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研究生:吳怡欣
研究生(外文):Yi-Hsin Wu
論文名稱:兒茶素微脂粒劑型之口服輸送系統研究
論文名稱(外文):Development of liposomal formulation of catechin for oral delivery system
指導教授:黃耀斌黃耀斌引用關係
指導教授(外文):Yaw-Bin Huang
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:藥學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:72
中文關鍵詞:兒茶素微脂粒藥物動力學
外文關鍵詞:catechinliposomespharmacokinetic
相關次數:
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有相當多研究報導說明自由基攻擊細胞所產生的代謝產物與老化速率以及許多疾病的形成有明顯的相關性。所以抗氧化劑是可對抗自由基且避免對人體的氧化性傷害,進而減少慢性疾病的產生。現今許多文獻指出兒茶素具有許多生理活性,不但可清除自由基,還可抗發炎、抗病毒、抗菌、抗突變與抗癌。然而兒茶素口服生體可用率非常低(2~5%),清除率也極高,因此本研究藉由liposomes之處方劑型來提高口服生體可用率,並延長藥物在血漿中之作用時間。
我們利用Epikuron 200之磷脂質,以及膽固醇(cholesterol)作為製備微脂粒處方之材料,並探討處方不加或分別加入表面修飾劑stearylaminem與dicetyl phosphate以及非離子界面活性劑Tween 80對處方之影響,且比較此三組微脂粒的物理性質,包含包埋率、粒徑大小以及處方在第0、14、21、30天之安定性的變化,進一步篩選出理想之處方。此三組所製備出的微脂粒平均粒徑大小為35~65 nm,包埋率在50~80%之間。體外釋放試驗顯示,其最佳處方在第12小時之累積釋放量可達55%。
在篩選出理想之處方後,以口服給予(+)-Catechin 30 mg/kg於健康大鼠,並與溶液劑做比較,觀察大鼠體內血漿中藥物動力學情形以及腦部組織的分佈。結果發現經過微脂粒製備化的(+)-Catechin,其曲線下面積與生體可用率皆比溶液劑來得好。
A lot of studies have proven that decay rate and many diseases obviously correlate with free radicals attack to living cells and its generated metabolic product. Hence antioxidants can against free radicals and avoid oxidative damages for human, and then decrease generation of chronic diseases. In recent years, numerous studies indicated that catechin not only remove free radicals but has various types of pharmacological properties such as anti-inflammatory, anti-viral, anti-bacterial, anti-mutagenic and anti-carcinogenic. However the oral bioavailability of catechin is known to be low (2~5%), and the systemic clearance is also high. The purpose of this experiment was to promote oral bioavailability of catechin with liposomal formulation and prolong duration time of drug in plasma.
We used phospholipid, Epikuron 200, and cholesterol as material to prepare liposome formulation, and discuss the effect of liposome formulation which contain phospholipid and cholesterol or the mix of ionic surfactants such as stearylamine, dicetyl phosphate and nonionic surfactant such as Tween 80. For the three group that physical properties of drug entrapment efficiency, particle size and stability of formulation on day 0, 14, 21 and 30 were respectively compared, and further, we were sieve out optimization of formulation. Preparation of three group of liposome that the mean diameters were 35~65 nm and the entrapment efficiency were 50~80%. According to in vitro release experiment result, the optimization of formulation, catechin accumulated amount was 55% at 12 hour.
After sieve out optimization of formulation, we using oral administration for health rats with dosage 30 mg/kg of catechin. Compared with catechin solution, we observed the pharmacokinetics and distribution in brain tissue. In vivo result, we found the area under the curve and bioavailability of catechin-liposome were better than solution.
附圖目錄.................................................4
附表目錄.................................................5
中文摘要.................................................7
英文摘要.................................................8
壹、緒論.................................................9
一、研究背景..........................................9
二、氧化(oxidation)以及自由基(free radical)..........10
三、類黃酮...........................................13
1.結構與分類.....................................13
2.兒茶素類(Catechins)............................13
四、微脂粒...........................................16
1.微脂粒結構.....................................16
2.微脂粒的分類...................................16
2.1多層微泡微脂粒(MLV, Multilamellar vesicles)...............................................16
2.2 單層大微泡微脂粒(LUV, Large unilamellar vesicles)...............................................17
2.3單層小微泡微脂粒(SUV, Small unilamellar vesicles)...............................................17
3.微脂粒之組成...................................20
3.1磷脂質(Phospholipids).........................................20
3.2膽固醇(Cholesterol).......................20
3.3電解質(Electrolytes)......................20
4.影響微脂粒之性質...............................22
4.1影響微脂粒穩定性的物理因素................22
4.2影響微脂粒穩定性的化學因素................22
5.提升微脂粒之安定性.............................22
6.微脂粒之應用及其優缺點.........................23
6.1微脂粒之優點..............................23
6.2微脂粒的限制與缺點........................23
7.微脂粒與細胞間之作用機轉.......................24
五、研究目的.........................................25
貳、材料與方法..........................................26
一、藥品與化學試劑...................................26
二、儀器設備.........................................27
三、實驗方法.........................................28
1.主成分進行UV-Scan以確定HPLC條件................28
2.體外定量分析...................................28
2.1HPLC層析條件..............................28
2.2體外檢量線製作............................28
2.3體外檢品檢量線之確效......................29
3.動物...........................................31
3.1生物檢體之萃取率..........................31
3.2體內檢量線製作............................32
4.劑型設計.......................................34
4.1微脂粒製備方法............................34
4.2微脂粒的粒徑大小、粒徑分散性及表面電位測量......................................................40
4.3微脂粒的包埋率(Encapsulation ratio, ER%)....................................................40
4.4微脂粒處方之穿透式電子顯微鏡觀察(TEM)...................................................41
4.5微脂粒處方凝集評估........................41
5.(+)-Catechin微脂粒處方之體外釋放試驗...........41
5.1(+)-Catechin溶解度試驗....................41
5.2(+)-Catechin微脂粒體外釋放................41
6.(+)-Catechin-solution與(+)-Catechin-liposomes於大鼠體內藥物動力學評估......................................42
6.1口服投予(+)-Catechin-solution與(+)-Catechin-liposomes在血液和腦部之藥物動力學研究...................42
参、結果與討論..........................................43
1.(+)-Catechin分析方法建立.......................43
1.1主成分進行UV-Scan.........................43
1.2HPLC分析條件..............................43
1.3體外檢品檢量線............................45
1.4體外檢品檢量線之確效......................45
1.5萃取溶媒評估..............................46
1.6從動物組之檢品中萃取(+)-Catechin之最佳條件..48
1.7體內檢品萃取率............................49
1.8體內檢品檢量線............................50
2.劑型設計.......................................51
2.1處方評估..................................51
2.2處方之粒子大小、粒徑分散性、表面電位及包埋率評估......................................................51
2.3含藥處方之穿透式電子顯微鏡(TEM)結構之觀察......................................................57
2.4微脂粒處方之安定性試驗....................58
3.(+)-Catechin體外釋放試驗.......................63
4. (+)-Catechin-solution與(+)-Catechin-liposomes血液之藥物動力學
研究評估.......................................65
4.1口服投予給藥..............................65
5. (+)-Catechin-solution與(+)-Catechin-liposomes腦部之藥物動力學研究評估....................................67
5.1口服投予給藥..............................67
肆、結論................................................68
伍、參考文獻............................................69

附圖目錄
圖一:類黃酮基本結構....................................14
圖二:綠茶中主要兒茶素之結構............................15
圖三:微脂粒之結構......................................18
圖四:微脂粒之構造與種類................................19
圖五:phosphatidic acid化學結構式.......................21
圖六:膽固醇結構式......................................22
圖七:微脂粒與細胞間之作用..............................24
圖八:(+)-Catechin與Caffeine之UV吸收圖譜................44
圖九:藥品Catechin及內部標準品Caffeine之HPLC層析圖譜....44
圖十:(+)-Catechin體外檢品之檢量線,濃度範圍為0.5~10
μg/mL..................................................45
圖十一:(A)以dichloromathane;(B)以ethyl acetate萃取(+)-Catechin之層析圖譜......................................47
圖十二:(A)為空白血液以及(B)空白腦之層析圖譜............47
圖十三:(+)-Catechin體內檢品檢量線......................50
圖十四:理想處方(liposomes含有1% Tween 80)經穿透式電子顯微鏡照像之外觀..............................................57
圖十五:微脂粒處方中添加stearylamine之粒徑大小隨時間變化圖......................................................62
圖十六:微脂粒處方中添加Tween 80之粒徑大小隨時間變化圖......................................................62
圖十七:(+)-Catechin體外釋出濃度與時間之關係......................................................64
圖十八:口服投予劑量30 mg/kg (+)-Catechin後之平均血漿濃度經時變化..................................................65

附表目錄
表一:自由基引發的臨床疾病..............................11
表二:UV/Vis分光光度儀條件..............................28
表三:製備總量40 g之微脂粒各種處方......................35
表四:表中合適的微脂粒處方水相中添加(+)-Catechin之濃度......................................................36
表五:表中合適的微脂粒處方添加表面電荷修飾劑Stearylamine之含量......................................................37
表六:表中合適的微脂粒處方添加表面電荷修飾劑Dicetylphosphate之含量..................................................38
表七:表中合適的微脂粒處方添加非離子性界面活性劑Tween 80之含量......................................................39
表八:(+)-Catechin同日間(intra-assay)體外檢品檢量線之精密度及準確性................................................46
表九:(+)-Catechin異日間(inter-assay)體外檢品檢量線之精密度及準確性................................................46
表十:比較各萃取液在組織中萃取(+)-Catechin的影響........48
表十一:(+)-Catechin於大鼠血液及組織中萃取率............49
表十二:處方型態評估結果................................53
表十三:微脂粒處方中含不同藥量之粒子大小、粒徑分散性、表面電位及包埋率變化..........................................54
表十四:含藥3 mg/mL (+)-Catechin之微脂粒處方於脂質組成比例14/86中加入不同劑量之stearylamine對微脂粒處方粒子大小、粒徑分散性、表面電位及包埋率的影響..........................55
表十五:含藥3 mg/mL (+)-Catechin之微脂粒處方於脂質組成比例14/86中加入不同劑量之dicetylphosphate對微脂粒處方粒子大小、粒徑分散性、表面電位及包埋率的影響......................55
表十六:含藥3 mg/mL (+)-Catechin之微脂粒處方於脂質組成比例14/86中加入不同劑量之Tween 80對微脂粒處方粒子大小、粒徑分散
性、表面電位及包埋率的影響..............................56
表十七:微脂粒處方中添加stearylamine之安定性評估........59
表十八:微脂粒處方中添加dicetylphosphate之安定性評估....59
表十九:微脂粒處方中添加Tween 80之安定性評估............60
表二十:各處方在第30天之粒徑大小與包埋率................61
表二十一:(+)-Catechin於各medium中的溶解度..............63
表二十二:口服投予劑量30 mg/kg (+)-Catechin後經時變化之平均血漿濃度................................................66
表二十三:口服投予劑量30 mg/kg (+)-Catechin後第8小時脂腦部分區濃度..................................................67
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