VD3脂质体的制备及其体内外抑制光老化活性初步研究

发布时间：2018-06-14 10:44

本文选题：维生素D3 + 脂质体　；参考：《吉林大学》2017年硕士论文

【摘要】：维生素D3(Vitamin D3,VD3)是一种脂溶性的维生素,属于一种作用于钙磷代谢的激素前体,又叫做“阳光维生素”。VD3具有很多方面的功效,如调节钙磷代谢,预防骨质疏松,改善心脑血管疾病,提高免疫力等作用。但是由于VD3是脂溶性的,不溶于水,限制了其药效。为了提高VD3的生物利用度,需要一种更为安全高效的VD3制剂。脂质体是一种单层或多层两性双分子层组成的结构,内部包含水溶性或脂溶性物质,可以作为药物载体,达到提高药效和降低副作用的目的。而本文主要利用VD3的高效抗氧化性,阻止皮肤细胞中自由基的过度产生,提高自由基清除酶的活性,将VD3与脂质体的结合,制备成维生素D3脂质体(L-VD3)来达到防止皮肤光老化的作用。本文主要包括如下内容:1.VD3体外分析方法学的建立。建立了VD3的体外分析方法-高效液相色谱法(HPLC),使用此方法得到的VD3的精密度和回收率均符合体外分析测定要求。HPLC测定L-VD3中的药物含量,并且该方法测定的L-VD3中药物的回收率符合体外分析要求。建立了用超滤离心法来分离游离VD3,进而测定L-VD3的包封率的方法。此方法可以很好的使游离药物分离,并且方便有效,且该法测定的VD3的精密度和回收率也均符合体外分析测定的要求。2.VD3常规脂质体制备及性能评价分别就乙醇注入法,薄膜均质法,以及反向蒸发法对L-VD3的制备方法进行了筛选,对比包封率和脂质体的粒径,得到乙醇注入法是制备L-VD3的最适合的方法。接着分别就L-VD3配方中磷脂含量、磷脂与胆固醇的比例、脂药比以及磷酸缓冲液的PH值进行单因素实验。筛选出来磷脂含量、磷脂与胆固醇的比例、脂药比作为主要的考察对象进行响应面优化实验,得到最优工艺配方为:磷脂含量为25mg,磷脂与胆固醇的比例为4.5:1,脂药比为2.5:l。以最优配方条件下得到vd3常规脂质体的包封率的理论值为87.83%,实际值为86.79%。3.l-vd3的稳定性研究本文在温度、光照、冻融的条件下进行了影响l-vd3稳定性因素的测试。其中温度在4-40℃的条件下,包封率基本不发生太大变化,稳定性较好,适宜保存l-vd3;光照对l-vd3的稳定性没有过多影响,因此可以说明vd3在制备成脂质体之后对光的稳定性大大提高了,可以不用避光保存;冻融条件对l-vd3的稳定性也没有过多影响,可以说明vd3制成脂质体后对温度的突变稳定性较好,也可以很好的适应温度的变化。另外,本文进行了vd3的体外释放试验,发现l-vd3在皮肤中的单位截留药量为182.13μg/cm2,而vd3溶液在皮肤中的单位截留药量为113.82μg/cm2,l-vd3的单位截留药量要远大于vd3溶液,并且l-vd3的透皮量也小于vd3溶液,因此将vd3做成脂质体会更有利于皮肤吸收。之后又进行了加速实验和长期实验,发现l-vd3的包封率和其外观形态并没有太大的变化,说明l-vd3的稳定性较好。4.l-vd3的药效研究本文建立了皮肤光老化模型。经过晒伤处理的大鼠皮肤都有明显的褶皱,红肿,斑点,白块等不同程度的光老化现象,说明模型建立成功。药物治疗了一段时间之后观察大鼠的皮肤状态,发现涂抹l-vd3和阳性对照药物后的大鼠皮肤基本上恢复正常,没有明显的白斑褶皱破损等现象,光洁有弹性;涂抹了vd3溶液的大鼠皮肤状况也有一定的改善,虽然仍有有一些黄斑红点,但不是很多;而涂抹pbs溶液的阴性对照组大鼠皮肤依旧有红斑出血白斑褶皱等光老化现象,没有得到改善。之后将各组大鼠的皮肤进行he染色,观察切片的电镜图片,发现涂抹l-vd3和阳性对照药物的大鼠表皮真皮结构基本完整,内部纤维组织、腺体等排布均匀,与正常的大鼠皮肤组织切片状态相差无几;涂抹了vd3溶液的大鼠表皮真皮结构略有改变,内部纤维组织、腺体个别变形,但整体上比较有序排列;而涂抹pbs溶液的阴性对照组大鼠表皮真皮严重分层,之间的界限模糊不清,内部腺体纤维也杂乱无章甚至变形,皮肤损伤状况没有改善。以上结果说明,L-VD3可以有效的防止皮肤的光老化现象,有比较显著的效果。
[Abstract]:Vitamin D3 (Vitamin D3, VD3) is a fat soluble vitamin. It belongs to a hormone precursor that acts on calcium and phosphorus metabolism. It is also called "sunshine vitamin".VD3, which has many effects, such as regulating calcium and phosphorus metabolism, preventing osteoporosis, improving cardiovascular and cerebrovascular diseases, and improving immunity. But because VD3 is fat soluble, insoluble. In order to improve the bioavailability of VD3, in order to improve the bioavailability of the VD3, a more safe and efficient preparation is needed. The liposome is a structure composed of a single layer or multilayer bisexual biolecular layer, which contains water-soluble or fat soluble substances, which can be used as a drug carrier to achieve high efficacy and reduce side effects. The effective antioxidant activity of VD3 is used to prevent the excessive production of free radicals in skin cells, improve the activity of free radical scavenging enzymes, and combine VD3 with liposomes to prepare vitamin D3 liposomes (L-VD3) to prevent skin photoaging. This article mainly includes the following contents: the establishment of the method of 1.VD3 in vitro analysis and the establishment of the body of VD3. The external analysis method - high performance liquid chromatography (HPLC), the precision and recovery of VD3 obtained by this method are all in accordance with the requirement for the determination of the drug content in L-VD3 by.HPLC in vitro, and the recovery rate of the drug in L-VD3 is in conformity with the requirements of the in vitro analysis. The separation of free VD3 is established by ultrafiltration centrifugation, and the determination of L-V is established. The method of encapsulation rate of D3. This method can make the separation of free drugs very good, and it is convenient and effective, and the precision and recovery of VD3 measured by this method also conform to the requirements of in vitro analysis and determination of.2.VD3. The preparation and performance evaluation of the conventional lipid system and performance evaluation on the ethanol injection method, the thin film homogenization method, and the reverse evaporation method for the preparation of L-VD3. The method was screened to compare the encapsulation efficiency and the particle size of liposomes. Ethanol injection was the most suitable method for preparing L-VD3. Then, the content of phospholipid, the proportion of phospholipid and cholesterol, the ratio of lipid to cholesterol, the pH of the phosphate buffer and the pH value of the phosphoric acid buffer were tested. The content of phospholipid, the proportion of phospholipid and cholesterol, the proportion of lipids were screened, and the proportion of phospholipid and cholesterol was screened. The response surface optimization experiment was carried out as the main object. The optimum formula was obtained: the content of phospholipid was 25mg, the proportion of phospholipid and cholesterol was 4.5:1, and the theoretical value of the encapsulation efficiency of VD3 conventional liposomes was 87.83% under the optimal formulation condition, and the stability of 86.79%.3.l-vd3 was studied in this paper. In the conditions of temperature, light and freezing and thawing, the stability factors of l-vd3 were tested. Under the condition of 4-40 C, the encapsulation efficiency did not change much, the stability was better, and the stability of the l-vd3 was better. The light has not too much effect on the stability of l-vd3, so it can explain the stability of VD3 to the light after preparing the liposome. As a result, the freezing and thawing conditions have no effect on the stability of l-vd3, which shows that the VD3 liposomes have a better temperature mutation and can adapt to the change of temperature. In addition, the release test of VD3 in vitro is carried out in this paper, and the unit interception of l-vd3 in the skin is 182.13. Mu g/cm2, and the unit of VD3 solution in the skin of unit interception of the amount of 113.82 u g/cm2, l-vd3 unit to intercept the dose is much larger than the VD3 solution, and l-vd3 transdermal volume is less than the VD3 solution, so the VD3 to make lipid experience is more beneficial to the skin absorption. Then, the accelerated experiment and long-term experiments were carried out, and the encapsulation rate and external appearance of l-vd3 were found. The state has not changed too much. It shows that the stability of l-vd3 is better than that of.4.l-vd3. The skin photoaging model has been established in this paper. The skin of rats treated with sunburn has obvious folds, redness, blotches, white patches and other light aging phenomena, indicating the success of the model. After a period of drug treatment, the rats were observed. The skin status, the skin of rats after smearing l-vd3 and positive control drugs was basically restored to normal, without obvious leukoplakia fold damage and other phenomena, smooth and flexible; the skin condition of rats smeared with VD3 solution was also improved, although there were some yellow spot red spots, but not many; and the negative control group smeared with PBS solution The skin of rats was still characterized by erythema hemorrhage and leukoplakia folds, which were not improved. Then the skin of rats in each group was stained with he, and the electron microscope pictures of the slices were observed. It was found that the dermal structure of the skin of rats smeared with l-vd3 and positive control drugs was basically complete, the internal fibrous tissue, glands, etc. were evenly distributed, with the normal rat skin. The tissue section of the tissue was different. The dermal structure of the rat epidermis with VD3 solution was slightly changed, the internal fibrous tissue and the gland were deformed, but on the whole, the epidermis of the negative control group smeared with PBS solution was seriously stratified, the boundary between the epidermis was not clear, the inner gland fibers were disorderly and even deformed. The results showed that L-VD3 could effectively prevent photoaging of skin and had a significant effect.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R96

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