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研究生:李家源
研究生(外文):Li Chia Yuan
論文名稱:紫色甘藍菜中抗發炎活性成分之研究
論文名稱(外文):Studies on the Anti-inflammatory Components of Purple Cabbage (Brassica oleracea L. var.)
指導教授:林金源林金源引用關係
指導教授(外文):Jin-Yuarn Lin
學位類別:碩士
校院名稱:國立中興大學
系所名稱:食品科學系
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:2005
畢業學年度:93
語文別:中文
論文頁數:105
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本論文之目的為利用活性碳、膠體層析等分離純化方法,經由細胞實驗並測定細胞激素的變化,來探討紫色甘藍菜中免疫調節活性成分的種類與其特性。
在免疫調節活性純化指標選取方面,以BALB/c雌鼠脾臟細胞進行實驗,在添加脂多醣(lipopolysaccharide, LPS)及紫色甘藍菜粗萃取樣品不同濃度的細胞培養模式下,測定促發炎細胞激素IL-1β、IL-6、TNF-α及抗發炎細胞激素IL-10的分泌情況,結果發現,在細胞培養48小時的條件下,IL-1β分泌量與樣品的添加沒有相關性,IL-6、IL-10及TNF-α等細胞激素在添加紫色甘藍菜樣品的情況下,相較於僅以LPS刺激的對照組有明顯的變化,使紫色甘藍菜整體呈現抗發炎的免疫活性,因此選取IL-6、IL-10及TNF-α三種細胞激素做為本實驗免疫調節活性的純化指標。
在免疫活性物質純化方面,紫色甘藍菜粗萃取樣品,利用活性碳處理,將樣品中的小分子成分吸附去除,再進行細胞培養,結果紫色甘藍菜粗萃取樣品原有抑制IL-6分泌的活性,在活性碳處理過後,此活性消失,故推測此抑制IL-6分泌的活性物質可能為小分子成分;粗萃取物中原有刺激IL-10分泌的活性,在活性碳處理過後,此活性仍存在,故推測此刺激IL-10分泌的活性物質可能為大分子物質。
利用膠體Sephadex LH-20處理粗樣品,以OD280及OD254吸收光圖譜來區分,結果共區分出五個區分物,其中第二區分物(F2)含有抑制IL-6分泌的活性物質,依其耐熱性質及以HPLC分析判斷,其活性成分可能包括花青素Oenin和Malividin及其醣基衍生物;F2中亦含有刺激TNF-α分泌的活性成分,但在加熱後此活性遭到破壞,故推此具有刺激TNF-α分泌的活性物質,可能為蛋白質;第四區分物(F4)中含有刺激IL-10分泌的活性物質,經加熱處理後活性更強,推測此具有刺激IL-10分泌的活性物質可能包含有多酚類。
利用硫酸銨沈澱分離出樣品中的蛋白質,蛋白質再進行細胞實驗,結果發現,F2區分物中原本具有抑制IL-6分泌的活性消失,證實其為非蛋白質;F4區分物原本具有刺激IL-10分泌的活性則仍存在,由SDS-PAGE電泳結果發現,此F4區分物中含有次單元分子量35kDa以下的蛋白質,但F4區分物中蛋白質沈澱物的活性仍不如原F4區分物,故推測F4區分物中尚有其他活性成分可刺激IL-10分泌;TNF-α分泌受到F4抑制,在F4區分物沈澱取得蛋白質後仍可以看到其活性,故推測其活性成分可能包括有蛋白質。
綜合本實驗結果,紫色甘藍菜確含有抗發炎的活性物質,推斷其中之花青素Oenin和Malvidin及其醣基衍生物可能為抑制IL-6分泌的主要活性物質;紫色甘藍菜中亦含有可能具有刺激IL-10分泌的蛋白質,此兩類活性物質對於發炎相關細胞激素具有調控作用,至於其調控機制,則仍有待後續更深入研究。
Abstract
This study was to investigate the anti-inflammatory components of purple cabbage (Brassica oleracea L. var.). To characterize the active components, the purple cabbage juice was partially purified by activated charcoal absorption and by gel filtration chromatography with sephadex LH-20. A lipopolysaccharide (LPS)-stimulated splenocyte culture model from female BALB/c mice was established to evaluate each fraction from activated charcoal absorption and gel filtration chromatography. The pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, and anti-inflammatory cytokine, such as IL-10, secreted by LPS-stimulated splenocytes were chosen as anti-inflammation purification markers of purple cabbage.
The results reveled no correlation between IL-1β secretion and purple cabbage addition to the LPS-stimulated splenocyte culture model. We peculated that the IL-1β cytokine is an earlier inflammation indicator; however, it was not a suitable marker of inflammation in our 48-hour cell culture model. According to the secretion profiles of IL-6, IL-10, and TNF-α cytokine, the IL-6 and TNF-α cytokines were chosen as inflammation markers and the IL-10 cytokine was chosen as an anti-inflammation marker in the purifying process of purple cabbage.
The activated charcoal might absorb and exclude the components with low molecular weight in purple cabbage, such as polyphenols, flavonoids, anthocyanins, pigments, and peptides etc. In the activated charcoal-absorbed study, the suppressing activity against IL-6 secretion disappeared after the purple cabbage juice was treated with activated charcoal. We conjectured that the active components of suppressing effect on IL-6 secretion in purple cabbage might be small components with low molecular weight. The increasing activity of IL-10 secretion was enhanced after purple cabbage juice was treated with activated charcoal. We peculated that the active components of increasing effect on IL-10 secretion in purple cabbage might be big components with high molecular weight, such as proteinaceous substances.
In the study of gel filtration chromatography with sephadex LH-20, purple cabbage juice was resolved into five fractions (F1-F5) according to the chromatographic spectra of OD280 and OD254. The F2 fraction showed the suppressing activity against IL-6 secretion. According to their heat-resistant characteristics and HPLC chromatograms, we convinced that active components of F2 fraction consisted of anthocyanins, Oenin , Malividin, and Malividin-glycosides. It was also found that the F2 fraction exhibited the suppressing activity against TNF-α secretion. As the suppressing activity disappeared after the F2 fraction was heated, we speculated the active components against TNF-α secretion in F2 fraction were proteinaceous substances. The F4 fraction demonstrated an increasing activity of IL-10 secretion. We speculate the active components of increasing activity of IL-10 secretion in F4 fraction are polyphenols based on its heat-resistant characteristic and composition.
The anti-inflammation active fractions, purple cabbage juice, activated charcoal-treated purple cabbage juice, F2, and F4 fractions, were further precipitated with 100% ammonium sulfate to obtain their proteinaceous substances. The precipitated proteins were subjected to SDS-PAGE electrophoresis. Referring to the SDS-PAGE electrophoresis chromatograms, we conjectured that proteinaceous substances and other components in the F4 fraction promoted the IL-10 secretion activity. The proteinaceous substances in F4 fraction also inhibited the TNF-α secretion of LPS-stimulated splenocytes.
In summary, there are some anti-inflammation components in purple cabbage juice. We speculate that some kinds of anthocyanins, such as Oenin, Malvidin and Malvidin-glycosides, may inhibit IL-6 secretion. Some proteinaceous substances in purple cabbage juice may enhance IL-10 secretion. However, the immunomodulatory mechanisms and the characteristics of anti-inflammation components in purple cabbage remain to be further clarified.
總 目 錄
表目錄 v
圖目錄 vii
中文摘要 ix
英文摘要 xi

第一章 緒言 1
第一節 前言 1
第二節 文獻整理 2
一、 蔬果中類黃酮及花青素 2
二、 細胞激素及發炎反應 8
第三節 研究動機及目的 14
第二章 紫色甘藍菜免疫活性物質純化指標之選擇 16
第一節 前言 16
第二節 材料與方法 16
一、 樣品製備 16
二、 動物飼養 16
三、 脾臟細胞取得及培養 16
四、 細胞激素測定 18
五、 統計方法 19
第三節 結果 20
一、 紫色甘藍菜榨汁與LPS共同培養對脾臟細胞分泌細胞激素之影響 20
第四節 討論 22
第五節 結論 23
第三章 紫色甘藍菜各區分處理物之抗發炎免疫調節功能 24
第一部分 以活性碳處理紫色甘藍菜榨汁樣品 24
第一節 前言 24
第二節 材料與方法 24
一、 樣品製備 24
二、 動物飼養 24
三、 脾臟細胞取得及培養 24
四、 細胞激素測定 24
五、 成分分析 25
六、 統計方法 27
第三節 結果 28
一、 紫色甘藍菜活性碳處理樣品回收率及成分分析 28
二、 紫色甘藍菜活性碳處理對BALB/c小鼠脾臟細胞分泌不同細胞激素之影響 31
第四節 討論 33
第五節 結論 34
第二部分 以SEPHADEX LH-20管柱區分紫色甘藍菜榨汁樣品 35
第一節 前言 35
第二節 材料與方法 35
一、 SEPHADEX LH-20管柱製備及其區分物處理 35
二、 動物飼養 36
三、 脾臟細胞取得及培養 36
四、 細胞激素測定 36
五、 成分分析 36
六、 高效液相層析儀(HPLC)分析 36
七、 統計方法 37
第三節 結果 38
一、 紫色甘藍菜榨汁樣品經管柱(Sephadex SEPHADEX LH-20)層析 38
二、 紫色甘藍菜Sephadex SEPHADEX LH-20管柱層析樣品之回收率及其成分分析 41
三、 紫色甘藍菜Sephadex SEPHADEX LH-20管柱層析樣品對BALB/c小鼠脾臟細胞分泌不同細胞激素之影響 46
四、 紫色甘藍菜Sephadex SEPHADEX LH-20管柱層析區分物加熱後對BALB/c小鼠脾臟細胞分泌不同細胞激素之影響 51
五、 紫色甘藍菜SEPHADEX LH-20管柱層析各區分物高效液相層析分析 56
第四節 討論 60
第五節 結論 65
第三部分 紫色甘藍菜區分物中蛋白質特性之探討 66
第一節 前言 66
第二節 材料與方法 66
一、 樣品製備 66
二、 動物飼養 66
三、 脾臟細胞取得及培養 66
四、 細胞激素測定 66
五、 SDS-PAGE電泳 67
六、 統計方法 70
第三節 結果 71
一、 紫色甘藍菜活性區分物中蛋白質對BALB/c小鼠脾臟細胞分泌不同細胞激素之影響 71
二、 紫色甘藍菜各區分物蛋白質之電泳分析 76
第四節 討論 78
第五節 結論 79
第四章 討論與總結 80
第一節 討論 80
第二節 總結 81
第五章 參考文獻 82








表 目 錄
表1-1 數種蔬果中主要的花青素種類及色澤 4
表1-2 數種常見的蔬果中花青素之含量 6
表1-3 數種細胞激素及其特性 10
表2-1 紫色甘藍菜榨汁對BALB/c雌鼠脾臟細胞在LPS刺激下分泌不同細胞激素之影響 21
表3-1 紫色甘藍菜榨汁樣品活性碳處理前後之成分變化 29
表3-2 紫色甘藍菜活性碳處理前後對BALB/雌鼠脾臟細胞在LPS刺激下分泌不同細胞激素之影響 32
表3-3 紫色甘藍菜Sephadex LH-20管柱層析區分物之成分分析 42
表3-4 紫色甘藍菜Sephadex LH-20管柱層析區分物加熱處理後之成分分析 43
表3-5 紫色甘藍菜榨汁經Sephadex LH-20管柱層析區分物對BALB/c雌鼠脾臟細胞在LPS刺激下分泌IL-6之影響 48
表3-6 紫色甘藍菜榨汁經Sephadex LH-20管柱層析區分物對BALB/c雌鼠脾臟細胞在LPS刺激下分泌IL-10之影響 49
表3-7 紫色甘藍菜榨汁經Sephadex LH-20管柱層析區分物對BALB/c雌鼠脾臟細胞在LPS刺激下分泌TNF-α之影響 50
表3-8 紫色甘藍菜榨汁及其Sephadex LH-20管柱層析區分物及加熱後對BALB/c雌鼠脾臟細胞在LPS刺激下分泌IL-6之影響 53
表3-9 紫色甘藍菜榨汁及其Sephadex LH-20管柱層析區分物加熱後對BALB/c雌鼠脾臟細胞在LPS刺激下分泌IL-10之影響 54
表3-10 紫色甘藍菜榨汁及其Sephadex LH-20管柱層析區分物加熱後對BALB/c雌鼠脾臟細胞在LPS刺激下分泌TNF-α之影響 55
表3-11 紫色甘藍菜各活性區分中之蛋白質對BALB/c雌鼠脾臟細胞在LPS刺激下分泌IL-6之影響 73
表3-12 紫色甘藍菜各活性區分中之蛋白質對BALB/c雌鼠脾臟細胞在LPS刺激下分泌IL-10之影響 74
表3-13 紫色甘藍菜各活性區分中之蛋白質對BALB/c雌鼠脾臟細胞在LPS刺激下分泌TNF-α之影響 75
表4-1 紫色甘藍菜各處理物對BALB/c雌鼠脾臟細胞在LPS刺激下分泌不同細胞激素之彙整 81




















圖 目 錄
圖1-1 類黃酮的化學結構 2
圖1-2 食物中類黃酮的分類、化學結構、食物來源及TEAC 3
圖1-3 酸櫻桃中花青素cyanidin及cyanidin於細胞培養液中主要裂解產物的結構 5
圖1-4 一氧化氮製造示意圖 9
圖1-5 輔助型T細胞的分類 11
圖1-6 實驗設計流程圖 15
圖3-1 紫色甘藍菜榨汁樣品及其活性碳處理物光譜掃瞄 30
圖3-2 紫色甘藍菜樣品管柱層析(Sephadex LH-20)之280nm吸光圖譜 39
圖3-3 紫色甘藍菜樣品管柱層析(Sephadex LH-20)之254nm吸光圖譜及區分物分劃 40
圖3-4 紫色甘藍菜管柱層析(Sephadex LH-20)各區分物全波長光譜掃瞄 44
圖3-5 紫色甘藍菜管柱層析(Sephadex LH-20)各區分物經加熱後全波長光譜掃瞄 45
圖3-6 紫色甘藍菜榨汁樣品其及Sephadex LH-20各區分物之HPLC圖譜 57
圖3-7 花青素delphinidin和malvidin 之HPLC圖譜 59
圖3-8 紫色甘藍菜經Sephadex LH-20處理之不同區分物中花青素Oenin (retention time=3.03 min)含量與抑制脾臟細胞IL-6分泌之相關性 61
圖3-9 紫色甘藍菜經Sephadex LH-20處理之不同區分物中花青素Malvidin glycoside (retention time=69.92 min)含量與抑制脾臟細胞IL-6分泌之相關性 62
圖3-10 紫色甘藍菜經Sephadex LH-20處理之不同區分物中花青素Malvidin glycoside (retention time=71.49 min)含量與抑制脾臟細胞IL-6分泌之相關性 63
圖3-11 紫色甘藍菜經Sephadex LH-20處理之不同區分物中花青素Malvidin (retention time=74.11 min)含量與抑制脾臟細胞IL-6分泌之相關性 64
圖3-12 紫色甘藍菜榨汁樣品、活性碳處理物及Sephadex LH-20區分物F2和F4之SDS-PAGE電泳圖譜 77
圖3-13 紫色甘藍菜榨汁樣品、活性碳處理物及Sephadex LH-20區分物F2和F4硫酸銨沈澱物之SDS-PAGE電泳圖譜 77
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