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論文名稱(中文) 黃耆與黃耆皂苷IV之含量分析及其於C3H/He小鼠之馬兜鈴酸引起腎炎模型的藥效評估
論文名稱(英文) 定量分析中黃芪提取物黃芪及其對馬兜鈴酸致腎病毛細血管密度
校院名稱 臺北醫學大學
系所名稱(中) 藥學研究所
系所名稱(英) Graduate Institute of Pharmacy
學年度 98
學期 2
出版年 99
研究生(中文) 王書藝
研究生(英文) Shu-I Wang
學號 M301097014
學位類別 碩士
語文別 中文
口試日期 2010-06-21
論文頁數 139頁
口試委員 指導教授-陳世銘
委員-莊聲宏
委員-陳振文
中文關鍵字 馬兜鈴酸腎病變  黃耆  黃耆皂苷  IV  乙型轉型生長因子  肝細胞生長因子  基質金屬蛋白分解酶  HPLC  ELSD 
英文關鍵字 Aristolochic acid nephropathy (AAN)  astragalus  astragaloside IV  TGF-β (transforming growth factor-β)  HGF (Hepatocyte Growth Factor)  MMP-9 (matrix metalloproteinase-9)  HPLC (High Performance Liquid Chromatography)  ELSD (Evaporative Light Scattering 
學科別分類
中文摘要 馬兜鈴酸(馬兜鈴酸機管局)在中草藥引起的腎病變中扮演重要的角色。本研究利用高效液相色譜法蒸發光散射檢測法(HPLC - ELSD法)針對黃耆的主要活性物質-黃耆皂苷四,進行含量分析,並藉由投予機管局引起之馬兜鈴酸腎病變(馬兜鈴酸腎病,公告) ,評估以蒸餾水抽提的黃耆濃縮劑(水提取物黃芪,自動曝光) ,以甲醇溶液抽提的黃耆濃縮劑(甲醇提取物黃芪,上午)及黃耆皂苷四(黃芪四,禽流感)對馬兜鈴酸腎病的改善效果。
本實驗選用反相®性能的蛋白質, 18封端作為層析管柱,以乙腈(乙腈)與水( 32:68 )的混合液作為移動相,流速為1毫升/分鐘。給予純系小鼠毛細血管密度( 6週的男)3.0微克/毫升機管局當飲用水連續56天,之後治療組分別經口投予聲發射(0.5克/公斤,1.0克/公斤,2.0克/公斤) ,下午( 0.5克/公斤,1.0克/公斤,2.0克/公斤)及禽流感病毒(10毫克/公斤,20毫克/公斤,40毫克/公斤)連續14天,對照組則投予等量蒸餾水,普通組則全程給予蒸餾水。借由測定尿蛋白,尿中的N -乙酰-β - D -氨基酶(NAG )與血中尿素氮(BUN )及血清肌酐(Scr ) ,以評估小鼠腎功能,腎組織使用考績染色觀察病理組織改變,並進行免疫螢光染色(轉化生長因子- β,肝細胞生長因子和MMP - 9) ,以辨識損傷部位之特異性抗原。
含量分析的結果顯示,聲發射,下午中皆含有黃耆皂苷四,含量為分別為427.4 ± 2.9微克/克及974.6 ± 36.9微克/克。而投予聲發射(0.5克/公斤,1.0克/公斤,為 2.0 g /公斤) ,下午( 0.5克/公斤,1.0克/公斤,2.0克/公斤)及禽流感病毒(10毫克/公斤,20毫克/公斤,40毫克/公斤)之治療組的尿蛋白,尿NAG ,血尿素氮,血肌酐值都有降低;組織學及免疫螢光染色觀察發現腎組織損傷的情形皆有緩解。根據上述結果發現,治療藥物之療效依序為1.0 ≧自動曝光自動曝光自動曝光2.0 ≧ 0.5, 1.0 ≒上午上午上午2.0 ≧ 0.5,禽流感病毒亞型禽流感病毒40 ≧ 20 ≧ 10禽流感病毒。由於自動曝光,上午中僅含有少量禽流感病毒,且其投予禽流感病毒的劑量低於以純物質禽流感病毒治療的組別,而其各組間各項腎功能指標緩解情形卻相似,因此推測黃耆中除了禽流感外可能還有其他有效成分可以緩解機管局所造成的腎損傷。總結來說,聲發射,上午,禽流感病毒皆顯示能緩解機管局所造成的腎損傷。
然而,由於本實驗僅為小型試驗,未來仍需要更多的樣本數以觀察證實黃耆於AAN的的療效。
英文摘要 馬兜鈴酸(AA )已被證明發揮了關鍵作用的中草藥腎病。本研究的目的是定量確定其內容黃芪四(禽流感,同田生物技術,上海,中國)的高效液相色譜蒸發光散射檢測法(HPLC - ELSD法)在水提取物黃芪( AE)和甲醇黃芪提取物(上午) 。此外,本研究還評估了療效AE和上午,在機管局和禽流感引起的腎病( AAN的) 。
®性能的蛋白質,反相柱18封端,用於分析AE和禽流感病毒在上午。禽流感病毒分離,以乙腈:水( 32:68 )為流動相。流速為 1毫升/分鐘。機管局溶解於蒸餾水(含3μg/ml )作為飲用水毛細血管密度(6週齡雄性)為56天。治療組口服與美國運通(0.5克/公斤,1.0克/公斤,2.0克/公斤) ,下午( 0.5g/kg的,1.0克/公斤,2.0克/公斤)和禽流感病毒(10毫克/公斤, 20毫克/公斤,40毫克/公斤) ,每日1次後,小鼠誘導AAN的。對照組用蒸餾水管理。正常組僅管理與蒸餾水整個實驗。尿蛋白(向上),尿N -乙酰-β - D -氨基酶(NAG ),尿素氮( BUN)和血肌酐(Scr )水平評估腎功能。腎組織遭到了組織學檢查( PAS染色和免疫熒光) 。該抗體,包括轉化生長因子-β(轉化生長因子- β) ,肝細胞生長因子(肝細胞生長因子) ,和MMP - 9(基質金屬蛋白酶9),被選為承認特異性抗原在損傷部位。
定量分析的基礎上,禽流感病毒是在這兩個AE和上午,禽流感病毒和內容黃芪提取物427.4 ± 2.9微克/克和974.6 ± 36.9微克/克,分別。與對照組相比,向上,尿NAG ,尿素氮,肌酐明顯降低所有治療組。此外,治療組改善AA誘導腎臟形態損害,降低染色強度轉化生長因子-β和肝細胞生長因子增加強度和MMP - 9的範圍內組織的損傷。在對療效的比較治療情況如下:自動曝光自動曝光1.0 ≧ 2.0 ≧聲發射0.5, 1.0 ≒上午上午上午0.5和2.0 ≧ 20 ≧禽流感病毒亞型禽流感病毒亞型禽流感病毒40 ≧ 10。總之,我們的研究表明,聲發射,上午,禽流感病毒能改善腎益的AAN的成果。除了禽流感病毒,有可能有其他有效成分黃芪對AAN的。更大規模的研究證實黃芪的腎臟保護作用的體內與 AA腎病是必要的。
論文目次 目錄我
圖目錄六
第九表目錄
縮寫表我
中文摘要三
摘要v
第一章緒言1
第二章文獻回顧2
第一節馬兜鈴酸腎病變的由來2
第二節馬兜鈴酸之基本特性4
2.1用途4
2.2含馬兜鈴酸的生藥及使用現況4
2.3結構與特性5
2.4馬兜鈴酸的代謝6
第三節馬兜鈴酸腎病變(馬兜鈴酸腎病,馬兜鈴酸腎病)10
3.1臨床表現及病理特徵10
3.2鑑別診斷11
3.3致病機轉15
3.4藥物治療17
第四節馬兜鈴酸腎病變的實驗模型19
4.1馬兜鈴酸的急性腎毒性19
4.2馬兜鈴酸的慢性腎毒性21
第五節腎小管間質性纖維化23
5.1轉化生長因子-β 29
5.2肝細胞生長因子(HGF )33
5.3基質金屬蛋白酶38
第六節黃耆及其藥效研究41
6.1生藥學考察41
6.2黃耆的藥效研究45
6.1黃耆皂苷的藥效研究50
6.2黃耆中其他成分之藥效研究53
第三章研究目的57
第四章材料與方法58
第一節黃耆濃縮劑中黃耆皂苷四之HPLC - ELSD測定含量分析58
1.1實驗試藥58
1.2儀器裝置59
1.3色譜條件59
1.4標準品配置與檢量線製作59
1.5檢品配置60
1.6分析方法之精密度(精密)與準確度(精度)試驗60
第二節黃耆濃縮劑與黃耆皂苷四在慢性AAN的之藥效評估61
1.1實驗動物61
1.2實驗藥物61
1.3實驗設計61
1.4尿液收集63
1.5動物犧牲法,血液及組織切片製作63
1.6尿蛋白,尿NAG含量測定64
1.7血清中尿素氮及肌酐的含量測定64
1.8 Periodoic酸希夫( PAS)染色組織染色64
1.9組織損傷程度的量化65
1.10免疫螢光染色(免疫)66
1.11統計方法66
第五章結果67
第一節黃耆濃縮劑中黃芪甲苷之HPLC - ELSD測定含量分析67
1.1耆濃縮劑中黃耆皂苷含量之四,高效液相色譜,蒸發光散射圖譜67
1.2線性(線性)67
1.3分析方法之精密度與準確度67
1.4黃耆濃縮劑之黃耆皂苷含量分析68第四節
第二節以蒸餾水抽提之黃耆濃縮劑對慢性馬兜鈴酸腎病的藥效評估75
2.1尿蛋白,尿NAG含量分析75
2.2血清中尿素氮分析78
2.3血清中肌酐分析78
2.4組織病理考績染色80
2.5組織損傷量化分析80
2.6免疫螢光染色及量化分析83
第三節以甲醇抽提之黃耆濃縮劑對慢性馬兜鈴酸腎病的藥效評估87
3.1尿蛋白,尿NAG含量分析87
3.2血清中尿素氮分析90
3.3血清中肌酐分析90
3.4組織病理考績染色92
3.5組織損傷量化分析92
3.6免疫螢光染色及量化分析95
第四節黃耆皂苷四對慢性馬兜鈴酸腎病的藥效評估99
4.1尿蛋白,尿NAG含量分析99
4.2血清中尿素氮分析第102
4.3血清中肌酐分析102
4.4組織病理考績染色104
4.5組織損傷量化分析第104
4.6免疫螢光染色及量化分析107
第六章討論112
第一節黃耆濃縮劑中黃耆皂苷四之HPLC - ELSD測定含量分析112
第二節黃耆濃縮劑及黃耆皂苷四在AAN的之藥效評估114
第七章結論122
參考文獻123
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