山胡椒果實精油對牙周炎患者口腔分離細菌之抑菌活性、細胞毒性和抗發炎活性的探討

山胡椒精油在過去多項研究中被指出其具有抗發炎、抗氧化、抗菌等特性，牙周疾病為一因細菌定殖於牙齦下，產生發炎反應致使牙齦組織受到破壞、牙周囊袋加深，提供致病菌更佳生長溫床，最終形成慢性發炎之口腔疾病。由於鮮少有研究將山胡椒萃取物應用於口腔細菌相關研究上，故本研究欲先鑑定分離出牙周炎相關的菌種，進而探討山胡椒果實精油 (essential oil of Litsea cubeba fruits, EOLCF) 1) 對口腔鑑定分離出的細菌之抑菌活性，2) 對口腔來源細胞的細胞毒性，以及 3) 對細菌脂多醣 (lipopolysaccharide, LPS) 刺激口腔細胞抑制 interleukin-6 (IL-6)分泌之抗發炎能力。我們從牙齦下牙菌斑生物膜的混合液經純種培養後，分離鑑定出有三株厭氧菌和三株需氧菌;其中五株菌種與牙周炎相關較密切，一株是口腔健康者常出現的菌種。EOLCF 利用無溶劑微波萃取法取得，經電子鼻 (E-nose) 分析測定並結合資料庫比對分析精油成分後，發現EOLCF主要成分是d-limonene(53.93%) 和citral(15.66%)。EOLCF 對口腔分離鑑定出來的六株細菌，由抑菌試驗結果可得知，對照組 0.2% chlorohexidine 的抑菌環的直徑大小介於 11.00±0.00mm ~26.00± 0.00mm，EOLCF 在兩倍稀釋濃度時，抑菌環的直徑大小介於 17.78± 1.54mm ~35.55±5.74mm之間，不同菌種間具有不同的抗菌活性，而對於需氧菌的最小抑菌濃度則是類似的，MIC50 皆為 1% EOLCF 稀釋濃度。經細胞培養模式下測試細胞毒性的結果顯示，EOLCF 稀釋 800 倍後，接近無細胞毒性，而 EOLCF 對口腔癌細胞與正常牙周組織細胞的毒性，精油稀釋倍率相差約 100 倍，在 IL-6 抗發炎活性試驗下，可以看出在 5x105 倍數稀釋之 EOLCF 仍具有抑制促發炎細胞激素 IL-6 的能力。這些結果表明 EOLCF 對牙周炎患者分離的口腔細菌具有顯著的抗菌作用，且在不同菌種間亦有明顯差異，對於經 LPS 刺激之細胞亦具有抑制發炎反應的能力。

關鍵字

抗菌；抗發炎；山胡椒；牙周炎

並列摘要

The essential oil of Litsea cubeba fruit (EOLCF) has been shown anti-inflammatory, anti-oxidant and anti-bacterial properties in several studies. Periodontitis is mainly caused by pathogenic bacteria infection in the subgingival tissues, causing an inflammatory reaction and damage to the gum tissue, and eventually forming a chronic and inflammatory oral disease. However, there are few studies focusing on the effects of EOLCF on oral bacteria. Our aims are to isolate the periodontitis-related bacterial strains and to evaluate the effects of EOLCF on antimicrobial activity of identified bacteria strains, cytotoxicity to oral cells and the ability to reduce IL-6 level by oral cancer cell (Ca9-22) stimulated by lipopolysaccharide (LPS). Three anaerobic and three aerobic bacteria strains were isolated and identified from subgingival plaque biofilms using MALDI-TOFMS method. After the verification with Pubmed data, five strains were closely related to periodontitis, and one strain was commonly found in normal oral cavity. The EOLCF components were analyzed using a GC/ FID device in combination with E-nose and its reference database. The main components were d-limonene (53.93%) and citral (15.66%). The results of anti-bacterial test using 2-fold dilution of EOLCF showed that diameters of the inhibition zone were varied between 17.78±1.54mm ~35.55±5.74 mm and positive control of 0.2% chlorohexidine were 11.00±0.00mm ~26.00±0.00mm. A significant difference in the diameters of inhibition zone was found between these six different bacteria strains. The minimum inhibitory concentration for aerobic bacteria is 1% EOLCF. When the EOLCF was 800-fold dilution, the EOLCF cytotoxicity to primary culture cells of normal periodontal tissue was almost equal to those without treatment. However, the cytotoxic effect of EOLCF on oral cancer Ca9-22 cell line was nearly 100-fold higher than that on primary culture cells. Further to test the anti-inflammatory ability, the effect of reduction on IL-6 cytokine level still could be detected using Ca9-22 cells treated with 5x105-fold dilution of EOLCF. These results suggested that EOLCF has a significant antibacterial effect on oral bacteria isolated from patients with periodontitis. Antibacterial effect of EOLCF can be varied among different bacteria strains. The EOLCF has an ability to inhibit IL-6 production under the oral cells stimulation with LPS.

並列關鍵字

antimicrobial ； anti-inflammation ； Litsea cubeba ； periodontal disease

參考文獻

Armitage, G. C. (1999). Development of a classification system for periodontal diseases and conditions. Ann Periodontol, 4(1), 1-6. doi:10.1902/annals.1999.4.1.1

何振隆、李淑靜、趙國評、花國鋒、蘇裕昌. (2016). 鑽石級的精油―