山苦瓜葉組成分抑制牙齦卟啉單胞菌誘導 之發炎反應

Abstract

牙周病（Periodontal disease）是全球性的主要口腔健康問題。嚴重的牙周病除了會導致成人掉齒，也與心血管疾病、動脈粥狀硬化、類風濕性關節炎、糖尿病等疾病呈正相關。 牙齦卟啉單胞菌( Porphyromonas gingivalis, P.gingivalis)是造成牙周病的主要致病菌之一，P.gingivalis屬於革蘭氏陰性菌，生長環境為厭氧，會潛藏在宿主牙齦下。P.gingivalis會藉由活化宿主細胞膜上的類鐸受體(TLRs)並啟動下游MyD88與MAPK等訊息傳遞，造成促發炎介質的生成。 山苦瓜(Momordica charantia Linn. var. abbreviata Ser.)的果實較比苦瓜小，苦味較強，具有降血糖、降血脂、抗發炎等作用。本研究以活性導向方式分離山苦瓜葉中具抑制P.gingivalis誘導發炎之成分。首先使用P.gingivalis刺激人類單核球THP-1細胞釋出IL-6和IL-8作為模式，分離山苦瓜葉萃取物、區分物並鑑定其有效成分為葫蘆烷型三萜類5β,19-epoxycucurbita-6,23-diene-3β,19,25-triol、 3β,7β,25-trihydroxycucurbita-5,23-dien-19-al能夠顯著抑制牙齦卟啉單胞菌誘導IL-6、IL-8生成，且顯著抑制MAPK路徑中蛋白質ERK和p38磷酸化。在in vivo，以P.gingivalis刺激小鼠牙齦組織，其組織中發炎介質COX-2、IL-6、TNF-α mRNA表現量顯著上升，給予5β,19-epoxycucurbita-6,23-diene-3β,19,25-triol和3β,7β,25-trihydroxycucurbita-5,23-dien-19-al均顯著抑制COX-2、IL-6、TNF-α基因表現。 故本研究結果證實fra.5211(5β,19-epoxycucurbita-6,23-diene-3β,19,25-triol)和fra.5212(3β,7β,25-trihydroxycucurbita-5,23-dien-19-al)為山苦瓜葉內具有抑制P.gingivalis誘導發炎之成分，未來可望應用於P.gingivalis誘導發炎相關疾病的治療與控制，例如牙周炎、心臟疾病等。

Periodontal disease is a one of the main global major oral health problem. Periodontitis causes tooth loss in adults and also increases the risk of cardiovascular disease and diabetes mellitus. Porphyromonas gingivalis is a gram-negative black pigmented anaerobe that colonizes the subgingival crevice, has been identified as one of the major periodontal pathogens. Wild bitter melon (Momordica charantia Linn. var. abbreviata Ser.) is a common vegetable and has been reported to possess hypoglycemic, antihyperlipidemic, antioxidant, anticancer, antibacterial, and anti-inflammatory properties.In this study, activity-directed fractionation and purification processes were employed to identify the anti-inflammatory active compounds using heat-killed P.gingivalis-stimulated human monocytic THP-1 cells in vitro. Total phenol extract (TPE) was prepared from leaves of wild bitter melon (WBM; Momordica charantia Linn. var. abbreviata Ser.). Five major fractions were collected from TPE and were evaluated for their inhibitory effect against P.gingivalis-induced IL-8 and IL-6 productions in vitro. Among the test fractions, the fraction 5 showed strong activity and was subjected to separation and purification by using chromatographic techniques. Two cucurbitane triterpenoids showing potent activity were identified by comparing spectraldata to be fra.5211 (5β,19-epoxycucurbita-6,23-diene-3β,19,25-triol)fra.5212(3β,7β,25-trihydroxycucurbita-5,23-dien-19-al). Treatments of both cucurbitane triterpenoids in vitro potently suppressed IL-6 and IL-8 levels in heat-killed P. gingivalis-stimulated THP-1 cells. Both cucurbitane triterpenoid attenuated heat-killed P. gingivalis-induced extracellular signal-regulated kinase, P38 and ERK.In addition, both cucurbitane triterpenoid effectively suppressed COX-2, IL-6, and TNF-α mRNA levels in P. gingivalis-stimulated gingival tissue of mice. Our data suggested that 5β,19-epoxycucurbita-6,23-diene-3β,19,25-triol) and fra.5212(3β,7β,25-trihydroxycucurbita-5,23-dien-19-al) significantly reduced P. gingivalis-induced inflammation via inhibiting MAPK signaling.