【作者】 刘星；

【导师】 张汝华； 陈大为； 张荣庆；

【作者基本信息】 沈阳药科大学 ， 药剂学， 2002， 博士

【摘要】 本文选用天然蜂毒作为模型药物，尝试开展了其体内外抗肿瘤作用及其体外抑制黑色素瘤分子机制的初步探讨，在此基础上进行了口服结肠定位释药系统的实验研究。研究的内容主要包括：（1）蜂毒体内外抗肿瘤作用及其机理研究；（2）蜂毒指标性成分体内外分析方法的建立；（3）蜂毒理化性质及其初步稳定性的考察；（4）蜂毒结肠定位释药系统的设计；（5）蜂毒口服包衣微丸体内过程的初步评价五个部分。 四甲基偶氮唑盐染色法实验结果表明，蜂毒显著抑制体外肿瘤细胞生长增殖，并具有浓度和时间依赖关系，IC₅₀为2.73～13.64μg／ml。流式细胞测量结果证明天然蜂毒可将黑色素瘤K1735M2细胞周期阻断于G₁期，并有亚二倍体峰出现。琼脂糖凝胶电泳可以看到DNA片断的分带，即产生DNA Ladder现象，蜂毒诱导黑色素瘤K1735M2细胞形态分化，形成长的树枝状结构，并有脂粒产生，实验结果均为细胞凋亡的显著特征。蜂毒体外抑制黑色素瘤K1735M2细胞生长可能是由于激活Caspase-3活性，最终导致细胞凋亡。园二色谱结果说明，蜂毒作用的黑色素瘤K1735M2细胞膜构象发生明显变化，另外，研究发现，蜂毒能降低黑色素瘤K1735M2细胞对基底膜和细胞外基质的粘附能力，提示蜂毒抑制肿瘤细胞增殖可能是多种生物学效应综合的结果。动物实验研究结果表明，蜂毒能明显抑制移植性B16鼠黑色素瘤生长，其作用机理有待于进一步研究。 采用高效液相凝胶层析定性鉴别方法，对蜂毒中指标性活性成分蜂毒肽进行定性鉴别，同时建立了相同条件的HPLC含量测定方法，其灵敏度高，分离度好，重现性强，结果平均回收率为98.39％，相对标准差为0.55％。研究建立了凝血酶原活化时间（PT）测定法测定蜂毒中活性组分抗凝血活性，结果表明凝血时间T与蜂毒浓度在0～6.0mg／ml范围内具明显的线性关系，相关系数为0.9722。以福林—酚试剂法进行含量及释放度测定，其在25～250μg／ml浓度范围内线性关系良好，相关系数为0.9994，平均回收率为100.59％，相对标准差为1.25％。用酶联免疫吸附法对制剂中蜂毒多肽的血药浓度的体内测定方法进行了考察，结果其方法的最低检测限为0.1 ng／ml，在0.0001～10μg／ml浓度范围内对数线性关系良好，相关系数为0.9981，平均回收率为100.65％，相对标准差为1.51％。上述方法简便、快速、灵敏、稳定、准确，可以满足本研究体内外样品分析要求。刘 星：沈阳药科大学博士研究生学位论文 摘 要 本文采用疏水凝胶层析和离于交换凝胶层析两步分离提纯，精制出天然蜂毒中活性组分，经过高效液相凝胶层析鉴定，其主要活性组分为指标性成分蜂毒肽。对精制蜂毒中活性组分的理化性质进行测定，结果分子量范围小于3500，在生理条件下荷正电。初步稳定性试验结果表明，蜂毒多肽抗凝血活性在生理pH条件、低温及光照下较稳定，但易被蛋白酶降解而夫活。在实验条件下其化学结构稳定。圆二色谱研究结果表明，蜂毒肽在水溶液中的构象随pH值及浓度而变化，井以。－螺旋构象与磷脂膜结人口 口 利用诲藻酸钠具有生物相容、pH依赖及对肠上皮粘膜粘附的特性，将含蜂毒脂质体均匀混悬于高浓度的海藻酸钠溶液中，然后滴入含有固凝剂的氯化钙溶液中交联固化并冷冻干燥，制成海藻酸钙凝胶小球（直径约lmm）测定凝胶小球中蜂毒多肽0叱内的释放速度，按Weibllll分布进行拟合，以包裹率、释药速度常数及相关系数等为判定指标，经正交设计卜O1试验，筛选出了最佳凝胶小球处方组成。通过对流化床包衣工艺的优化，采用肠溶性材料 Eudragit S 100水分散体包衣，制备了天然蜂毒多肽口服结肠粘附定位释药微丸。处方因素的影响试验结果表明，蜂毒多肽与磷脂的比例、海藻酸钠的浓度、氯化钙的浓度、含药脂质体的量及固凝剂的量等对小球的粒径大小。包裹率、重新溶胀时间及药物释放度有显著影响；干燥条件、包衣膜用量、释放介质pH值以及桨转速等工艺因素对药物释放有显著影响。D服制剂中蜂毒多肽的释药动力学特征符合Weibllll分布模型，相关系数为0．9529。 本试验采用Y－闪烁显像技术对包衣微丸在人胃中的排空以及在肠道中的转运和滞留情况进行了初步的研究。试验结果说明，包衣微丸升结肠端微丸滞留，基本达到了海藻酸钙凝胶小球包衣微丸在结肠粘附定位释药的设计要求。通过测定大鼠单剂量静脉注射及日服给药后的血药浓度，对蜂毒多肽海藻酸钙凝胶小球包衣微丸体内过程进行了科学的评价，其在体内的绝对生物利用度为79石9％，而口服蜂毒冻干粉在体内的绝对生物利用度为5二2％。更多还原

【Abstract】 In the present study, bee venom was selected as a model drug and used to study the effects on the proliferation of cancer cells in vitro and in vivo and to determine its possible mechanisms of action in cancer cells. Lastly, an oral colon delivery system (OCDS) was developed to deliver bee venom. The scope of this thesis research includes: (1) the study of the anticancer activity of the bee venom in vitro and in vivo and its possible mechanisms of action; (2) the establishment of analytical methods of typical principles in bee venom in vitro or i/i vivo; (3) the study of the physicochemical properties and the stabilities of the active constituents in bee venom; (4) the design of the OCDS; (5) the evaluation of the OCDS in vivo.In this study MTT test showed that the proliferation of various tumor cells in vitro was inhibited by bee venom in a concentration- and time- dependent manner. The ICso ranged from 2.73 to 13.64 jj,g/ml. The inhibition of K1735M2 melanoma cells was due to the arrest at the GI stage of the cell cycle with a sub-Gi peak as analyzing a DNA histogram by flow cytometer. The bee venom induced apoptosis-like cell death as identified by histological observations and by DNA fragmentation (DNA laddering on the agarose gel electrophoresis), and long dendrite-like projections were observed and lipid and dark granules accumulated. All these results suggested that bee-venom-induced cytostatic effects caused the induction of apoptotic events, which might activate Caspase-3 to induce cells apoptosis. Circular dichroism study showed that an increased order of membrane conformation was induced by bee venom. In addition, the study showed that the adhesion of cancer cells on the extracellular matrix was decreased when treated with bee venom. The results suggested that it might be a connection of various bio-effectiveness by which bee venom inhibited tumor growth. The in vivo experiments indicated that bee venom successfully inhibited the growth of implanted B16 solid tumor proliferation and growth. Further research on the mechanism by which bee venom inhibited the growth and proliferation of the B16 melanoma in vivo is on going.A quantitative GF-HPLC method was developed to analyze melittin, the typical activeingredient in bee venom. It was also used as a sensitive and reproducible method to determine the content of melittin in bee venom, with a recovery of better than 98% and RSD of 0.55%. Prothrombin time (PT) test was also performed to determine the anti-prothrombin activity of the polypeptides in bee venom. The result showed that at concentrations 0 to 6.0 mg/ml, bee venom prolonged the coagulation times in a dose-dependent manner with a coefficient of 0.9722. The Folin-phenol reagent method was adopted to determine the content of active constituents of bee venom in the preparations. The method was validated and linear response range was 25 to 250 M,g/ml, and the recovery was 100.59% with RSD of 1.25%. The ELISA method was adopted to determine the plasma bee venom concentration validated range from 0.0001-10 fxg/ml (r2=0.9981) in rats with the limit of determination at 0.1 ng/ml and the drug recovery at 100.65%, which indicated this ELISA method was suitable to analyze the samples produced in vitro and in vivo experiments.In this study, the polypeptiedes were concentrated from bee venom through a two-step chromatographic separation method using a hydrophobic HPLC and an ion-exchange HPLC columns. Afterwards, pure melittin (59.0%) was separated using GF-HPLC. A study of physicochemical properties of polypeptides in bee venom showed that the molecular weight were below 3500 with positive charge under physiological conditions. The anti-prothrombin activities of the active constituents in bee venom were independent on physiological pH condition, low temperature and light but sensitive to protease. The chemical structure was stable during the experiment. The conformation of melittin in solution changed with pH value and concentration, and a-helix bound to PC membrane.Utilizing sodium alginate fo更多还原