Synthesis and Natural Distribution of Anti-inflammatory Alkamides from Echinacea

The synthesis of the alkamides 2Z,4E-undeca-2,4-dien-8,10-diynoic acid isobutyl amide (1) and 2Z,4E-undeca-2,4-dien-8,10-diynoic acid isobutyl amide (5) was accomplished by organometallic coupling followed by introduction of the doubly unsaturated amide moiety. The distribution of these two amides in accessions of the nine species of Echinacea was determined

Echinacea increases arginase activity and has anti-inflammatory properties in RAW 264.7 macrophage cells indicative of alternative macrophage activation

The genus Echinacea is a popular herbal immunomodulator. Recent reports indicate that Echinacea products inhibit nitric oxide (NO) production in activated macrophages. In the present study we determined the inhibitory effects of alcohol extracts and individual fractions of alcohol extracts of Echinacea on NO production, and explored the mechanism underlying the pharmacological anti-inflammatory activity. The alcohol extracts of three medicinal Echinacea species, E. angustifolia, E. pallida and E. purpurea, significantly inhibited NO production by lipopolysaccharide (LPS)-activated the RAW 264.7 macrophage cell line, among them E. pallida was the most active. The Echinacea-mediated decrease in NO production was unlikely due to a direct scavenging of NO because the extracts did not directly inhibit NO released from an NO donor, sodium nitroprusside. An immunoblotting assay demonstrated that the extract of E. pallida inhibited inducible nitric oxide synthase (iNOS) protein expression in LPS-treated macrophages. The enzymes iNOS and arginase metabolize a common substrate, L-arginine, but produce distinct biological effects. While iNOS is involved in inflammatory response and host defense, arginase participates actively in anti-inflammatory activation. Arginase activity of RAW 264.7 cells stimulated with 8- bromo-cAMP was significantly increased by alcohol extracts of all three Echinacea species. The polar fraction containing caffeic acid derivatives enhanced arginase activity, while the lipophilic fraction containing alkamides exhibited a potential of inhibiting NO production and iNOS expression. These results suggest that the anti-inflammatory activity of Echinacea might be due to multiple active metabolites, which work together to switch macrophage activation from classical activation towards alternative activation

Echinacea tennesseensis ethanol tinctures harbor cytokine- and proliferation-enhancing capacities

Background—Members of the genus Echinacea are used medicinally to treat upper respiratory infections such as colds and influenza. The aim of the present investigation was to characterize the phytomedicinal properties of the American federally endangered species Echinacea tennesseensis. Methods—Fifty-percent ethanol tinctures were prepared from roots, stems, leaves, and flowers and tested separately for their ability to influence production of IL-1β, IL-2, IL-10, and TNF-α as well as proliferation by young human adult peripheral blood mononuclear cells (PMBC) in vitro. Tincture aliquots were stored at three different temperatures (4°, −20°C, and −80°C) for 21 h before testing. At one-month post-extraction, tinctures stored at −20°C were tested again for cytokine modulation. Phytochemical analyses were performed using HPLC. Results—Fresh root, leaf, and flower tinctures stimulated PBMC proliferation. Fresh root tinctures alone stimulated IL-1β, IL-10, and TNF-α production. No tinctures modulated IL-2 production. Stem tinctures showed no activity. Storage temperature did not influence any outcomes. Root tinctures maintained their ability to modulate IL-1β, IL-10, and TNF-α production after one month of storage at −20°C. Conclusions—These results suggest E. tennesseensis harbors phytomedicinal properties that vary by plant organ, with roots demonstrating the strongest activities

Alcohol extract of Echinacea pallida reverses stress-delayed wound healing in mice

Healing of open skin wounds begins with an inflammatory response. Restraint stress has been well documented to delay wound closure, partially via glucocorticoid (GC)-mediated immunosuppression of inflammation. Echinacea, a popular herbal immunomodulator, is purported to be beneficial for wound healing. To test the hypothesis, an alcohol extract of E. pallida was administrated orally to mice for 3 days prior to, and 4 days post wounding with a dermal biopsy on the dorsum. Concominantly, mice were exposed to 3 cycles of daily restraint stress prior to, and 4 cycles post wounding. Echinacea accelerated wound closure in the stressed mice, but had no apparent wound healing effect for the non-stressed mice when compared to their respective controls. To test if the positive healing effect is through modulation of GC release, plasma corticosterone concentrations were measured in unwounded mice treated with restraint stress and the herbal extract for 4 days. Plasma GC in restraint stressed mice gavaged with Echinacea was not different from mice treated with restraint only, but was increased compared to the vehicle control. This data suggests that the improved wound healing effect of Echinacea in stressed mice is not mediated through modulation of GC signaling

Enhancement of Innate and Adaptive Immune Functions by Multiple Echinacea Species

Echinacea preparations are commonly used as nonspecific immunomodulatory agents. Alcohol extracts from three widely used Echinacea species, Echinacea angustifolia, Echinacea pallida, and Echinacea purpurea, were investigated for immunomodulating properties. The three Echinacea species demonstrated a broad difference in concentrations of individual lipophilic amides and hydrophilic caffeic acid derivatives. Mice were gavaged once a day (for 7 days) with one of the Echinacea extracts (130 mg/kg) or vehicle and immunized with sheep red blood cells (sRBC) 4 days prior to collection of immune cells for multiple immunological assays. The three herb extracts induced similar, but differential, changes in the percentage of immune cell populations and their biological functions, including increased percentages of CD49+ and CD19+ lymphocytes in spleen and natural killer cell cytotoxicity. Antibody response to sRBC was significantly increased equally by extracts of all three Echinacea species. Concanavalin A-stimulated splenocytes from E. angustifolia- and E. pallida-treated mice demonstrated significantly higher T cell proliferation. In addition, the Echinacea treatment significantly altered the cytokine production by mitogenstimulated splenic cells. The three herbal extracts significantly increased interferon-γ production, but inhibited the release of tumor necrosis factor-α and interleukin (IL)-1β. Only E. angustifolia- and E. pallida-treated mice demonstrated significantly higher production of IL-4 and increased IL-10 production. Taken together, these findings demonstrated that Echinacea is a wide-spectrum immunomodulator that modulates both innate and adaptive immune responses. In particular, E. angustifolia or E. pallida may have more anti-inflammatory potential

Metabolic Profiling of Echinacea Genotypes and a Test of Alternative Taxonomic Treatments

The genus Echinacea is used as an herbal medicine to treat a variety of ailments. To better understand its potential chemical variation, 40 Echinacea accessions encompassing broad geographical and morphological diversity were evaluated under controlled conditions. Metabolites of roots from these accessions were analyzed by HPLC-photo diode array (HPLC-PDA), GC-MS, and multivariate statistical methods. In total, 43 lipophilic metabolites, including 24 unknown compounds, were detected. Weighted principal component analysis (WPCA) and clustering analysis of the levels of these metabolites across Echinacea accessions, based on Canberra distances, allowed us to test two alternative taxonomic treatments of the genus, with the further goal of facilitating accession identification. A widely used system developed by McGregor based primarily on morphological features was more congruent with the dendrogram generated from the lipophilic metabolite data than the system more recently developed by Binns et al. Our data support the hypothesis that Echinacea pallida is a diverse allopolyploid, incorporating the genomes of Echinacea simulata and another taxon, possibly Echinacea sanguinea. Finally, most recognized taxa of Echinacea can be identified by their distinct lipophilic metabolite fingerprints

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

Because of the popularity of Echinacea as a dietary supplement, researchers have been actively investigating which Echinacea constituent or groups of constituents are necessary for immunemodulating bioactivities. Our prior studies indicate that alkylamides may play an important role in the inhibition of prostaglandin E2 (PGE2) production. High-performance liquid chromatography fractionation, employed to elucidate interacting anti-inflammatory constituents from ethanol extracts of Echinacea purpurea, Echinacea angustifolia, Echinacea pallida, and Echinacea tennesseensis, identified fractions containing alkylamides and ketones as key anti-inflammatory contributors using lipopolysaccharideinduced 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. E. pallida showed significant inhibition of PGE2 with a first round fraction, containing gas chromatography-mass spectrometry (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

Constituents and receptor-mediated calcium signaling underlying bioactivities of Echinacea

With a long history of traditional medicinal use for various ailments including pain relief, Echinacea is currently widely used as an herbal immunomodulator. Despite the popularity of the herb and many pharmacological and clinical studies, it is still uncertain which specific compound(s) are responsible for the reported bioactivities, and the molecular mechanism of action of Echinacea remains poorly understood. Using a combination of HPLC-PDA, GC-MS and HPLC-ESI-MS/MS, we evaluated the phytochemical variation between roots of forty Echinacea accessions with broad geographical and morphological diversity grown under controlled conditions. A dendrogram was generated from these metabolite data and was compared with the two existing taxonomic treatments of the Echinacea genus. We also provided the first examination of the Echinacea-activated calcium-signaling pathway. Three models, rat hippocampal cells, Xenopus laevis oocytes and human HEK293 cells, have been established. Electrophysiological techniques and intracellular calcium imaging were used to study the effect of Echinacea constituents on intracellular Ca2+. In all three systems, Echinacea mediates an increase in cytosolic Ca2+. In cultured rat astrocytes Echinacea extracts stimulated elevation of cytosolic Ca2+ and this activation is related to the CB1 receptor. In frog oocytes, Echinacea-induced current is associated indirectly with transfected TRPV1 pain receptor. Treatment of HEK293 cells with Echinacea extract resulted in an increase in cytosolic Ca2+, and investigation of this Ca2+ signaling indicated the involvement of PLC activation, IP3 receptor and SOCE pathway. Six bioactive fractions of Echinacea were identified, and metabolite analysis indicates lipophilic constituents are associated with this bioactivity. Interestingly, the constituents thought to be responsible for the major bioactivity of Echinacea (i.e., alkamides/ketones, caffeic acid derivatives) cannot explain the observed shifts in Ca2+. So far, the identity of the receptor(s) that bind(s) to Echinacea and activate(s) the observed calcium elevation in HEK293 remains unknown. CB1 and TRPV1 are not involved as neither is present in HEK293 cells. We propose that Echinacea compounds activate specific receptors, including CB1 and TRPV1, in cells of the immune and central nervous system, and perturb intracellular Ca2+ signaling, and that this signaling underlies anti-inflammatory and pain-modulating activities of Echinacea.</p

Synthesis and Natural Distribution of Anti-inflammatory Alkamides from Echinacea

The synthesis of the alkamides 2Z,4E-undeca-2,4-dien-8,10-diynoic acid isobutyl amide (1) and 2Z,4E-undeca-2,4-dien-8,10-diynoic acid isobutyl amide (5) was accomplished by organometallic coupling followed by introduction of the doubly unsaturated amide moiety. The distribution of these two amides in accessions of the nine species of Echinacea was determined