蘭花活性成分白楊素與蝴蝶蘭水萃物抗發炎功效之探討

慢性發炎是導致自身免疫性系統失調與大多數疾病發生的共同原因之一，有效抑制發炎將能提高疾病的治癒能力。蘭花除觀賞用途外，近年來有研究指出蝴蝶蘭水萃物可以美白，抗 UVA、UVB 所造成的皮膚傷害；而白楊素是一種存在於蘭花中的天然類黃酮類化合物，自古為中國的傳統藥材之一，具有抗發炎特性和神經保護作用。過去文獻指出白楊素可抑制經由脂多醣 (Lipopolysaccharide, LPS) 誘導的發炎反應，阻斷活化神經微膠細胞 (Microglia) NF-κB 和 JNK 的表現量，減少發炎反應。本研究探討使用白楊素 (chrysin, 0.5-10 M) 與蝴蝶蘭水萃物 (water extract of Phalaenopsis orchid flower, WEPF, 25-200 ng/mL) 對 LPS 誘導的小鼠 RAW 264.7 巨噬細胞抗發炎作用與參與機制。利用ELISA、RT-PCR、Western blot 和 Gelatin Zymography 方法測定相關發炎因子水平的變化。結果顯示，白楊素在濃度10 M，蝴蝶蘭水萃物在濃度 200 ng/mL 時對小鼠 RAW264.7 巨噬細胞產生之發炎介質具有顯著抑制作用，藉由減少促炎細胞激素，如:介白素(interleukin, IL)-1, -6、腫瘤壞死因子(tumor necrosis factor, TNF)-的分泌，降低基質金屬蛋白酶 (Matrix metalloproteinase, MMP)-9 的表現，同時抑制 I激酶活性，使 p65 進入細胞核內，進行基因轉錄與調控下游促發炎細胞激素。綜合以上發現，白楊素與蝴蝶蘭水萃物可顯著抑制 LPS 誘導小鼠 RAW 264.7 巨噬細胞的發炎反應，並透過阻斷通過 NF和 MAPK 信號通路，使 MMP-9 表現量減少，以及降低 TNF-、IL-1與IL-6 分泌，達到抗小鼠巨噬細胞發炎的功效。

關鍵字

蝴蝶蘭水萃物、白楊素、發炎反應、介白素、基質金屬蛋白酶

並列摘要

Inflammation is one of the mechanisms for autoimmune disorders and a common feature of most diseases. Effective suppression of inflammation is a crucial means for disease treatment. Orchids, a group of angiosperms belonging to the Orchidaceae family, are traditionally used as an herbal medicine and many constituents obtained from different parts of orchid with biological activities including anti-inflammation. In the present study, we used water extract of Phalaenopsis aphrodite subsp. Formosana (WEPF) and Chrysin, a flavonoid in Cymbidium, to evaluate and compare their anti-inflammatory properties in a lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophage cell line. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay was used to determine the toxicity of compounds on cells. Nitric oxide (NO) and inducible NO synthase (iNOS) were assayed by commercial kits. The levels of several inflammation-related factors were determined by enzyme-linked immunosorbent assay (ELISA), gelation zymography and RT-PCR. The molecular signaling was analyzed by Western blot. The results showed that the levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1 in RAW 264.7 cells were significantly induced by LPS at a concentration of 100 ng/mL; the induced proteins were then depressed by treating chrysin and WEPF at a concentration higher than 10 M, 200 ng/mL, respectively. The gelatinolytic activity and mRNA expression of matrix metalloproteinase (MMP)-9 were inhibited by chrysin and WEPF higher than 10 M and 100 ng/mL, respectively. The levels of NO and iNOS were both increased by the treatment of WEPF or chrysin. Additionally, MAPKs and NF-B signaling were inactivated by chrysin and WEPF. In conclusion, chrysin and WEPF both could inhibit the LPS-stimulated inflammation in RAW264.7 cells by suppressing the levels of NO, iNOS, IL-6, IL-1β, TNF- and MMP-9 through inactivating MAPKs and NF-B. Chrysin and WEPF might use to improve the treatment of inflammation-related diseases.