以痤瘡桿菌刺激單核球細胞為模式探討山苦瓜葉萃取物之區分物抑制IL-8生成

Abstract

痤瘡桿菌(P. acnes)，屬於革蘭氏陽性厭氧菌(gram-positive anaerobic bacterium)，是存在人類皮膚毛囊、口腔、大腸、結膜的正常菌群之一，為尋常性痤瘡皮膚毛囊中的主要致病菌，也與眼內炎、心內膜炎、角膜炎、前列腺發炎癌化等發炎性疾病相關。痤瘡桿菌活化細胞膜上類鐸受體(TLR)，啟動下游MyD88與MAPK等訊息傳遞，增加NF-κB與AP-1等轉錄因子活性，造成促發炎介質的生成。 苦瓜是常見的蔬菜，文獻指出苦瓜(Momordica charantia L.)具有降血糖、降血脂、抗氧化、抗腫瘤、抗菌、抗發炎等作用，其活性成份也陸續被分離出來，像是類黃酮、酚酸、三萜、共軛次亞麻油酸等。先前研究指出，山苦瓜葉甲醇萃取物能夠抑制THP-1人類單核球細胞受痤瘡桿菌刺激之IL-8 (CXCL8)、TNF-α、IL-1β產生，並能降低細胞核內轉錄因子NF-κB，在動物實驗中亦能減緩ICR小鼠耳朵受痤瘡桿菌刺激所引起之紅腫發炎現象。 本篇研究分析山苦瓜葉之營養成分並接續分離山苦瓜葉甲醇萃取物，以抑制化學趨化激素IL-8為指標，希望找出具有抗發炎活性之山苦瓜葉區分物與成份。 實驗結果顯示，花蓮一號山苦瓜葉含有水份81.22%、粗蛋白6.26%、粗脂肪1.22%、粗纖維1.92%、灰份2.85%、維生素C 1920.14 μg/g drybasis。另外，山苦瓜葉甲醇萃取物之正已烷層區分物Hex-2m能夠顯著抑制痤瘡桿菌誘導之IL-8生成，抑制ERK、JNK、p38蛋白質磷酸化，並且能降低小鼠耳朵受痤瘡桿菌刺激所引起之紅腫發炎現象。透過化學呈色法、NMR、GC-FID、LC-MS分析出山苦瓜葉活性區分物Hex-2m中含有三萜類化合物，脂肪酸部分以α-次亞麻油酸(α-linolenic acid, C18:3 n-3)為主要成份。除此之外，GC-MS資料庫比對到squalene、phytol、維生素K1、維生素E (α-tocopherol、γ-tocopherol)、β-ionone與dihydroactinidiolide等化合物亦是活性區分物Hex-2m中之成份。Propionibacterium acnes (P. acnes) is a gram-positive bacterium that forms part of the normal flora of the human skin, oral cavity, large intestine, and conjunctiva. P. acnes plays an important role in acne vulgaris and other diseases, including endophthalmitis, endocarditis, keratitis, and inflammation of the prostate leading to cancer. P. acnes can stimulate toll-like receptor (TLR) on the cell membrance, active MyD88 and MAPK signaling, and increase the activaction of transcription factors NF-κB and AP-1. The signal transduction of TLR leads to the expression of pro-inflammatory mediators. Bitter melon (Momordica charantia L.) is a common vegetable and has been reported to possess hypoglycemic, antihyperlipidemic, antioxidant, anticancer, antibacterial, and anti-inflammatory properties. The bioactive compounds in bitter melon, such as flavonoids, phenolic acids, triterpenoids, and conjugated linolenic acids, have been identified. Previous study showed that methanolic extracts from wild bitter melon leaf inhibited P. acnes-induced IL-8 (also called CXCL8), TNF-α, IL-1β, and NF-κB in THP-1 cells and suppressed the inflammatory response in vivo. In this study, we analyzed the nutrient contents of Hualien-1 wild btiier melon leaf (H1L) and tried to find the active subfractions and compounds followd by the inhibitory effects on P. acnes-induced IL-8. Our resultes showed the nutrient contents of H1L were 81.22% moisture, 6.26% crude protein, 1.23% crude fat, 1.92% crude fiber, 2.85% ash, and vitamin C 1920.14 μg/g dry basis, respectively. The subfraction, Hex-2m, inhibited the level of P. acnes-induced IL-8 and suppressed ERK, JNK, p38 protein phosphorylation in THP-1 cells. This subfraction also showed an anti-inflammatory activity in P. acnes-stimulated ears of ICR mice. Triterpenoids, fatty acids, including α-linolenic acid, were measured in this subfraction by using colorimetric method, NMR, GC-FID and LC-MS. Squalene, phytol, vitamin K1, α-tocopherol, γ-tocopherol, β-ionone, and dihydroactinidiolide were identified by GC-MS with the library data.