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研究生:李淑琪
研究生(外文):Shu-Chi Li
論文名稱:三七或丹蔘利用紅麴菌或牛樟菌發酵炮製後對於凝血作用及血脂之影響
論文名稱(外文):Effects of Monascus purpureus or Antrodia camphorate-fermented Panax notoginseng or Savia miltiorrhiza on coagulation and lipid profile
指導教授:謝淑玲謝淑玲引用關係
指導教授(外文):Shu-Ling Hsieh
口試委員:王志傑吳志忠游宜屏張月萍
口試委員(外文):Jyh-Jye WangChih-Chung WuYi-Ping YuYueh-Ping Chang
口試日期:2014-07-25
學位類別:碩士
校院名稱:國立高雄海洋科技大學
系所名稱:水產食品科學研究所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:144
中文關鍵詞:三七丹蔘紅麴牛樟凝集因子動脈斑塊
外文關鍵詞:Panax notoginsengSavia miltiorrhizaMonascus purpureusAntrodia camphorateantithromboticantiatherosclerosis
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丹蔘及三七在中醫藥之應用上已知其具有活血、化瘀之作用;過去的研究亦發現此等材料對於心血管疾病具有良好之保健功效。另外,在近年來利用微生物發酵中草藥所產生之炮製產品,因具有增強療效或改變藥理特性之作用,於是此一技術逐漸成為藥物與保健食品研究開發之新領域。因此,本研究擬探討三七或丹蔘利用紅麴菌或牛樟菌發酵炮製後對於抗血栓及降血脂作用之影響。本研究首先利用EA. hy 926細胞株培養之模式,以腫瘤壞死因子 (tumor necrosis factor alpha, TNF-α) 誘發細胞間粘附因子-1 (intercellular adhesion molecule-1, ICAM-1) 表現上升後,再給予不同劑量之三七紅麴菌發酵炮製物 (Monascus purpureus-fermented Panax notoginseng, MPPN)、丹蔘紅麴菌發酵炮製物 (Monascus purpureus-fermented Savia miltiorrhiza, MPSM)、紅麴 (Monascus purpureus, MP)、三七牛樟菌發酵炮製物 (Antrodia camphorate-fermented Panax notoginseng, AP)、丹蔘牛樟菌發酵炮製物 (Antrodia camphorate-fermented Savia miltiorrhiza, AS) 及牛樟 (Antrodia camphorate, AC),以初步瞭解此等材料降低心血管疾病之潛力。由EA. hy 926細胞實驗模式之結果可知在牛樟發酵物部分,以AS對於ICAM-1之表現具有最佳之IC50;而紅麴發酵物則以MPPN之抑制效果最佳。進一步本研究利用AS及MPPN進行動物實驗,分別探討AS及MPPN對抗血栓及降血脂之影響。首先以高脂飲食餵食SD大鼠為期三週以誘發其產生高血脂症後,再以不同濃度之MPPN餵食十週,以探討MPPN對血栓形成以及血脂質之影響。實驗結果顯示給予大鼠MP、PN、低劑量之MPPN (low-dose MPPN, MPPN-L) 和高劑量之MPPN (high-dose MPPN, MPPN-H) 後其流血時間 (bleeding time) 皆顯著高於控制組 (P<0.05);當給予大鼠PN時其凝血酶原時間 (prothrombin time, PT) 顯著高於控制組 (P<0.05);當給予大鼠MP、PN、MPPN-L和MPPN-H後其蛋白C (protein C) 含量顯著高於控制組 (P<0.05);而當給予大鼠PN、MPPN-L和MPPN-H後,其第三抗凝血酶 (anti-thrombin III) 含量顯著高於控制組 (P<0.05);另外,當給予大鼠MP、PN、MPPN-L和MPPN-H後其動脈斑塊面積 (atherosclerotic plaque area) 及三酸甘油脂 (triglycerides, TG) 含量皆顯著低於控制組 (P<0.05);而當給予PN、MPPN-L和MPPN-H時,低密度脂蛋白膽固醇 (low density lipoprotein cholesterol, LDL-C) 的含量也顯著下降 (P<0.05)。由上述結果可知以紅麴發酵後之三七(MPPN) 具有抗血栓及降血脂之能力。接下來,本研究利用高脂飲食餵食SD大鼠三週,誘發其產生高血脂症後,再以不同濃度之AS餵食十週以探討AS對血栓形成以及血脂質之影響。實驗結果顯示給予大鼠AC、低劑量之AS (low-dose AS, AS-L) 和高劑量之AS (high-dose AS, AS-H) 後其流血時間顯著高於控制組 (P<0.05),且以高劑量AS效果最佳;當給予大鼠AC、SM、AS-L和AS-H後,其活化部分凝血活酶時間 (activated partial thromboplastin time, APTT) 皆顯著高於控制組 (P<0.05);而給予大鼠AS-L和AS-H後其PT時間顯著高於控制組 (P<0.05);當給予大鼠AC和AS-H,抗凝血因子蛋白C含量顯著高於控制組;而給予大鼠AS-H時第三抗凝血酶含量也顯著高於控制組 (P<0.05);另外,在動脈斑塊面積的結果中也發現給予大鼠AC、SM、AS-L和AS-H後,斑塊面積顯著下降 (P<0.05),而且當給予大鼠AS-H後,其斑塊面積顯著低於其他組別 (P<0.05)。在血液脂質方面的變化,則可得知給予大鼠AC、SM、AS-L和AS-H後,其TG及LDL-C皆顯著低於控制組,而且當給予大鼠AS-H後,其TG含量顯著低於其他各組 (P<0.05)。顯示AS-H具有較佳之抗凝血、抗血栓及降血脂效果,且呈劑量依賴性關係。綜合上述結果顯示三七或丹蔘經微生物發酵後皆具有抗血栓及降血脂之能力。
Panax notoginseng (PN) and Savia miltiorrhiza (SM) have been show that they can improve blood stasis and prevent cardiovascular and cerebrovascular diseases. On the other hands, traditional Chinese medicine will be regulated pharmacological effects after microbial fermentation, so its become a new trend on medicine pharmacological research. The aims of this study were investigated the effect of Monascus purpureus or Antrodia camphorate-fermented Panax notoginseng or Savia miltiorrhiza on antithrombotic and improve hyperlipidemia. Firstly, suppression of Monascus purpureus-fermented Panax notoginseng (MPPN) and Monascus purpureus-fermented Savia miltiorrhiza (MPSM), Monascus purpureus (MP), Antrodia camphorata-fermented Panax notoginseng (AP), Antrodia camphorate-fermented Savia miltiorrhiza (AS) and Antrodia camphorate (AC) on intercellular adhesion molecule-1 (ICAM-1) expression on human umbilical vein endothelial cells (EA. hy 926 cells) stimulated by tumor necrosis factor (TNF-α) were investigated in this study. Results show that AS and MPPN groups have the lowest IC50 for ICAM-1 levels on EA. hy 926 cells induce by TNF-α. To study the effects of AS and MPPN on the anti-antithrombotic and anti-hyperlipidemia, Sprague-Dawley (SD) rats fed with a high fat diet was as an animal experimental. In this study, to induce thrombosis and hyperlipidemia, SD rats were fed a high-fat diet for three weeks then 0.5 g/kg BW MP, 0.1 g/kg BW PN, 0.1 g/kg BW MPPN (MPPN-L) or 1.1 g/kg BW (MPPN-H), respectively, were added to their high-fat diet for ten weeks. From bleeding times analysis, MP, PN, MPPN-L or MPPN-H groups have a significantly longer than that of the control group (P<0.05). In PN group rats, the prothrombin times (PT) was significantly higher than that of the control group rats (P<0.05). After treated with PN, MPPN-L and MPPN-H to SD rats, their anti-thrombin III level were also significantly higher than the control group (P<0.05). In MP, PN, MPPN-L or MPPN-H groups, the protein C levels were significantly higher than that of the control group (P<0.05). The atherosclerotic plaque area assay results also found a significant decrease in plaque area (P<0.05) after treated with MP, PN, MPPN-L and MPPN-H in experimental rats. From the blood lipid content analysis, there are significant decrease in triglyceride (TG) level (P<0.05) after treatment with MP, PN, MPPN-L or MPPN-H. In addition, the low density lipoprotein cholesterol (LDL-C) levels of PN, MPPN-L and MPPN-H groups were significantly higher than that of the control group (P<0.05). These results indicate that MP, PN and MPPN with a enhance anti-coagulant, anti-thrombotic and improve hyperlipidemia effects, among which P. notoginseng had the best regulation. On the other study, to induce thrombosis and hyperlipidemia, SD rats were fed a high-fat diet for three weeks then 2.4 g/kg BW AC, 0.1 g/kg BW SM, 2.5 g/kg BW AS (AS-L) or 12.5 g/kg BW (AS-H), respectively, were added to their high-fat food for ten weeks. From bleeding times analysis, AC, AS-L or AS-H groups have a significantly longer than that of the control group (P<0.05). After experimental rats were treated AC, SM, AS-L or AS-H, their activated partial thromboplastin times (APTT) were significantly higher than that of the control group (P<0.05). After giving SD rats AS-L and AS-H, their PT were significantly higher than that of the control group (P<0.05). Antithrombin III level of AS-H group was also significantly higher than that of the control group (P<0.05). In AC and AS-H groups rat, protein C levels were significantly higher than that of the control group. From atherosclerotic plaque area assay results show a significant decrease in plaque area (P<0.05) after treatment with AC, SM, AS-L or AS-H, among which AS-H had the best affect. The results of the blood lipid examination, a significant decrease in TG and LDL-C level (P<0.05) in AC, SM, AS-L, or AS-H groups, among which AS-H in TG had the best improved. These results indicate that AS can enhance experimental rats anti-coagulant, anti-thrombotic and anti-hyperlipidemia. Moreover, it exhibited a dose-dependent relationship which indicates that AS has the potential on cardiovascular disease prevention. Our study indicate PN and MPPN can have anti-thrombotic and anti-hyperlipidemia affects. And, AS can enhance the anti-coagulant, anti-thrombotic and improve hyperlipidemia as compare AP along group.
摘要 I
Abstract IV
誌謝 VII
表目錄 XVI
圖目錄 XVIII
縮寫對照表 XXI
壹、前言 1
貳、文獻回顧 3
一、中草藥炮製 3
(一) 中草藥炮製之目的 3
(二) 中草藥炮製之種類及應用 3
(三) 微生物發酵炮製之原理及作用 4
(四) 微生物發酵炮製之應用及現況 5
二、微生物於中草藥發酵方面之應用 5
(一) 紅麴菌 5
1. 紅麴菌之簡介 6
2. 紅麴菌之功能 6
3. 紅麴菌在中草藥發酵之研究 11
(二) 牛樟菌 11
1. 牛樟菌之簡介 11
2. 牛樟菌之功能 13
3. 牛樟菌在中草藥發酵之研究 16
三、中草藥於食品中之應用 17
(一) 三七 17
1. 三七之簡介 17
2. 三七之功能 17
3. 三七主要活性成分及研究 22
4. 三七發酵物之研究 24
(二) 丹蔘 24
1. 丹蔘之簡介 24
2. 丹蔘之功能 24
3. 丹蔘主要活性成分及研究 28
4. 丹蔘發酵物之研究 29
四、心血管疾病之發生與調控 29
(一) 心血管疾病之罹患率 29
(二) 血管巨噬細胞黏附機制 30
(三) 血栓形成機制 31
1. 血管內皮細胞與抗血栓之作用關係 32
2. 血小板與血栓形成之作用關係 33
3. 血液凝集機制與血栓形成之作用關係 33
4. 血液抗凝集系統與血栓形成之作用關係 35
(四) 血液脂質與血栓形成之作用關係 37
(五) 中草藥與心血管疾病之關係 38
參、研究目的 40
肆、材料與方法 41
一、實驗儀器 41
二、實驗藥品 41
三、實驗設計 44
(一) 三七或丹蔘之紅麴菌或牛樟菌發酵物之製備 44
1. 三七或丹蔘之紅麴菌之固態發酵物之製備 44
2. 三七或丹蔘之牛樟菌之液態發酵物之製備 44
(二) 細胞實驗模式 47
1. EA. hy 926細胞株之培養、解凍、繼代與保存 48
2. 細胞存活率試驗分析及形態觀察 50
(三) 動物實驗模式 53
1. 動物試驗設計 54
2. 血管主動脈斑塊分析 57
3. 斷尾流血時間分析 57
4. 血漿凝集因子分析 58
5. 血漿抗凝集因子分析 60
6. 血液脂質分析 62
7. 數據整理及統計分析 62
伍、結果 63
一、利用細胞實驗探討三七或丹蔘之紅麴菌或牛樟菌對於細胞生存力及血液凝集分子之影響 63
(一) 三七紅麴發酵物對於EA. hy 926細胞株細胞生存力之影響 63
(二) 丹蔘紅麴發酵物對於EA. hy 926細胞株細胞生存力之影響 66
(三) 單獨紅麴發酵物對於EA. hy 926細胞株細胞生存力之影響 66
(四) 三七牛樟發酵物對於EA. hy 926細胞株細胞生存力之影響 71
(五) 丹蔘牛樟發酵物對於EA. hy 926細胞株細胞生存力之影響 71
(六) 單獨牛樟發酵物對於EA. hy 926細胞株細胞生存力之影響 76
(七) 三七紅麴發酵物對於EA. hy 926細胞株黏附蛋白ICAM-1表現之影響 79
(八) 丹蔘紅麴發酵物對於EA. hy 926細胞株黏附蛋白ICAM-1表現之影響 79
(九) 單獨紅麴發酵物對於EA. hy 926細胞株黏附蛋白ICAM-1表現之影響 79
(十) 三七牛樟發酵物對於EA. hy 926細胞株黏附蛋白ICAM-1表現之影響 80
(十一) 丹蔘牛樟發酵物對於EA. hy 926細胞株黏附蛋白ICAM-1表現之影響 84
(十二) 單獨牛樟發酵物對於EA. hy 926細胞株黏附蛋白ICAM-1表現之影響 84
(十三) 三七及丹蔘之紅麴菌及牛樟菌發酵炮製物ICAM-1表現之IC50之結果 88
二、利用動物實驗探討三七之紅麴菌發酵物對於血液凝集及血脂質變化之研究 89
(一) MPPN對高脂飲食下SD大鼠生長特性之影響 89
(二) MPPN對高脂飲食SD大鼠臟器相對重量之影響 89
(三) MPPN對高脂飲食SD大鼠肝腎功能之影響 89
(四) MPPN對高脂飲食SD大鼠流血時間之影響 93
(五) MPPN對高脂飲食SD大鼠血液凝集因子之影響 93
(七) MPPN對高脂飲食SD大鼠血液指標之影響 95
(八) MPPN對高脂飲食SD動脈斑塊面積之影響 95
(九) MPPN對高脂飲食SD大鼠血液脂質之影響 95
三、利用動物實驗探討丹蔘之牛樟菌發酵物對於血液凝集及血脂質變化之研究 102
(一) AS對高脂飲食SD大鼠生長特性之影響 102
(二) AS對高脂飲食SD大鼠臟器相對重量之影響 102
(三) AS對高脂飲食SD大鼠肝腎功能之影響 102
(四) AS對高脂飲食SD大鼠流血時間之影響 103
(五) AS對高脂飲食SD大鼠血液凝集因子之影響 103
(六) AS對高脂飲食SD大鼠抗血液凝集因子之影響 109
(七) AS對高脂飲食SD大鼠血液指標之影響 109
(八) AS對高脂飲食SD大鼠動脈斑塊面積之影響 112
(九) AS對高脂飲食SD大鼠血液脂質之影響 112
陸、討論 115
柒、結論 121
捌、參考文獻 122

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