Echinacea species and alkamides inhibit prostaglandin E(2) production in RAW264.7 mouse macrophage cells

Abstract

Inhibition of prostaglandin E(2) (PGE(2)) production in lipopolysaccharide-stimulated RAW264.7 mouse macrophage cells was assessed with an enzyme immunoassay following treatments with Echinacea extracts or synthesized alkamides. Results indicated that ethanol extracts diluted in media to a concentration of 15 microg/mL from E. angustifolia, E. pallida, E. simulata, and E. sanguinea significantly inhibited PGE2 production. In further studies, PGE2 production was significantly reduced by all synthesized alkamides assayed at 50 microM, by Bauer alkamides 8, 12A analogue, and 14, Chen alkamide 2, and Chen alkamide 2 analogue at 25 microM and by Bauer alkamide 14 at 10 microM. Cytotoxicity did not play a role in the noted reduction of PGE2 production in either the Echinacea extracts or synthesized alkamides. High-performance liquid chromatography analysis identified individual alkamides present at concentrations below 2.8 microM in the extracts from the six Echinacea species (15 microg/mL crude extract). Because active extracts contained <2.8 microM of specific alkamide and the results showed that synthetic alkamides must have a minimum concentration of 10 microM to inhibit PGE2, it is likely that alkamides may contribute toward the anti-inflammatory activity of Echinacea in a synergistic or additive manner.

Figure 1

Inhibition of PGE₂ production by Soxhlet ethanol extracts of medicinal species of Echinacea treated at their highest concentration (6 g of extract diluted initially in DMSO and then prepared as 0.1% of the media). Each bar represents three replicates ± standard deviation. Dry material was obtained from the 2005 harvests grown by NCRPIS. Johnny’s Selected Seeds accessions are denoted JS on the graph, and PI is indicative of accessions from NCRPIS.*, p < 0.05, and **, p < 0.001, for comparison of extract to control. Media + DMSO and media + DMSO + LPS treatments are represented by gray and black bars, respectively.

Figure 2

Inhibition of PGE₂ production by extracts of three medicinal and three nonmedicinal species of Echinacea (diluted to 15 μg/mL of extract in DMSO) obtained from NCRPIS with different harvest dates and accession numbers. Each species mean is represented by a bar (two to six replicates each), and variability is represented as 95% confidence intervals of the mean. Treatments analyzed without the addition of LPS did not affect PGE₂ levels with values for PGE₂ as percent of media + DMSO control <20% (data not shown). One E. angustifolia extract, from the 2004 harvest, treated without LPS, was excluded from the analysis on the basis of the optical density data point being outside the standard curve.*, p < 0.05, and **, p < 0.001, for comparison of extract to control (media + DMSO + LPS).