臺灣博碩士論文加值系統

本研究將探討超臨界二氧化碳萃取大花咸豐草與小金英之有效成份對於抗氧化、抗發炎和抗癌成效。實驗以總多酚、總類黃酮測定、DPPH自由基清除率、TEAC當量測定、鐵離子螯合和還原力測定等化學方法測試細胞外抗氧化能力，並以過氧化氫( H2O2) 毒殺作用的保護能力測試細胞內抗氧化效果。另外以一氧化氮分析( NO assay) 和硝基藍四氮還原試劑( NBT assay) 評估萃取物抗發炎能力。接著測試大花咸豐草萃取物( BP) 與小金英萃取物( IX) 老鼠正常肝細胞( Clone 9) 與人類肝腫瘤細胞( HepG2和Huh-7) 的毒殺效果，並以細胞免疫化學法探討HepG2毒殺機制，評估萃取物的抗癌能力。此外，利用高效液相層析儀( HPLC) 與紫外光( UV) 吸收輔以質譜儀( Mass) 質量鑑定，進行BP與IX之主成份定量分析。實驗結果顯示BP的總多酚當量為79 ± 0.25GAE (mg)/100g ，總類黃酮當量為24.1± 0.36 RE(mg)/100g，DPPH 50%自由基清除濃度( IC 50) 為25μg/mL，該濃度的TEAC當量為74± 2.88μM。當BP濃度在350μg/mL時，對於H2O2的傷害有最大保護能力，約有70%的存活率，NO生成當量約38μM，硝基藍四氮( NBT) 還原測試，發現可抑制25%細胞產生發炎反應。經由HPLC和Mass分析BP主成份，得到槲皮素1.97mg/g、芹菜素0.45mg/g。
IX的總多酚當量為45.4± 0.5GAE(mg)/100g，總類黃酮當量為36.6± 0.7RE(mg)/100g，DPPH自由基清除率的IC 50為12.5μg/mL，當濃度為250μg/mL時和人造抗氧化劑丁基羥基甲苯( BHT) 的清除率相當，而該濃度的TEAC當量為157± 2.82μM。在濃度為350μg/mL對H2O2傷害具有最大保護能力，約有62%的存活率，NO生成當量約46μM，硝基藍四氮( NBT) 還原測試，發現可抑制32%的細胞產生發炎反應。經由HPLC和Mass分析IX主成份，得到芹菜素9.56mg/g、木犀草素4.24 mg/g。細胞毒殺作用的結果顯示各種濃度的BP和IX皆會對人類肝癌細胞( HepG2) 產生毒殺作用，藉著細胞免疫化學方法發現BP和IX會使cleaved Caspase- 8, Caspase- 3, PARP顯著( p<0.05)提升，而且表現量與萃取物濃度有關，因此認為BP和IX會使細胞經由外在路徑凋亡程序造成細胞凋亡。綜合上述實驗結果，證實超臨界二氧化碳萃取出的大花咸豐草與小金英有效成份在抗氧化、抗發炎與抗癌方面有顯著功效，因此超臨界二氧化碳萃取會使雜草的實用性提高。

Bidens Pilosa L. var. (Composite) and Ixeris Chinensis Nakai (Composite) are Asteraceae perennial plant found widely throughout Taiwan. It is used as a folk medicine for the treatment of bronchitis, pneumonia, pharyngitis, dysentery, and the ingestion of poisons, on the basis of its antifebrile, antidotal, and analgesic effects. Supercritical Carbon Dioxide Extraction was used to obtain effective extracts from Bidens Pilosa and Ixeris Chinensis. The total phenolic content, total flavonoid content 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity, Trolox equivalent anti-oxidizing capacity (TEAC) assay, ferrous ion chelating and reducing power were used to evaluate the overall antioxidative ability of the extracts. Nitric oxide and nitro blue tetrazolium (NBT) assays were used to evaluate the anti-inflammatory properties, cytotoxic effect and immunohistochemical methods were used to evaluate the antitumor potential of the extract. Qualitative and quantitative analysis of the effective properties of the B. Pilosa( BP) and I. Chinensis extracts (IX) was performed using high-pressure liquid chromatography (HPLC) with a UV spectrophotometer and mass detector. The results revealed that BP has a total phenolic content of 79±0.25 gallic acid equivalents (GAEs) mg/100g, total flavonoid content of 24.1±0.36 rutin equivalents (RE) mg/100g, IX has a total phenolic content of 45.4±0.5 gallic acid equivalents (GAEs) mg/100g, total flavonoid content of 36.6±0.7 rutin equivalents (RE) mg/100g DPPH radical scavenging activity of 50% (IC50) was 25 g/mL, and an antioxidant activity of 74 ± 2.88 μM Trolox equivalents. BP at a concentration of 350 g/mL could prevent oxidative stress, induced by hydrogen peroxide (H2O2), and inhibit 25% of Phorbol esters (PMA) induced THP-1 monocyte differentiation. DPPH radical scavenging activity of IX similar to butylated hydroxyanisole (BHA) at 250 g/mL and an antioxidant activity of 157 ± 2.88 μM Trolox equivalents. IX at a concentration of 350 g/mL could prevent oxidative stress, induced by hydrogen peroxide (H2O2), and inhibit 64.3% of Phorbol esters (PMA) induced THP-1 monocyte differentiation. HepG2 cells treated with IX at a concentration of 350 g/mL caused a rapid induction of caspase3 activity and stimulated proteolytic cleavage of poly (ADPribose) polymerase (PARP) and induced apoptosis. Quantitative analysis showed that the content of apigenin and luteolin in the IX was 9.56 mg/g and 4.24 mg/g, respectively.

第一章 緒論
1.1前言…………………………………………………………………..1
1.2研究動機……………………………………………………………..2
第二章 文獻回顧………………………………………………………..2
第三章 材料與方法
3.1化學實驗器材……………………………………………………….10
3.2生物實驗器材……………………………………………………….10
3.3化學實驗藥品與試劑……………………………………………….11
3.4生物實驗藥品與試劑……………………………………………….12
3.5植物………………………………………………………………….13
3.6超臨界二氧化碳萃取方法………………………………………….14
3.7細胞株和細胞培養………………………………………………….14
3.7.1 Clone 9………………………………………………………….14
3.7.2 Raw 264.7………………………...…………………………….14 3.7.3 HepG2……………………………………………………………..14
3.7.4 Huh-7…………………………………………………..……….15
3.7.5 THP-1………………………………………………...……….15
3.7.6 NIH3T3……………………………………………………….15
3.8細胞株冷凍保存…………………………………………………….17
3.9細胞株解凍程序…………………………………………………….17
3.10細胞培養液配置…………………………………………………..17
3.10.1配置DMEM培養基…………………………………………17
3.10.2配置RPMI-1640培養基…………………………………….18
3.11磷酸鹽緩衝液配置………………………………………………..18
3.12細胞數目計算……………………………………………………..18
3.13細胞存活率測試方法……………………………………………..20
3.14細胞外抗氧化測試
3.14.1總多酚當量測試……………………………………….…..21
3.14.2總類黃酮當量測試…………………………………………21
3.14.3清除DPPH自由基測定……………………………………21
3.14.4 TEAC當量測試……………………………………………21
3.14.5亞鐵離子螯合能力測定……………………………………22
3.14.6還原力測定…………………………………………………22
3.15細胞內抗氧化測試……………………………………………..…22
3.16大花咸豐草與小金英萃取物對於輻射傷害之細胞保護試驗…..23
3.17細胞抗發炎測試
3.17.1 NO‧ assay…………………………………………………23
3.17.2 NBT assay……………………..……………………………24
3.18正常細胞和癌細胞的毒性測試…………………………………...24
3.19西方點墨法
3.19.1蛋白質定量…………………………………………………25
3.19.2 SDS-PAGE and Western blot analysis…………………...…25
3.20成份分析…………………………………………………………..26
3.21統計分析…………………………………………………………..26
第四章 結果與討論
4.1細胞外抗氧化能力
4.1.1總多酚當量和總類黃酮當量測定……………………………27
4.1.2 DPPH自由基清除測試………………………………………28
4.1.3 TEAC當量測試………………………………………………29
4.1.4亞鐵離子螯合能力測試………………………………………31
4.1.5還原力測試……………………………………………………32
4.2細胞內抗氧化試驗………………………………………………….33
4.3大花咸豐草與小金英萃取物對於輻射傷害之細胞保護試驗……33
4.4細胞內抗發炎測試
4.4.1 NO‧assay…………………………………………………….37
4.4.2 NBT assay……………………………………………………..38
4.5細胞毒性實驗………………………………………………………40
4.6大花咸豐草與小金英萃取物毒殺癌細胞機制的探討……………44
4.7 LC/MS分析結果……………………………………………………53
第五章 結論與未來展望………………………………………………60

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