桂花粗萃物及其成分於腸道發炎免疫功能之探討

Study on Intestinal Mucosa Immunomodulatory Effect of Compounds and Extract from Osmanthus fragrans

王淨瑤(Jing-Yao Wang)

指導教授 ： 葉怡玲

國立虎尾科技大學/文理學院/生物科技研究所/碩士(2014年)

摘要

桂花(Osmanthus fragrans flower) 具有特殊香氣，除了可作為食品，亦可入藥。根據中國醫書《本草匯言》記載，桂花具「散冷氣，消瘀血，止腸風血痢」。許多文獻指出， 腸道炎症 (Inflammatory bowel disease；IBD)會使腸道上皮細胞受損並導致嚴重發炎，因此維持腸道免疫調節在改善腸道炎症上扮演重要角色。
桂花中含有許多成分，已有數種成分被發現具有抗發炎功效。洪等人藉由小鼠動物模式之研究，發現口服桂花乙醇粗萃物具有改善過敏性呼吸道發炎之能力，但其免疫調節機制仍不清楚。故本論文將評估探討桂花粗萃物及其相關成分對於腸道免疫調節之功效。
首先利用H2O2刺激腸道上皮細胞模擬腸道發炎，再以桂花乙醇粗萃物(OFE)及相關成分探討是否可抑制發炎細胞激素IL-8的分泌，且評估此抑制機制是否為NF-κB訊息依賴型途徑。更進一步進行動物實驗，包含兩個方向，(1)先將 OFE或Verbascoside (VB)與小鼠骨髓衍生樹突細胞共培養，觀察其成熟程度和細胞激素的變化。(2)再藉由口服方式評估OFE或VB的毒性並觀察是否會影響BALB/c小鼠全身性及腸道的免疫活性。
由結果得知OFE、VB、rutin、phillyrin、methyl 4-hydroxycinammate以及eutigoside A可藉由NF-κB依賴型路徑抑制IL-8分泌，但phillygenin無法抑制IL-8分泌。動物實驗結果顯示 (1) OFE及VB 不會促使樹突細胞成熟，甚至在OVA與天然物共同的作用下也能抑制樹突細胞的成熟。(2) 連續14天餵食VB，發現小鼠脾臟中CD4+ CD25+ T細胞有和CD4+ Foxp3+ T細胞顯著增加，並於腸道沖洗液中發現其具有促使TSLP分泌之能力；連續14天餵食OFE，發現小鼠脾臟中CD4+Foxp3+ T細胞有顯著增加，但TSLP則無顯著差異。未來對於OFE及VB是否能扮演免疫調節佐劑成分的角色來協助治療發炎性及過敏性疾病，還要再進一步探討。

關鍵字

桂花 ； 抗發炎 ； NF-κ B ； 樹突細胞 ； IBD

並列摘要

Owing to its special aroma, the flower of Osmanthus fragrans, called Kwai-fah in China, has been used as a beverage and as an additive for foods. Ben Cao Hui Yan, traditional Chinese medical literature, describes the usefulness of these flowers for phlegm and stasis reduction, arrest of dysentery with blood in the bowel, and stomachache and diarrhea treatment. Many studies have reported that intestinal inflammation that damage intestinal epithelial cells is the major cause for inflammatory bowel disease (IBD). The immunomodulatory activity maintenance in intestinal environment will be the important issue in IBD therapy.
Previously, anti-allergic effect of OFE (ethanol extract of O. fragrans flowers) has been reported, but the immunomodulatory mechanism has not been studied. This study is focused on the intestinal immune modulatory activity of O. fragrans extract and its related compound.
At the first, we evaluated the IL-8 inhibitory activities of OFE and its related compounds in vitro after H2O2 administration. The NF-κB signal pathway dependent or independent by this inhibition is also been evaluated. In vivo study, two parts including (1) Using mouse bone marrow derived dendritic cells, the maturation and cytokine expressing pattern were discussed. (2) The immunomodulatory effect after oral feeding OFE and Verbascoside (VB) to BALB/c mice was also be studied.
In vitro results showed OFE and it’s related components (verbascoside, rutin, phillyrin, methyl 4-hydroxycinammate or eutigoside A but not phillygenin) can suppress IL-8 secretion after 0.5 hour H2O2 treatment. And their IL-8 suppression is through NF-κB pathway. In the other hand, the IL-8 inhibition by phillygenin is through NF-κB pathway. OFE and verbascoside could not promote the maturation of dendritic cells. Furthermore, OFE and VB also can suppress the maturation of ovalbumin treated dendritic cells. Cell numbers of CD4+ CD25+ T cells increased in mouse spleen after oral feeding with VB for 14 days. Cell numbers of Foxp3+ Treg cells increased in mouse spleen after oral feeding withOFE or VB for 14 days.The concentration of TSLP increased in intestinal lavage fluid. But, these phenomena can not be found for 28 days. Whether OFE and VB can assist in the treatment of inflammatory , more investigation is needed in the future.

並列關鍵字

Osmanthus fragrans ； anti-inflammatory ； NF-κ B ； dendritic cells ； IBD

參考文獻

[1] M. R. Neutra, N. J. Mantis, and J. P. Kraehenbuhl, "Collaboration of epithelial cells with organized mucosal lymphoid tissues," Nat Immunol, vol. 2,no. 11, pp. 1004-9, 2001.

[2] S. Ahrne and M. L. Hagslatt, "Effect of lactobacilli on paracellular permeability in the gut," Nutrients, vol. 3,no. 1, pp. 104-17, 2011.

[3] 吳季瑾, "大鼠腸阻塞之黏膜組織受損及上皮屏障喪失的機制: 腸道上皮肌凝蛋白輕鏈激酶之角色," 臺灣大學生理學研究所學位論文, no. 2007 年, 2007.

[4] 金紅芝 and 李?寶, "人腸道微生態系統的研究進展 Progress in the Study of Human Intestinal Microecosystem," 自然雜誌, vol. 26,no. 2, pp. 88-91, 2004.

[5] E. S. Vega, Y. R. Patin, and L. Llorente, "El intestino: mas alla de la inmunologia," Reumatologia clinica, vol. 1,no. 2, pp. 121-128, 2005.