【作者】 王志钢；

【导师】 张保献； 李慧；

【作者基本信息】 中国中医科学院 ， 中药学， 2011， 硕士

【摘要】 由于眼部结构复杂并存在许多屏障,传统眼用制剂在疗效和患者顺应性等方面有诸多问题,为了更好地使治疗药物进入眼内发挥疗效,新型眼部给药系统的研究逐渐成为药剂学研究的热点之一。中药治疗眼部疾病历史悠久,特别是在治疗内眼疾病,如青光眼、白内障、玻璃体出血、糖尿病所致视网膜病变、视网膜动静脉阻塞等方面具有明显的优势特色。常用中药当中,葛根和丹参经药理研究和临床使用证明均有扩张微血管,改善微循环的作用,对眼底病、眼外伤、慢性炎症性眼病等具有很好的疗效。传统给药形式主要为口服,而眼部给药比全身给药对进入眼内达到靶区更具优势与可行性,为了更好地体现与发挥中医药防治眼病的优势,有必要结合现代制药技术与给药系统的理论,对中药眼部给药系统进行研究和开发。非离子表面活性剂囊泡(non-ionic surfactant vesicles),简称为类脂囊泡(niosomes),是一种与脂质体类似的新型给药系统。其作为眼用制剂载体的优势在于：作为药物的贮库,不仅可以延长药物在角膜和玻璃体内的滞留时间,还可以保护药物分子免受泪液或角膜上皮中代谢酶的破坏,使药物在角膜表面产生缓控释增效作用；并且囊泡对外眼组织,结膜和巩膜具有很强的亲和力,滴眼后可迅速分散,增强了药物对角膜的穿透性。眼用即型凝胶是近来研究较多的一种新型眼部给药载体,其在非生理状态下为自由流动的液体,给药后在眼部生理条件下形成凝胶,能够延长药物在眼内的滞留时间,从而提高生物利用度。为了改善葛根素滴眼液临床上使用存在眼部滞留时间短患者顺应性差的问题,本课题尝试将葛根素制成非离子囊泡以研究囊泡在眼部应用的可行性；同时选用高分子聚合物泊洛沙姆407与188作为温度敏感型眼用凝胶的基质,对葛根和丹参复方眼用即型凝胶给药系统进行了初步研究,为今后研制开发中药复方眼用新型制剂奠定基础。本课题主要研究内容分为以下四部分：第一部分葛根及丹参的提取与纯化研究。通过文献资料与预实验,对制剂所用中药葛根及丹参分别进行了提取与纯化。其中葛根以葛根素为指标,采用70%乙醇回流提取然后正丁醇溶解萃取冰醋酸重结晶的方法,得到纯度约89%的葛根素；丹参中有效成分分为丹参总酮和总酚酸两部分,分别以丹参酮II A、丹参总酮和丹酚酸B、丹参总酚酸为指标,对丹参进行醇提和水提,得到有效部位纯度大于50%的丹参总酮和总酚酸提取物。第二部分葛根素非离子表面活性剂囊泡的制备研究。对葛根素非离子囊泡的制备及理化表征进行了初步研究,实验中对比发现注入法所形成的囊泡外观分散性较好,粒径较小且较为集中。通过比较离心、透析及葡聚糖凝胶柱层析等分离方法,最后优选葡聚糖凝胶柱层析法为葛根素非离子囊泡的有效分离方法。但含量测定结果显示葛根素的包封率很低,是本实验留待解决的一个主要问题。第三部分丹葛明目即型凝胶的处方筛选及制备研究。以泊洛沙姆溶液经模拟泪液稀释前后的相转变温度为考察指标,采用均匀设计法优选了温度敏感型眼用凝胶基质处方,建立了泊洛沙姆浓度与相变温度的拟合方程,结果表明凝胶相变温度随着泊洛沙姆407的浓度增加而下降；而泊洛沙姆188在一定范围内能够提高泊洛沙姆407的相变温度。在符合条件的结果中选择了3个处方,分别对其在模拟泪液稀释前后的黏度进行了测定,最后确定以浓度较低的19%泊洛沙姆407与1%泊洛沙姆188合用作为温度敏感眼用凝胶的基质。在此基础上,对丹葛明目即型凝胶的处方作进一步筛选,确定处方中需要添加的其它附加剂,包括助溶剂(吐温20)、防腐剂(苯扎氯铵)及pH调节剂(NaOH)等,然后对药物及辅料加入后的胶凝温度及黏度的变化进行了考察,并作了相应调整,最后制备得到符合要求的丹葛明目温度敏感眼用凝胶。第四部分丹葛明目即型凝胶的体内外初步评价研究。建立了丹葛明目即型凝胶中药物的含量测定方法：以葛根素和丹酚酸B为制剂的指标成分,采用高效液相色谱法进行测定。采用无膜溶出法对制备的丹葛明目即型凝胶进行了体外溶蚀及药物释放的考察。结果表明：凝胶中药物的释放受溶蚀控制,且二者均遵循零级动力学过程。家兔眼部的滞留时间实验显示眼用即型凝胶能显著延长药物在眼部的滞留时间。经兔眼单次给药和多次给药后观察,结果丹葛明目即型凝胶对兔眼无刺激性反应。经豚鼠皮肤多次涂抹给药,也未见皮肤过敏反应。本课题的创新在于采用新技术新方法对中药眼部给药系统进行了尝试,对葛根素非离子囊泡及丹葛明目即型凝胶进行的相关研究均未见有报道。课题的研究探索结果可以为解决中药眼用制剂一些共性技术问题提供一定的借鉴思路和方法,有助于推动中药眼用新制剂更进一步的发展。更多还原

【Abstract】 Owing to the complex structure and many barrier in the eye,traditional ophthalmic preparation have a lot of problems in patient compliance,healing effect et al. In order to make therapeutic drugs exert better curative effect on intraocular, novel ophthalmic drug delivery system gradually become the hot spot in pharmaceutics researches.It has been a long history to treat ocular diseases by using chinese herbal medicine.TCM has obvious characteristic advantage especially in the treatment of interna eye diseases such as glaucoma,cataracts,vitreous hemorrhage,retinopathy caused by diabetes,retinal vein occlusion and retinal artery occlusion.Among the commonly used chinese herbal medicine, By pharmacological research and clinical use,puerarin and Salvia miltiorrhiza are proved that they can expand capillaries and improve microcirculation,have good curative effect in retina,ocular trauma,chronica inflammatory disease.The main administration form of chinese herbal medicine was oral,but ophthamic drug administrtion had more advantages and feasibility to enter in the target areas of eye.In order to better reflect and develop the advantage of preventing ocular disease by TCM,it is necessary to combine the theory of modern pharmaceutical technology and drug delivery system for research and development of TCM ophthamic delivery system.Non-ionic surfactant vesicles,for short niosomes, is a new drug delivery system similar with liposomes.As ophthalmic drug carrier,its advantage lies in:prolong the drug retention time in cornea and vitreous body as drug storage library, protect the drug molecules from destroy by metabolic enzymes in tears or corneal epithelium, and exert the drug effect in corneal surface slowly; it also has a strong affinity with eyes organization,conjunctiva and sclera, and can disperse rapidly in the eye after droping, hence to enhance the cornea penetrability. In situ forming eye gel is another kind of ophthalmic drug delivery system well-studied recently, in physiological condition it is free flow liquid, in physiological conditions it will form a gel.This can prolong drug residence time in the eye, so as to improve the drug bioavailability.The aim of this study is to improve low bioavailability and potential risk of clinical pue eyedrops through pue niosomes, and then we study the feasibility of its application in the eye. Meanwhile we choose poloxamer407 and poloxamer188 as temperature sensitive gel matrix, to study pueraria lobata and salvia miltiorrhiza compound in situ forming eye gel, in order to lay foundation for new herbal ophthalmic preparations in the future.In this study,the main investigation focused on four parts as follows:The first part, the extraction and Pueification of Puerarin and Salvia miltiorrhiza. through the literature material and prediction of preparation,we extract and Pueify pueraria lobata and salvia miltiorrhiza respectively. Among them pueraria lobata with pue for index, the 70% ethanol refluxing extraction and then dissolved n-buoh extraction method of recrystallization glacial acetic acid, get the Pueity of 89% pue; Salvia miltiorrhiza are divided into the extraction and Pueification of total ketone and total phenolic acid two parts, respectively by ketoneⅡA,total and phenolic acids B, total phenolic acids for effective component index to the dan-shen root ethanol-extraction and water extraction, get more than 50% effective parts Pueity of salvia miltiorrhiza total ketone and total phenolic acids extract.The second part, Puerarin nonionic surfactant vesicles preparation research. After the comparison of the centrifugation, dialysis and glucan gel column chromatography method, we determine glucan gel column chromatography as the effective separation methods. Then the preparation of puerarin niosome and its characterizition is studied, experiment contrast found Injection method formed into law vesicles appearance dispersivity is good, small and is relatively concentrated particle. But the content determination results showed that the puerarin entrapment rate is very low, this is one of the main problems need to be solved.The third part, the research on prescription and preparation of Dange mingmu in situ forming eye gel. The phase transition temperature before and after diluted by STF is observed as index, we use the uniform design method to optimum the temperature sensitive type ophthalmic gel matrix prescription, established fitting equation between poloxamer concentration and phase-change temperatures, the results showed that the gel phase-change temperatures with concentrations of P407 decreases; And in a certain range P188 can improve the phase-change temperatures. Then we choose three prescription in the results, the viscosity of them before and after diluted by STF are conducted, finally we determine the lower concentration:19% P407 and 1% P188 as temperature sensitive ophthalmic gel matrix. On this basis, the other additional agents in prescription of Dange mingmu in situ eye-gel is screened, including help solvent (tween 20), preservative (benzene pierced ammonium chloride) and pH regulator (NaOH), etc, and then after accession of drug and accessories, gelation temperature and viscosity was investigated, to meet the requirements,we make corresponding adjustment to get Dange mingmu temperature sensitive in situ forming eye gel.The last part, the preliminary evaluation research on Dange mingmu in situ forming eye gel. Established the content determination methods of Dange mingmu in situ forming eye gel:using HPLC to analyse pue and Salvianolic acid B (preparation index component). According to the in vivo characteristic of ocular administration,a memebraneless model was used to study the gel erosion and drug release simultaneously.Correlation analysis demonstrated that drug release,which followed zero-order kinetics,from poloxamer vehicle was completely controlled by gel erosion.The retention time of rabbit eye experiment shows that in situ eye-gel significantly prolong drug retention time in the eye. The rabbit eye single dose and many times experiment demonstrate that Dange mingmu in situ forming eye gel have no irritant reaction to rabbit eyes. There was also no allergic reaction after spraying on the skin of guinea pigs many times.The novelty in our studies lies in we attemp to use new technology, new method on ophthalmic drug delivery system of TCM,in order to make the drugs and carrier achieve an complementary effection,there are no essays established about relative researches on pue niosomes and Dange mingmu in situ forming eye gel.The study results can provide certain reference and ideas to solve some of the TCM technical issues in common, which is benefit to promote further development on ophthalmic new preparations of TCM and its compounds.更多还原