Study On The Pharmacokinetics Of Xiaoaiping And Chlorogenic Acid In Rats And The Quality Standard Of Xiaoaiping Preparations

Xiaoaiping isolated from the stems of Marsd enia tenacissima, an Asclepiadaceae plant, has been documented to possess functions against inflammation and cancers. The quality control method of Xiaoaiping was investigated.The pharmacokinetics of chlorogenic acid and constituent chlorogenic acid in Xiaoaiping in rats were compared.The quality control method was studied for the evaluation.The results of this study can provide reference for clinic trial and further studies.Part one The content determination of chlorogenic acid in xiaoaiping tablet and injection by HPLCObjective:To establish a HPLC method for the content determination of chlorogenic acid in Xiaoaiping Tablet and injection.Methods:The Diamonsil C₁₈ column(250mm×4.6mm,5μm) was used.The mobile phase was consisted of 0.2% phosphoric acid with methyl alcohol (65:35). The detective wave length was at 327nm, the velocity of flow was 1.0ml·min^-1 , the temperature of the column was ambient temperature,and injection volume was 20μl.Results:The linear ranges of Chlorogenic acid was 2.22⁹6.2μg,the linear regression equation was Y=57382X+28096,r = 0.9999, The Precision of chlorogenic acid was good and RSD was 1.72%. The recoveries of high, medium and low concentration were 99.9%¹03.7%.When the Signal-to-Noise was 3,LOD(limit of detection) of Chlorogenic acid was 0.00148μg.When the Signal-to-Noise was 10,LOQ (limit of quantitation) of chlorogenic acid was 0.00592μg. The stabiliy of samples was good within 24 hours.Conclusion:The method is simple, rapid, accurate . It can be used for the quantitative analysis of xiaoaiping tablet and injection.Part two Studies on the HPLC fingerprints of Xiaoaiping InjectionObjective:To establish the chromatographic fingerprint for the quality control of Xiaoaiping injection.Method:Analysis on a Diamonsil C18(250mm×4.6mm,5μm)column eluted with mobile phases containing Methanol and Phosphate buffer ingradient mode(0.00～10.00 min, 0â†'5% B;12.00～30.00 min;5%â†'20% B;32.00～80.00min,20%â†'45%B).The flow rate was 1.0 mL·min^-1,and the detection wavelength was at 327 nm.The temperature of column was 25℃.And the data of 10 batches of Xiaoaiping injection were analysed by"similarity evaluation for chromatographic Fingerprint of Traditional Chinese Medicine"software.Result:Nine common peaks were selected in chromatograms,and all the common peeks were separated effectively.Chlorogenic acid peak as the reference peak, the relative retention time and relative peak areas RSD are in line with requirements.Conclusion:The precision,stability,and repeatability of this method were satisfying.The method developed can be used to control the quality of Xiaoaiping injection.Part there Analytical methods of chlorogenic acid in biological samples by HPLCObjective:According to the structural characteristics of chlorogenic acid, establish a HPLC analytical methods for serum in rats. so as to provide detecting technology for pharmacokinetic studies .Methods: (1)Using Diamonsil C18 chromatographic column (250mm x 4.6 mm, 5μm),mobile phase:0.2%phosphate-methanol(65:35) and velocity of 1.0ml?min^-1.The internal standard is tinidazole.Uses photodiode array detector. Detected wave length:327nm; (2) Basic methods of biological samples treatment: take 100μl samples and 100ul tinidazole, add 700μl of methanol for extraction and protein deposition, then filtered by 0.22μm microporous membrane, drying by distillation of 37℃water bath, solid residues dissolved with 100μl mobile phase, 20μl for injection.Results:Chlorogenic acid work curve of linear range for 0.45μg～112μg(the ratio of CGA and TNZ), Y=0.4058X+0.9881, r=0.9997. the limit of detection was 0.0018μg and the limit of quantification was 0.0062μg. The relative recovery for the analysis of CGA in three different concentrations (high, medium, low) were98.55%,104.95%,83.24%. The precision(RSD) of injection in three different concentrations (high, medium, low) were 3.83%,2.29% and 3.07%,the precision(RSD) of repeatabilit y for the analysis were 4.64%,1.26% and1.58%.The chlorogenic acid in serum has no significant change in 30 days when it was stored in-20℃.Conclusions:The measurement of chlorogenic acid in biological samples was achieved by HPLC method. The method was sensitive,specific and simple enough to determine the concentration of chlorogenic acid and to obtain pharmacokinetic parameters.Part four The Pharmacokinetic study of chlorogenic acid and xiaoaoping in ratsObjective:To study The pharmacokinetics of chlorogenic acid and constituent chlorogenic acid in Xiaoaiping in rats.Method:(1) Chlorogenic acid group: Sixty Wistar rats were divided into 6 groups randomly, each group has 10 rats (♂5,♀5 ) . One group ig. chlorogenic acid 40mg·Kg^-1 ,20mg·Kg^-1and 10mg·Kg^-1,about 0.5ml blood samples were collected from tail vein before administration and at10min,30min,1h,1.5h,2h,4h,6h,8h,12h,24h after administration. another group iv. chlorogenic acid 40mg·Kg^-1 ,20mg·Kg^-1and 10 mg·Kg^-1 ,about 0.5ml blood samples were collected from tail vein before administration and at 5min,10min,20min,30min,45min,60min,90min,120min,180min,240min after administration. (2) Xiaoaiping group: Twenty Wistar rats were divided into 2 groups randomly, each group has 10 rats(♂5,♀5). One group ig. Xiaoaiping solution 20ml·kg^-1(Equivalent to CGA 20 mg·kg^-1),about 0.5ml blood samples were collected from tail vein before administration and at10min,30min,1h,1.5h,2h,4h,6h,8h,12h,24h after administration. another group iv. Xiaoaiping injection 2.5ml·kg^-1 ( Equivalent to CGA20mg·kg^-1 ) about 0.5ml blood samples were collected from tail vein before administration and at 5min,10min,20min,30min,45min,60min,90min,120min,180min,240min after administration. (3) The data was automatic handled with 3P97 pharmacokinetic software, calculating the main pharmacokinetic parameters of each rat with the best compartment model as the standard, using statistical moment method for calculating AUC and MRT values and calculating the absolute bioavailability.Results:(1)The three dose groups (high, medium, low) failed to detect the CGA in the 0-24h after rats taked the chlorogenic acid and Xiaoaiping. (2) the mean serum concentration-time curves of chlorogenic acid after iv. with three doses(high, medium, low) were confirmed to open two-compartment model. The main pharmacokinetic parameters were:â‘ Group iv.40mg·kg^-1:t_{1/2 alpha}=19.781 min,t_{1/2 beta}=68.377min,V_c= 0.847L·kg^-1·min^-1,CL_s= 0.029L·kg^-1·h^-1;â‘¡Group iv.20 mg·kg^-1:t_{1/2 alpha}=15.221min, t1/2 beta=59.458min,V_c= 0.446L·kg^-1·min^-1,CL_s= 0.017L·kg^-1·h^-1;â‘¢Group iv.10 mg·kg^-1:t_1/2alpha=7.878min,t_1/2beta=56.783min,V_c=0.257L·kg^-1·min^-1,CL_s=0.012L·kg^-1·min^-1. (3) the mean serum concentration-time curves of Xiaoaiping after iv. was confirmed to open two-compartment model. The main pharmacokinetic parameters were: t_1/2alpha=12.559min,t_{1/2 beta}=48.283 min,V_c= 0.403L·kg^-1·min^-1,CL_s= 0.018L·kg^-1·h^-1.Conclusions:The chlorogenic acid can not be detected in the serum after rats taked the chlorogenic acid and Xiaoaiping. The serum concentration of chlorogenic acid declined quickly after iv. in rats, thet_{1/2 alpha} was about 20 min and the t_{1/2 beta} was about 1h, the t_{1/2 beta} of high dosage group was higher than low dosage group significantly. Apparent volume of distribution (V_c) and total clearance (CL (s)) was positively correlated with dose. When dose increases, the concentration in tissue and cell increases, the body's ability to clear drugs enhances. The other components of Xiaoaiping injection have a synergistic effect on the metabolism of chlorogenic acid, apparent volume of distribution and total clearance of drug did not change significantly.