Endogenous Levels ofEchinaceaAlkylamides and Ketones Are Important Contributors to the Inhibition of Prostaglandin E2 and Nitric Oxide Production in Cultured Macrophages

Abstract

Due to the popularity of Echinacea as a dietary supplement, researchers have been actively investigating which Echinacea constituent or groups of constituents are necessary for immune modulating bioactivities. Our prior studies indicate that alkylamides may play an important role in the inhibition of prostaglandin E2 (PGE2) production. HPLC fractionation, employed to elucidate interacting anti-inflammatory constituents from ethanol extracts of E. purpurea, E. angustifolia, E. pallida, and E. tennesseensis identified fractions containing alkylamides and ketones as key anti-inflammatory contributors using lipopolysaccharide induced PGE2 production in RAW264.7 mouse macrophage cells. Nitric oxide (NO) production and parallel cytotoxicity screens were also employed to substantiate an anti-inflammatory response. Echinacea pallida showed significant inhibition of PGE2 with a first round fraction, containing GC-MS peaks for Bauer Ketones 20, 21, 22, 23, and 24, with 23 and 24 identified as significant contributors to this PGE2 inhibition. Chemically synthesized Bauer Ketones 21 and 23 at 1 μM each significantly inhibited both PGE2 and NO production. Three rounds of fractionation were produced from an E. angustifolia extract. GC-MS analysis identified the presence of Bauer Ketone 23 in third round Fraction 3D32 and Bauer Alkylamide 11 making up 96% of third round Fraction 3E40. Synthetic Bauer Ketone 23 inhibited PGE2 production to 83 % of control and synthetic Bauer Alkylamide 11 significantly inhibited PGE2 and NO production at the endogenous concentrations determined to be present in their respective fraction, thus each constituent partially explained the in vitro anti-inflammatory activity of their respective fraction. From this study two key contributors to the anti-inflammatory properties of E. angustifolia were identified as Bauer Alkylamide 11 and Bauer Ketone 23.

Echinacea purpurea root extract inhibits TNF release in response to Pam3Csk4 in a phosphatidylinositol-3-kinase dependent manner

“…Multiple researchers have reported on the anti-inflammatory effects of Echinacea extracts especially with regard to Echinacea derived alkylamides and ketones [8,[20][21][22]. Few studies however have reported on the anti-inflammatory properties of Echinacea derived polysaccharides such as what we describe here.…”

“…Echinacea-derived alkylamides have been shown to inhibit COX1 and COX2 in in vitro enzyme assays, [28], and to inhibit LPS stimulated production of PGE 2, NO, chemokines, and TNFa from RAW264.7 macrophages. [8,20,29]. Alkylamides also induce expression of arginase in RAW264.7 cells which suggests an ability to polarize macrophages toward an alternatively activated, anti-inflammatory phenotype [30].…”

“…Like the inconclusive clinical results on Echinacea, these in vitro studies are also complicated as Echinacea contains a number of chemical components including polysaccharides, flavonoids, and alkylamides and ketones [4][5][6][7][8][9][10]. To further confound the issue, a number of different solvents have been used to generate extracts containing various amounts of these phytochemicals.…”

Echinacea and its alkylamides: Effects on the influenza A-induced secretion of cytokines, chemokines, and PGE2 from RAW 264.7 macrophage-like cells

“…De novo transcription of COX-2 is necessary for PGE 2 production in RAW 264.7 cells [39]. Alternatively, the alkylamides could be mediating their effects on PGE 2 through the induction of nitric oxide [40]. Nitric oxide has been shown to enhance COX-1-dependent, constitutive production of PGE 2 and at the same time inhibit COX-2-dependent, inducible production of PGE 2 [41].…”

Echinacea purpurea (L.) Moench modulates human T-cell cytokine response

“…Generally ethanol extracts are expected to contain higher levels of alkylamides and phenolic compounds, while aqueous extracts contain more hydrophilic compounds such as polysaccharides and glycoproteins. Polysaccharides, glycoproteins, and cichoric acid have been identified as having immune-stimulating activity [11,20,21] in contrast to alkylamides and ketones that appear to be anti-inflammatory in vitro and in vivo [22-24]. Purified polysaccharide extracts from Echinacea increased macrophage chemotaxis, production of reactive oxygen intermediates (ROS), and inhibited growth of fungi in vivo [25].…”