綜合上述結果顯示，小本山葡萄的萃取物 miyabenol A，能夠抑制由 LPS 引起的一氧化氮生成。此種抑制作用並非經由藥物對細胞產生毒性或 抑制 iNOS 酵素活性，亦不是 miyabenol A 對 NO 的捕捉能力。miyabenol A 抑制一氧化氮生成的機轉，是經由抑制轉錄因子 NF-κB 的活化，進而 抑制 iNOS mRNA 表現。抑制 NF-κB 的活性，主要是經由抑制 p38 MAPK 開始，經過 Akt、 IKK 、IκB，最後影響 NF-κB 的活化（圖十五）。
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NO Production
Miy aben ol A
Ami nogu anid ine
Resv erat rol LPS contro l
N it ri te C o n cen tra ti o n
（圖一） Miyabenol A 、 resveratrol 與 aminoguanidine 對 LPS 誘發 RAW 264 巨噬細胞釋放 NO 之影響
RAW 264 巨噬細胞在 LPS（1 μg/ml）刺激下同時給予的 miyabenol A (0.1 μM、
0.3 μM、1 μM、3 μM、5 M、10 μM、30 μM) resveratrol （ 10 μM、15 μM、20 μM、 30 μM ）與 aminoguanidine （ 1 M、3 μM、10 μM、30 μM ）作用 18 小時，測量 nitrite 濃度。*P < 0.05，與 LPS 組作比較（n=3）。
Miy ab en ol A
Am ino gu an idi ne
Re sve rat rol LP S
con tro l
MTT Assay OD 570 nm
0.0
（圖二） Miyabenol A、resveratrol 與 aminoguanidine 對 RAW 264 巨噬細胞存 活率之影響
利用MTT 偵測方法測量 miyabenol A (0.1 μM、0.3 μM、1 μM、3 μM、5 μM、
10 μM、30 μM) 、resveratrol （ 10 μM、15 μM、20 μM、 30 μM ）與
aminoguanidine （ 1 μM、3 μM、10 μM、30 μM ）對細胞存活率的影響。（n=3）
Miyabenol A
（圖三）a､ Miyabenol A、aminoguanidine 在 RAW 264 巨噬細胞對於 LPS 誘 導生成之 iNOS 酵素活性影響及細胞毒性分析
M iya be no l A
Am in og ua ni di ne SN P
Nitri te Concentrati o n( μM)
0 10 20 30
40
0.1 μΜ 0.5 μΜ1 μΜ 3 μΜ 5 μΜ 10 μΜ 30 μΜ
（圖四）Miyabenol A、aminoguanidine 對於 SNP 產生一氧化氮的捕捉能力 100 μM SNP 溶於 10 mM Tris（PH 7.5）中，於室溫狀態下靜置 3 小時後，加 入不同濃度的 miyabenol A（0.1 μM、0.5 μM、1 μM、5 μM、10 μM、50 μM、
100 μM）及 aminoguanidine（1 μM、 3 μM 、10 μM、30 μM），於 12 小時後 測量 nitrite 濃度。
control LPS 3uM 5uM 10uM
－ －
3 10 －
－3 10 μM
iNOS
GAPDH
0 hr 2 hr 4 hr 8 hr
－ ＋ － ＋ － ＋ － ＋
Miyabenol A
－ ＋ － ＋ － ＋ － ＋
LPS
（圖七） Miyabenol A 對 LPS 誘發 RAW 264 巨噬細胞 iNOS mRNA 穩定之 影響
RAW 264 細胞以 LPS（1 μg/ml）刺激細胞 5 小時，同時加入 actinomycin-D（0.1 μg/ml）以及 miyabenol A（10 μM），作用 0、2、4、8 小時後，取下細胞萃取 RNA，
進行 RT-PCR 分析。以 GAPDH 的表現量作為比較基準。（n=2）
NF-κB Actin
C LPS 5 10 μM
Miyabenol A
IkB
p-Ikk
Actin
Actin
C LPS 5 10 μM
Miyabenol A
（圖八） Miyabenol A 對 LPS 誘發 RAW 264 巨噬細胞 NF-κB、IκB、IKK 表 現的影響
RAW 264 細胞以 miyabenol A (5μM、10μM ) 預處理 1 小時，加入 LPS ( 1 μg/ml )分別作用 10 分鐘（ IkB ）、30 分鐘（ Ikk ）及 1 小時（ NF-κB ），利 用 Western Blot 方法來分析。
C LPS 5 10 μM
c-fos
Miyabenol A
Actin
p-c-Jun
C LPS 5 10 μM
Miyabenol A
c-Jun
p-ATF-2 ATF-2
（圖九）Miyabenol A 對 LPS 誘發 RAW 264 巨噬細胞 AP-1 活化的影響 RAW 264 細胞以 miyabenol A (5μM、10μM ) 預處理 1 小時後，加入 LPS ( 1 μg/ml ) 分別作用 10 分鐘（p-c-Jun）及 30 分鐘（c-fos、p-ATF-2），利用 Western Blot 方法來分析。
C LPS 5 10 μM
Miyabenol A
p-p38 p38
C LPS 5 10 μM
Miyabenol A
p-ERK ERK
p-JNK JNK
（圖十）Miyabenol A 對 LPS 誘發 RAW 264 巨噬細胞 MAPKs 活化的影響 RAW 264 細胞以 miyabenol A (5 μM、10 μM )預處理 1 小時，同時加入 LPS ( 1 μg/ml )作用 10 分鐘，利用 Western Blot 方法來分析。
C LPS 5 10 μM
p-Akt Actin
Miyabenol A
（圖十一）Miyabenol A 對 LPS 誘發 RAW 264 巨噬細胞 Akt 活化的影響 RAW 264 細胞 以 miyabenol A (5μM、10μM ) 預處理 1 小時，同時加入 LPS ( 1 μg/ml )作用 30 分鐘，利用 Western Blot 方法分析之。
IkB Actin
C LPS 10 30 μM
SB203580
p-Ikk Actin
p-Akt Akt
C LPS 10 30 μM
SB203580
（圖十二） P38 抑制劑 SB203580 對 LPS 刺激 RAW 264 造 成 IKK、IκB、
Akt 磷酸化的影響
RAW 264 細胞加入 SB203580 (10 μM、30 μM )預處理 1 小時，分別以 LPS ( 1 μg/ml )作用 10 分鐘（ IkB ）、30 分鐘（ Ikk、p-Akt ）利用 Western Blot 方法 來分析。
p-P38 P38
C LPS 10 30 μM
Wortamanin
IkB Actin
C LPS 10 30 μM
Wortamanin
p-Ikk Actin
（圖十三）Akt 抑制劑 wortamanin 對 LPS 刺激 RAW 264 造成 IKK、IκB、
p-P38 磷酸化的影響
RAW 264 細胞加入 wortamanin (10 μM、30 μM ) 預處理 1 小時後，以 LPS ( 1 μg/ml )作用 10 分鐘利用 Western Blot 方法來分析。
COX-2
DMEM LPS 3uM 5uM 10uM
COX II protein band intensity
0.0
LPS receptor LPS
IKK
IkB P
NF-kB P
P38
NF-κB
iNOS , COXΠ
Akt
Miyabenol A
(圖十五)Miyabenol A 抑制 RAW 264 巨噬細胞受到 LPS 誘導活化之訊號傳遞路 徑
O OH HO
HO
OH
O OH
HO
O
HO OH
OH
OH H
H
H
H H
H
附圖一：Miyabenol A（國立中國醫藥研究所黃鈺玲老師提供）
附圖二：LPS 活化 MAPKs、PI3K、IKK/Iκ-B/NF-κB 調控 transcription factors 路 徑圖(摘錄自 Guha and Mackman, 2001)