葛根素微脂體於大鼠腎系膜細胞之體外抗氧化評估

In Vitro Antioxidative Properties of Liposomal Puerarin in Rat Mesangial Cells

陳竺音(Chu-Yin Chen)

指導教授 ： 謝明發

中原大學/工學院/生物醫學工程研究所/碩士(2013年)

https://doi.org/10.6840/cycu201300958

摘要

隨著飲食不均衡及久坐不運動的生活方式，罹患糖尿病的人口顯著增加，葛根素是從藤葛莖塊中提取所得之主要有效成分，以被用於抗高血糖及抗氧化之製劑，然而其口服利用率與溶解度不佳是藥物治療上最主要的障礙，因此，必須以藥物載體劑型有效的遞送葛根素，本研究目的為製備具穩定性及生物相容性之微脂體劑型用於防治糖尿病腎病變。利用乙醇於室溫、50 oC、70 oC 提煉葛根素後再以雙溶劑分層萃取，其純度分別為 83.4、70.5 及 55.6 % ，以 1,1-二苯基-2-三硝基苯肼自由基評估葛根素抗氧化能力，結果顯示 1.25 mg/mL 葛根素於 10 分鐘內清除 22.1 % 以上自由基。以水浴式超音波或濾膜擠壓法製備微脂體 (E70C30T0、E65C30T5、E60C30T10)，評估其粒徑大小以及於蔗糖及大白兔血漿溶液中的穩定度，於蔗糖保存液組別中，利用超音波震盪法製備微脂體其五天的粒徑變化率為 11.97、1.07 及 1.94 % ，以濾膜擠壓製備微脂體其粒徑變化率為 1.88、1.78 及 1.55 % ，結果發現以濾膜擠壓法製備 E60C30T10 組成之微脂體可得到穩定的粒徑大小，然而在血漿溶液中則顯著的受到羰酸酶攻擊產生磷酸水解；葛根素包覆率分別為40.45、49.24 及 53.79 %。體外腎系膜細胞毒性研究顯示，葛根素微脂體相較於單純葛根素溶液可增加 1.19 倍之細胞存活率， 33 mM 葡萄糖培養基可顯著增加細胞內部活性氧物種 (ROS) 含量，並且促進細胞增生，此為糖尿病腎系膜細胞模型。此外細胞增生、TGF-β1 基因表現以及 Smad 2/3 蛋白核轉入現象可藉由添加葛根素微脂體達到抑制的效果，並且呈現劑量依賴性。本研究成功製備可於高糖環境之下保護細胞免於毒性影響之葛根素微脂體劑型，並且具有潛力用於防治糖尿病腎病變。

關鍵字

微脂體 ； 葛根素 ； 氧化壓力 ； 糖尿病

並列摘要

Along with unbalance diet and sedentary lifestyle, the population of diabetes mellitus is increasing significantly. Puerarin, a major active ingredient extracted from Pueraria lobata, is known as antihyperglycemic and antioxidantive agents. However, the poor oral availability and aqueous solubility are major barriers for drug formulation. Therefore, to effectively deliver puerarin, a liposomal dosage form of puerarin is used. This study aimed to develop a stable and biocompatible liposomal formulation for combating diabetic nephropathy disease. Two-phase solvent were used to extract the puerarin at room temperature、50 oC and 70 oC, the purity was found to be 83.4、70.5 and 55.6 % respectively. The anti-oxidative capacity of puerarin were evaluate by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical experiment, and found 1.25 mg/mL extracted puerarin can eliminate up to 22.1 % of free radical (DPPH) in the first ten minutes of incubation. Liposomes (E70C30T0、E65C30T5、E60C30T10) was prepared by water-bath sonicaion or membrane extrution methods and characterized for the particle size and its stability in sucrose and rabbit serum solution. In sucrose solution for 5 days, increase ratio of the particle was 11.97、1.07 and 1.94 % by sonication method, and 1.88、1.78 and 1.55 % by extrution method, respectively. It was found that membrane extrution of E60C30T10 formulation could produce the stable particles in sucrose solution. However, the phosphate groups of liposomes formula in serum was significantly hydrolyzed by serum carboxyesterase. In the third part, liposomal puerarin were prepared using extrution method (E70C30T0、E65C30T5、E60C30T10). The drug loading efficiency were 40.45、49.24 and 53.79 %, respectively. The study of in vitro cytotoxicity showd the puerarin encapsulated by liposomes could increased 1.19 times of cell viability of mesangial cell line compared with free puerarin. The 33 mM glucose medium significantly encanced the reactive oxygen species (ROS) level, and improve the cell proliferation, which was used as an in vitro model of diabetic nephropathy. The cell proliferation、gene expression of TGF-β1 and nuclear translocation of Smad 2/3 proteins in 33 mM glucose medium were suppressed in a concentration dependent manner by liposomal puerarin. In summary, this study successfully prepare the liposomal puerarin wich can provide cytoprotective effect in high glucose condition, and is of potential in combating diabetic nephropathy.

並列關鍵字

Liposome ； Oxidative stress ； Diabetic mellitus ； Puerarin

參考文獻

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