Studies on the Anti-inflammatory Components of Purple Cabbage (Brassica oleracea L. var.)

Abstract

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.