鸦胆子油交联环糊精包合物的制备及其药效学考察

【作者】 陈琳；

【导师】 胡荣； 卜平；

【作者基本信息】 扬州大学 ， 药剂学， 2016， 硕士

【摘要】 鸦胆子油(Brucea javanica oil)是从苦木科植物鸦胆子成熟果实中提取而得的油脂类物质。现代药理学研究表明鸦胆子油具有抗癌、抗病毒、抑菌、抗胃溃疡、降脂降压、抑制血栓形成等药理作用。临床上使用鸦胆子油乳剂作为辅助抗肿瘤药物,主要用于治疗消化系统肿瘤。但乳剂属于多相动力学不稳定分散体系,在受热、受冷及长期储存过程中会引起乳析和破裂,而渗漏的鸦胆子油毒性大,对胃肠粘膜和血管壁的刺激性也很严重。因此亟需开发出良好的鸦胆子油新剂型尤其是口服剂型。本实验以环氧氯丙烷(EPI)为交联剂制备交联环糊精(CDP)并包合鸦胆子油(BJO),考察BJO-CDP包合物的口服给药的安全性和有效性,并与市售鸦胆子油乳剂进行比较,主要实验内容及结果如下：1 BJO-CDP包合物的制备①以p-CD单体为实验材料,EPI为交联剂制备CDP,通过扫描电镜法、红外光谱法、核磁共振法以及熔程的改变验证了CDP的形成。②通过均质法制备了鸦胆子油交联环糊精包合物(BJO-CDP),并采用扫描电镜法、红外光谱法、核磁共振法以及熔程的改变对BJO-CDP进行表征,验证了包合物的形成。③以鸦胆子苦醇为指标成分,建立HPLC分析包合物中鸦胆子苦醇含量的方法,并进行方法学考察。实验结果表明：鸦胆子苦醇浓度(X)与峰面积(Y)线性关系良好,鸦胆子苦醇回归方程为：Y=1×107X+987534,R2=0.999。鸦胆子苦醇的专属性、精密度及稳定性良好,可用于包合物含量测定。④以包封率为考察指标,采用单因素考察对制备中的主要工艺条件进行筛选,包括CDP与BJO投料比、包合温度、均质时间及稀释BJO的溶剂量等,以进行正交试验。⑤以包封率为考察指标,对CDP与BJO的投料比、包合温度、均质时间及溶解BJO的溶剂量四种因素进行正交试验设计,确定制备包合物的最佳工艺为：CDP与BJO投料比(质量比)为10：1,包合温度为20℃,均质时间为6min,稀释BJO的溶剂的量为0.5g。2 BJO-CDP包合物的质量考察①根据2015版《中国药典》凡例项下溶解度测定方法,对BJO-CDP在不同介质中的溶解度进行测定,结果表明：BJO-CDP在蒸馏水、人工胃液及人工肠液中溶解度良好,但在甲醇、乙醇中几乎不溶。②采用显微熔点测定仪,对β-CD、CDP及BJO-CDP包合物的熔程进行测定,结果表明：β-CD熔程为298-305℃,CDP熔程为271-278℃,BJO-CDP熔程为267-274℃。③在光照、高温及高湿条件下对BJO-CDP稳定性进行考察,结果表明：BJO-CDP在光照、高温条件下指标成分鸦胆子苦醇含量未发生明显变化,稳定性良好；在高湿条件下,包合物吸湿后产生粘稠、结块,提示包合物的吸湿性较强,在制备、储存及运输过程中应尽量保持密封干燥。3 BJO-CDP包合物的药效学考察①从鸦胆子油包合物对7721人肝癌细胞的体外抑制试验结果可以看出,鸦胆子油包合物的IC50为0.9518 mg/ml,鸦胆子油乳剂的IC50为1.219mg/ml,与市售鸦胆子油乳剂相比,鸦胆子油包合物对7721人肝癌细胞的体外抑制率有所提高。通过流式细胞仪检测BJO-CDP包合物对细胞凋亡影响的结果可以看出,随着药物浓度增加,包合物能明显诱导7721细胞凋亡。②从包合物对小鼠CT26结肠癌细胞移植性肿瘤抑制作用的实验结果中可以看出,鸦胆子油交联环糊精包合物中、高剂量组与鸦胆子油乳剂高剂量组对小鼠结肠癌细胞的生长均有显著抑制作用(p<0.05),抑瘤率分别为23.86%、42.61%、35.91%,且鸦胆子油交联环糊精包合物高剂量组对小鼠的体重影响较同剂量的鸦胆子油乳剂要小,说明鸦胆子油被制成交联环糊精包合物后体内抑癌效率提高,且毒性降低。③从急性毒性试验结果可以看出,鸦胆子油交联环糊精包合物的安全性显著高于市售鸦胆子油乳剂。BJO-CDP包合物的LD5o未能测出,市售鸦胆子油乳剂的LDso为9.780g/kg。本实验成功制备BJO-CDP包合物,并对其制备工艺及相关性质进行研究,结果表明：该包合物制备工艺可行,可显著改善BJO溶解度,提高药物稳定性及生物利用度。且制成包合物后,与临床使用乳剂相比,安全性提高,体内外抑制癌细胞的能力也有所提高,同时毒性降低。更多还原

【Abstract】 Brucea javanica oil (BJO) is extracted from the nucleoli of Brucea javanica (L) Merr (Simaroubaceae) which is widely distributed from Southeast Asia to northern Australia Brucea javanica is characterized as an antipyretic and detoxifying plant, and widely used in the treatment of lung, prostate, and gastrointestinal cancer, and has potent antimalarial, anti-inflammatory, and antiviral effects, with low toxicity. But application of BJO is restricted by its poor solubility, and BJO is administered as an emulsion (BJOE) for intravenous use. However, due to the thermodynamic instability of emulsions, BJOE easily delaminates, flocculates, and rancidifies during the storage. Additionally, because BJO is a traditional Chinese medicine of bitter and cold, it is bad for the stomach and intestines. To alleviate its negative effects on the digestive tract and improve its curative effects, it is essential to develop new formulation.CDP was obtained by reaction of β-CD with epichlorohydrin (EPI). And a new water-soluble inclusion complex of Brucea javanica oil (BJO) with β-cyclodextrin polymer (CDP) was prepared by saturated aqueous method.1 Preparation of BJO-CDP① CDP was obtained by reaction of β-CD with epichlorohydrin (EPI) and characterized by SEM,1H NMR, FT-IR and the change of melting range.② BJO-CDP was prepared by saturated aqueous method and characterized by SEM,1H NMR, FT-IR and the change of melting range.③ A HPLC method was established by using Brusatol as the index components and methodological investigation was carried out to evaluate the method. The results of HPLC showed that the peak area of Brusatol had a good linear relationship. The regression equation of Brusatol was Y=1 ×107X+987534, R2=0.999. The specificity, precision and stability all meet the requirement which showed that the HPLC method can be used to determine the content of the inclusion complexes.④Use single factor experiment to filter apposite mass ratio of BJO and CDP, temperature, time and concentration of solvent for orthogonal test.⑤The multiple factors including mass ratio of BJO and CDP, time, temperature and concentration of solvent were investigated in the inclusion process to optimize the preparation conditions of BJO-CDP. The result showed that the optimum preparation conditions for inclusion were established as follows:the mass ratio of BJO and CDP was 1:10, time was 6 min, temperature was 20 ℃ and the amount of ether was 0.5g.2 Quality Inspection①Research the solubility according to the legend items of the solubility determination in China Pharmacopoeia (version 2015) in different media of the lyophilized powder was determined. The BJO-CDP has good solubility in distilled water, artificial gastric juice and artificial intestinal fluid but almost insoluble in methanol, ethanol.②Melting range was also detected. The melting range of -CD was 298～305 ℃, CDP was 271～278℃, BJO-CDP was 267～274℃.③The stability of the BJO-CDP inclusion complexes was detected by putting them under the condition of illumination, high temperature and high humidity. The results showed that BJO-CDP has good stability under illumination and high temperature. The inclusion complexes becomes toughness and agglomeration at high humidity condition which indicated that we should try to keep dry at the procedure of preparation、storage and transport of BJO-CDP.3 Pharmacodynamics of BJO-CDP①The pharmacodynamics of BJO-CDP inclusion complexes was investigated using MMC-7721 cell lines. The commercial emulsion of BJO (BJOE) was used as a reference. The IC50 values obtained in BJO-CDP group and emulsions group were 0.9518 and 1.219mg/ml respectively. Flow cytometry revealed that medium and high dosage of drugs had significant role in inducing apoptosis of 7721 cells②In the experiment that the inhibition effects on mice colon cancer, significant inhibition effects on colon cancer was observed in BJO-CDP groups with middle dose (p<0.05) and high dose (p<0.05), whereas in BJOE groups, significant effect was only observed with high dose (p<0.05). Moreover, the BJO-CDP groups had less weight loss than BJOE groups. These results indicates that BJO-CDP had stronger antitumor activity with a lower toxitity than BJOE.③In the acute toxicity test, the median lethal dose (LD50) of BJOE was 9.78 g/kg. In contrast, all mice treated with inclusion complexes survived even at the highest dosage (15.36 g/kg).In conclusion, the present study suggests that BJO-CDP polymer significantly decreased toxicity of BJO and enhance the antitumor activity. Therefore, β-cyclodextrin plomer (CDP) may be a potential effective delivery vehicle for this poorly soluble antitumor drug.更多还原