Anti-inflammatory effect of pomegranate flower in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages

Figures & data

Figure 1. Total ion chromatograms of PFE in negative ESI mode.

Table 1. Characterisation of compounds in PFE by HPLC-QTOF-MS/MS.

Peak	tR (min)	[M-H]^−	Major and important MS^2 ions	Identification
1	8.66	633.1440	481,301, 275, 247, 203, 175, 169	Galloyl-HHDP-glucoside
2	9.01	799.1565	781, 479, 301, 273, 257	Granatin A
3	9.57	483.1295	301,275,169,125	Digalloyl-glucoside
5	11.35	635.1635	465,313,271, 169 , 125	Tri-O-galloyll-glucoside
6	12.14	633.1528	481, 301, 275, 247, 203, 175, 169	Galloyl-HHDP-glucoside
7	13.45	951.1904	933,463,301,275,229,167	Granatin B
8	18.44	301.0272	284, 229 ,185 145, 129	Ellagic acid
9	19.22	491.1155	453,301,275,209,177,169	Ellagic acid derivative
10	19.81	477.1152	301,275,187,151,125	Ellagic acid- rhamnoside
11	21.35	301.0651	255,179,151,121,107	quercetin
12	21.43	463.1409	301, 271 ,255, 229, 179 ,151	Quercetin-O-glucoside
13	23.74	285.0703	71,255,227,151	Kaempferol
14	25.41	285.6710	285,241,175,151,133	Luteolin

t_R: retention time; HHDP: hexahydroxydiphenoyl.

Figure 2. Cytotoxicity of PFE in RAW 264.7 cells. Cells were treated with different concentrations of PFE for 24 h, and viability was assayed by the MTT assay. Data represent mean values of triple determinations ± SEM. PFE at 100 μg/mL was not cytotoxic.

Figure 3. Effects of PFE on LPS-induced NO, PGE2 production and iNOS, COX-2 protein expression levels in LPS-induced RAW264.7 cells. Cells were incubated in the presence of PFE or in combination with 1 μg/mL LPS for 18 h. The culture supernatant was analyzed for NO (A), PGE₂ (B) production. The iNOS and COX-2 (C) expression levels were determined by Western blotting. Data show mean ± SEM values of three independent experiments. *p < 0.05 and **p < 0.01 indicate significant differences from LPS-stimulation value.

Figure 4. Effects of PFE on TNF-α (A), IL-6 (B) and IL-1β (C) in LPS-induced RAW264.7 cells. The cells were pretreated with the different concentrations of PFE for 1 h and then exposed to 1 μg/mL LPS for 18 h. The levels of TNF-α, IL-1β and IL-6 in the supernatant were determined by ELISA. Data show mean ± SEM values of three independent experiments. *p < 0.05 and **p < 0.01 indicate significant differences from LPS stimulation value.

Figure 5. Effects of PFE on NF-κB p65 and IκBα activity in LPS-stimulated RAW 264.7 cells. The cells were pretreated with the different concentrations of PFE for 1 h and then exposed to 1 μg/mL LPS for additional 30 min. Cytoplasm and nuclear extracts proteins of cells were harvested for measurements of NF-κB p65 and IκB-α protein by Western blotting. β-Actin and Lamin B were used as the internal control. Data show mean ± SEM values of three independent experiments. *p < 0.05 and **p < 0.01 indicate significant differences from LPS-stimulation value.

Figure 6. Effects of PFE on phosphorylation of MAPKs activity in LPS-stimulated RAW 264.7 cells. The cells were pretreated with the different concentrations of PFE for 1 h and then exposed to LPS for 30 min. Total cellular proteins of cells were harvested for measurements of total or phosphorylated ERK1/2, JNK, and p38 by Western blotting. Data show mean ± SEM values of three independent experiments. *p < 0.05 and **p < 0.01 indicate significant differences from LPS-stimulation value.