摘要
葛根素(Puerarin)是一种从中药豆科植物野葛、甘葛藤根中提取的异黄酮类活性成分。研究显示,葛根素具有抗氧化、降低胆固醇、降血糖、降血压、解酒保肝等药理活性,广泛用于心脑血管疾病和糖尿病等的治疗。体内体外研究表明,葛根素可以通过对炎症反应、细胞凋亡和中性粒细胞活化等的抑制作用对脑缺血造成的组织损伤有保护作用。但是溶解度小、体内半衰期短、脑组织中浓度低、口服生物利用度低等特点限制了其临床应用。
近年来,纳米(nanoparticles, NP)载药系统被用于将药物传输入脑和改善生物利用度。聚合纳米粒作为药物载体,可以提高体内药物浓度、控制药物释放并且可以减少毒副作用,是当今研究的热点。聚氰基丙烯酸丁酯纳米粒(polybutylcyanacrylate nanoparticles, PBCN)载药系统可以通过表面修饰吐温80(PS80)运载药物穿过血脑屏障(blood brain barrier, BBB),同时该载药系统还可以提高药物的口服生物利用度。
本研究以葛根素为模型药物,制备葛根素聚氰基丙烯酸丁酯纳米粒(puerarin polybutylcryanoacrylate nanoparticle, PUE-PBCN),通过静脉给药小鼠药代动力学和体内组织分布研究,大鼠口服给药体内药代动力学研究,以及脑缺血再灌注大鼠模型药效学研究,从药动学和药效学角度评价PBCN作为脑靶向给药载体的优势,并对其作为口服给药载体进行初步研究。
选用乳化聚合法制备PUE-PBCN,在单因素考察的基础上,采用正交设计优化、筛选最佳处方和制备工艺,制备的工艺参数为：pH2, PUE0.3%(w/v), BCA0.6%(v/v), F68/Dex70(0.5%/0.5%, v/v),乳化温度25℃,搅拌速度400r/min,搅拌时间4h。依据优化处方制备的PUE-PBCN制剂的外观为白色带乳光胶体溶液,电镜下观察呈圆形,无粘连,平均包封率为48.75%,平均载药量为15.02%,粒径分布范围较窄,平均粒径159.4nm, zeta电位-15mV。对PUE-PBCN进行差示扫描量热法、红外光谱和X-射线衍射分析,发现制成纳米粒后PUE已被PBCN包裹或吸附,不再以晶体结构存在,而是以非晶态存在。利用透析法考察了PUE-PBCN的体外释药行为,结果表明体外释药符合双相动力学过程,前期药物快速释放,后期缓慢释放,与葛根素对照溶液相比,PUE-PBCN具有显著的缓释作用。稳定性考察实验显示PUE-PBCN4℃条件下保存较为稳定。
小鼠体内PUE-PBCN的药代动力学和组织分布研究表明,小鼠尾静脉注射PUE-PBCN (10mg/kg)与相同剂量PUE注射液相比,药代动力学显著改变,药物体内滞留时间增加,血药浓度升高,AUC为后者的1.53倍。同时药物在各组织中的MRT显著增加,各组织AUC均较PUE注射液组增大,特别是脑组织AUC为PUE原药组的2.63倍；大鼠口服PUE-PBCN与PUE混悬液后体内药代动力学过程研究表明,与混悬液相比PBCN能够显著提高PUE的生物利用度,相对生物利用度为556.33%。
以神经功能学评分、实验动物体重、脑含水量、脑梗死体积百分率、脑组织病理形念学变化及氧化应激为主要评价指标,从药效学方面观察了PUE-PBCN静脉注射给药对脑缺血再灌注损伤的保护作用。实验结果显示：假手术组大鼠无明显神经功能缺损症状,体重减轻最小,左右脑含水量相似,脑梗塞面积最小,脑组织病理组织学观察正常,脑组织和血清SOD活性均为各组中最高；模型组大鼠较假手术组观察到明显的神经功能缺陷,体重减轻最大,右侧缺血大脑的脑含水量显著高于左侧正常大脑,梗塞面积显著增大,病理组织学检查显示脑组织病变侧海马回及齿状回组织结构均消失,间质大面积高度水肿,脑组织和血清SOD活性显著最低,MDA含量增高；PUE及PUE-PBCN组均能够改善大鼠各项评价指标,其中PUE-PBCN组与同剂量PUE溶液组相比,药理活性显著提高。
本研究结果证实了PBCN有助于PUE透过血脑屏障,显著增加PUE在脑组织的分布,改善PUE在胃肠道的吸收,提高了相对生物利用度,增强了PUE对局灶性脑缺血再灌注损伤的保护作用。本课题研究为探讨和总结葛根素脑靶向给药系统提供了实验依据,为缺血性脑卒中治疗探索新思路和新方法。
Puerarin is an isoflavone C-glycoside found in Traditional Chinese Medicinal herbs such as Pueraria lobata, Ohwi, and P.thomsonni Benth. During the last three decades, puerarin has become known for its profound pharmacological activities, including antioxidation, cholesterol-lowering, anti-diabetic, anti-hypertension, anti-alcoholism and hepatoprotection, and has been proved to be helpful in the treatment of cardiovascular, cerebralvascular and hyperglycemic diseases. It has been shown that Puerarin has the pharmacologic action of protecting against cerebral ischemia mediated by the inhibition of inflammatory responses, apoptosis, and neutrophil activation in vitro and in vivo. However, the poor solubility, short elimination half-life lower the drug concentration in brain and poor oral bioavailability limits the clinical advantages of puerarin.
In recent years, nanoparticles (NP) technology was used to deliver drugs to the brain and improve the bioavailability. Polymeric nanoparticles was the focus of attention as potential drug systems currently, which could enhance the concentration and slow the release of drug as well as decrease the peripheral toxicity. As one promising delivery system, polybutylcyanacrylate nanoparticles (PBCN) have attracted considerable attention. PBCN coated with polysorbate80(Ps80) is able to deliver drug to cross the blood brain barrier (BBB) as well as improve the oral bioavailability.
In the study,puerarin loaded polybutylcryanoacrylate nanoparticle (PUE-PBCN) was prepared, and the pharmacokinetics and tissue distribution in mice by intravenous administration of PUE-PBCN, and pharmacokinetic studies in rats by oral administration of PUE-PBCN, as well as the pharmacological effect of PUE-PBCN on the ischemia/reperfusion injury model in rats were studied. The results showed the possibility of PBCN as the drug delivery carriers of targeting brain and oral administration.
PUE-PBCN was prepared by emulsion polymerization. Optimizing formulation was designed by orthogonal experiment on the basis of single factor study, and showed asfollows:pH2, PUE0.3%(w/v), BCA0.6%(v/v), F68/Dex70(0.5%/0.5%, v/v), the temperature of emulsification was at25℃, the speed and time of stirring were400r/min and4h, respectively.
PUE-PBCN preparation was the white opalescent colloidal solution, exhibited a spherical shape under transmission electron microscopy with an average size of159.4nm, and the zeta potential was-15.0mV. Physicochemical state of PUE in PBCN was investigated by differential scanning colorimetry, X-ray diffraction and Fourier transform infrared spectroscopy. The in vitro release of PUE loaded PBCN showed an initial burst release followed by a sustained release. The preliminary stability study showed that the storage of PUE-PBCN at4℃could keep a good stability.
The results of pharmacokinetic and biodistribution to brain performed in mice after intravenous administration showed that the drug concentrations in blood and brain for PUE-loaded PBCN were both greater than that for the free drug. The oral pharmacokinetic study in rats showed that the relative bioavailability of PUE encapsulated PBCN to the crude PUE was more than550%.
Compared with free drug, the intravenous injection of puerarin loaded nanoparticles exerted the better neuroprotective effect on rats with focal cerebral ischemiec injury via significantly decreasing neurological deficit scores, increasing body weight, lowing brain water content, reducing the infarct volume, relieving brain tissue lesions, increasing SOD activity and decresing MDA level of brain tissue and serum.
The conclusions of this study confirm that PBCN can help PUE through the blood-brain barrier, significantly increase the drug distribution in brain tissue, improve the gastrointestinal absorption of PUE and increase the relative bioavailability, and enhance the protective effect of puerarin on focal cerebral ischemia/reperfusion injury. The results of this project could explore and summarize puerarin brain targeted delivery system, provide a theoretical basis, and provide a new idea and method for the treatment of ischemic stroke.
引文
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