The Research Of Praeparatum Of Catalpol By Gelsiccation On Its Prescription And Technology And The Study On Its Pharmacokinetics

Background:Cerebral ischemia disease is serious harmful to human's healthy. How to use drugs to prevent and cure cerebral ischemia became the international focus. The catalpol in rehmannia can cure the cerebral ischemia disease. But now, there is no report about the praeparatum of catalpolObjective:To determine the prescription and the forming technology for the praeparatum of catalpol by lyophillization. To establish the HPLC methods for determination of catalpol in serum and cerebrospinal fluid (CSF) of rats to study the pharmacokinetics of praeparatum of catalpol and to detect catalpol in CSF for authenticating the rationalities of the prescription and forming technology.Methods:1. The research of praeparatum of catalpol on its prescription and technology 1.1The assaying of catalpol in water-solutionThe column: Hypersil C18 (250mm×4.0mm.5um); percolumn: C18 (10mm×4.6mm, 5um); mobile phase: solution of 0.1% phosphoric acid -acetonitrile (99:1, V/V); flow rate: 1.0ml/min; sample size: 20μl; detection wave length: 210nm. Use this condition to measure standard curve, the degree of precision, reproducibility, method recovery, and stability test.1.2Research of prescription and technologyPrincipal agent: catalpol; adjuvants: mannicol, aminoacetic acid, mycose; pH conditioner: solution of 10% NaOH solution; solvent: water for injection. According to the eutectic point, choose different gelsiccations and draw freeze drying curve to determine the best lyophillization. Choice indicators: color of samples, forming, water-solubility, clarity, content of catalpol.2. The study of catalpol in pharmacokinetics and in CSF of rats2.1The analytical method of catalpol in serum and CSFThe column: Hypersil C18 (250mm×4.0mm.5um); percolumn: C18 (10mm×4.6mm, 5um); mobile phase: water-acetonitrile (99.5:0.5, V/V); flow rate: 1.0ml/min; sample size: 20μl; column temperature: 40℃; detection wave length: 210nm. Use this condition to measure standard curve, the degree of precision, absolute recovery, method recovery, and stability test of the samples.2.2The study on pharmacokinetics Divided 10 healthy rats into catalpol group and catalpol praeparatum group randomly, 5 for each group. Catalpol and its praeparatum were injected by vena caudalis 5.0mg/kg to the 2 different 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 fossa 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 and catalpol praeparatum in rats.2.3Detection of the concentration of catalpol in CFSDivided 10 healthy rats into catalpol group and catalpol praeparatum group randomly, 5 for each group. Catalpol ande its praeparatum were injected by vena caudalis 5.0mg/kg to the 2 different groups. After injection 40 min, take suction 0.1 ml CFS from cavitas subarachnoidealis to measure the concentration of drug.Results:1. The research of praeparatum of catalpol on its prescription and technology1.1The assaying of catalpol in water-solution 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 5.9 min, the resolution with the closed chromatographic peak was more than 1.5. From drawing the standard curve, it was found that the linear relationship was well when the concentration of catalpol was between 5.0⁷0μg/ml. The linear regression equation is: y=4.0139x+1.8369(r=0.9999).The lowest delectability is 0.3μg/ml.The recovery is 100.22%. The derivation and reproducibility RSD% were less than 2%.Catalpol praeparatum was stable in water for 8 hours.1.2Research of prescription and technologyPrescription and preparation: catalpol 10g, manicol 100g, mycose 50g, 10% NaOH solution 150ml for making the solution pH6.8～7.0, add water for injection to 2000 ml for 1000 bottles. Lyophillization: sample's eutectic point is -20℃. Freeze the samples under -40℃for 3 hours. Lyophylization: evacuation to reach 1～3Pa, rise the temperature to -24℃in 3 hours, keep 9 hours. Resolution drying: rise the temperature to 40℃in 3 hours, keep 8 hours, cryodesiccation is end. The finished products were consistent with the requests.2. The study of catalpol in pharmacokinetics and in CSF of rats2.1The analytical method of catalpol in serum and CSFThe analytical method of catalpol in 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 8000, the retention time was about 10.3 min, the resolution with the closed chromatographic peak was more than 1.5. From drawing the standard curve, it was found that the linear relationship was well when the concentration of catalpol was between 0.5⁶0μg/ml. The linear regression equation is: y=5376.4x+859.1(r=0.9996). The lowest delectability is 0.3μg/ml. The absolute recoveries of the three drug's concentrations are (88.02±2.21)%, (87.36±3.14)%, (89.80±0.08)%.The method recoveries are (101.36±4.32)%, (99.75±2.78)%, (100.05±1.89)%. The with-day and between-day derivation RSD were less than 6%. Catalpol in serum was stable in a refrigerator of -20℃for 15 days(P>0.05).The analytical method of catalpol in CSF: the number of theoretical plates of catalpol in CSF sample was more than 8000, the retention time was about 10.3 min, and the resolution with the closed chromatographic peak was more than 1.5. From drawing the standard curve, it was found that the linear relationship was well when the concentration of catalpol was between 0.5⁴0μg/ml. The linear regression equation is: y=5214.1x+1499.4(r=0.9997). The lowest delectability is 0.3μg/ml. The absolute recoveries of the three drug's concentrations are (99.22±1.71)%, (89.11±1.17)%, (86.87±0.98)%.The method recoveries are (99.82±1.98)%, (101.11±3.04)%, (100.12±2.30)%. The with-day and between-day derivation RSD% were less than 4%. Catalpol in CSF was stable in a refrigerator of -20℃for 15 days(P>0.05). 2.2The study on pharmacokinetics Catalpol and its praeparatum were injected by vena caudalis 5.0mg/kg to the 2 different groups. The action of kinesics was consistent with the feature of the two-compartment model. The important parameters of pharmacokinetic are: catalpol: the disposition half life of the serum(T_1/2α)are (27.47±5.10)min;the elimination half life of the serum (T_1/2β) are:(165.13±12.47) min; the apparent volumes of distribution of the central compartment (Vc) are:(0.1983±0.0159mg/kg)/(μg/ml). The clearance rate (cl) are:(0.0014±0.0001)mg/kg/min/(μg/ml); area under curve (AUC) are:(3500.95±240.41)(mg/ml) . min. catalpol praeparatum: catalpol: the disposition half life of the serum(T_1/2α)are:(30.52±5.81);the elimination half life of the serum (T_1/2β) are:(182.25±36.32)min;the apparent volumes of distribution of the central compartment (Vc) are: (0.1919±0.0162)(mg/kg)/(μg/ml).The clearance rate (cl) are: (0.0015±0.0001)mg/kg/min/(μg/ml); area under curve (AUC) are: (3451.66±210.41) (mg/ml) / min.There is no obviously variance between catalpol and catalpol praeparatum(P> 0.05).Detection of the concentration of catalpol in CSF: catalpol and its praeparatum were injected by vena caudalis 5.0mg/kg to the 2 different groups. After injection 40 min, the concentration of catalpol in CSF of catalpol group is (5.09±1.03)μg/ml, the concentration of catalpol in CSF of catalpol praeparatum is (6.19±0.93)μg/ml.Conclusion: 1. In the research, it determined the prescription and the forming technology for the praeparatum of catalpol by lyophillization and supplied clinic a new praeparatum of catalpol to cure cerebral ischemia.2.In the research, the reversed phase high performance liquid chromatography (RP-HPLC) was used to measure the concentration of catalpol in water solution, serum and CSF, this method is good in linearity and easy to carry on, can measure the concentration of catalpol precisely.3. According the pharmacokinetics, results indicated that the adjuvants in praeparatum of catalpol didn't impact the elimination axiom of catalpol in vivo. The adjuvants were reasonable. The result of concentration of catalpol in CSF indicated that the adjuvants couldn't advance catalpol into blood brain barrier (BBB).