海芙蓉藥材之抗氧化、保肝、鎮痛、抗發炎活性研究

中國醫藥大學機構典藏 China Medical University Repository, Taiwan > 中醫學院 > 中國藥學暨中藥資源學系暨碩博班 > 博碩士論文 > Item 310903500/41246

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題名:	海芙蓉藥材之抗氧化、保肝、鎮痛、抗發炎活性研究 Studies on the Anti-oxidant, Hepatoprotective, Analgesic and Anti-inflammatory Activity of Hai-Fu-Rung
作者:	張恬寧
貢獻者:	中國藥學暨中藥資源學系博士班
關鍵詞:	海芙蓉;抗氧化;保肝;鎮痛;抗發炎 Hai-Fu-Rung;Anti-oxidant Activity;Hepatoprotective Activity;Analgesic Activity;Anti-inflammatory Activity
日期:	2011-01-21
上傳時間:	2011-10-17 16:00:18 (UTC+8)
出版者:	中國醫藥大學
摘要:	本研究目的首先以高效液相層析質譜儀分析法，建立芙蓉菊的水層抽出物(CCW)和烏芙蓉的甲醇抽出物(LWM)之指紋圖譜全圖譜，也透過高效液相層析質譜儀分析法得知芙蓉菊水層抽出物含有有效成分rutin和scopoletin。其次比較芙蓉菊跟烏芙蓉的水層及甲醇抽出物其萃取物之體外抗氧化活性，也比較芙蓉菊和烏芙蓉不同溶劑分層的萃取物之體外抗氧化活性，最後探討芙蓉菊的水層抽出物和烏芙蓉的甲醇抽出物對肝臟保護和鎮痛、抗發炎活性，以及抗細胞增殖活性。本研究的研究方法如下︰在體外抗氧化方面有ABTS自由基清除測定、測定總酚類含量、類黃酮含量、黃酮醇含量、FRAP測試、以TLC片進行快速DPPH呈色反應、DPPH清除自由基活性、還原力、螯合亞鐵離子測定、清除超氧陰離子之能力測定、清除一氧化氮NO (nitric oxide)能力測定和抑制脂質過氧化之能力等項目。在急性肝炎方面，檢驗血清中GOT (glutamate oxaloacetate transaminase)，GPT (glutamate pyruvate transaminase)，肝臟組織SOD (superoxide dismutase)，GPx (glutathione peroxidase)，CAT (catalase)數據、肝臟組織切片H.E.染色 (hematoxylin-eosin stain)、血清NO含量之測定、血清總抗氧化力測定、血清之腫瘤壞死因子含量測定(tumor necrosis factor-α, TNF-α)、肝臟GSH (glutathione)之活性、肝臟脂質過氧化之含量測定，而Zymography結果顯示MMP-9 (matrix metalloproteases)的抑制。在鎮痛與抗發炎方面以醋酸誘導扭體試驗和福馬林注射足蹠之舔足時間測試及λ-角叉菜膠 (λ-carrageenan)誘導足蹠浮腫測試，以及深入探討機制實驗，包括血清及蹠組織NO含量之測定、血清及蹠組織脂質過氧化測定、血清及蹠組織之腫瘤壞死因子含量測定和蹠組織切片及染色。本研究結果，在高效液相層析質譜儀分析法中，rutin與芙蓉菊的水層抽出物顯示出有相似的波峰與相同的8分鐘的滯留時間；scopoletin與芙蓉菊的水層抽出物顯示出有相似的波峰與相同的13分鐘的滯留時間；gallic acid與烏芙蓉的水層抽出物顯示出有相似的波峰與相同的8分鐘的滯留時間。本研究在體外抗氧化方面，整體而言，以水層抽出物CCW和LWW的效果比甲醇抽出物CCM和LWM好，分層萃取的部份以EtOAC及n-BuOH分層的抗氧化活性較強。在急性肝炎方面，血清中GOT，GPT，肝臟組織SOD，GPx，CAT，GSH數據，血清NO含量、血清總抗氧化力、血清之腫瘤壞死因子含量、肝臟脂質過氧化之含量及Zymography結果，依給藥劑量的增加而減少數值，及肝臟組織切片H.E.染色方面，給藥組傷害有減輕的趨勢。在鎮痛與抗發炎方面，依給藥劑量的增加對小鼠醋酸扭體反應次數有顯著抑制作用，對福馬林誘導的後期疼痛有明顯的抑制效果，在抗發炎方面，會抑制λ-角叉菜膠誘導的足部浮腫。CCW和LWM均會減少小鼠發炎足蹠組織中丙二醛(malondialdehyde, MDA)及一氧化氮NO的含量。且CCW和LWM亦會減少腫瘤壞死因子的含量的含量。在iNOS (inducible nitric oxide synthase)和COX-2 (cyclo-oxygenase- 2)的western blotting 實驗中，看見CCW可以抑制iNOS和COX-2的表現。綜合上述研究結果顯示，芙蓉菊水層抽出物含有活性成分rutin和scopoletin，烏芙蓉水層抽出物含有活性成分gallic acid，且其EtOAc及n-BuOH分層具有抗氧化活性。芙蓉菊的水層抽出物和烏芙蓉甲醇抽出物對四氯化碳誘發的急性肝損傷有保護作用，並有鎮痛、抗發炎的活性，其活性成分分別為rutin，scopoletin及gallic acid。 In this study, the chemical profile of water extract of Crossostephium chinensis (L.) Makino (CCW) and methanol extract of Limonium wrightii (Hance) Ktze.(LWM) was analyzed by LC-MS-MS spectrometer (high-performance liquid chromatography tandem mass). Chromatographic pattersn from HPLC analysis (254 nm) of rutin and scopoletin were detected in CCW. We then compared the results in anti-oxidant experiment with water and methanol extract of Crossostephium chinensis (L.) Makino (CCW, CCM) and Limonium wrightii (Hance) Ktze.(LWW, LWM). We also compared the anti-oxidant capability of different partition layers of CCM and LWM.Eventually, the effects of CCW and LWM on hepatoprotectic, analgesic and anti-inflammation activity were evaluated. Further assessment of the anti-carcinogenic activity and the mechanisms of its chemopreventive action were also performed. The antioxidative assays included: ABTS ([2,2’-azinobis (3-ethylbenzothiazoline)-6-sulfonic acid] diammonium salt) free radical scavenging assay, TPC (determination of total phenolics contents) , (FRAP) ferric reducing antioxidant power assay, rapid screening of antioxidant by dot-blot DPPH (1,1-diphenyl-2-picrylhydrazyl) staining, DPPH radical-scavenging activities, reducing power measurement, Chelating ability, superoxide radical scavenging, nitric oxide (NO) radical scavenging and lipid peroxidation assay. In the hepatoprotective studies, serum levels of GOT (glutamate oxaloacetate transaminase) , GPT (glutamate pyruvate transaminase), NO production, antioxidant capacity, TNF-α (tumor necrosis factor-α) concentration in serum, the liver SOD (superoxide dismutase), and GPx (glutathione peroxidase), the CAT (catalase) activities, GSH (glutathione) contents, TBARS (thiobarbituric acid reactive substances) formation and the liver tissue examinations were determined. Activities of MMP-9 (matrix metalloproteinases) in SDS-PAGE zymography of serum and liver were also determined. The full time lick induction test and the λ-carrageenan paw edema induction test were also performed. Light microscopy observations of mice paw sections stained with Van Gieson of iNOS (inducible nitric oxide synthase) and COX-2 (cyclo-oxygenase- 2) were performed. The expression of iNOS and COX-2 were inhibited by western blotting. NO concentration, tissue MDA (malondialdehyde) concentration, tissue TNF-α concentration of foot and serum in mice were determined. The results showed that CCW did contain the active contents rutin and scopoletin, with the retention time 8 min between CCW and rutin, and retention time 13 min between CCW and scopoletin, respectively. And LWM did contain the active content gallic acid, with the retention time 8 min between LWM and gallic acid. In the anti-oxidant capability of this study, water extract of Crossostephium chinensis (L.) Makino and Limonium wrightii (Hance) Ktze.(CCW, LWW) are better than methanol extract of Crossostephium chinensis (L.) Makino and Limonium wrightii (Hance) Ktze.(CCM, LWM). Significant in vitro oxidative resistance phenomena by EtOAC and n-BuOH fractions were observed. Liver functional, inflammation, morphological and mechanism studies GOT, GPT, SOD, GPx, CAT, GSH, NO, antioxidant capacity, TNF-α concentration, TBARS formation, zymography showed reduction of liver damage by administration of CCW. Accordingly, analgesia and foot edema reduction together and MDA, Nitric Oxide concentration and TNF-α concentration were also significantly observed in CCW and LWM. In conclusion, CCW contained active compounds, rutin and scopoletin whereas LWM contained active compound gallic acid, both have the strong anti-oxidation. Significant in vitro oxidative resistance phenomena by EtOAC and n-BuOH fractions were observed to be better than other partition layer. CCW and LWM both have liver protection, analgesic, anti-inflammative and anti-hepatoma activities.
顯示於類別:	[中國藥學暨中藥資源學系暨碩博班] 博碩士論文

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