以NF-kB或IFN-r轉錄活性篩選具免疫調節中草藥－穿心蓮活性成份之單離與鑑定

Abstract

本研究首先利用轉染 (transfection)，分別建構能表現TH-1 的細胞激素IFN-γ 或NF-κB 啟動子連接報導基因luciferase 作為檢測之細胞，RAW 264.7 巨噬細胞 株、EL-4 T 淋巴球細胞株等，以作迅速篩選之工具，再分別以初代腹腔細胞和 脾臟細胞利用in vitro 給予中草藥材方式，及以in vivo 餵食動物方式探討腹腔細 胞、脾臟細胞分泌發炎性介質之變化，最後再利用腹腔注射LPS 誘發急性發炎 動物模式，試圖以系統性之篩選模式找尋具有調節免疫反應之中草藥。結果顯示 穿心蓮（Andrographis paniculata (Burm. F.) Nees）可顯著降低LPS/IFN-γ刺激 RAW264.7 巨噬細胞NF-κB luciferase 活性。另外；將10 ~ 12 週大之BALB/cJ 雌 鼠分為LPS 組、PDTC 組、穿心蓮EtOAc 萃取物高劑量組 (3.12 mg/kg BW)、中 劑量組 (1.56 mg/kg BW)、低劑量組 (0.78 mg/kg BW) 管餵一週後，以LPS (15 mg/kg BW) 進行腹腔注射，6 小時後犧牲，分析血清、腹腔巨噬細胞中促發炎性 細胞激素含量，以及腦部組織中促發炎性細胞激素mRNA 表現量。結果顯示穿 心蓮萃取物可顯著降低發炎小鼠血清、腹腔巨噬細胞中促發炎性細胞激素 TNF-α、IL-12p40 和化學趨化物質MIP-2 (macrophage inflammatory protein-2)、 Nitric oxide (NO)等含量，並降低腦組織中MIP-2 mRNA 表現量。 更進一步將穿心蓮乙酸乙酯層區分物以不同極性流洗，以分離純化活性成 份，實驗結果經1H-NMR、13C-NMR、2D NMR、EI-Mass 測定已分離、純化鑑 定出26 個化合物， 結果發現5-hydroxy-7,8-dimethoxyflavone (1) 、 5-hydroxy-7,8-dimethoxyflavanone (2) 和一個混合存在的化合物β-sitosterol and stigmasterol (3)、ergosterol peroxide (11)、14-deoxy-14,15-dehydroandrographolide (15) 、19-O-Acetyl-14-deoxy-11,12-didehydroandrographolide (16) 、 3,19-O-diacetylanhydroandrographolide (21) 、19-O-acetylanhydroandrographolide (22) 、hexahydro-14-dehydroandrographolide (23) 、 3,19-dioxolabda-8(17),11E,13-trien-16,15-olide (24) 其對於LPS/IFN-γ活化刺激 RAW 264.7 巨噬細胞，可顯著抑制NF-κB luciferase 活性及降低促發炎性細胞激 素分泌量。本研究由穿心蓮26 個化合物中篩選出，對於腸病毒71 型感染RD 細 胞 (human embryonal rhabdomysarcoma cells) 產生細胞病變 (cytopathic effect, CPE) 效應具有保護效果之化合物，主要可分為(1) flavonoids 類：如化合物2, 9。 (2) 固醇類：如化合物11。(3) diterpenoids 類：如化合物10, 15, 17, 19, 20, 23, 24, 25。(4) 其他類如化合物5 等。由實驗結果發現，穿心蓮中同時具有抗發炎和抗 腸病毒71 型之化合物為5-hydroxy-7,8-dimethoxyflavanone (2)、ergosterol peroxide (11)、新化合物14-deoxy-14,15-dehydroandrographolide (15)、新合成衍生物 hexahydro-14-dehydroandrographolide (23) 及 3,19-dioxolabda-8(17),11E,13-trien-16,15-olide (24) 為其功能性成份因子。

The purpose of this project is to establish a screening system for Traditional Chinese medicine herbs (TCMHs), which may exert regulatory effects on immune and inflammatory responses. Therefore, the effects of TCMHs on transient transfection were compared with those on BALB/c primary cells. The scheme for the screening system is to construct plasmids containing reporter gene luciferase with promoter sequence either of nuclear factor-κ B (NF-κB) or interferon-γ (IFN-γ). These reporter plasmids were transiently transfected into RAW 264.7 cell or EL-4 T cell to test for luciferase activity. TCMHs were also added to lipopolysaccharide/interferon-γ (LPS/IFN-γ)-activated peritoneal macrophages and Concanavalin A (Con A)-activated spleen cells. LPS has been known to induce endotoxin shock. The effect of TCMH, Andrographis paniculata on septic shock in vivo was explored. BALB/cJ mice were orally received the treatment of EtOAc fraction extract of A. paniculata at 0.78, 1.56 and 3.12 mg/kg BW for one week, then were i.p. injected with LPS (15 mg/kg BW). Mice serum were detected cytokines level. The results revealed that A. paniculata EtOAc fraction extract can inhibit serum and peritoneal exudate cells TNF-α, IL-12p40, MIP-2 and NO production (p < 0.05) and inhibition brain MIP-2 mRNA expression. The EtOAc soluble fraction as further separated by silica gel column chromatography to give total of 26 fractions. Compounds 5-hydroxy-7,8-dimethoxyflavone (1), 5-hydroxy-7,8-dimethoxyflavanone (2) and β-sitosterol and stigmasterol (3), ergosterol peroxide (11), 14-deoxy-14,15-dehydroandrographolide (15), 19-O-Acetyl-14-deoxy-11,12-didehydroandrographolide (16), 3,19-O-diacetylanhydroandrographolide (21), 19-O-acetylanhydroandrographolide (22), hexahydro-14-dehydroandrographolide (23), 3,19-dioxolabda-8(17),11E,13-trien-16,15-olide (24) can decreased NF-κB luciferase activity in RAW 264.7cells activated by LPS/IFN-γ. On the other hand, compounds 2, 5, 9, 10, 11, 15, 17, 19, 20, 23, 24, 25 can inhibition of cytopathic effect in human embryonal rhabdomysarcoma cells (RD cells) challenged with enterovirus 71 (EV71). The study show that 5-hydroxy-7,8-dimethoxyflavanone (2)、ergosterol peroxide (11) 、14-deoxy-14,15-dehydroandrographolide (15) 、 hexahydro-14-dehydroandrographolide (23) 、 3,19-dioxolabda-8(17),11E,13-trien-16,15-olide (24) showing the inhibition effects on LPS/IFN-γ induced NF-κB activation in murine cell line RAW 264.7, but also anti-EV71 infection in RD cells.