咖啡及其多酚成分綠原酸對於牙周炎致病因子牙齦卟晽單胞菌的影響

The effects of coffee and chlorogenic acid on the virulence factor of periodontitis - Porphyromonas gingivalis.

鄒欣樺(Sing-Hua Tsou)

指導教授 ： 林育誼

中山醫學大學/口腔醫學院/口腔科學研究所/博士(2020年)

https://doi.org/10.6834/CSMU201900276

摘要

牙周病是由於細菌的侵襲，導致宿主免疫系統反應。牙周組織發炎反應持續，會導致牙齦萎縮、牙周附著喪失與齒槽骨流失，最終導致牙齒脫落。牙齦卟晽單胞菌為牙周病致病菌之一，為革蘭氏陰性菌。咖啡作為日常攝取多酚成分的主要飲食來源，咖啡目前被發現具有抑制細菌生長及減緩發炎反應的能力。本研究針對牙齦卟晽單胞菌ATCC 33277進行實驗，檢測咖啡提取物及其主要酚酸-綠原酸對牙齦卟晽單胞菌生長和蛋白酶活性的影響。先使用連續稀釋法去製備咖啡和綠原酸溶液。以目測濁度法與抹盤菌落計數法檢測綠原酸對牙齦卟晽單胞菌的抑制效果。之後使用時間殺滅法去測定牙齦卟晽單胞菌經過綠原酸溶液分別處理不同時間點後，牙齦卟晽單胞菌的菌落單位的變化。本實驗使用顯色底物經牙齦卟晽單胞菌分解的程度來測定牙齦卟晽單胞菌蛋白酶的活性，以光密度分析儀使用OD490 nm進行分析。最後使用抑菌圈實驗去檢測牙齦卟晽單胞菌對於咖啡的敏感程度差異。實驗結果表明，綠原酸對抗牙齦卟晽單胞菌的最小抑菌濃度為4 mg/mL，最低殺菌濃度為16 mg/mL。而以高於最小抑菌濃度的綠原酸處理牙齦卟晽單胞菌後，在時間殺滅試驗可發現綠原酸對於牙齦卟晽單胞菌具有長時間的抑制效果，且以高於最小抑菌濃度的綠原酸處理的牙齦卟晽單胞菌的蛋白酶活性皆顯著減少。通過紙錠擴散試驗可發現，咖啡具有抑菌活性，而不同烘焙度的咖啡之間的抑菌效果沒有顯著差異，且咖啡濃度越濃抑菌圈越明顯。我們的研究表明綠原酸對於牙齦卟晽單胞菌除了具有抗菌活性也可減少該菌的蛋白酶的活性，同時咖啡也被證實具有抑菌效果。

關鍵字

綠原酸 ； 時間殺滅試驗 ； 牙周致病菌 ； 細菌蛋白酶紙錠擴散試驗

並列摘要

Periodontal disease is a result of the host immune response to bacterial invasion. This disease will lead to gingiva recession, periodontal attachment loss and alveolar bone destruction, eventually leading to the exfoliation of the tooth. Coffee is a major dietary source of polyphenols, and coffee has shown its abilities of anti-bacterium and anti-inflammation. In this study, we aimed to investigate whether coffee extract and its primary phenolic acid, chlorogenic acid, affect the growth and protease activity of a periodontopathogen Porphyromonas gingivalis. Coffee extract and chlorogenic acid were prepared by two-fold serial dilution. Turbid metric test and plate count method were used to examine the inhibitory effects of chlorogenic acid on P. gingivalis. Time-kill assay was used to measure changes in the viability of P. gingivalis after exposure to chlorogenic acid for 0–24 h. The protease activity of P. gingivalis was analyzed using the optical density of a chromogenic substrate. As a result, the minimum inhibitory concentration (MIC) of chlorogenic acid was 4 mg/mL, and the minimum bactericidal concentration was 16 mg/mL. Chlorogenic acid at concentrations above MIC resulted in a longer lasting inhibitory effect on P. gingivalis viability and significantly reduced associated protease activity. Coffee extract showed antibacterial activity as observed by disk diffusion test, whereas these inhibitory effects were not affected by different roast degrees of coffee. Collectively, our novel findings indicate that chlorogenic acid has antimicrobial activity against P. gingivalis, and this activity was also confirmed present in the coffee extract.

並列關鍵字

chlorogenic acid ； Porphyromonas gingivalis ； time-kill test ； protease activity ； disk-diffusion test

參考文獻

1. Petersen, P.E.; Baehni, P.C. Periodontal health and global public health. Periodontol. 2000 2012, 60, 7-14, doi:10.1111/j.1600-0757.2012.00452.x.

2. Ezzo, P.J.; Cutler, C.W. Microorganisms as risk indicators for periodontal disease. Periodontology 2000 2003, 32, 24-35, doi:10.1046/j.0906-6713.2003.03203.x.

3. Hajishengallis, G.; Darveau, R.P.; Curtis, M.A. The keystone-pathogen hypothesis. Nat. Rev. Microbiol. 2012, 10, 717-725, doi: 10.1038/nrmicro2873.

4. Cohen, J. The immunopathogenesis of sepsis. Nature 2002, 420, 885.

5. Imamura, T. The role of gingipains in the pathogenesis of periodontal disease. Journal of periodontology 2003, 74, 111-118, doi:10.1902/jop.2003.74.1.111