臺灣博碩士論文加值系統

黑蒜是由生蒜經多日高溫高濕熟成，使辛辣味轉為甘甜，除口感改善外， 許多研究發現黑蒜具有抗氧化、抗肝病、降血脂等多種生理活性，本實驗室先前研究發現黑蒜熱水萃取物具有抗發炎潛力，但熟成過程中的活性成份變化與抗發炎機制仍未知，因此本研究進一步探討黑蒜熟成過程中熱水萃取物成份之變化，及其多醣與富含多酚萃取物對BALB/c雌鼠腹腔巨噬細胞之抗發炎作用。結果顯示經熟成10、17及20天的多醣萃取物皆有抑制TNF-α分泌的作用，在熟成20天的樣品觀察到劑量依賴性，且在濃度40及200 ug/ml顯著降低TNF-α /TL-10比值。富含多酚萃取物組，在不同熟成天數的黑蒜和控制組相比皆有抑制TNF-α分泌的作用，經整體評估，以熟成20天的黑蒜熱水萃取物抗發炎功效最佳，因此選取熟成20天的黑蒜萃取物分離純化並探討作用機制。結果黑蒜多醣經Sepharose 6B膠體過濾層析後，得到三個區分物，區分物1的平均分子量為1.40 x 106 Da, 單醣組成主要為半乳糖、木糖和甘露糖，區分物2的分子量為2.39 x 106 Da, 單醣主要為半乳糖、半乳糖醛酸和阿拉伯糖，區分物3的分子量為4.27 x 103 Da, 單醣主要為葡萄糖、半乳糖和甘露糖。三個區分物皆能降低發炎腹腔巨噬細胞的TNF-α分泌，其中以區分物3分子量最小卻抗發炎功效最佳，為了解作用機制，以RT-qPCR方法研究多醣區分物對發炎相關基因表現影響，包括TLR-2、TLR-4及NF-κB，發現區分物3降低TLR-2和NF-κB基因表現，推測區分物3可能是作用於TLR-2及下游NF-κB表現而達到抗發炎功效，黑蒜多醣及區分物1, 2則非經由此路徑。富含多酚萃取物經Sephadex LH-20膠體過濾層析後，得到三個區分物，但皆無抗發炎功效，推測其抗發炎作用須透過區分物的協同或加成作用。

Black garlic is produced resulting from chemical and biochemical reactions in the fresh garlic bulb (Allium sativum L) in a high temperature and controlled humidity condition for a few days. In particular, black garlic has strong anti-oxidant, anti-liver cancer and hypolipidemic effects as compared with fresh raw garlic. Previous studies in our laboratory had found that hot water extracts of black garlic had anti-inflammatory potential. However its anti-inflammatory ingredients and mechanisms during aging process still remain unclear. Therefore, the purpose of this study was to investigate changes in the composition of hot water extracts and anti-inflammatory effects of polysaccharides and polyphenol-rich extracts from black garlic during aging process LPS-stimulated primary peritoneal macrophages from female BALB/c mice. The results showed that polysaccharides in the black garlic aging for 20 days, (20dBGPS) inhibited the secretion of TNF-α dose-dependently. Polysaccharide in 10, 17 and 20 days significantly decreased the secretion ratio of TNF-α/TL-10 at a concentration of 40 and 200 ug/ml. The polyphenol-rich extracts with different aging days also inhibited the secretion of TNF-α. Overall of the results that revealed the black garlic aging for 20 days had the best anti-inflammatory effects. Therefore black garlic aging for 20 days was selected to investigate anti-inflammatory ingredients and mechanisms produsted of 20dBGPS and 20dBGEE were purified using gel filtration. Black garlic polysaccharides, 20dBGPS, were resolved into three factions including BPSF1, BPSF2 and BPSF3 using Sepharose 6B gel filtration column. The mean molecular weight of BPSF1 was 1.40 x 106 Da, and its monosaccharide composition was mainly galactose, xylose and mannose. The mean molecular weight of BPSF2 was 2.39 x 106 Da, and its monosaccharides were mainly composed of galactose, galacturonic acid and arabinose. The mean molecular weight of BPSF3 was 4.27 × 103 Da, and its monosaccharides mainly consisted of glucose, galactose and mannose. All three fractions had anti-inflammatory effects for they significantly reduced TNF-α secretion by LPS-stimulated macrophages. BPSF3 had the lowest mean molecular weight which the best anti-inflammatory effect. To unravel possible and anti-inflammatory mechanisms, real time quantitative polymerase chain reaction was used to evaluate changes in relative expression amounts of inflammation-related molecules. We found that BPSF3 down-regulated TLR-2 and NF-κB gene expression, suggesting that BPSF3 achieved its anti-inflammatory effects by down-regulating of NF-κB gene expression in TLR signaling pathways. However 20dBGSP and BPSF1, 2 did not share the anti-inflammation pathway. The purification of 20dBGEE using Sephadex LH-20 gel filtration resolved into three factions, BGEE1, BGEE2 and BGEE3, but they did not have anti-inflammatory effects, respectively. It is speculated that the anti-inflammatory effect of polyphenol in black garlic might through be synergistic or additive manners.

摘要 i
Abstract ii
目次 iv
表目次 vii
圖目次 viii
縮寫對照表 x
緒言 1
第一章 文獻回顧 2
第一節、免疫系統簡介 2
一、免疫反應 2
二、巨噬細胞 4
三、類鐸受體 5
第二節、發炎免疫反應 7
第三節、多醣、多酚之免疫調節 9
一、多醣之免疫調節作用 9
二、多酚之免疫調節作用 9
第四節、黑蒜 10
第五節、研究動機與目的 12
第二章 不同天數熟成黑蒜多醣及其富含多酚萃取物對小鼠初代免疫細胞之抗發炎功效 13
第一節、前言 13
第二節、實驗架構 14
第三節、材料與方法 15
一、不同天數熟成黑蒜萃取物之樣品製備 15
二、光譜掃描分析 17
三、黑蒜萃取物熟成期間組成份變化測定 18
四、不同天數熟成黑蒜萃取物對BALB/c 雌鼠初代脾臟免疫細胞生長之影響 19
五、熟成不同天數之黑蒜萃取物對初代腹腔巨噬細胞發炎反應之影響 23
六、統計分析 28
第四節、結果與討論 28
一、不同天數熟成黑蒜萃取物之萃取產率及組成份測定 28
二、光譜掃描分析 29
三、不同天數熟成黑蒜萃取物對BALB/c 雌鼠初代脾臟免疫細胞生長之影響 30
四、熟成不同天數之黑蒜萃取物對初代腹腔巨噬細胞發炎反應之影響 30
五、結論 46
第三章 黑蒜多醣與富含多酚萃取物之純化及區分物鑑定 47
第一節、前言 47
第二節、實驗架構 47
第三節、材料方法 48
一、熟成20天黑蒜多醣之純化區分 48
二、熟成20天黑蒜富含多酚萃取物之區分物純化 48
三、熟成20天黑蒜多醣與富含多酚萃取物及膠體過濾區分物對BALB/c 雌鼠初代脾臟免疫細胞生長之影響 50
四、熟成20天黑蒜多醣與富含多酚區分物及膠體過濾區分物對BALB/c 雌鼠初代腹腔巨噬細胞發炎反應之影響 51
五、熟成20天黑蒜多醣區分物之分子量測定 51
六、熟成20天黑蒜多醣及膠體過濾區分物的單醣組成分析 52
七、統計分析 55
第四節、結果與討論 56
一、熟成20天黑蒜多醣之區分物純化 56
二、熟成20天黑蒜富含多酚萃取物之區分物純化 56
三、熟成20天黑蒜多醣與富含多酚萃取物及膠體過濾區分物對BALB/c 雌鼠初代脾臟免疫細胞生長之影響 59
四、熟成20天黑蒜多醣與富含多酚萃取物及膠體過濾區分物對BALB/c 雌鼠初代腹腔巨噬細胞發炎反應之影響 59
五、熟成20天黑蒜多醣區分物之分子量測定 65
六、熟成20天黑蒜多醣及其膠體過濾區分物的單醣組成分析 66
第五節、結論 76
第四章 熟成20天黑蒜多醣及其區分物對初代腹腔巨噬細胞之抗發炎機制之研究 77
第一節、前言 77
第二節、材料與方法 77
一、初代腹腔巨噬細胞之取得 77
二、初代腹腔巨噬細胞之培養與總RNA抽取 77
三、逆轉錄反應 (Reverse transcription reaction)-將RNA逆轉錄成cDNA 79
四、即時定量聚合酶連鎖反應Real-time Quantitative Polymerase Chain Reaction (real-time qPCR) 81
五、統計分析 85
第三節、結果與討論 86
熟成20天黑蒜多醣及膠體過濾區分物對以LPS誘導發炎的BALB/c 雌鼠初代腹腔巨噬細胞訊息傳遞相關基因表現之影響 86
第四節、結論 88
第五章 總討論與總結 90
第六章 參考文獻 91

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四季農漁產品, 行政院農委會https://www.coa.gov.tw/theme_data.php?theme=product&id=34查詢年份2019
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