【作者】 陈秀杰；

【导师】 毕开顺； 陈晓辉； 于治国；

【作者基本信息】 沈阳药科大学 ， 药物分析学， 2010， 博士

【摘要】 本研究选择具有显著舒张血管作用的黄酮类化合物---木犀草素为研究对象,为提高木犀草素的生物利用度,研究了木犀草素乙酰化衍生物的定向合成工艺、药理活性和药动学行为,为木犀草素的开发提供依据。1.采用乙酰化定向合成方法,以木犀草素和乙酰氯为原料合成了三乙酰化木犀草素(TRALT)和四乙酰化木犀草素(TEALT).反应条件为：木犀草素和乙酰氯的物料比为1：4,溶剂为吡啶和丙酮(1：1,v/v),常温反应6h,反应产物混合物经柱层析分离得到两个化合物,经HPLC归一化法检测纯度均达到98%以上。经IR,1HNMR,MS鉴定,确定为三乙酰化木犀草素和四乙酰化木犀草素。2.为了研究木犀草素、三乙酰化木犀草素和四乙酰化木犀草素的药理活性强弱,对3个化合物进行了大鼠胸主动脉环舒张作用和大鼠常压密闭抗缺氧作用的对比研究。在舒张血管实验中,考察了木犀草素、三乙酰化木犀草素和四乙酰化木犀草素在内皮完整和内皮不完整情况下对去甲肾上腺素(NA)诱导的大鼠胸主动脉收缩的舒张作用。结果表明木犀草素对NA诱导的血管收缩具有舒张作用,且这种作用具有浓度依赖性,无内皮依赖性。三乙酰化木犀草素和四乙酰化木犀草素呈现相同的作用趋势,其中四乙酰化木犀草素的舒张血管作用在三者中最显著。在动物常压密闭抗缺氧实验中,考察了木犀草素、三乙酰化木犀草素和四乙酰化木犀草素对小鼠耐缺氧作用的影响。结果表明3个受试化合物的平均存活时间(min)均长于阳性药物地奥心血康(22.9±2.5)(P<0.05),且四乙酰化木犀草素组小鼠的平均存活时间(29.3±3.0)显著长于木犀草素组(23.0±3.5)和三乙酰化木犀草素组(24.9±3.4)(P<0.05)。根据上述体内、体外药理活性实验结果,进一步选择四乙酰化木犀草素作为研究对象,进行四乙酰化木犀草素抗心肌缺血作用、木犀草素和四乙酰化木犀草素在大鼠体内药动学比较研究。3.采用静脉注射垂体后叶素致大鼠急性心肌缺血和冠脉结扎致大鼠心肌梗塞药理模型从血流动力学、心肌收缩和舒张性能等方面考察了四乙酰化木犀草素对心肌缺血的防治作用,并辅助以调节血脂和活血化瘀实验,评价四乙酰化木犀草素的药理作用。实验结果表明：其一,四乙酰化木犀草素能够明显降低心肌缺血大鼠Ⅱ导联心电图ST段及T波的异常抬高；减慢心率(HR),降低平均动脉压(MAP)和左心室收缩峰压(LVSP)升高幅度,改善心肌缺血时的血流动力学状态；并且降低左心室舒张末期压力(LVEDP),升高左室内压最大下降速率(-dp/dtmax),对左室内压最大上升速率(+dp/dtmax)无明显影响。其二,四乙酰化木犀草素能降低冠脉结扎致心肌大鼠ST段和T波的异常抬高；减慢心率(HR),降低平均动脉压(MAP)和左心室收缩峰压(LVSP);对左室内压最大上升速率(+dp/dtmax)无明显影响,降低左心室舒张末期压力(LVEDP),升高左室内压最大下降速率(-dp/dtmax);能降低冠脉结扎致心肌缺血大鼠心肌梗塞范围和心肌耗氧指数(MVO2I,降低血清中CK, LDH, MDA含量,升高SOD活性和NO水平；其三,四乙酰化木犀草素能降低高血脂大鼠血清中总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)水平,同时升高高密度脂蛋白(HDL)水平。其四,四乙酰化木犀草素能明显降低急性血瘀大鼠高、中切全血粘度(ηb),高切全血还原粘度(ηrv),红细胞聚集指数(AI)。以上结果表明,四乙酰化木犀草素能改善心肌缺血时的血流动力学状态,减少回心血量,改善心肌舒张性能,不影响心肌的收缩性能,降低心肌耗氧量；提高心肌抗氧化酶活性来对抗自由基损伤,保护和稳定心肌细胞,改善心肌功能；同时它还具有调血脂作用和降低急性血瘀模型动物的血液粘稠度,改善血液流变状态,从多方面发挥抗心肌缺血作用。4.建立了测定大鼠血浆中木犀草素和四乙酰化木犀草素的HPLC方法。木犀草素和四乙酰化木犀草素均在0.04-1.6μg.mL-1浓度范围内线性关系良好(r=0.9997和r=0.9990),方法回收率分别为96.1%(RSD%=2.4%)和93.3%(RSD%=2.3%)。采用本研究建立的方法,对木犀草素和四乙酰化木犀草素在大鼠血浆中的药动学进行了比较研究。结果表明大鼠灌胃木犀草素(3Omg.kg-1)后,其血浆浓度经时变化过程符合二室模型,Tmax为0.47 h, Cmax为895 ng.mL-1,AUC0→t为3443ng.h.mL-1,t1/2为3.7 h。大鼠灌胃四乙酰化木犀草素(30mg.kg-1)后,其血浆浓度经时变化过程符合二室模型,Tmax为0.51 h, Cmax为1666ng.mL-1,AUC0→t为4937ng.h.mL-1,t1/2为4.38 h。结果表明四乙酰化木犀草素吸收时间无显著变化,但吸收量显著增加,体内排泄趋于减慢,呈现良好的生物利用度。综上所述,本研究将药物化学、药理学、药物分析学相结合,考察了四乙酰化木犀草素的抗大鼠心肌缺血作用和体内药动学行为,为四乙酰化木犀草素的开发提供依据。更多还原

【Abstract】 Tri-acetyl-luteolin (TRALT) and tetra-acetyl-luteolin (TEALT) were oriented synthesized to improve the pharmacokinetics of luteolin and were examined their pharmacological effects by observing the relaxation effects of luteolin(LT), TEALT and TRALT on norepinephrine (NA)-induced contractions in the aorta rings isolated from rats with or without endothelium. The results showed that they all attenuated the contraction in a concentration-dependent manner and in an endothelium-independent manner.Effect of vasodilation of tetra-acetyl-luteolin is the strongest one among them.Moreover, the anti-anoxia activity of luteolin and its acetylated derivates were observed. the mean survival times of rats in three treated groups is longer than the positive drug control group(22.9±2.5), and the survival time of the rats treated by tetra-acetyl-luteolin(29.3±3.0) was significantly longer than luteolin(23.0±3.5) and its tri-acetyl-luteolin groups(24.9±3.4)(P< 0.05). Hence, we selected tetra-acetyl-luteolin to investigate the pharmacokinetics and pharmacodynamics of anti-myocardial ischemia of rats in vivo.To investigate the pharmacodynamic efficacy and mechanism of Tetra-acetyl-luteolin on the experimental animal model of acute myocardial infarction (AMI) through hemodynamics, myocardial contractility and dilatity. The rat model of acute myocardial infarction (AMI) was induced by Piturin (Pit) injection, the influence on blood-lipids in hyperlipidemia rats and hemorheological parameters in acute hypostasis rats were also studied. The results are①In rats with AMI by Piturin injection, Tetra-acetyl-luteolin desent significantly the raised ST segment and T wave in ECG leadⅡ, slowed down significantly HR and lowered significantly the raised BP, LVSP and LVEDP, increased-dp/dtma-, but no obvious influence on+dp/dtmaX.②In rats ligated the left desend branch of cornary artery, Tetra-acetyl-luteolin desent significantly the raised ST segment and T wave in ECG lead II, slowed down HR and lowered BP, LVEDP, LVSP and MVO2I, increased-dp/dtmax, but no obvious influence on+dp/dtmax.It reduced the myocardial infarction size and lowered serum LDH, CK, MDA and increased serum SOD, NO.③Tetra-acetyl-luteolin lowered significantly serum TC, TG, LDL and increased HDL in hyperlipidemia rats fed with hyperlipidemia food.④Tetra-acetyl-luteolin lowered whole blood viscosity, whole blood reduction viscosity and AI of acute hypostasis rats. The results indicated Tetra-acetyl-luteolin could improve hemodynamics and myocardial dilatity in AMI animal model, did not influence myocardial contractility maredly and decreased myocardial oxygen consumption. It could improve myocardial enzyme activity and energy metabolism in AMI. Those mentioned above are the main mechanism of Tetra-acetyl-luteolin against AMI,simultaneously it could regulate blood lipids in acute hyperlipidemia rats and lower whole blood viscosity, improve hemorheology in acute hypostasis rats, and tetra-acetyl-luteolin may protect myocardium from ischemia injury.Accurate and reproducible HPLC methods were developed and validated for the determination of concentrations of luteolin (LT) and tetra-acetyl-luteolin (TALT) in rat plasma. The linear calibration curves were obtained in the concentration range of 0.04-1.6μg.mL-1 (r=0.9997 and r=0.9990) with a lower limit of quantification (LLOD) of 0.04μg.mL-1. The average recoveries were 96.1%(RSD%=2.4%) and 93.3%(RSD%= 2.3%), respectively.The pharmacokinetics of luteolin and tetra-acetyl-luteolin was investigated by HPLC method. The plasma concentration-time curves of luteolin and tetra-acetyl-luteolin could both be evaluated by the two compartment model. Luteolin’s Tmax, Cmax,AUC, t1/2 were 0.47 h,895ng.mL-1. and 3443 ng.h.mL-1,3.70 h, respectively; tetra-acetyl-luteolin’s Tmax, Cmax,AUC, t1/2 were 0.51 h,1666ng.mL-1,4937 ng.h.mL"1,4.38h respectively.Above all, tetra-acetyl-luteolin showed potent effect of anti-myocardial ischemia of rats in vivo, and had good pharmacokinetic profiles, which provide the basis for developing it further.更多还原