Extract activates AMPK in C2C12 cells. (A) Extract increased AMPK phosphorylation. C2C12 cells were treated with aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (1 mmol/l), extract (5 μg/ml), or macelignan (10 μM) for 24 hours. Phosphorylated AMPK was examined by Western blot analysis, (B) extract increased the mRNA expression of ACS, CPT-1. The expression was estimated using quantitative real-time RT-PCR. Data represent are shown as mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001)

Extract attenuates the induction of ER stress. (A) Extract prevented the increase of thapsigargin-stimulated luciferase activity in thapsigargin-treated HepG2. HepG2 cells were transfected with ERSE-Luc reporter and then treated with extract (5 μg/ml), rosiglitazone (10 μM), or macelignan (10 μM) for 24 hours in the presence of thapsigargin (10 ng/ml). (B) Extract increased the levels of GRP78 and peIF. HepG2 cells were preincubated with extract (5 μg/ml), rosiglitazone (10 μM), or macelignan (10 μM) for 24 hours and then treated with thapsigargin (10 ng/ml) for 24 hours. GRP78 p-eIF were measured by Western blot analysis. Data represent are shown as mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001)

Extract improved (A) postprandial glucose and (B) insulin sensitivity in db/db mice. After a 12-hourfast, male mice (12 weeks-old) were intraperitoneally injected with glucose (0.5 g/kg body weight) and insulin (2 units/kg body weight). The blood glucose concentration was then measured at the indicated times and was presented as a percentage of the glucose injection zero time. Values are mean ± SD (n = 7). ^abcd Data not sharing a common letter are significantly different (P < 0.05) after one-way ANOVA and Duncan's multiple-range test.

Extract functions as a PPARγ agonist. (A) Extract increased the ligand-binding activity of PPARγ. HEK293 cells were transfected with pFA-PPARγ and pFR-Luc (UAS-Gal4-luciferase) and then treated with extract (5 μg/ml), rosiglitazone (10 μM), or macelignan (10 μM) for 24 hours. (B) Extract induced transcriptional activity of PPARγ. Differentiated 3T3-L1 adipocytes were transfected with 3 × PPREs-tk-Luc and treated with extract (5 μg/ml), rosiglitazone (10 μM), or macelignan (10 μM) for 24 hours. (C) Extract induced adipogenesis. Oil red O staining was measured after differentiation of 3T3-L1 cells in medium containing 0.1% DMSO (control), extract (5 μg/ml), rosiglitazone (1 μM), or macelignan (10 μM) for seven days. (D) Extract increased PPARγ target gene (aP2) expression in 3T3-L1 adipocytes. Differentiated 3T3-L1 cells were treated with extract (5 μg/ml), rosiglitazone (10 μM), or macelignan (10 μM) for 24 hours. Expression of mRNAs was estimated using quantitative real-time RT-PCR, and the results were expressed as mRNA levels relative to 0.1% DMSO (control). Data represent are shown as mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001)

Extract inhibits NFkB signaling in HepG2 cells. (A) extract prevented the increase of TNF-α-stimulated luciferase activity in TNF-α treated HepG2. HepG2 cells were transfected with NFkB-Luc reporter and then treated with extract (5 μg/ml), rosiglitazone (10 μM), or macelignan (10 μM) for 24 hours in the presence of TNF-α (10 ng/ml) (B) extract increased the IkB level. HepG2 cells were preincubated with extract (5 μg/ml), rosiglitazone (10 μM), or macelignan (10 μM) for 24 hours and then treated with TNF-α (10 ng/ml) for one hour. IκBα was measured by Western blot analysis. Data represent are shown as mean ± SD of three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001)

Extract beneficial effects on (A) body weight and (B) fasting blood glucose level in db/db mice after 3-week treatment. Values shown are mean ± SD (n = 7). ^abcd Data not sharing a common letter are significantly different (P < 0.05) after one-way ANOVA and Duncan's multiple-range test. NS: non-significance