Effects Of MCAO On Pharmacokinetics Of Geniposide In Rat Brain And Blood By Enzyme - Linked Immunosorbent Assay

Objective1.To develop an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for concentration measurement and pharmacokinetic analysis of geniposide in Plasma and rat brain homogenates using and monoclonal antibodies (MAb) against GEN (anti-GEN MAb), validating icELISA method to determine GEN concentration in Plasma and rat brain homogenates, And compared with HPLC, providing an new method of medicine concentration analysis and pharmacokinetic studies of traditional Chinese medicine in Plasma and rat brain homogenates.2. Geniposide is Qingkailing Xingnaojing drugs and other anti-cerebral ischemia important active ingredient. The use of middle cerebral artery occlusion model (middle cerebral artery occlusion, MCAO) investigate whether cerebral ischemia pathological processes affecting glycosides gardenia through the blood-brain barrier, applied research for the treatment of ischemic stroke Geniposide provide more realistic the pharmacokinetic parameters.Methods1.Indirect competition ELISA (ic-ELISA) methods were developed and validated for the measurements and pharmacokinetic analysis of GEN in in Plasma and rat brain homogenates, standard curve were prepared and its methodology including assay linearity and sensitivity of detection, precision and accuracy, recovery, stability and specificity of anti-GEN MAb were validated. Determination of GEN concentration in rat Plasma using ic-ELISA compare with HPLC.2. Preparation of MCAO rat model:SD rats 176, according to the table of random numbers were randomly divided into two groups, sham operation group and model group. N= 88,11 according to randomization time point,8 rats at each time point. Before modeling all the animals in the tail vein intravenous catheter. MCAO model modeling method with reference to literature, Plug 1.5 hours after infusion, starting time for the recording.3. Plasma and brain tissue samples drawn.Begin taking the starting time, sham group and model group were injected intravenously Geniposide 100mg·kg-1, After administration 1,5,10,15,20,40,60,90,120,150,180 min (8 rats per time point), blood was collected from the abdominal aorta, placed in Anticoagulant vacuum tube, taken brain homogenates after perfusion.①Samples, a standard curve comparing concentration of puerarin with its absorbance measured by ic-ELISA was plotted. These pharmacokinetic parameters were calculated in Kinetica version 5.0 and SPSS 21.0. ② concentration of GEN in plasma samples was detected by HPLC and draw plasma drug concentration-time curve. These pharmacokinetic parameters were calculated in Kinetica version 5.0 and SPSS 21.0. By indirect competitive ELISA method for detecting the concentration of Geniposid in brain homogenate samplese, brain tissue protein concentration in each Samples was detected with a protein concentration assay kit, And treated with the sham group, the average protein content of each sample is this standard brain geniposide content, geniposide= standard concentration detector geniposide concentration/test protein concentration x standard protein concentration. Painting the concentration of in brain homogenate concentration standard Geniposide-time curve. These pharmacokinetic parameters were calculated in Kinetica version 5.0 and SPSS 21.0.Results1. An indirect competition ELISA method was developed and validated for the measurements and pharmacokinetic analysis of GEN in in rat Plasma. We obtained a linear regression coefficient of 0.9903, and the linear regression equation, y=-0.198 ln(x)+1.206. A linear relationship was found within the range of 52.59ug/mL-200ug/mL. The sensitivity (IC50) of the assay was determined as 35.36ug/mL. The limit of detection was2.59ug/mL (mean±S.D x 3, n=20). Both intra-day and inter-day repeatability and precision was achieved, with relative standard deviation (RSD) lower than 15%. A mean recovery of 95-115% was obtained, with RSD lower than 15%. Stability studies indicated that geniposide is stable during sample preparation and analysis. We did not detect cross-reactivity against any of the other related compounds.2. An indirect competition ELISA method was developed and validated for the measurements and pharmacokinetic analysis of GEN in in rat brain homogenates. We obtained a linear regression coefficient of 0.9948, and the linear regression equation, y=-0.198 ln(x)+1.206. A linear relationship was found within the range of 2.59ug/mL-200ug/mL. The sensitivity (IC50) of the assay was determined as 29.73ug/mL. The limit of detection wasl.81ug/mL (mean±S.D×3, n=20). Both intra-day and inter-day repeatability and precision was achieved, with relative standard deviation (RSD) lower than 15%. A mean recovery of 95-115% was obtained, with RSD lower than 15%. Stability studies indicated that geniposide is stable during sample preparation and analysis. We did not detect cross-reactivity against any of the other related compounds.3.By HPLC was developed and validated for the measurements and pharmacokinetic analysis of GEN in in rat Plasma.in order Geniposide peak area (Y) of Geniposide concentration (x) linear regression. We obtained a linear regression coefficient of 0.9989, and the linear regression equation, y= 0.0554x-0.7462. A linear relationship was found within the range of 1ug/mL-lOOug/mL. The limit of detection was2ug/mL (mean±S.D ×3, n=20) RSD was 7.53%. Both intra-day and inter-day repeatability and precision was achieved, with relative standard deviation (RSD) lower than 15%. A mean recovery of 95-115% was obtained, with RSD lower than 15%.4. From the measured by ELISA model group and sham-operated group rat plasma GEN concentration-time curves show, Cmax model group= 1530.87±298.22ug/mL, Tmax= 4.25 ±3.95min, AUC0-4= 46583.75±11767.14 ug/mL ·min, T1/2= 53.91±18.84min, MRT 76.65±13.21min; sham group:Cmax= 1294.45±371.08ug/mL, Tmax= 5.5±.50min, AUCo-t= 45066.25±14540.23ug/mL-min,T1/2=46.31±16.05min,MRT=77.51±16.11min. No significant differences were calculated.5. Indirect competitive ELISA method study MCAO model Geniposide timetable concentration rats plasma, with the statistical software SPSS21.0 statistical analysis, t= 15min Geniposide in concentration model group were higher than sham group, p= 0.03; t= 20min:less than model group, sham group, p= 0.02; no difference in other time points, suggesting that t= 15-20min during geniposide plasma concentrations decrease rapidly, possible reasons related to geniposide into the brain.6. Measured by HPLC from the model group and the sham group rat plasma Geniposide concentration-time show:Cmax model group=1419.625±414.96ug/mL, Tmax= 37.45± 9.33min,AUCo-t=25412.99±3718.59ug/mL-min,T1/2=26.61±6.44min,MRT=76.65±13.21 min;sham group:Cmax= 1067.88±8.35ug/mL, Tmax=1.5±.414min, AUC0-t= 24120.26 ±6859.672ug/mL·min, T1/2= 23.07±9.08min, MRT= 48.467±12.17min. No significant differences were calculated.7.Pharmacokinetic studies tudy of MCAO model in rats Geniposide analysised by statistical software SPSS21.0 statistics masured by HPLC, t= 15min Geniposide concentration model group were higher than sham group, p= 0.002; t= 20min:model group less than sham group, p= 0.007; t= 40min Geniposide concentration model group greater than the sham group, p= 0.02; no difference in the remaining time. Blood Gardenia curve occurs when the glycoside drug bimodal, the second peak time faster. Model group Geniposide metabolism faster,Geniposide may related to the brain.8. When the ELISA and HPLC measured Geniposide in model group rat plasma concentration-time curve. The trend and bimodal behavior Geniposide concentration-time curves are the same as reported, in the model group, t= 15,40,180min ELISA method and HPLC were significantly different p<0.05, and the rest by ELISA and HPLC were no significant differences. Sham group t= 15, t= 60min, ELISA method and HPLC were significantly different p<0.05. No significance exist in rest by ELISA and HPLC. ELISA and HPLC measured result is statistically significant at individual time points, the trend of two sets of data are the same in general.9. From the GEN concentration-time table and curves, the drug from the blood into the brain Geniposide have double peaks, the first peak should be around 15min, a second peak at about 60min. at t=40,60min the right side of the model group and the sham group were significantly different; data analyzed by SPSS21.0 statistical software at t= 10,60, the left side of the model group and sham group were significantly different, p<0.05; t= 1,5,10,40,60,90 when the left and right sides of the model group were significantly different p <0.05. The left side of the model group Cmax= 379.45±112.91ug/g, Tmax= 41±0min, AUC0-t= 36932.5±3607.51ug/g±min, T1/2= 66±31, MRT= 125±3min. The right of the model group, Cmax= 420.25 ±79.03ug/g, Tmax= 43±33min, AUC0.t= 44606.88± 7209.59ug/g±min, T1/2= 84±56, MRT= 141±4min. Sham group Cmax=391.21± 50.17ug/g, Tmax=33 ±27min, AUC0-t= 36407.16 ±5765.45ug/g±in, T1/2=135±122, MRT= 194±51min. After statistical analysis, Mode right AUC0-t> Mode left AUC0-t, P= 0.009; Mode right AUC0-t> Sham AUC0-t, P= 0.012434; Mode left AUC0-tand Sham AUC0t, P= 0.4. Geniposide concentration in the brain:the right of the model group (plug side)> the left side of the model group, P= 0.009; the model group on the right (plug side)> sham group, P= 0.012 left side of the model group and the sham group no difference, P= 0.4Conclusions1. Quick, specific and sensitive indirect competitive ELISA assay methods for the measurement and pharmacokinetic analysis of geniposide in Plasma and rat brain homogenates, were developed for the first time. Method validations Plasma and rat brain homogenates,were satisfactory and meet the criteria specified for the analysis of biological samples. Stability studies indicated that geniposide are stable during sample preparation and analysis.2.1n this experiment, indirect competitive ELISA and HPLC method are used to study plasma Geniposide pharmacokinetics in MCAO model dynamics rats, ELISA and HPLC assay data a the trend of two sets of data are the same in general.3.Drugs curve trend of wo groups of rats of re the same as reported, bimodal phenomenon are found in the blood concentration of Geniposide-time curves. MCAO model was slightly higher than sham group at 0-10min, model group, plasma concentrations decline rapidly in 15-20min. The second peak time faster, but the second peak of the lower number.4. MCAO model rats left side, the right side of the model group and the sham group brain Geniposide content:the model group on the right (plug side)> the left side of the model, model on the right (plug side)> sham group. Reperfusion injury, brain tissue absorption Geniposide increased after cerebral ischemia indicate Geniposide into the brain tissue to speed, which may be blood-brain barrier damage after cerebral ischemia related.5. Compared with the traditional method,use of a normal animal, MCAO model of cerebral ischemia anti-rat drug pharmacokinetics, derived drugs may differ in pharmacokinetic characteristics of the blood and brain, indicating the disease state, especially blood-brain barrier destruction, the pharmacokinetic parameters of the drug will have an impact. Therefore, in the study of neuroprotective effects of drugs, the use of animal models of cerebral ischemia observed pharmacokinetics, more in line with clinical practice, clinical practice more instructive.