Anti-inflammatory potential of ellagic acid, gallic acid and punicalagin A&B isolated from Punica granatum

Abstract

Background: Punica granatum (pomegranate), an edible fruit originating in the Middle East, has been used as a traditional medicine for treatment of pain and inflammatory conditions such as peptic ulcer. The numerous risks associated with nonsteroidal anti-inflammatory drugs (NSAIDs) for treatment of pain and inflammation give rise to using medicinal herbs as alternative therapies. This study aimed to evaluate the anti-inflammatory effect of isolated compounds from the ethyl acetate (EtOAc) fraction of P. granatum by determination of their inhibitory effects on lipopolysaccharide (LPS), stimulated nitric oxide (NO), prostaglandin E2 (PGE-2), interleukin-6 (IL-6) and cyclooxxgenase-2 (COX-2) release from RAW264.7 cells.

Methods: The compounds ellagic acid, gallic acid and punicalagin A&B were isolated from EtOAc by high performance liquid chromatography (HPLC) and further identified by mass spectrometry (MS). The inhibitory effect of ellagic acid, gallic acid and punicalagin A&B were evaluated on the production of LPS-induced NO by Griess reagent, PGE-2 and IL-6 by immunoassay kit and prostaglandin E2 competitive ELISA kit, and COX-2 by Western blotting.

Results: Ellagic acid, gallic acid and punicalagin A&B potentially inhibited LPS-induced NO, PGE-2 and IL-6 production.

Conclusion: The results indicate that ellagic acid, gallic acid and punicalagin may be the compounds responsible for the anti-inflammatory potential of P. granatum.

Keywords: Cytokines; Cytotoxicity; Ellagic acid; Gallic acid; Inflammation; Punica granatum; Punicalagin.

Fig. 1

HPLC chromatogram of ethyl acetate fraction showing the presence of punicalagin A&B collected at 14.1 min

Fig. 2

HPLC chromatogram of ethyl acetate fraction showing the presence of ellagic acid collected at 22.6 min

Fig. 3

Mass spectrum showing the presence of punicalagin A&B [M-H] -m/z 1083.43

Fig. 4

Mass spectrum showing the presence of ellagic acid [M-H] -m/z 301

Fig. 5

Cell viability was determined from the 24 h culture of cells stimulated with LPS (1 μg/mL) in the presence of ellagic acid, gallic acid and punicalagin The data represent mean ± SD of n = 3, *p < 0.05,**p < 0.01 vs LPS treated control

Fig. 6

Effect of ellagic acid, gallic acid and punicalagin on nitrite production in LPS-stimulated RAW264.7 cells. The cells were stimulated with 1 μg/mL of LPS only or with LPS plus various concentrations (50, 100, 150, 200 μg/mL) of gallic acid for 24 h. Nitrite production was determined by the Griess reagent method. The data represent the mean ± SD of n = 3, *** p < 0.001, vs #LPS treated control* means significant difference by Tukey Kramer test

Fig. 7

Effect of ellagic acid, gallic acid and punicalagin on PGE-2 production in LPS-stimulated RAW264.7 cells. The cells were stimulated with 1 μg/ml of LPS only or with LPS plus various concentrations (50, 100, 150, 200 μg/mL) of ellagic acid, gallic acid and punicalagin n = 3, ***p < 0.001 vs #LPS treated control* means significant difference by Tukey Kramer test

Fig. 8

Effect of ellagic acid, gallic acid and punicalagin on IL-6 production in LPS-stimulated RAW264.7 cells. The cells were stimulated with 1 μg/ml of LPS only or with LPS plus various concentrations (50, 100, 150, 200 μg/mL) of for ellagic acid, gallic acid and punicalagin 6 h. IL-6 produced and released into the culture medium was assayed by ELISA method. The data represent the mean ± SD of n = 3, *** p < 0.001 vs #LPS treated control* means significant difference by Tukey Kramer test

Fig. 9

Effect of ellagic acid, gallic acid and punicalagin on the activation of COX-2 in the LPS-stimulated RAW264.7 cells. RAW264.7 cells were stimulated with LPS (1 μg/mL) in ellagic acid, gallic acid and punicalagin A&B (50, 100, 150, 200 μg/mL) for 24 h. Whole-cell lysates were subjected to 10% SDS-PAGE and expression of COX-2 and β-actin were determined by western blotting. Band intensities were expressed as relative density compared to LPS control. The figure is representative of three similar experiments