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研究生:劉吉豐
研究生(外文):Chi-Feng Liu
論文名稱:生藥川芎口秦及天然物蜂膠之藥理作用的研究
論文名稱(外文):Pharmacological Research of Tetramethylprazine and Propolis
指導教授:林松洲林松洲引用關係
指導教授(外文):Song-Chow Lin
學位類別:博士
校院名稱:臺北醫學大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:91
語文別:中文
論文頁數:124
中文關鍵詞:川芎口秦蜂膠脂質過氧化細胞色素c
外文關鍵詞:TetramethylprazinePropolislipid peroxidantioncytochrome c
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為了探討瞭解中國大陸常用民間生藥,川芎口秦(Tetramethylpyrazine)的保肝作用,本實驗室以動物實驗模式來研究此生藥的保護功能。急性肝細胞傷害的誘導是以具有肝毒性的抗黴菌藥物-Econazole來進行:以300 mg/kg的劑量口服投與至大白鼠體內,而後再以生藥川芎口秦於肝毒性藥物投與後的第二、第四和第八小時後各自口服投與生藥 10 mg/kg和 50 mg/kg的劑量,以觀察川芎口秦的保肝作用。
  在保肝效果的評估中,除了藉由檢測實驗動物的血清轉胺酵素值(SGOT和SGPT)之外,實驗同時就病理學上的變化,以評估肝細胞組織學上的損傷,諸如:脂肪變性(fatty degeneration)、氣球樣變性(ballooning degeneration)、肝小葉中心壞死現象(centrilobular necrosis)、炎症細胞(kupffer cells)、淋巴球(lymphocytes)、嗜中性白血球(neutrophils)與嗜伊紅性白血球(eosinophils)之增殖性發炎浸潤(inflammatory infiltration)等項目的觀察來綜合判定。並以確實被證實對肝炎有效的乳薊草成分silymarin作為對照藥物。
  由實驗結果顯示,在川芎口秦的保肝作用實驗中,雄性大白鼠經口投與Econazole (300 mg/kg)後24小時,會造成血清轉胺酵素值急遽升高(SGOT:1955.6±63.4 IU/L;SGPT:348.6±17.8 IU/L),而於econazole經口投與後的第二、第四和第八小時經口投與川芎口秦的生理食鹽水溶液,SGOT與SGPT值明顯抑制為:(1)10 mg/kg:SGOT:766.3±44.9 IU/L;SGPT:221.3±20.6 IU/L,(2)50 mg/kg:SGOT:635.0±24.0 IU/L;SGPT:122.8±9.5 IU/L。
為了探討瞭解中國大陸常用民間生藥,川芎口秦(Tetramethylpyrazine)的保腎作用,本實驗室以動物實驗模式來研究此生藥的保護功能。急性腎傷害的誘導是以具有腎毒性的物質-酒精來進行:預先口服生藥川芎口秦(10、25和50 mg/kg)三十分鐘後,再以濃度95%之酒精口服於小白鼠體內,以觀察川芎口秦的保腎作用。
在保腎效果的評估中,除了藉由檢測實驗動物的尿素氮及肌酐酸(BUN和Creatinine)之外,實驗同時就川芎口秦對於酒精所引起之脂質過氧化病理學上的變化,以評估腎細胞的損傷。
以實驗的結果顯示,在川芎口秦的保腎作用實驗中,酒精(10 ml/kg)口服投與雄性WISTAR小白鼠後,造成BUN及Creatinine升高(BUN:27.9 ±1.7 gm/dl;Cr:0.21 ±0.01 gm/dl),而於事先三十分鐘口服投予川芎口秦生理食鹽水溶液後,其BUN與Creatinine值明顯抑制為:(1)10 mg/kg :BUN:18.1 ±1.3;Cr:0.2 ±0.01,(2)25 mg/kg:BUN:13.6 ±1.2;Cr:0.06 ±0.03,(3)50 mg/kg:BUN:13.4 ±1.3;Cr:0.06 ±0.03。
此外,在川芎口秦的抗氧化活性研究方面,於脂質過氧化試驗及細胞色素C還原比色法測試中,川芎口秦均顯現有抗脂質過氧化及清除超氧自由基的能力。
由於川芎口秦的保腎及抗氧化活性研究結果,我們推測生藥之抗氧化活性與其保腎活性確實有關,抗氧化活性越佳則保腎活性越好,但是使用過量也可能造成體內之腎毒性。
蜂膠(Propolis)為蜜蜂採蜜時,同時採集了某些樹木(例如:白楊木、七葉樹、柳樹及橡樹等)的樹皮及嫩芽內的樹脂,這些物質和蜜蜂的唾液結合成一種具黏性的棕綠色芳香性膠狀物,蜜蜂將此種膠狀物吐在蜂巢內,人類因而能由蜂巢內採集到此種自然界中最寶貴之生藥──蜂膠。至於蜂膠對於腸胃的保護功能及機轉,至今尚未清楚。本實驗主要是在探討蜂膠是否對於急性無水酒精(Absolute ethanol)造成胃潰瘍有保護作用,並深入了解其作用機轉。由急性無水酒精胃傷害模式中,蜂膠(1、5、10%,P.O.)的投與可以呈現劑量依賴性(Dose-dependent)地減少胃黏膜由無水酒精(1.0 ml/kg,P.O.)投與所造成胃黏膜損傷的程度,此一保護結果可由大體照相(Gross)中觀察得知。此外在體外(in vitro)及體內(in vivo)實驗的TBA實驗中亦發現蜂膠可直接與無水酒精之毒性代謝物質或代謝所產生之自由基(Free radical)結合,減少這些毒性物質對細胞的傷害。
實驗證明,投與蜂膠可以減少無水酒精胃傷害。其可能之保護機轉為透過抑制自由基的效應而產生。
Abstract: 2,3,5,6-tetramethylpyrazine(TMP)is well known as a true calcium antagonist. The aim of this study was to investigate the hepatoprotective and therapeutic effects of TMP on acute econazole-induced liver injury. The hepatological effect of various concentrations of TMP was first assessed by the biochemical assay of SGOT and SGPT and then by hepatohistological microscopic examination. The dose-response relationship of liver injury induced by various doses of econazole was observed simultaneously from serum biochemical assay of SGOP and SGPT, and also from hepatohistological microscopic examination, in the determination of the hepatoprotective effects of various concentrations of TMP on SGOT and SGPT elevation induced by hepatotoxic dose of econazole (300 mg/kg). The inhibitory effect of various concentration of TMP or vitamin E (as positive control, 0.5mM: in vitro, 0.69 mM: in vivo) on FeCl2-induced (in vitro)or econazole-induced (in vivo) lipid peroxidation was also investigated. The superoxide scavenging activity of various concentrations of TMP in econazole-damaged rat liver homogenate was assessed by cytochrome C reduction method. Results showed that the hepatoprotective effect of TMP might be, at least in part, due to its inhibitory ability on membrance lipid peroxidation and free radical formation, or due to its free radical scavenging ability. The improvement of the serum transaminases and MDA levels in rat liver homogenate, the hepatohistological microscopic examination, and the assessment of free radical scavenging activity by cytochrome C reduction method can be used to detect the hepatoprotective and therapeutic effects of TMP on acute econazole-induced liver injury.
Acute administration of absolute ethanol (10 ml/kg) per os to fasted mice produced extensive renal failure as measured by a rise in blood urea nitrogen and creatinine. Pretreatment with oral administration of tetramethylpyrazine (TMP) prevented such failure. The maximal effect against absolute ethanol-induced renal failure could be observed 1 h after TMP administration .In order to further investigate the renal protective mechanism of TMP, experiments on lipid peroxidation and superoxide scavenging activity were conducted. Renal homogenates made from mice treated with ethanol showed that TMP pretreatment had an antioxidant effect. Mice in acute renal failure had higher malonic dialdehyde concentrations than those pretreated with TMP. The renal protective mechanism of TMP was attributed, in part, to its prominent superoxide scavenging effect, which protects the kidney from superoxide-induced renal damage.
Reactive oxygen species have been implicated in the pathogenesis of a variety of acute injury models, including ischemia-reperfusion injury and ethanol-induced renal failure. Acute ingestion of absolute ethanol (5 g/kg) has been reported to lead to an accelerated increase in lipid peroxidation, an index of oxidative stress. Protection against renal injury can be achieved by a variety of agents, including scavengers of hydroxylation and superoxide dismutase, which converts superocide to hydrogen peroxide. Previous studies have shown that renal failure is often associated with ischemic injury and nephrectomy. Absolute ethanol-induced renal failure is a useful model in mice, but the detailed mechanism of its pathogenesis is still not fully clear.
Oxygen is essential for life, but it may also be dangerous. Reduction of oxygen in tissue produces an number of oxygen free radicals which may induce cellular damage and even cell death. Oxygen-handling cells have different systems, e.g. superoxide dismutase, peroxidases and catalases, which protect them against the toxic effects of oxygen free radicals.
Tetramethylpyrazine (TMP) is a constituent of Ligusticum wallichii French. It not only blocks the entry of extracellular calcium through calcium channels but also inhibits the release of intracellular stored calcium in vascular smooth muscle cells. In 1997, Feng et al. reported that pretreatment with YMP in hypoxic isolated rat heart enhances prostaglandin I2 outflow and attenuates the release of thromboxane A2 in rat heart during normoxia, hypoxia and reoxygenation, and hence could significantly protect the myocardium from hypoxic injury. Actually, TMP could be useful as a therapeutic agent in ischemic heart disease with coronary artery disease by suppressing coronary vasoconstriction and ischemic changes in the tissues produced by endothelin-1.
Since TMP has been confirmed to be a true calcium antagonist, it may play very important roles in the area of tissue protection and preservation. In addition, in its use as a highly potent antihypertensive drug, it may exert favorable effects on renal hemodynamics related to the reversal of renal vasoconstrictors. Although the mechanisms of action of TMP in the setting of chronic renal failure are not yet fully established, its beneficial effects may be related to protective actions such as the reduction of renal hypertrophy, modulation of mesangial cell uptake of macromolecules, changes in the permselectivity of the glomerulus and decreased free radical formation.
Acute administration of absolute ethanol often leads to tissue damage, especially in the renal system. The aims of the present study were to investigate whether TMP administration per os in mice could protect the kidney from absolute ethanol-induced lesions, and if TMP did offer protection, what its mechanism of action might be.
Acute p.o. administration of absolute ethanol (1.0 ml/kg) to fasted rats produced extensive necrosis of gastric mucosa. Pretreatment with p.o. administration of propolis ethanol extract (PEE) could effectively and dose-dependently prevent such necrosis. This protective effect is called “cytoprotection”. The maximal cytoprotective effect against absolute ethanol (AE)-induced gastric mucosal lesion was observed at 1 hour after PEE administration. A gross examination of the gastric mucosa showed a marked improvement in groups receiving PEE. In order to further investigate the gastric protective mechanism of PEE, lipid peroxidation (LPO) levels in vivo and in vitro were estimated. PEE exhibited dose-dependent superoxide scavenging activity and antioxidant effect in absolute ethanol induced lipid peroxidation in rat gastric mucosal homogenate. It was concluded that the gastric protective mechanism of PEE contributed, at least in part, to its ability to inhibit lipid peroxidation, and hence indirectly protect the gastric mucosa from oxidative stress.
目 錄
頁 次
謝 誌 Ⅰ
目 錄 Ⅱ
圖 次 Ⅶ
表 次 Ⅷ
縮寫字元 Ⅹ
第 一 部 份: 生藥川芎嗪的介紹與研究 1
第一章: 藥理學研究 3
第二章: 川芎嗪的化學研究 4
第三章: 川芎嗪的藥物代謝動力學 5
第 二 部 份: 生藥川芎嗪對於抗黴菌藥物
Econazole引起肝傷害的肝保護作用
及治療效果的研究 11
中文摘要 12
英文摘要 14
第一篇: 川芎嗪的保肝作用 15
第一節: 肝炎模型 15
第二節: 緒論 16
第三節: 材料與方法 18
第四節: 肝功能檢查 20
第五節: 細胞組織病理變化 21
第六節: 統計學上的分析 24
流程圖一 25
流程圖二 26
第七節: 結果 27
第二篇: 川芎嗪的抗氧化作用 29
第一章: 抗脂質過氧化作用之研究 29
第一節: 自由基之敘述 29
第二節: 材料與方法 32
第三節: 結果 35
第二章: 自由基清除作用之研究 36
第一節: 實驗原理 36
第二節: 材料與方法 37
第三節: 結果 40
第四節: 討論
第 三 部 份: 生藥川芎嗪在急性酒精腎傷害
的治療應用與機轉研究 43
中文摘要 44
英文摘要 45
第一篇: 川芎嗪的保腎作用 47
第一節: 腎炎模型 47
第二節: 緒論 48
第三節: 材料與方法 49
流程圖一 51
第四節: 統計學上的分析 52
第五節: 結果 53
第二篇: 川芎嗪的抗氧化作用 54
第一章: 抗脂質過氧化作用之研究 54
第一節: 材料與方法 54
第二節: 結果 57
第二章: 自由基清除作用之研究 58
第一節: 實驗原理 58
第二節: 材料與方法 58
第三節: 結果 58
第四節: 討論 59
第 四 部 份: 天然物蜂膠在急性酒精性
胃黏膜病變的細胞保護作用 63
中文摘要 64
英文摘要 65
第一篇: 蜂膠的胃黏膜保護作用 66
第一節: 消化性潰瘍模型 66
第二節: 緒論 67
第三節: 材料與方法 68
第四節: 血紅素和血球比容分析 70
第五節: 統計學上的分析 70
流程圖一 71
第六節: 結果 72
第二篇: 蜂膠的抗氧化作用 73
第一章: 抗脂質過氧化作用之研究 73
第一節: 材料與方法 73
第二節: 結果 76
第二章: 自由基清除作用之研究
第一節: 實驗原理 77
第二節: 材料與方法 77
第三節: 結果 77
第四節: 討論 78
參 考 文 獻 85
APPENDIX 1. Publication
APPENDIX 2. Publication
APPENDIX 3. Publication
APPENDIX 4. Publication
APPENDIX 5. Publication
圖 次
一. Econazole 各投與劑量下, 對誘發急性肝損傷模式之肝組織切片圖. 98
二. 川芎嗪於各投與劑量下, 對 Econazole 誘發急性肝損傷模式之肝組織切片圖. 99
三. 利用無水酒精一豪升產生老鼠胃傷害巨觀圖. 100
四. 利用不同濃度的蜂膠酒精萃取液治療胃部由於給予一毫升的無水酒精傷害巨觀圖. 101
表 次
表1. 川芎嗪於各投與劑量下, 對 Econazole 誘發大白鼠急性肝損傷 模式之 SGOT及 SGPT 值. 102
表2. 川芎嗪於體外及體內測試之脂質過氧化之丙二酸雙醛值. 103
表3. 不同濃度之川芎嗪及標準品維生素E於細胞色素C測試中,清除超養自由基活性之吸光值及抑制百分比. 104
表4. 利用三種不同劑量的川芎嗪, 以口服三十分鐘後, 其BUN及Creatinine 值和僅投與生理食鹽水和無水酒精之小白鼠腎功能檢查值之比較. 105
表5. 口服三種不同劑量的川芎嗪 ( 10, 25, 50 mg/kg ) 對於小白鼠腎臟均質液抗脂質過氧化的情形. 106
表6. 口服川芎嗪 10, 25, 50 mg/kg 三種劑量, 對抗因無水酒精造成腎臟均質液脂質過氧化的情形. 107
表7. 利用三種不同濃度的生藥川芎嗪溶液, 對於無水酒精在腎均質液中, 引起自由基產生的清除作用. 108
表8. 利用 Photoshop 軟體影像分析胃潰瘍之損傷情形. 109
表9. 不同濃度的蜂膠酒精萃取液對於血紅素( Hb)和血比容 ( Hct)的影響程度. 110
表10. 利用不同濃度的蜂膠酒精萃取液, 在體外實驗中,對於氯化亞鐵 ( Fecl2) 或氯化亞鐵加上絕對酒精所引起胃均質液脂質過氧化的抑制作用. 111
表11. 利用不同濃度的蜂膠酒精萃取液對於絕對酒精再體內引起胃均質液脂質過氧化的抑制作用. 112
表12. 利用各種不同濃度的蜂膠酒精萃取液, 對於無水酒精在胃均質液中, 引起自由基產生的清除作用. 113
縮寫字元
1. tetramethylpyrazine, TMP
2. Malonic dialdehyde, MDA
3. SGOT, serum glutamate oxaloacetate transaminase
4. SGPT, serum glutamate pyruvate transaminase
5. LPO, lipid peroxidation
6. BUN, blood urin nitrogen
7. Cr, creatinine
8. SOD, superoxide dismutase
9. Hb, hemoglobin
10. Hct, hematocrit
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