【作者】 江志强；

【导师】 涂瑶生；

【作者基本信息】 广州中医药大学 ， 中药学， 2012， 博士

【摘要】 肝癌是最常见的恶性肿瘤之一,由于其高发病率及高死亡率,成为影响人类健康的最大威胁,据统计,目前我国肝癌发病人数占全球的55%,而死亡人数约占全世界肝癌死亡人数的45%[1]。因此,肝癌的治疗成为我国医药工作者迫切需要攻克的难题。穿心莲内酯(andrographolide)系自爵床科植物穿心莲Andrographis paniculata (Burm. f)Nees中提取得到的二萜内酯类化合物,是中药穿心莲的主要有效成分之一,具有清热、解毒、凉血、消肿等功能。现代研究表明,穿心莲内酯具有抗肿瘤和免疫调节等方面的活性,在治疗胃癌、肝癌、肺癌、乳腺癌等多种肿瘤上效果明显,使其成为近年来在治疗癌症方面较有发展前途的药物。相关研究表明,穿心莲内酯具有较明显的抗肝癌活性,但给药后穿心莲内酯主要分布于肾脏,肝靶向不明显,这在很大程度上影响了其抗肝癌作用的发挥；另一方面,穿心莲内酯属于二萜内酯类,结构不稳定,易受外界环境(温度、pH值)的影响阳]。针对上述问题,研究和开发-种能够增强穿心莲内酯肝靶向性、提高其稳定性的药物传递系统,具有十分重要的临床指导意义。类脂质囊泡(Non-ionic surfactant vesicle, Niosome,以下简称囊泡)是一种优良的药物载体,其以各种非离子表面活性剂为载体材料,在水合介质中通过自身闭合形成的双分子层囊泡状载体。囊泡具有类似生物膜结构的双分子层膜,与细胞膜有高度亲和性,可促进药物跨膜转运；囊泡包裹药物后,一方面可以减少药物在达到作用部位前被破坏,增加药物稳定性,同时药物透过双分子层向外扩散,呈现缓慢释放规律,另一方面通过控制囊泡粒径实现被动靶向,改变药物的体内分布,在人体内主要被网状内皮系统吞噬,增加药物对肝、脾、肺和骨髓等组织的靶向性,达到增效减毒的作用,是靶向给药系统研究的良好载体。针对穿心莲内酯肝靶向性不明显、稳定性差等缺陷,将其制成囊泡,使穿心莲内酯富集于肝脏,缓慢释药,从而很好地发挥抗肝癌作用。本文系统地考察了穿心莲内酯的理化性质、穿心莲内酯囊泡的制备工艺、质量标准、组织分布特点及体外抗肝癌活性等,主要研究内容及结果如下：(1)对穿心莲内酯的相关理化性质进行了考察,为穿心莲内酯囊泡的研制奠定基础。结果如下,穿心莲内酯的油水分配系数为1.42；在各有机溶剂中穿心莲内酯溶解度大小依次为甲醇(13.54mg·mL-1)、丙二醇(9.84mg·mL-1)、无水乙醇(8.02mg·mL-1)、正辛醇(1.36mg·mL-1)、三氯甲烷(0.36mg·mL-1)；在不同pH值磷酸盐缓冲液中穿心莲内酯的溶解度在20-70μg·mL-1范围内；25℃各pH值下穿心莲内酯96小时内含量变化较小,但pH9.4下累积降解了15%左右；80℃各pH值下穿心莲内酯24小时内降解均较明显：穿心莲内酯在20%丙二醇生理盐水溶液中随温度的升高,含量有一定程度地减少。以上试验结果显示,穿心莲内酯呈脂溶性,略溶于甲醇、无水乙醇、丙二醇,极微溶于三氯甲烷,在水中几乎不溶；穿心莲内酯在25℃下稳定,在80℃下稳定性较差；在20%丙二醇生理盐水溶液中温度对其有一定的影响。(2)穿心莲内酯囊泡制备工艺研究。主要包括处方因素优化、工艺条件优化、超声工艺优化等方面。通过单因素试验与正交试验得到穿心莲内酯囊泡最佳制备条件为：采用薄膜分散法制备囊泡,司盘60用量50mg(81.50%),胆固醇用量7.35mg(10.35%),穿心莲内酯用量5mg(8.15%),加有机溶剂溶解,混合均匀,置茄形瓶中混匀,减压除去有机溶剂(50℃,100r·min-1,-0.1Mpa,1h),在瓶壁上形成-层薄膜,加20%丙二醇生理盐水溶液10mL,玻璃球5粒,于45℃下旋转水合(125r·min-1)50min,冰水浴冷却,得穿心莲内酯囊泡混悬液,采用细胞超声破碎仪对其超声处理(100W)10次,5秒／次,即可。按以上最佳工艺条件制备的穿心莲内酯囊泡包封率高(72.36%)、载药量大(5.90%)、分散性良好,粒径符合肝靶向要求,且工艺条件稳定、合理、可行。(3)穿心莲内酯囊泡质量评价。结果表明,穿心莲内酯囊泡多为球形及类球形粒子,外观圆整,大小较均一,表面光滑、分散性良好,粒径主要分布在400nm以下范围内,平均粒径为206nm,包封率为72.36%,载药量为5.90%,pH值为8.47,4℃下囊泡稳定,24小时囊泡中药物累积释放度为65%,符合Higuchi动力学方程。分别采用红外光谱法(IR)和差示量热分析法(DSC)对穿心莲内酯囊泡形成进行了鉴定。红外光谱图显示,穿心莲内酯囊泡与原辅料相比有较大差别,穿心莲内酯囊泡指纹区产生了两个强而宽的新吸收峰,穿心莲内酯12,13位双键振动吸收峰1646.94cm-1消失,差示量热分析图谱显示,与原辅料比较,穿心莲内酯囊泡形成了两个吸热峰,且单位吸热量明显减小。从红外光谱图和差示量热分析图可推测囊泡结构已经形成,穿心莲内酯被顺利包入囊泡内。(4)穿心莲内酯囊泡小鼠体内组织分布研究。首先对囊泡组与溶液组在小鼠体内组织分布数据及药-时曲线图进行了分析,然后运用DAS2.1.1药动学软件进行拟合,确定两给药组中每种器官最佳房室模型,并求算出相关药动学参数AUC(0→∞)、Cmax和MRT(0→∞)及靶向评价参数Te与Ce。结果显示,两给药组中的每种器官最佳房室模型为二室开放模型(C=Ae-αt+Be-βt),肝靶向效率Te穿心莲内酯囊泡为3.66,穿心莲内酯溶液为0.62,肝峰浓度之比Ce为2.45,穿心莲内酯囊泡与穿心莲内酯溶液相比,MRT(0-∞)明显增大。以上结果表明,与穿心莲内酯溶液相比,穿心莲内酯囊泡具有明显的肝靶向性及较好的缓释作用。(5)穿心莲内酯囊泡体外抗肝癌细胞实验研究。通过MTT法考察了穿心莲内酯囊泡对HepG2肝癌细胞存活率的影响,结果显示,其能明显降低HepG2肝癌细胞存活率(n=6,P<0.01),空白囊泡对HepG2肝癌细胞存活率基本无影响；通过荧光定量RT-PCR法检测了穿心莲内酯囊泡对细胞Be1-2mRNA和Bax mRNA两种基因表达的影响,结果显示,穿心莲内酯囊泡能明显提高HepG2肝癌细胞中Bax mRNA表达量,降低Be1-2mRNA的表达量,降低Bel-2/Bax比值,空白囊泡组对细胞Be1-2mRNA和Bax mRNA两种基因的表达影响不明显。以上结果表明,穿心莲内酯囊泡对HepG2肝癌细胞具有较好的抑制作用。综上所述,本文采用薄膜分散法,成功研制了穿心莲内酯囊泡,制备工艺条件稳定可行、重现性良好,穿心莲内酯囊泡质量稳定可控,具有较明显的肝靶向性及较好的缓释作用,抗肝癌活性明显,达到了研究目的,为治疗肝癌药物的开发奠定了良好的实验基础。本文在制备穿心莲内酯囊泡的过程中,展开了以下几方面的探索与研究,并且获得了良好的效果。1.针对穿心莲内酯肝靶向性不明显、稳定性差等不足,首次成功研制了穿心莲内酯囊泡。本文研制的穿心莲内酯囊泡质量稳定可控、包封率较高(72.36%)、载药量较大(5.90%),具有较明显的肝靶向作用和较好的缓释作用,为治疗肝癌药物的开发提供了一种新的思路和方法。2.对中药囊泡研制中水合介质的选择进行了有益的探索。实验研究中,结合囊泡形成条件与穿心莲内酯的溶解特性,探索性地选用20%丙二醇生理盐水溶液作为水合介质,能最大程度的将穿心莲内酯包入囊泡内,提高了囊泡对药物的包封率与载药量。更多还原

【Abstract】 Hepatocarcinoma which is a malignant tumour has seriously affected the human health because of the high incidence and the high mortality rate. According to statistics, in our country the hepatocarcinoma cases take55%of the global cases and the death toll from hepatocarcinoma is about45%of the total number of people die from hepatocarcinoma all over the world. So the treatment of hepatocarcinoma becomes a fiendish problem that is in dire need to be overcome. Andrographolide with the abilities of clearing heat, expelling miasma, cooling blood and detumescence, which belongs to a diterpenoid Lactone composition, is one major active ingredient extracted from Andrographipaniculata（Burm. f.）Nees. In the recent research, andrographolide has been found to posses multi-pharmacological effect including anti-tumor and immuno-modulating and shows the remarkable curative effect on hepatocarcinoma, gastric cancer, lung cacer, breast cancer, at el. So andrographolide is a kind of promising medicine in preventing or treating all kinds of cancers. Some studys showed andrographolide takes an remarkable anti-hepatoma activity. But the curative effect is affect by the lack of hepatic targeting effect. On the other hand, andrographolide is unstability and be apt to be influenced by some environmental factors such as temperature, pH value, at el. Therefore it will play a important role in the emergency treatment to develop a medicine delivery systems to enhance andrographolide hepatic targeting effect and maintain its stability.Niosome is a good kind of drug carrier, nonionic surfactants in hydration medium could be self-assembled into the mini-bilayer vehicles with single cell or multicell. It has the bilayer membrane which is similar to biological membrane, thus it has high affinity with cell membrane and can enhance medicine across membrane transport. Niosome as the medicine release system enfold medicine, on one hand it can increase medicine stability and make medicine release slowly, on the other hand it can take passive targeting effect by controlling its particle size, and then change medicine distribution in vivo. Niosome which is phagocytized chiefly in reticulo-endothelial system in vivo can increase the targeting ability of medicine in liver, spleen, lung and bone marrow and can reach the aim of improving effect and decreasing toxicity.Thus it is the good carrier of targeting drug delivery system.For the drawback of andrographolide inapparent hepatic targeting effect, we construct the administration system of andrographolide niosome. After being encapsulated in the small size vesicles, andrographolide can be targeted passively to the liver, at the same time, the vesicles can make it release slowly and increase its bioavailability.In this paper we do some work on andrographolide niosome, Including:to determine the physics and chemistry character of andrographolide, to optimize preparatian procedure of andrographolide niosome, to study quality standard of andrographolide niosome, to study the tissue distribution characteristics of andrographolide niosome, to determine anti-hepatoma activity of andrographolide niosome in vitro and so on. The whole paper contains the following parts:To study the physics and chemistry character of andrographolide to get ready for preparatian of andrographolide niosome. The partition coefficient of andrographolide in oil phase and aqueous phase is1.42, the order of andrographolide solubility in several organic solvents:Methanol （13.54mg· mL-1）> propylene glycol (9.84mg· mL^-1)>absolute alcohol (8.02mg· mL^-1)>n-octanol (1.36mg·mL^-1)>chloroform (0.36mg·^-1), the solubility of andrographolide in the different pH-values phosphate buffer is in the range from20μg·^-1 to70μg·mL^-1, the content of andrographolide at different pH values（1.8-9.4） at25℃in96h had a less reduction expect pH9.4, the content of andrographolide at different pH values（1.8-9.4） at80℃in24h had a apparent reduction, with a rise of temperature the amount of andrographolide in20%propylene glycol-normal saline solution decrease a little. The above results show that andrographolide is lipophilic, soluble in methanol, absolute alcohol and propylene glycol, hardly in water, stable at25℃and instable at80℃, more stable in20%propylene glycol-normal saline solution. The study of preparatian procedure of drographolide noisome, mainly including, the optimization of prescription and preparation conditions of andrographolide niosome. Using entrapment efficiency, medicine loading rate or the average diameter as evaluation indexes, through a single factor experiment and orthogonal test, the optimized preparation conditions are as following:the film dispersion method was used, span6050mg, cholesterol7.35mg, andrographolide5mg,20%propylene glycol-normal saline solution10mL, hydration rotational Speed125r·min¹, hydration time50min, hydration temperature45℃. and then andrographolide niosome is coped with ultrasonic wave under such conditions as ultrasonic power100W,10times,5seconds each time. The andrographolide niosome which is made under the optimized preparation conditions is with the high entrapment efficiency （72.36%）, the high medicine loading （5.90%） and the suitable diameter, and comes up to the requirement of hepatic targeting effect. Preparatian procedure of andrographolide niosome is stable, reasonable and feasible.The quality evaluation of andrographolide niosome in some ways. It can been seen by the results, the niosome was intact spherical, uniform and good dispersion, the mean particle diameter is206nm, entrapment efficiency, medicine loading rate and pH value are72.36%,5.90%and8.47respectively, it is stable at4℃, the release kinetics in vitro of andrographolide niosome meets Higuchi kinetic equation, about65%of andrographolide release in24h. Andrographolide niosome is in accordance with quality request and hepatic targeting request. To identify andrographolide niosome by Infrared spectrum and DSC. The IR pictures show, there is some difference between andrographolide niosome and raw materials, two obvious new absorption peak appears in the Fingerprint area of andrographolide niosome, the absorption peak of andrographolide1646.94cm^-1 is disappear. The DSC pictures show, there are the double endothermic peaks for andrographolide niosome, the heat-absorbing capacity per gram fall markedly. These indicate the niosome has formed and andrographolide is enclosed into the bilayer well.Research on the tissue distribution characteristics of andrographolide niosome injected via mice caudal vein. To analyze the medicine concentration and medicine concentration-time curve in each tissue between andrographolide niosome group and andrographolide solution group. To fit the best compartment model and pharmacokinetic parameters AUC（0→00）, Cmax and MRT（0→∞） of each tissue by DAS2.1.1pharmacokinetic program, to calculate the targeting parameters Te and Ce. The results are shown, each tissue matches up to two-compartment open mode(C=Ae^-αt+Be^βt, hepatic targeting efficiency Te of andrographolide niosome and andrographolide solution is3.66and0.62respectively, comparing andrographolide niosome group and andrographolide solution group, the proportionality of peak Concentration in liver Ce is2.45, MRT（0-∞） increase obviously. A conclusion is as following, niosome can improve andrographolide hepatic targeting effect markedly and make medicine release slowly.An experimental study of anti-HepG2cell of andrographolide niosome in vitro. To determine the effect of andrographolide niosome on the survival rate of HepG2cell by MTT test and on the expression of Bcl-2mRNA and Bax mRNA by RT-PCR. Results show that andrographolide niosome can lower the survival rate of HepG2cell, increase Bax mRNA expression and decrease Bcl-2mRNA expression. Results reveal that andrographolide niosome can inhibit HepG2cell obviously.In summary, following the optimized preparation conditions we develop andrographolide niosome successfully, the preparation process is steady, feasible and with good reproducibility, the quality of andrographolide niosome is stable and controlled, andrographolide niosome shows the obvious effect of hepatic targeting and slow-releasing medicine. So it is successful to achieved the object of the present study, at the same time it makes a good foundation for exploitation of correlative new medicine.In this paper, in the course of andrographolide niosome preparation, some innovation and explore are as follow:For the drawback of inapparent hepatic targeting effect and instability of andrographolide, It is the first time to develop andrographolide niosome. andrographolide niosome with the high entrapment efficiency （72.36%） and the high medicine loading （5.90%） shows the obvious effect of hepatic targeting and slow-releasing medicine, it is the new train of thought to develop new drug of treating liver cancer.To choose20%propylene glycol-normal saline solution as hydration medium. First,20%propylene glycol-normal saline solution is low toxicity. Secondly, andrographolide niosome can form in the20%propylene glycol-normal saline solution. Thirdly, andrographolide can dissolve a little in20%propylene glycol-normal saline solution, the part of andrographolide which is not encapsulated can be embeded in the bi-layer in molecular form continuously, so it is benefit to increase the entrapment efficiency.更多还原