新育種苦瓜之抗發炎研究及其分子機制探討

Anti-inflammatory effect of new variety selection of bitter melon and its molecular mechanism

陳宜芬(Yi-Fen Chen)

指導教授 ： 徐慶琳

中山醫學大學/健康管理學院/營養學研究所/碩士(2012年)

摘要

苦瓜 (Momordica charantia L.) 之生理活性包含有抗菌、抗病毒、降血糖、降血壓、抗發炎和抗癌等功能。然而，新品系苦瓜在抗發炎的影響目前仍不明確。因此，本研究以LPS誘導RAW 264.7巨噬細胞產生發炎反應後，同時處理蘋果苦瓜、苦瓜明道選72和苦瓜沖繩選33，進行新育種苦瓜之抗發炎研究及其分子機轉探討。本研究內容分成二部分：(一) 將蘋果苦瓜、苦瓜明道選72和苦瓜沖繩選33之果肉和種子以不同溶劑萃取後，以LPS誘導RAW 264.7巨噬細胞試驗模式評估抗發炎影響。(二) 具抑制潛力之苦瓜所分離之苦瓜分離物A、B、C、D和E對LPS誘導RAW 264.7巨噬細胞發炎反應之影響。(一) 以總抗氧化能力和總多酚分析評估其抗氧化能力。此分析證實苦瓜乙酸乙酯萃取物具有較高抗氧化能力。接著，以LPS誘導RAW 264.7巨噬細胞產生發炎反應後處理苦瓜明道選72果肉乙酸乙酯萃取 (EAEFMCMDS72)，發現其具有最佳抑制一氧化氮 (NO)、前列腺素E2 (PGE2)、白介素 (IL-6)、腫瘤壞死因子-α (TNF-α) 和單核球趨化蛋白質 (MCP-1) 之分泌。在蛋白質表現上，苦瓜明道選72果肉乙酸乙酯萃取物可抑制 LPS誘導RAW 264.7巨噬細胞產生一氧化氮合成酶 (iNOS) 和環氧化酶-2 (COX-2) 之發炎蛋白質表現。在基因表現上，苦瓜明道選72果肉乙酸乙酯萃取物亦具有抑制iNOS、COX-2、IL-6和TNF-α表現。(二) 苦瓜明道選72果肉可分離出五種苦瓜分離物A、B、C、D和E，其中苦瓜分離物A為最具有抑制LPS誘導RAW 264.7巨噬細胞分泌一氧化氮 (NO)、前列腺素E2 (PGE2)、、腫瘤壞死因子-α (TNF-α)、單核球趨化蛋白質 (MCP-1) 和白介素-1β (L-1β) 之分泌。在蛋白質表現上，苦瓜分離物A可抑制 LPS誘導RAW 264.7巨噬細胞產生一氧化氮合成酶 (iNOS) 和環氧化酶-2 (COX-2)、磷酸化-ERK (p-ERK)、磷酸化-JNK (p-JNK)、c-Jun和細胞核轉錄因子-κB (NF-κB) 等蛋白質表現。在基因表現上，苦瓜分離物A亦可抑制LPS誘導RAW 264.7巨噬細胞產生iNOS、COX-2、IL-1、IL-6、IL-8、TNF-α、類鐸接受器4 (TLR4)、janus kinase 2 (JAK2)、轉錄訊息傳遞及活化子蛋白3(STAT3)，血管內皮生長因子(VEGF) 和基質金屬蛋白酶9 (MMP9) 之基因表現。本研究結果顯示，以RAW264.7巨噬細胞為研究模式下，苦瓜分離物A可經由抑制p-ERK、p-JNK以及JAK2-STAT3等路徑，抑制NF-κB活化進而影響下游NO、PGE2、TNF-α、MCP-1和IL-1β等相關發炎細胞激素之分泌。

關鍵字

苦瓜 ； 抗發炎 ； RAW 264.7巨噬細胞 ； 蛋白質表現 ； 基因表現

並列摘要

Pharmacological actions of bitter melon have been investigated for various uses, including antibacterial, antiviral, antihyperglycemic, antihypertensive, anti-inflammatory and anticancer actions. However, anti-inflammatory effects of new variety selection of bitter melon are unclear. Therefore, the aim of this study was investigate the effects of Momordica charantia, Momordica charantia MDS72, and Momordica charantia ONS33 on lipopolysaccharide (LPS)-induced inflammatory responses in RAW 264.7 macrophages and its molecular mechanism. There are two topics included in this study, (1) Effects of different solvent extracts from the fruit and seed of Momordica charantia, Momordica charantia MDS72, and Momordica charantia ONS33 on LPS-induced inflammatory responses in RAW 264.7 macrophages. (2) Effects of compounds A, B, C, D, and E isolated from the potential bitter melon on LPS-induced inflammatory responses in RAW 264.7 macrophages. (1) The evaluation of antioxidant activity was determined by trolox equivalent antioxidant capacity (TEAC) and total phenolics assays. These assays demonstrated a relatively high antioxidant activity for ethyl acetate extract of bitter melon. These results revealed that the ethyl acetate extract of the fruit of Momordica charantia MDS72 (EAEFMCMDS72) had the most prominent inhibitory effect on LPS-stimulated nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemotactic protein-1 (MCP-1) productions in RAW 264.7 macrophages. In protein expression, EAEMCMDS72 inhibited LPS-stimulated protein expressions of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in RAW 264.7 macrophages. EAEFMCMDS72 also inhibited LPS-stimulated gene expressions of iNOS, COX-2, IL-6, and TNF-α in RAW 264.7 macrophages. (2) Compounds A, B, C, D, and E were isolated from the fruits of Momordica charantia MDS72. These results indicated that the compound A had the most prominent inhibitory effect on LPS-stimulated nitric oxide (NO), prostaglandin E2 (PGE2), TNF-α, MCP-1, and interleukin-1 β (IL-1β) productions in RAW 264.7 macrophages. In protein expression, compound A inhibited LPS-stimulated protein expressions of iNOS, COX-2, phosphor-ERK (p-ERK), phosphor-JNK (p-JNK), c-Jun, and nuclear factor kappa B (NF-κB) in RAW 264.7 macrophages. In gene expression, compound A also inhibited LPS-stimulated gene expressions of iNOS, COX-2, IL-1, IL-6, IL-8, TNF-α, toll-like receptor 4 (TLR4), janus kinase 2 (Jak2), signal transducer and activator of transcription 3 (STAT3), vascular endothelial growth factor (VEGF), and matrix metallopeptidase 9 (MMP9) in RAW 264.7 macrophages. These results demonstrate that compound A isolated from the fruits of Momordica charantia MDS72 suppress LPS-induced NO, PGE2, TNF-α, MCP-1, and IL-1β productions by inhibiting expressions of p-ERK, p-JNK, and JAK2-STAT3 as well as reducing NF-κB in nucleus of RAW 264.7 cells.

並列關鍵字

bitter melon ； anti-inflammation ； RAW 264.7 macrophages ； protein expression ； gene expression

參考文獻

台灣省農業試驗所和中國種苗改進協會。

Arnao, M.B., Cano, A., Hernández-Ruiz ,J., García-Cánovas, F., and Acosta, M. (1996). Inhibition by L-ascorbic acid and other antioxidants of the 2.2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) oxidation catalyzed by peroxidase: a new approach for determining total antioxidant status of foods. Analytical Biochemistry 236, 255-261.

Alam, S., Asad, M., Asdaq, S.M.B., and Prasad V.S. (2009). Antiulcer activity of methanolic extract of Momordica charantia L. in rats. Journal of Ethnopharmacology 123, 464-469.

Altman, R., and Rutledge, J.C. (2010). The vascular contribution to Alzheimer's disease. Clinical Science 119, 407-421.

Bartok, B., and Firestein, G.S. (2010). Fibroblast-like synoviocytes: key effect or cells in rheumatoid arthritis. Immunological Reviews 233, 233-255.