Lankun Wu

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

ETHNOPHARMACOLOGICAL RELEVANCE The genus Echinacea is a popular herbal immunomodulator. Recent reports indicate that Echinacea products inhibit nitric oxide (NO) production in activated macrophages. AIM OF THE STUDY 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. MATERIALS AND METHODS Alcohol extracts of three medicinal Echinacea species, Echinacea angustifolia, Echinacea pallida and Echinacea purpurea, were prepared using Soxhlet apparatus and fractionated using HPLC. NO production by LPS activated RAW 264.7 macrophage cells was measured using a Griess reagent and iNOS detected using immunoblotting. In addition, effects on arginase activity were measured in RAW 264.7 cells stimulated with 8-bromo-cAMP +/- LPS. RESULTS Alcohol extracts of all three Echinacea species significantly inhibited NO production by lipopolysaccharide (LPS)-activated the RAW 264.7 macrophage cell line; among them Echinacea 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 Echinacea 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. CONCLUSIONS 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.

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. Concomitantly, 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.

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 immune-modulating bioactivities. Our prior studies indicate that alkylamides may play an important role in the inhibition of prostaglandin E2 (PGE(2)) 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 lipopolysaccharide-induced PGE(2) 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 PGE(2) 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 PGE(2) inhibition. Chemically synthesized Bauer ketones 21 and 23 at 1 microM each significantly inhibited both PGE(2) 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 PGE(2) production to 83% of control, and synthetic Bauer alkylamide 11 significantly inhibited PGE(2) 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.

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.

The Synthesis and Natural Distribution of the Major Ketone Constituents in Echinacea pallida

The first synthesis of a series of ketones naturally occurring in E. pallida is described. The natural distribution of these ketones among different Echinacea species is also reported.

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-1beta, IL-2, IL-10, and TNF-alpha 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, and -80 degrees C) for 21h before testing. At 1-month post-extraction, tinctures stored at -20 degrees 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-1beta, IL-10, and TNF-alpha 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-1beta, IL-10, and TNF-alpha production after 1month of storage at -20 degrees C. CONCLUSIONS These results suggest E. tennesseensis harbors phytomedicinal properties that vary by plant organ, with roots demonstrating the strongest activities.

Hypericum Gentianoides Produces Bioactive Compounds in Schizogenously Formed Glands

The genus Hypericum is home to a number of plant species that have promising medicinal properties. Hypericum gentianoides is a North American species formerly utilized medicinally by Cherokee Native Americans [1]. Several unusual acylphloroglucinols that may induce immune responses in mammalian macrophages have recently been discovered in this species [2]. Here, we report several bioactivities of H. gentianoides extracts on mammalian cells and describe the ontology of secretory glands accumulating these phytochemicals in planta. Hypericum gentianoides extracts alter calcium homeostasis in HEK293 cells and rat astrocytes. In addition extracts from H. gentianoides inhibit the infectivity of human immunodeficiency virus (HIV) on HeLa37 cells in infectivity assays.

Echinacea-induced cytosolic Ca2+ elevation in HEK293

BackgroundWith a traditional medical use for treatment of various ailments, herbal preparations of Echinacea are now popularly used to improve immune responses. One likely mode of action is that alkamides from Echinacea bind to cannabinoid type 2 (CB2) receptors and induce a transient increase in intracellular Ca2+. Here, we show that unidentified compounds from Echinacea purpurea induce cytosolic Ca2+ elevation in non-immune-related cells, which lack CB2 receptors and that the Ca2+ elevation is not influenced by alkamides.MethodsA non-immune human cell line, HEK293, was chosen to evaluate E. purpurea root extracts and constituents as potential regulators of intracellular Ca2+ levels. Changes in cytosolic Ca2+ levels were monitored and visualized by intracellular calcium imaging. U73122, a phospholipase C inhibitor, and 2-aminoethoxydiphenyl borate (2-APB), an antagonist of inositol-1,4,5-trisphosphate (IP3) receptor, were tested to determine the mechanism of this Ca2+ signaling pathway. E. purpurea root ethanol extracts were fractionated by preparative HPLC, screened for bioactivity on HEK293 cells and by GC-MS for potential constituent(s) responsible for this bioactivity.ResultsA rapid transient increase in cytosolic Ca2+ levels occurs when E. purpurea extracts are applied to HEK293 cells. These stimulatory effects are phospholipase C and IP3 receptor dependent. Echinacea-evoked responses could not be blocked by SR 144528, a specific CB2 receptor antagonist, indicating that CB2 is not involved. Ca2+ elevation is sustained after the Echinacea-induced Ca2+ release from intracellular Ca2+ stores; this longer-term effect is abolished by 2-APB, indicating a possible store operated calcium entry involvement. Of 28 HPLC fractions from E. purpurea root extracts, six induce cytosolic Ca2+ increase. Interestingly, GC-MS analysis of these fractions, as well as treatment of HEK293 cells with known individual and combined chemicals, indicates the components thought to be responsible for the major immunomodulatory bioactivity of Echinacea do not explain the observed Ca2+ response. Rather, lipophilic constituents of unknown structures are associated with this bioactivity.ConclusionsOur data indicate that as yet unidentified constituents from Echinacea stimulate an IP3 receptor and phospholipase C mediation of cytosolic Ca2+ levels in non-immune mammalian cells. This pathway is distinct from that induced in immune associated cells via the CB2 receptor.