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http://libir.tmu.edu.tw/handle/987654321/36469
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| 題名: | 白朮炮製之研究 |
| 作者: | 王坤謄 |
| 貢獻者: | 藥學系(博士班) |
| 關鍵詞: | 白朮 炮製 atractylon atractylenolide III 酒精引發胃黏膜損傷 自由基清除 MMPs |
| 日期: | 2010 |
| 上傳時間: | 2010-10-21 09:57:33 (UTC+8) |
| 摘要: | 白朮為經常用於補脾健胃之中藥，且根據傳統醫書記載利用不同輔料進行加工炮製後可減去燥性與增強補脾益氣效用。但炮製後的品質至今仍無標準化規範。因此本研究將針對白朮炮製前後成分及藥理變化製訂出白朮之標準規範。研究方法共分為三個部分: 第一部分先建立白朮指標成分快速分離流程，結果顯示: 利用n-hexane冷浸萃取法再經矽膠管柱層析，可快速分離得到atractylon、atractylenolides I、II、III與biatractylolide等這五個指標成分。第二部分收集白朮市售品，分析其指標成分的含量，並利用自行炮製之白朮樣品建立標準白朮炮製流程，以了解白朮炮製後化學成分之變化。結果顯示: 台灣所販賣的白朮市售品中以紅土及灶心土炒製品比例較高(32/38)，而其中指標成分含量相差甚多，差異將近50%。此外，市售白朮生品中atractylon含量較高，而炮製品則是以atractylenolide III含量較高。利用恆溫炫風式滾筒炒鍋進行藥材炮製，結果顯示atractylon隨加熱時間延長而含量下降，atractylenolides II及III則隨加熱時間延長而含量上升。在重金屬檢測中，白朮經輔料炮製後其鉛及銅含量隨之上升。第三部分則利用體內外胃黏膜損傷試驗與抗氧化試驗來評估白朮炮製前後藥理活性之改變，結果顯示: 白朮炮製前後樣品皆無降低胃酸總酸度之功效，而在體外與體內的實驗中皆能顯著的抑制酒精所引發之胃黏膜損傷，但組間無差異。Atractylenolide III為白朮中主要活性成分，其調控機制為促進TIMP-1及TIMP-2蛋白質表現，進而抑制MMP-2及MMP-9酵素活性及蛋白質表現，減緩酒精性胃潰瘍之發生。抗氧化功效上，白朮生品精油具有顯著的DPPH自由基清除、抑制脂質過氧化與提升肝臟過氧化氫酵素功效，但炮製後其抗氧化效用隨之下降，而白朮生品與炮製品精油對H9C2, 3T3及WI-38細胞株皆不具有顯著細胞毒性。綜合以上結果，本研究建議白朮使用於胃黏膜保護功效時以清炒較佳，因白朮清炒後其胃黏膜保護功效無顯著下降，但可降低生品中水分含量，更易於保存。此外，白朮樣品內atractylenolide III含量在1.08-1.84 mg/g之間，即有顯著的胃黏膜保護功效。
Atractylodes Rhizoma, the dried rhizome of Atractylodes ovata De Candolle, is widely used for strengthening the stomach functions and usually underwent the processing procedure before usage. Nowadays, the standard operation procedure (SOP) for using Atractylodes Rhizoma is not well-established. Hence, the SOP of Atractylodes Rhizoma was set up according to the variations of chemical contents and pharmacological effects after processing. The study was divied into 3 parts. Firstly, atractylon, atractylenolides I, II, III and biatractylolide were isolated through silica gel chromatography. Secondly, the commercial Atractylodes Rhizoma was collected for analyzing the contents of atractylon, atractylenolides II and III. Results showed that almost all commercial Atractylodes Rhizoma was processed samples (32/38), including stir-frying with red soil or burnt clay. Besides, contents of these 3 pure components in commercial Atractylodes Rhizoma varied a lot (almost reaching 50%). Contents of atractylon were higher in raw materials, but contents of atractylenolides were higher in processed samples. In addition, following the prolonged processing time, contents of atractylon were decreased, but atractylenolides II and III increased. In the heavy metal analysis, contents of Pb and Cu were increased following stir-frying with assistant substrates. Thirdly, we used in vitro and in vivo gastroprotective assays and anti-oxidative effects to evaluate the pharmacological variations after processing. Results showed that raw and processed Atractylodes Rhizoma did not significantly decrease the total acid of gastric juice. However, raw and processed Atractylodes Rhizoma significantly protected the ethanol-induced gastric mucosal damage in vitro and in vivo, but no significant changes between each group were found. Atractylenolide III was the principal gastroprotective component of Atractylodes Rhizoma and the possible mechanism was through regulating the expressions of MMP-2, MMP-9, TIMP-1 and TIMP-2. In the anti-oxidative effects, raw materials displayed significant DPPH-scavenging, anti-lipid peroxidative and enhanced catalase activities effects. However, the anti-oxidative effects of raw materials were reduced after processing. Both raw and processed Atractylodes Rhizoma essential oils did not show the cytotoxicities in H9C2, 3T3 and WI-38 cell lines, Taken together, we suggested that Atractylodes Rhizoma which was stir-fried without assistant substrates was better for the gastroproective usages. In addition, the atractylenolide III contents in Atractylodes Rhizoma should range from 1.08 to 1.84 mg/g. |
| 關聯: | 151頁 |
| 描述: | (一)論文目次
目錄…………………………………………………………………………………………………………………………1
圖目錄………………………………………………………………………………………………………………………9
表目錄……………………………………………………………………………………………………………………11
中文摘要…………………………………………………………………………………………………………………12
英文摘要…………………………………………………………………………………………………………………14
縮寫表………………………………………………………………………………………………………………………16
緒論……………………………………………………………………………………………………………………………18
白朮之文獻回顧………………………………………………………………………………………………………23
第一部分 白朮指標成分分離 36
壹、前言…………………………………………………………………………………………………………………37
貳、實驗材料與方法………………………………………………………………………………………………41
1. 白朮指標成分分離標準流程制定…………………………………………………………………41
1-1. 實驗材料………………………………………………………………………………………………………41
1-2. 化學試劑………………………………………………………………………………………………………41
1-3. 實驗儀器…………………………………………………………………………………………………………41
1-4. 分離流程…………………………………………………………………………………………………………42
叁、實驗結果………………………………………………………………………………………………………………44
1. Atractylon之圖譜解析………………………………………………………………………………44
2. Atractylenolide I之圖譜解析………………………………………………………………45
3. Atractylenolide II之圖譜解析……………………………………………………………46
4. Atractylenolide III之圖譜解析……………………………………………………………47
5. Biatractylolide之圖譜解析……………………………………………………………………48
肆、討論…………………………………………………………………………………………………………………………49
第二部分 白朮炮製前後成分變化 51
壹、前言…………………………………………………………………………………………………………………………52
貳、實驗材料與方法……………………………………………………………………………………………………55
1. 白朮市售品之收集.………………………………………………………………………………………………55
2. 白朮標準炮製流程建立…………………………………………………………………………………………55
2.1 炮製所需之材料……………………………………………………………………………………………………55
2-1.1. 中藥材白朮………………………………………………………………………………………………………55
2-1.2. 炮製之輔料………………………………………………………………………………………………………55
2-1.3. 炮製之設備………………………………………………………………………………………………………55
2-2. 炮製之方法……………………………………………………………………………………………………………55
3. 白朮炮製樣品之成分變化………………………………………………………………………………………56
3-1. 化學試劑………………………………………………………………………………………………………………56
3-2. 實驗儀器…………………………………………………………………………………………………………………56
3-3. 炮製炮製樣品之品質管制……………………………………………………………………………………57
3-3.1. 白朮炮製樣品外觀顏色分析…………………………………………………………………………57
3-3.1.1. 分析原理…………………………………………………………………………………………………………57
3-3.1.2. 分析方法…………………………………………………………………………………………………………57
3-3.2. 白朮炮製樣品水分含量分析…………………………………………………………………………58
3-3.2.1. 樣品製備…………………………………………………………………………………………………………58
3-3.2.2. 分析方法………………………………………………………………………………………………………58
3-3.3. 白朮炮製樣品重金屬含量分析………………………………………………………………………58
3-3.3.1. 樣品製備…………………………………………………………………………………………………………58
3-3.3.2. 分析方法…………………………………………………………………………………………………………58
3-3.4. 白朮炮製樣品指標成分分析……………………………………………………………………………58
3-3.4.1. 樣品製備…………………………………………………………………………………………………………58
3-3.4.2. 高效液相層析儀分析……………………………………………………………………………………59
3-3.4.3. 標準檢量線製備……………………………………………………………………………………………59
3-3.4.4. 分析方法之確效………………………………………………………………………………………………60
3-3.5. 白朮炮製樣品水萃取物指標成分含量分析……………………………………………………61
3-3.5.1 樣品製備………………………………………………………………………………………………………………61
3-3.5.2. 標準檢量線製備…………………………………………………………………………………………61
3-3.6. Atractylon於受熱後之變化………………………………………………………………………61
3-3.6.1. 樣品製備……………………………………………………………………………………………………………61
3-3.6.2. 標準檢量線製備………………………………………………………………………………………………61
3-3.7. 白朮炮製樣品精油含量變化………………………………………………………………………………61
3-3.7.1. 精油萃取……………………………………………………………………………………………………………61
3-3.7.2. 高效液相層析儀分析………………………………………………………………………………………62
3-3.7.3. 氣相層析質譜儀分析………………………………………………………………………………………63
叁、實驗結果……………………………………………………………………………………………………………………………64
1. 白朮市售品種類整理…………………………………………………………………………………………………64
2. 白朮樣品炮製結果………………………………………………………………………………………………………65
3. 白朮炮製樣品之品質管制……………………………………………………………………………………………65
3-1. 白朮炮製前後樣品之顏色………………………………………………………………………………………65
3-2. 白朮炮製前後樣品之水分含量……………………………………………………………………………66
3-3. 白朮炮製前後樣品之重金屬含量………………………………………………………………………67
3-4. 白朮市售品及炮製樣品指標成分之分析…………………………………………………………67
3-4.1. 檢量線製備…………………………………………………………………………………………………………67
3-4.2. 分析方法之確效性……………………………………………………………………………………………68
3-4.3. 市售品指標成份含量………………………………………………………………………………………71
3-4.4. 白朮炮製前後樣品指標成分之含量………………………………………………………………73
3-4.5. 白朮炮製前後樣品之水萃取物中指標成分含量分析…………………………………74
3-4.6. 白朮炮製前後樣品之精油中指標成分含量 分析……………………………………74
3-4.7. Atractylon受熱後氧化之情形……………………………………………………………………75
3-4.8. 白朮炮製前後樣品精油組成之變化…………………………………………………………………76
肆、討論……………………………………………………………………………………………………………………………80
第三部分 白朮炮製前後藥理活性變化 84
壹、前言………………………………………………………………………………………………………………………………85
貳、實驗材料與方法…………………………………………………………………………………………………………89
1. 胃酸總酸度試驗…………………………………………………………………………………………………………89
1-1. 實驗動物…………………………………………………………………………………………………………………89
1-2. 實驗樣品…………………………………………………………………………………………………………………89
1-3. 實驗材料與儀器……………………………………………………………………………………………………89
1-4. 實驗方法………………………………………………………………………………………………………………89
2. 胃黏膜損傷保護試驗……………………………………………………………………………………………90
2-1. 胃黏膜細胞試驗……………………………………………………………………………………………………90
2-1.1. 胃黏膜細胞分離及培養………………………………………………………………………………90
2-1.1.1. 實驗動物…………………………………………………………………………………………………90
2-1.1.2. 胃黏膜細胞分離與培養所需試劑………………………………………………………90
2-1.1.3. 胃黏膜細胞分離流程……………………………………………………………………………90
2-1.1.4. 胃黏膜細胞鑑定流程……………………………………………………………………………91
2-1.2. 白朮炮製前後樣品與指標成分對胃黏膜細胞之毒性評估…………………91
2-1.2.1. 實驗樣品……………………………………………………………………………………………………91
2-1.2.2. 化學試劑…………………………………………………………………………………………………92
2-1.2.3. 實驗儀器…………………………………………………………………………………………………92
2-1.2.4. 實驗方法……………………………………………………………………………………………………93
2-1.3. 酒精濃度對胃黏膜細胞之影響……………………………………………………………………93
2-1.4. 白朮炮製前後樣品與指標成分保護酒精所引發胃黏膜細胞毒性之作用…………………………………………………………………………………………………………………………………………93
2-1.5. 白朮炮製前後樣品與指標成份保護酒精誘導胃黏膜細胞膜損傷之作用…………………………………………………………………………………………………………………………………………93
2-1.5.1. 實驗原理…………………………………………………………………………………………………94
2-1.5.2. 實驗方法…………………………………………………………………………………………………94
2-2. 大鼠急性胃潰瘍試驗………………………………………………………………………………………94
2-2.1. 實驗樣品…………………………………………………………………………………………………………94
2-2.2. 實驗動物…………………………………………………………………………………………………………94
2-2.3. 化學試劑…………………………………………………………………………………………………………94
2-2.4. 實驗儀器………………………………………………………………………………………………………95
2-2.5. 實驗所需之抗體……………………………………………………………………………………………95
2-2.6. 實驗方法………………………………………………………………………………………………………95
2-2.6.1. 酒精濃度誘導大鼠急性胃潰瘍之試驗………………………………………………95
2-2.6.2. 白朮炮製前後樣品與指標成分保護酒精誘導大鼠急性胃潰瘍之試驗……………………………………………………………………………………………………………………………………96
2-2.6.3. 胃潰瘍之病理切片…………………………………………………………………………………96
2-2.6.4. 受質凝膠電泳法………………………………………………………………………………………96
2-2.6.5. 西方點墨法…………………………………………………………………………………………………97
3. 抗氧化能力試驗…………………………………………………………………………………………………………98
3-1. DPPH自由基清除試驗…………………………………………………………………………………………98
3-1.1. 實驗樣品……………………………………………………………………………………………………………98
3-1.2. 化學試劑……………………………………………………………………………………………………………98
3-1.3. 實驗儀器……………………………………………………………………………………………………………98
3-1.4. 實驗方法……………………………………………………………………………………………………………98
3-2. 電子自旋共振法之自由基偵測法……………………………………………………………………99
3-2.1. 實驗樣品……………………………………………………………………………………………………………99
3-2.2. 化學試劑……………………………………………………………………………………………………………99
3-2.3. 實驗儀器……………………………………………………………………………………………………………99
3-2.4. 實驗方法……………………………………………………………………………………………………………99
3-3. 脂質過氧化之試驗………………………………………………………………………………………………100
3-3.1. 實驗樣品……………………………………………………………………………………………………………100
3-3.2. 實驗動物……………………………………………………………………………………………………………100
3-3.3. 化學試劑……………………………………………………………………………………………………………100
3-3.4. 實驗儀器……………………………………………………………………………………………………………100
3-3.5. 實驗方法……………………………………………………………………………………………………………100
3-4. 過氧化氫酵素試驗………………………………………………………………………………………………100
3-4.1. 實驗樣品……………………………………………………………………………………………………………101
3-4.2. 化學試劑……………………………………………………………………………………………………………101
3-4.3. 實驗儀器……………………………………………………………………………………………………………101
3-4.4. 實驗步驟……………………………………………………………………………………………………………101
4. 細胞毒性試驗………………………………………………………………………………………………………………101
4-1. 實驗樣品…………………………………………………………………………………………………………………101
4-2. 化學試劑…………………………………………………………………………………………………………………101
4-3. 實驗儀器…………………………………………………………………………………………………………………101
4-4. 實驗方法………………………………………………………………………………………………………………102
叁、實驗結果………………………………………………………………………………………………………………………103
1. 白朮炮製前後樣品水萃取物對於胃酸總酸度之影響………………………………………103
2.白朮炮製前後樣品與指標成分保護酒精誘導胃黏膜細胞毒性之作用……………103
2-1. 胃黏膜細胞之分離及鑑定…………………………………………………………………………………103
2-2. 白朮炮製前後樣品與指標成分對大鼠胃黏膜細胞毒性評估……………………104
2-3. 建立酒精誘導大鼠胃黏膜細胞毒性模式………………………………………………………104
2-4. 白朮炮製前後樣品保護酒精誘導大鼠胃黏膜細胞毒性之作用………………105
2-5. 白朮指標成分保護酒精誘導大鼠胃黏膜細胞毒性之作用………………………106
3. 白朮炮製前後樣品與指標成分保護酒精誘導大鼠急性胃潰瘍之作用…………107
3-1. 酒精引發大鼠急性胃潰瘍模式建立…………………………………………………………………108
3-2.白朮炮製前後樣品水萃取物與指標成分保護酒精誘導大鼠急性胃潰瘍之作用……………………………………………………………………………………………………………………………………………108
3-3. 大鼠急性胃潰瘍之病理分析………………………………………………………………………………109
3-4. 潰瘍部位之MMP-2及MMP-9活性分析………………………………………………………………109
4. 白朮炮製前後樣品抗氧化作用…………………………………………………………………………………116
4-1. 白朮炮製前後樣品清除DPPH自由基之作用……………………………………………………116
4-2. ESR偵測白朮炮製前後樣品精油清除自由基之作用………………………………………117
4-3. 白朮炮製前後樣品精油抑制脂質過氧化之作用……………………………………………118
4-4. 白朮炮製前後樣品精油增加過氧化氫酵素活性之作用………………………………119
5. 白朮炮製前後樣品與指標成分對於正常細胞之細胞毒性…………………………………120
肆、討論……………………………………………………………………………………………………………………………122
總結……………………………………………………………………………………………………………………………………127
參考文獻………………………………………………………………………………………………………………………………127
致謝………………………………………………………………………………………………………………………………………140
論文發表………………………………………………………………………………………………………………………………141
附錄一…………………………………………………………………………………………………………………………………142
附錄二…………………………………………………………………………………………………………………………………144
附錄三…………………………………………………………………………………………………………………………………146
附錄四…………………………………………………………………………………………………………………………………148
附錄五…………………………………………………………………………………………………………………………………
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