observed that the rats'recovery capability in catalpol dosage groups and citicoline dosage groups are both better than physiological saline groups.There were evidently different between the catalpol groups, citicoline groups and physiological saline groups (P<0.05); from the muscular energy evaluation, the muscular energy of rats in catalpol dosage groups and citicoline dosage groups are improved significantly, catalpol dosage groups have evidently different with model groups and physiological saline groups (P<0.05); from the beam-walking test , it was found that the balanced function of rats in each group is improved, and there were no evidently different between each group (P>0.05). Pathohistological observation: from observed the HE staining of brain tissue sections in each group, at the 7th day after the operation, the change of pathohistology was improved in each treated groups compared with pMCAO model group, the pathological changes like cellula nervosa swelling, the change of vacuolar will be improved; from observed the Nissl's staining of brain tissue sections in each group, the loose of tigroid bodies and the nerve cells in each treated group were improved compared with the pMCAO model group. the remnant nerve cells arranged for polarity and the arrangement of the model group was disordered.The effect on infarcted volume: it was found that after TTC staining of brain tissue sections in each group, the infarcted volume of pMCAO model group was larger than others. On the other hand, the infarcted volume in each catalpol dosage groups conspicuously contracted. Compared with the model groups the catalpol dosage groups has evidently different (P<0.01). There were no evidently different between physiological saline group and model group (P>0.05). Moreover, the diminution of infarct size in catalpol high dosage group was more conspicuous than middle and low groups.2. The result of catalpol's pharmacokinetic in rats.The establishment of the method that measured the concentration of catalpol in rats'serum: followed the above-mentioned chromatographic condition, the shape of the catalpol was well, no other impurities to interfere the result, the number of theoretical plates of catalpol in serum sample was more than 9000, the retention time was about 10.3 min. From drawing the standard curve, it was found that the linear relationship was well when the concentration of catalpol was between 0.5~70.1ug/ml. The linear regression equation is : Y=6996.6C-826.89(r=0.9992,n=5). The average absolute recovery of the drug's concentration is 86.04%~90.00% (n=5). The average method recovery is 100.56%~99.00% (n=5). RSD in the day is 5.76%~3.43% (n=5). RSD between the day is 4.02%~6.86% (n=5).The chemical parameters of catalpol of pharmacokinetic in rats: catalpol was injected in vein(the injection dosage were 3.5mg/kg, 7.0mg/kg and 14.0mg/kg).the action of kinesics was consistent with the feature of the model. The important parameters of pharmacokinetic list as follow.The elimination half life of the serum (T1/2β) are 165.46±12.35 min, 0.21±0.01 min, 185.74±13.69 min.The apparent volumes of distribution of the central compartment (Vc) are 0.19±0.03(mg/kg)/(ug/ml), 0.21±0.01(mg/kg)/(ug/ml) and 0.25±0.01(mg/kg)/(ug/ml). The clearance rate(Cl) are 0.0014±0.0001mg/kg/min/(ug/ml), 0.0016±0.0001mg/kg/min/(ug/ml) and 0.0018±0.0002mg/kg/min/(ug/ml).Area under curve (AUC) are 2450.50±169.22(mg/ml)×min, 4347.94±399.45(mg/ml)×min and 7918.80±793.61(mg/ml)×min.Conclusion:1.In this study, it shows that catalpol can promote the nerve functional recovery of rats that was in permanent middle cerebral artery occlusion, and it can improve the muscle energy of paralyzed rats that were caused by cerebral ischemia.Meanwhile, the catalpol plays an important role on the protection of brain tissue and avoidance of the loose of nerve cells, moreover, the caltalpol can reduce the infarct size in brain of rats that were in cerebral ischemia, and can reduce the loss of ischemic perim cerebral mantle neuron and the ischemic patho-change of brain tissue, it plays ischemic neuroprotection effect in cerebral ischemia.2.In this research, the reversed phase high performance liquid chromatography (RP-HPLC) was used to measure the concentration of catalpol in serum, this method is good in linearity, precision and reproducibility.On the other hand, this method is easy to carry on. These parameters above-mentioned are consistent with the two-compartment model, and can be used to guide and consult in clinical research.
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