中文摘要
C-藻藍素(C-Phycocyanin, C-PC)是一種膽色素蛋白質(biliphycoprotein)，由α及β兩個次單位組成之單體結構，為藍綠藻中螺旋藻(Spirulina platensis)之主要成份之一。
本研究將針對C-PC進行藥理活性之篩選，包括動物實驗探討其鎮痛作用，抗發炎作用及肝臟急性毒性之保護作用，另外對於細胞生長之調節能力及抗氧化能力亦一併測試，以瞭解C-PC之藥理活性及可能之相關作用機轉。
以小白鼠進行C-PC之鎮痛試驗發現對於紅外線引起的閃尾反應時間，二組均比控制組延長，並具統計上的差異(ANOVA) (p<0.001)。
C-PC對於λ-carrageenan誘發大白鼠足蹠浮腫試驗，顯示注射之劑量的C-PC (0.001、0.01、0.1、1.0 mg/kg)在5小時內大白鼠足蹠浮腫反應之當劑量為0.001 mg/kg時，抑制浮腫反應效果不顯著。
本研究以四氯化碳(CCl4)在誘發大白鼠急性肝炎反應並注射C-PC，發現其肝臟保護作用(hepatoprotective effect)，分別於背部投與C-PC 0.0001、0.001、0.01、0.1 mg/kg時四者均能明顯地降低血清中SGOT、SGPT數值，在光學顯鏡下觀察肝臟切片，發現藥物組比CCl4組有更輕微的病變效果。
以cytochrome c 還原比色試驗測得C-PC抗氧化能力之IC50值為6.10 ± 0.74 mg/ml，另外以xanthine oxidase方法測試而得知C-PC抗氧化能力IC50值為7.23 ±0.21 mg/ml，而以電子自旋共振(electron spin resonance, ESR方法檢測則其清除超氧自由基之活性各為1.08 × 103 unit/g，清除氫氧自由基之活性為7.11 unit/g，而三者中以ESR之方法較為精準。

電子自旋共振方法為利用電子捕捉技術(spin-trapping) 來測定以評估C-PC是否具有對活性氧自由基包括·O2-及·OH-具直接捕捉之抗氧化能力。結果測得C-PC之超氧離子(·O2-)的清除能力之IC50為3.28 mg/ml，而氫氧基離子(·OH-)清除能力IC50為4.75 mg/ml，顯示C-PC對超氧自由基與氫氧自由基均有顯著的清除能力，。
在以人類肝癌細胞(Hepatocarcinoma cell包括HepG2及2.2.15細胞)與大白鼠神經膠質瘤C6細胞(glioma, C6 cells)經加入不同劑量之C-PC (1.0、10 μg/ml)反應 72小時後，以流式細胞儀分析C-PC對細胞週期的影響，發現C-PC使HepG2和2.2.15.細胞均呈現出停滯於G0/G1期的現象，C6細胞之作用較不明顯。
由以上之研究結果顯示C-PC為一具有潛力之鎮痛、抗炎和細胞週期調節作用之試劑。

Abstract
C-Phycocyanin (C-PC), a water-soluble protein pigment, is one of the major constituents of Spirulina platenisi,. Which is a blue green algae and used in many countries as dietary supplements.The C-PC used in present study is a phycobiliprotein, and consist of two distinguishable protein subunits designated as α and β subunits.
The aim of this study was to screen pharmacological effects including analgesic, anti-inflammatory and hepatoprotective effects of C-PC in an animal model. Concomitantly, the antioxidant effect of C-PC was measured to confirm its free radical scavenger activity.
The analgesic effect of C-PC measured by tail-flick test shown the latency (sec) of control mice is approximately 4-5 sec. After administration different doses of C-PC (0.1, 1.0, 10 mg/kg), the latency resulted in a dose-dependent phenomenon and prolong from 4 sec to 4.5, 5.6, and 6.9 sec, respectively.
The anti-inflammatory effect of C-PC was analyzed in the λ –carrageenan injected rat. The hind paw edema percentage change by injection of different doses (0.1~1.0 mg/kg) of C-PC, our results have indicated that C-PC possessed a dose-dependent inhibitory effect on carrageenan-induced edema formation.
Intraperitoneal (i.p.) injection of different dose (0.01,0.1 1, 1.0 mg/kg) of C-PC on CCl4 (0.3 ml/kg) challenged rats have indicated that C-PC possessed a significant hepatotoprotective effect and significant decrease the serum levels of glutamate pyruvate transaminase (SGPT) and serum glutamate oxyloacetic transaminase (SGOT).
Antioxidant effect of C-PC was measured by both xanthine oxidase method, and cytochrome c method. The IC50 of C-PC measured by xanthine oxidase method is 7.23 ±0.21 mg/ml. The IC50 value of C-PC estimated by cytochrome c method is approximately 6.1±0.74 mg/ml.
Moreover, the direct active oxygen (·O2-) and hydrogen oxide (·OH) radical scavenging (SOD-like) activity was also confirmed by using advance electron spin resonance (ESR) method. We used the spin-trapping technique to evaluate free radical scavenging ability of C-PC. The IC50 (·O2- ) and (·OH) scavenging activities are 1.08 × 103 unit/g and 7.11 unit/g.
Our results have shown the superoxide radical (·OOH) scavenging activity of C-PC is approximately 3.28 mg/ml. By contrast, IC50 of the hydroxyl radical (·OH) scavenging activity of C-PC is 4.75 mg/ml.
Human hepatoma cells (HepG2 and 2.2.15 cells) and rat glioma C6 cells were cocultured with different concentration of C-PC (1.0, 10 and 20 μg/ml)for 72 hours, the cell cycle regulation of C-PC was measured by flow cytometer. We found the C-PC possessed a G0/G1 phase-arrest effect on all of the three kind cancer cell line. Whereas, the cell cycle modulations of C-PC are more significant in human hepatoma cells.
In conclusion, both our pharmacological screening tests and antioxidant bioactivity assay have indicated that C-PC is a potential antitumor, anti-inflammatory and analgesic agent.

目 錄
頁次
中文摘要-------------------------------------------------- 1
英文摘要-------------------------------------------------- 3
緒論-------------------------------------------------------- 5
材料與方法----------------------------------------------- 9
結果-------------------------------------------------------- 17
討論-------------------------------------------------------- 19
參考文獻-------------------------------------------------- 28
圖表-------------------------------------------------------- 35
附表-------------------------------------------------------- 72

表目錄
頁次
表 1. 藻藍素抗氧化能力之IC50
表 2. Mn2+ 活性的ESR信號及超氧自由基在各種 SOD的濃度
表 3. SOD like Superoxide Radical Scavenger 活性分析Mn2+及
hydroxyl radical 在各種抗壞血酸濃度的ESR 信號強度
表 4. 抗壞血酸對 Hydroxyl Radical Scavenger 活性分析
表 5. C-PC對 Hydroxyl Radical Scavenger 活性分析
表 6. 藻藍素對引發人類肝癌細胞Hep G2 細胞生長週期之影響
表 7. 藻藍素對引發人類肝癌細胞株 2.2.15. 細胞生長週期之影響
表 8. 藻藍素對引發老鼠神經癌瘤細胞株 C6 細胞生長週期之影響
附表1. 藻藍素以及台灣民間常用抗衰老防癌生藥及治療中風方劑
之抗氧化活性之比較
附表2. 藻類分類和俗名
附表 3. 植物學家根據藻類所含的色素種類、儲藏物質、
細胞壁成分、鞭毛數目和著生位置、細胞及細胞
構造的特徵
附表 4. 脂質自由基對生體內標的分子引起之障礙

圖目錄
頁次
圖 1. 螺旋藻(Spirulina platensis) 顯微鏡下的外觀
圖 2. 藻藍素化學結構圖
圖 3. 閃尾測定儀
圖 4. 藻藍素對紅外線引起老鼠閃尾反應的影響
圖 5. 藻藍素在各種濃度對大白鼠足蹠浮腫
圖 6. 藻藍素各種濃度對大白鼠足蹠浮腫抑制
圖 7. 藻藍素對肝臟保護作用抑制CCl4誘發血清SGOT上升
圖 8. 藻藍素對肝臟保護作用抑制CCl4誘發血清SGPT上升
圖 9. 大白鼠注射藻藍素72小時後肝臟之外觀
圖 10.大白鼠肝臟組織切片
圖 11.藻藍素處理由CCl4引起大白鼠肝臟組織病理組織切片
圖12. Superoxide radical 產生的ESR 圖譜
圖13.各種濃度的SOD產生的ESR 圖譜
圖14. C-PC 的 ESR 圖譜
圖15. [SOD] 和 [Io/I –1]間之關係
圖16.抗壞血酸抑制hydroxy radical的ESR圖譜


頁次
圖 17. Mn2+及 hydroxyl radical 在各種抗壞血酸濃度的ESR圖譜
圖 18. C-PC抑制hydroxy radical 的ESR圖譜
圖 19.藻藍素對人類肝癌HepG2細胞於細胞週期的影響.
圖 20.藻藍素對人類肝癌2.2.15.細胞於細胞週期的影響
圖 21.藻藍素對老鼠神經細胞瘤C6細胞於時細胞週期的影響
圖 22.藻藍素對人類肝癌G2細胞細胞週期的影響
圖 23.藻藍素對人類肝癌2.2.15.細胞細胞週期的影響
圖 24.藻藍素對老鼠神經細胞瘤C6細胞細胞週期的影響
圖 25.藻藍素對G2細胞細胞顆粒的影響
圖 26.藻藍素對人類肝癌2.2.15. 細胞細胞顆粒的影響
圖 27.藻藍素對老鼠神經細胞瘤C6細胞細胞顆粒的影響
圖 28.本研究室中藻藍素(C-PC)相關之藥理篩選試驗
圖 29.藻藍素(C-PC) 對CCl4誘發之肝毒性保護作用可能作用機轉
圖 30.藻藍素(C-PC) 對於癌細胞之細胞週期調節圖

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