臺灣博碩士論文加值系統

間質型膀胱炎是一種膀胱壁發炎的疾病，其臨床徵狀有疼痛、頻尿、尿急和尿失禁等；間質性膀胱炎的致病原因目前還沒有很清楚，推測可能是膀胱壁上皮發炎，使離子不正常的輸移，導致膀胱不正常收縮。類黃酮為常見的植物二次代謝產物，研究發現類黃酮具有抵抗一氧化氮產生或是抗氧化等功能；有研究顯示，間質性膀胱炎的膀胱壁上會產生一氧化氮，造成膀胱壁發炎，因此若有類黃酮能抗一氧化氮產生，應可治療間質性膀胱炎。本實驗目的為挑選出一系列12個類黃酮，以Raw264.7細胞株來篩選出最佳抗一氧化氮產生的藥物以及對細胞毒殺能力最小的藥物，以MTT來篩選最低毒殺能力的藥物，再以濃度為20 μg/mL的12種類黃酮作用於細胞，以0.5 μg/mL的脂多醣(LPS) 誘導Raw264.7細胞產生一氧化氮，取細胞培養液來分析當中的一氧化氮含量。實驗結果發現山奈酚具有較低毒殺細胞的能力以及較好的抗一氧化氮產生效果，在山奈酚濃度為50 μM時，其細胞生存率為85%，而細胞的一氧化氮產生率相較於LPS組為36.72%。進一步以西方點漬法來證明山奈酚抗發炎機轉，發現山奈酚對iNOS或COX-2蛋白質皆有抑制效果。藥物輸送則以實驗設計法mixture design設計20個微乳劑處方來運輸，處方組成以Tween80、Span20為界面活性劑，二次水為水相，乙醇為輔助界面活性劑，IPM為油相。製成處方進行體外穿透試驗，實驗結果發現這20個處方的12小時累積穿透量介於13.02~256.44 μg/cm2；粒徑大小在45.2~108.7奈米之間；導電度在12.47~183.63 ?嵞/cm之間；黏滯度在12.87~42.53 cps之間。以魚精蛋白/氯化鉀模式誘導間質性膀胱炎的膀胱收縮間隔(ICI)為3.2±0.8 分鐘，若以靜脈注射投予山奈酚溶液或皮下投予山奈酚微乳劑後其膀胱收縮間隔延長為7.9 ± 0.9 分鐘與8.9 ± 1.5分鐘。若是皮下給予山奈酚溶液，膀胱收縮間隔延長為4.0±1.3分鐘。

Interstitial cystitis (IC) is a inflammation condition of bladder. Its clinical syndrome characterized by bladder pain, irritative voiding symptoms, and sterile urine. Although the pathogenesis of IC is uncertain, it has been proposed that a dysfunctional epithelium allows the transepithelial migration of solute, such as potassium, that can depolarize sub-epithelial afferent nerves and provoke sensory symptoms. Flavonoids are common secondary metabolites in plant kingdom. Studies have showed that flavonoids inhibit oxidation or nitric oxide producing. iNOS induced NO overproduction cause epithelial barrier dysfunction and increase epithelial permeability. According to these studies, flavonoid might improve the symptom of interstitial cystitis. The aim of this study was to evaluate the anti-inflammatory effect of twelve flavonoid compounds and the inhibition contractive effect of the chosen flavonoid on bladder. Raw264.7 macrophage cell was chose as model cell. The anti-inflammatory effect was induced by lipopolysaccharide (LPS) in this study. The cells were treated at the concentration of 20 μg/mL for two hours, and then the LPS was added 0.5 μg/mL while the drugs still remained in the medium. The result showed that flavonoid kaempferol had lower cell toxicity and higher anti-inflammatory effect than other flavonoids. The cell viability and the inhibition of NO production were 85.8% and 53.57%, respectively while the concentration of kaempferol was 50 μM. The mechanism of anti-inflammatory effect of kaempferol is prove by western blot. It is found that kaempferol inhibit iNOS and COX-2 production. Drug delivery was designed by mixture design of experimental design. 20 microemulsion formulations were designed. Tween80 and Span20 were mixed together as surfactant. Isopropyl myristate (IPM) is oil phase. Ethanol is used as co-surfactant. Pure water is water phase. In vitro permeation experiment was tested. The Q of these formulations are between 13.02~256.44 μg/cm2. Size are between 45.2~108.7 nm. Conductivity are between 12.47~183.63 ?嵞/cm. Viscosity are between 12.87~42.53 cps. In this study, protamine/KCl were used to induce interstitial cystitis model. The ICI of hyperactivity bladder induced by protamine/KCl is 3.2±0.8 minute. The ICI value was prolonged to 4.0±1.3 min and 8.9±1.5 min for subcutaneous injected kaempferol solution and microemulsion treated. The ICI was prolonged to 7.9 ± 0.9 min by intravenous injected kaempferol solution.

壹、緒論 1
一、研究背景 1
二、類黃酮 5
三、以類黃酮治療間質性膀胱炎的機轉探討 10
四、微乳劑型 (Microemulsion) 13
五、實驗設計法 (Experimental design) 16
貳、研究目的 18
參、實驗材料及儀器設備 19
一、 材料 19
(一)、 藥品（Drugs） 19
(二)、 化學試藥（Chemical Reagents） 19
二、 儀器設備 21
肆、實驗方法 22
一、挑選類黃酮 22
(一)、細胞培養 22
(二)、各種類黃酮毒性評估 24
(三)、各類黃酮對發炎物質Nitro Oxide (NO)之抑制效果 27
(四)、與專一性/非專一性 NO synthase inhibitor比較抑制NO的效果 27
(五)、與市售NSAID對發炎物質NO之抑制效果 28
(六)、Kaempferol濃度與抑制NO效果的相關性實驗 28
(七)、Cox-2、iNOS蛋白質分析 29
二、Kaempferol分析方法建立 31
(一)、Kaempferol 之HPLC分析方法 31
(二)、Kaempferol之定量方法 32
(三)、溶解度試驗 33
三、Kaempferol處方製備 34
(一)、處方製備 34
(二)、體外穿透試驗( in vitro permeation test) 36
(三)、皮膚含量測定 40
(四)、處方性質測定 40
四、動物體內實驗 42
(一)、實驗動物 42
(二)、膀胱壓力容積檢測 (Cystometrography, CMG) 42
(三)、動物模式 44
(四)、投藥方式 45
(五)、藥效評估方法 47
五、數據資料及統計方法 49
(一)、體外試驗 49
(二)、體內試驗 49
伍、結果與討論 50
一、各種類黃酮對Raw264.7細胞之毒性結果 50
二、各種類黃酮對經由LPS誘發發炎之Raw264.7的抗NO效果 52
(一)、0.5小時與2小時之藥物對結果之影響 52
(二)、12種類黃酮之NO產量結果 55
(三)、結構與抑制NO生物活性之探討 56
(四)、山奈酚 (Kaempferol)簡介 58
(五)、Kaempferol 與iNOS inhibitor比較其抑制NO效果 59
(六)、Kaempferol 與市售NSAID藥物比較其抑制NO效果 59
三、Kaempferol 分析條件 63
(一)、Kaempferol的HPLC層析條件 63
(二)、Kaempferol體外分析檢量線 63
(三)、溶解度評估 66
四、Kaempferol 對iNOS、COX-2蛋白質產量之抑制效果 67
五、處方與穿皮試驗 70
(一)、微乳劑處方性質 70
(二)、體外穿透實驗(in vitro permeation experiment) 74
六、評估Kaempferol溶液對膀胱收縮的作用 85
(一)、藥效評估方式 85
(二)、動物模式 85
(三)、投藥比較 85
陸、結論 94
柒、參考文獻 96
捌、附錄 101

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