【作者】 付晓秀；

【导师】 齐永秀；

【作者基本信息】 泰山医学院 ， 药理学， 2011， 硕士

【摘要】 消癌平片和消癌平注射液均为植物通关藤的单味制剂,具有抗癌、消炎、平喘等功效,绿原酸为其中的有效成分之一。研究消癌平制剂的质量控制方法,比较绿原酸单体及制剂在大鼠体内的药物代谢动力学规律,可以为评价消癌平制剂的质量提供方法,并为深入研究消癌平的药效学提供理论依据。第一部分HPLC法测定消癌平片和消癌平注射液中绿原酸的含量目的:建立测定消癌平片和消癌平注射液中绿原酸含量的HPLC法方法:色谱柱为Diamonsil（?）（钻石）C18柱（250mm×4.6mm,5μm）;流动相为0.2%磷酸-甲醇（65:35）;流速为1ml·min^-1 ;内标为替硝唑;检测波长为327nm;室温;20μl进样。结果:在2.22μg·ml^-1～96.2μg·ml^-1的范围内绿原酸浓度与绿原酸峰面积与内标峰面积的比值呈现良好的线性关系,线性方程为Y=0.0892X+0.0539（n=5）, r = 0.9999,方法精密度好,RSD（n=5）为1.72%;方法的回收率为99.9%¹03.7%;供试品溶液在分析浓度下,24小时内基本稳定。结论:建立了测定消癌平片和消癌平注射液中绿原酸含量的HPLC法,该法灵敏度高,专属性强,适用于消癌平制剂的含量测定。第二部分消癌平注射液指纹图谱的建立目的:建立消癌平注射液HPLC指纹图谱分析方法。方法:色谱柱:Diamonsil（?）（钻石）C₁₈柱（250mm×4.6mm,5μm）,以A（甲醇）:B（磷酸盐缓冲液）为流动相,梯度洗脱（0. 00～10. 00 min,0→5% B;12.00～30.00 min, 5%→20% B;32.00～80.00min,20%→45%B）,流速1.0mL·min^-1 ,检测波长327nm,柱温25℃,进样量20μL。采用《中药色谱指纹图谱相似度评价》（2004年A版）软件,计算10批消癌平注射液HPLC指纹图谱的相似度。结果:在消癌平注射液中检测到9个共有指纹峰,各峰分离度良好,以绿原酸峰为参照物峰,各共有峰相对保留时间和相对峰面积的RSD均符合要求。结论:所建立的HPLC指纹图谱方法具有较好的精密度、稳定性和重现性,可用于消癌平注射液的质量控制。第三部分绿原酸血药浓度的HPLC测定法目的:建立大鼠血清中绿原酸的HPLC分析方法,为绿原酸的药代动力学研究提供检测技术。方法:采用迪马DiamonsilC₁₈色谱柱（250mm×4.6mm,5μm）;流动相为0.2%磷酸-甲醇（65:35）,流速为1.0ml-min^-1;替硝唑为内标;检测波长为327 nm。取大鼠血清样品,加入内标,以甲醇去蛋白,0.22μm微孔滤膜过滤,37℃水浴挥干,固体残留物用100μl流动相溶解,20μl进样。结果:线性方程为Y=0.4058X+0.9881（r=0.9997）,在0.45μg^-112μg范围内,线性关系良好,该分析方法检测限为0.0018μg,最低定量限为0.0062μg,高、中、低三种不同浓度血清样品平均回收率分别为98.55%、104.95%、83.24%,精密度实验RSD值分别为3.83%,2.29%,3.07%,重复性实验RSD值分别为4.64%,1.26%,1.58%。血清中绿原酸在-20℃冷冻避光条件下30天内稳定性较好。结论:建立了测定大鼠血清中绿原酸含量的HPLC分析方法,该法灵敏度好,专属性强,能够为绿原酸的药动学研究提供方法支持。第四部分消癌平制剂及其绿原酸单体的药动学研究目的:研究大鼠在灌胃和尾静脉注射绿原酸单体和消癌平制剂后绿原酸在大鼠体内的药动学特征,探讨消癌平制剂中其他成分对绿原酸代谢的影响,阐明绿原酸的药动学变化规律。方法:（1）绿原酸组: Wistar大鼠60只,体重180～220g,随机分为6组,每组10只（♂5只,♀5只）。灌胃单次给绿原酸40mg·kg^-1、20 mg·kg^-1、10 mg·kg^-1,于给药后10min、30min、1h、1.5h、2h、4h、6h、8h、12h、24h尾尖取血0.5ml;尾静脉单次注射绿原酸40mg·kg^-1、20 mg·kg^-1、10mg·kg^-1,于给药后5min、10min、20min、30min、45min、60min、90min、120min、180min、240min于尾尖取血约0.5ml,测定绿原酸含量。（2）消癌平组: Wistar大鼠20只,体重180～220g,随机分为2组,每组10只（♂5只,♀5只）,其中一组灌胃单次给消癌平溶液20ml·kg^-1(相当于CGA 20 mg·kg^-1),于给药后10min、30min、1h、1.5h、2h、4h、6h、8h、12h、24h尾尖取血0.5 ml。另外一组尾静脉单次给消癌平注射液2.5ml·kg-(1相当于CGA20mg·kg^-1),于给药后5min、10min、20min、30min、45min、60min、90min、120min、180min、240min于尾尖取血约0.5 ml,测定绿原酸含量。（3）采用3P97药动学软件处理数据,以最佳房室模型为标准计算每只大鼠的主要药代动力学参数。结果:（1）大鼠灌胃绿原酸和消癌平片后,三个剂量组在0-24h的血清中均未能检测出CGA。（2）尾静脉注射高、中、低三种剂量后,绿原酸在大鼠体内的药动学行为均符合二室模型。主要药动学参数为:①iv.40mg·kg^-1组:t_{1/2 alpha}=19.781 min,t_1/2beta=68.377min,V_c= 0.847L·kg^-1·min^-1,CL_s= 0.029L·kg^-1·h^-1;②iv.20 mg·kg^-1组: t_{1/2 alpha}=15.221min,t_{1/2 beta}=59.458min,V_c= 0.446L·kg^-1·min^-1,CL_s= 0.017L·kg^-1·h^-1;③iv.10 mg·kg^-1组: t_{1/2 alpha}=7.878min,t_1/2beta=56.783min,V_c= 0.257L·kg^-1·min^-1,CL_s= 0.012L·kg^-1·min^-1。（3）静脉注射消癌平注射液后药动学行为符合二室开放模型,主要药动学参数为: t_{1/2 alpha}=12.559min,t_1/2beta=48.283 min,V_c= 0.403L·kg^-1·min^-1,CL_s= 0.018L·kg^-1·h^-1。结论:大鼠灌胃给绿原酸和消癌平片后,血清中均未能检测出CGA。大鼠静脉注射绿原酸后,起始阶段血药浓度下降迅速,分布相半衰期t_{1/2 alpha}小于20min,消除相半衰期t_{1/2 beta}约为1h,高剂量组的消除半衰期t_{1/2 beta}明显高于低剂量组,表观分布容积V_c和总清除率CL_s与给药剂量呈正相关,随给药剂量的增加,药物在组织与细胞中的浓度增大,机体对药物的清除能力增强。静脉注射消癌平注射液后,制剂中的其他成分对绿原酸的代谢消除有协同作用,表观分布容积与药物总清除率没有明显变化。更多还原

【Abstract】 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, 0→5% B;12.00～30.00 min;5%→20% B;32.00～80.00min,20%→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:①Group 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;②Group 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;③Group 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.更多还原