Pharmacokinetics Of Dihydromyricetin In Animal

Objectives:1. To establish a method for determination of dihydromyricetin ( DMY ) in biosamples.2. To study the pharmacokinetics character and the toxicokinetics character of DMY by determining the plasma concentration after intragastric administration in Beagle dog with the RP-HPLC-UV method.3. To study the pharmacokinetics and tissue distribution of DMY in rats following intragastric administration with the RP-HPLC-UV method.Methods:1. Determination method of DMY in biosamples: The biological samples extracted by Methyl Cyanide ( EtAc ) were separated on a ZORBAX Eclipse CDB-C18 ( 4.6 mm × 150 mm, 5 ?m) for 20 minutes by gradient elution with mobile phase contained EtAc and phosphoric acid ( 0.1 %) at flow rate of 1.0 mL/min and column temperature of 25 ?. The elution mode was: EtAc -phosphoric acid (0.1%): 0?13 min: 15 % : 85 %?38 % : 62 %, 13?16 min: 38 % : 62 %, 16?20 min: 38 % : 62 %?15% : 85 %. The sample volume was 10 ?L. Detecting with the wavelength of 292 nanometer. Carbamazepine (car) was used as internal standard (IS ). Use the peak area ratio of DMY and IS ( y) and DMY concentration(x ) for regression analysis to get the standard linear regression equation. Calculate the samples' concentration according to the standard linear regression equation.2. Preparation of plasma sample for DMY: Take arterial blood from the animal with heparin for anti-coagulation ( 8 units/ml of whole blood ), centrifugate for 10 minutes at the speed of 4000 r/min and draw plasma sample. Take plasma sample of 500 ?L, add 5 ?L of car working solution and 10 ?L of phosphoric acid solution, centrifuge for one minute at 4000 r/min and mix them up completely, and operate in the ice water bath. Respectively use 1.0 mL, 0.5 mL and 0.5 mL of EtAc to extract 3 times by vortex oscillation, 3 minutes each time, centrifuge for 5 minutes at 4000 r/min each time, take 0.75 mL, 0.5 mL and 0.5 mL of the supernatant liquor respectively, compound and dry them by nitrogen flow at of 0.7 L/min each tube and at 25 ?. Dissolve all the samples with 100 ?L of carbinol by vortex oscillation for 30 seconds and centrifuge for 3 minutes at 4000 r/min. Draw the clear solution.3. Preparation of tissue sample for DMY: Take tissue homogenate sample of 500?L, add 5 ?L of car working solution and 10 ?L of phosphoric acid solution,centrifuge for one minute at 4000 r/min and mix them up completely, and operate in the ice water bath. Respectively use 1.0 mL, 0.5 mL and 0.5 mL of EtAc to extract 3 times by vortex oscillation, 3 minutes each time, centrifuge for 8 minutes at 4000 r/min for the first time, 5 minutes for the second and the third time, take 0.75 mL, 0.5 mL and 0.5 mL of the supernatant liquor respectively, compound and dry them by nitrogen flow at of 0.7 L/min each tube and at 25 ?. Dissolve all the samples with 100 ?L of carbinol by vortex oscillation for 30 seconds and centrifuge for 3 minutes at 4000 r/min. Draw the clear solution.4. Comparing the pharmacokinetics character of DMY between single and multiple dosage orally administrated in Beagle dog : Beagle dogs were administrated orally with single or multiple dosage of DMY at 150 mg/kg and blood samples were collected following a serials of set time points. The concentration of DMY in plasma was determined using the validated RP-HPLC-UV method . Pharmacokinetic parameters were caculated by Pharmacokinetic Module of DAS2.1 software. Making statistical analysis of pharmacokinetic parameters for DMY gained by the two administration roughts.5. Pharmacokinetics of DMY in rats: Rats were administrated orally with single dosage of DMY at 150,100,200 mg/kg,respectively. Then blood samples were collected following a serials of set time points. The concentration of DMY in plasma was determined using the validated RP-HPLC-UV method. Pharmacokinetic parameters were caculated by Pharmacokinetic Module of DAS2.1 software. Making statistical analysis of pharmacokinetic parameters for 3 dosage groups of DMY.6. Tissue distribution of DMY in rats: After a single oral dosage of 200 mg/kg of DMY,the rats were sacrificed by cervical dislocation at 0, 0.33,0.67, 1.5 h. Liver,brain and kidney were removed and homogenized with ultrapure water to prepare 0.2 g/mL homogenates. DMY in tissue homogenates were determined using the validated RP-HPLC-UV method. Concentration-time curve were studied.7. Toxicokinetics of DMY in Beagle dog: Beagle dogs were administrated orally with single dosage of DMY at 100 mg/kg,500 mg/kg, 2500 mg/kg,respectively. Then blood samples were collected at the first time and the last time of administration following a serials of set time points. The concentration of DMY in plasma was determined using the validated RP-HPLC-UV method. Pharmacokinetic parameters were caculated by Pharmacokinetic Module of DAS2.1 software. Making statistical analysis of pharmacokinetic parameters both the first and the last administration for 3 dosage groups of DMY.Results:1. Preparation of plasma sample for DMY and the sample stability: Extratction rate of DMY from plasma was significantly higher by ethyl acetate than acetonitrile. The extratction rate was nearly to 80 % by ethyl acetate for three times and it was higher at a low temperature. Recovery rate of DMY in plasma was higher at lower temperature and in shorter time according to drying way by nitrogen gas flow. DMY plasma samples were stable at room temperature for 1 hour and in refrigerator for 2 weeks,but unstable at room temperature for 2 hours and freezing-thawing.2. Determination method of DMY in biosamples: The RP-HPLC-UV method for determinating DMY of plasma, liver, brain and kidney has great systemic suitability and specificity. The extraction percentage was more than 70 %. The intra- and inter-day precisions ( RSD % ) were less than 10 % for the method,Calibration curves of Beagle dog and rat plasma were linear from 20 to 1000 ?g/L for DMY, with correlation coefficients more than 0.996, and the Lower Limit of Quantification ( LLOQ ) was 0.5 ng. Calibration curves of rat tissue, including liver, brain and kidney,were linear from 20 to 1000 ?g/g for DMY,with correlation coefficients more than 0.999,and the LLOQ was 0.5 ng.3. The pharmacokinetics of DMY in Beagle dog: In the detection of Beagle dog plasma samples, the quality control samples of high, medium and low concentrations, the lower limits of accuracy are above 85 % and the upper limits are within 115 %. So it is considered that the plasma samples have high accuracy of DMY detection. The pharmacokinetic research of administering DMY to the Beagle dogs shows that the procedure in vivo is coincident with the two-compartmental model with great individual variance, at both single-dose and multiple-dose of 150 mg/kg. DMY AUC0-t of single-dose was 4223 ?g/L·h, and the AUC0-t of multiple-dose was 253 ?g/L·h. Compared with the single-dose administration, the absorption of DMY decreased, while the rate of elimination accelerated obviously. DMY plasma concentration did not reach steady-state concentration ( CSS ) and there was no accumulation by multiple-dose oral administration.4. Pharmacokinetics of DMY in rats: In the detection of rat plasma samples, the quality control samples of high, medium and low concentrations, the lower limits of accuracy are above 85 % and the upper limits are within 115 %. So it is considered that the plasma samples have high accuracy of DMY detection. After oral administration of DMY to the rats at the dose of 50 mg/kg, 100 mg/kg, and 200 mg/kg, the peak value of the drug concentration decreased with the increasing of DMY dose. And to 200 mg/kg group it fell to 1/5 ? 1/9 of the peak value 6 ? 8 hours later, while DMY did not detected in the other 2 groups. After oral administration of DMY to the rats, the drug concentration in the blood and kinetic process varied greatly among individuals, and the dose was an important factor. According to the analysis of statistical moment parameters, AUC, Cmax,Clearance in plasma ( CL ) and apparent volume of distribution (V) for DMY rose obviously with DMY dose increased(p<0.05). increased as DMY increased. While the MRT, t1/2 and Tmax for DMY have no correlative relationship with the dose among the rats of 3 groups.5. Tissue distribution of DMY in rats: In the detection of rat tissue samples, the quality control samples of high, medium and low concentrations, the lower limits of accuracy are above 85 % and the upper limits are within 115 %. So it is considered that the plasma samples have high accuracy of DMY detection. DMY was detected in liver and kidney, at the time of 0.33 h after the rats oral administration for DMY with the dose of 200 mg/kg. DMY concentration both in liver and kidney have no great variance during the set research time, and concentration of DMY in kidney higher than which in liver, but the concentration of DMY in liver and kidney were lower than that in plasma. No DMY was detected in brain during the research time.6. Toxicokinetics of DMY in Beagle dog: The pharmacokinetic research of administering DMY to the Beagle dogs at the dose of 0.1 g/kg, 0.5 g./kg and 2.5 g/kg shows that the metabolic procedure is coincident with the two-compartmental model. After a single ( on the 1st day) intravenous oral administration, the CL for the three doses was 41 L/kg/h, 89 L/kg/h and 276 L/kg/h and the V was 82 L/kg,218 L/kg and 706 L/kg, respectively. The CL and V were significantly different among the three doses. The t1/2 for the three doses were 1.51 h, 1.79 h and 1.87 h,the t1/2 increased with the dose. The AUC for three doses were respectively 2494?g/L·h, 5546 ?g/L·h and 9096 ?g/L·h, which increased as DMY increased but there was no linear relationship between AUC and dose. After 3 months(on the 90th day)of oral administration, the CL for the three doses was 57 L/kg/h, 76 L/kg/h and 199 L/kg/li, respectively. CL in high group was great higher than that in low and medium group, with the statistic differencep<0.01. The V was 197 L/kg,170 L/kg and 325 L/kg, respectively. V were not significantly different among low and middle dose group. The t1/2 for the three doses was 2.18 h, 1.17 h and 1.17 h,respectively. The t1/2 decreased with dose. The AUC for the three doses were respectively 1758 ?g/L-h, 6528 ?g/L-h and 12935 ?g/L-h, and there was correlative relationship between AUC and dose (p<0.05 ) . When compared with those in the first single administration, the repeated administration resulted in an increase of V in low, while both the medium and high dose group it decreased and reduction in V and CL in high dose group. There was difference in t1/2 among the three groups.Conculsions:1. Temperature could be a important factor affecting stability of DMY in plasma samples. DMY plasma samples should be treated at lower temperature and stored in refrigerator after collection.2. It was a sensitive and accurate RP-HPLC-UV method for the quantitative determination of DMY in biosamples and suitable for the pharmacokinetic study of DMY.3. There was statistical difference of DMY in dogs between single-dosage and multiple-dosage at 150 mg/kg. The absorption of DMY decreased and the rate of elimination accelerated.4. Drug plasma concentration and metabolic process differed greatly among individuals after oral administration of DMY to rats. The absorption of DMY increased obviously, as well as the rate of elimination accelerated.5. Drug tissue concentration of DMY were low following orally administrated to rats, and there were difference for tissue distribution among liver, brain and kidney.6. After DMY orally administrated to Beagle dogs by once or for long time at large dose, the absorption of DMY increased and the rate of elimination accelerated obviously with the dose.7. Both the pharmacokinetic stduy of DMY for rat and the toxickinetic study of DMY for Beagle dog showed no statistical correlation between the absorption rate and the dose.