Echinacea Abstracts - 2

Extracts of Echinacea purpurea are among the most widely used herbal medicines throughout Europe and North America for the prevention or treatment of common cold, coughs, Bronchitis and other Upper Respiratory infections.

Popular preparations include expressed juice from the aerial parts of the plant (which contain PolySaccharides) and alcoholic tinctures from roots (containing Caffeic Acid derivatives and Alkylamides).

Since Immune modulation has been reported for similar extracts, Cytokine AntiBody arrays were used to investigate the changes in the ProInflammatory Cytokines and Chemokines.

Released from a cultured line of human Bronchial Epithelial Cells exposed to Rhinovirus 14 and two different chemically characterized Echinacea extracts.

Virus infection stimulated the release of at least 31 Cytokine-related molecules, including several important Chemokines known to attract inflammatory cells.

Most of these effects were reversed by simultaneous exposure to either of the two Echinacea extracts, although the patterns of response were different for the two extracts. These results could explain the AntiInflammatory properties of Echinacea extracts.

Furthermore, a number of these Cytokines were stimulated by the same Echinacea preparations in uninfected cells. These observations therefore provide support for the alleged beneficial uses of Echinacea extracts.

Copyright 2006 John Wiley & Sons, Ltd.

This paper reviews the chemistry, pharmacology and clinical properties of Echinacea species used medicinally.

The Echinacea species Echinacea angustifolia, Echinacea pallida and Echinacea purpurea have a long history of medicinal use for a variety of conditions, particularly infections, and today Echinacea products are among the best-selling herbal preparations in several developed countries.

Modern interest in Echinacea is focused on its ImmunoModulatory effects, particularly in the prevention and treatment of upper respiratory tract infections.

The chemistry of Echinacea species is well documented, and several groups of constituents, including Alkamides and Caffeic Acid derivatives, are considered important for activity.

There are, however, differences in the constituent profile of the three species.

Commercial Echinacea samples and marketed Echinacea products may contain one or more of the three species, and analysis of samples of raw material and products has shown that some do not meet recognized standards for pharmaceutical quality.

Evidence from preclinical studies supports some of the traditional and modern uses for Echinacea, particularly the reputed ImmunoStimulant (or ImmunoModulatory) properties.

Several, but not all, clinical trials of Echinacea preparations have reported effects superior to those of placebo in the prevention and treatment of upper respiratory tract infections.

However, evidence of efficacy is not definitive as studies have included different patient groups and tested various different preparations and dosage regimens of Echinacea.

On the basis of the available limited safety data, Echinacea appears to be well tolerated. However, further investigation and surveillance are required to establish the safety profiles of different Echinacea preparations.

Safety issues include the possibility of allergic reactions, the use of Echinacea by patients with autoimmune diseases and the potential for Echinacea preparations to interact with conventional medicines.

Alcohol tinctures prepared from aged Echinacea roots are typically taken for preventing or treating Upper Respiratory infections, as they are purported to stimulate immunity in this context.

The effects of long-term (> 1 year) dry storage on the capabilities of Echinacea spp. roots from mature individuals to modulate Cytokine production are unknown.

Using an older human adult model of influenza vaccination, we collected Peripheral Blood Mononuclear Cells from subjects 6 months post-vaccination.

And, stimulated them in vitro with the two Type A influenza Viruses contained in the trivalent 2004-2005 vaccine with a 50 % alcohol tincture prepared from the roots of one of seven Echinacea species: E. angustifolia, E. pallida, E. paradoxa, E. purpurea, E. sanguinea, E. simulata, and E. tennesseensis.

Before being processed into extracts, all roots had been stored under dry conditions for sixteen months.

Cells were cultured for 48 hours; following incubation, supernatants were collected and assayed for InterLeukin-2, InterLeukin-10, and Interferon-γ production, Cytokines important in the Immune response to Viral infection.

Four species ( E. angustifolia, E. purpurea, E. simulata, E. tennesseensis) augmented IL-10 production, diminished IL-2 production, and had no effect on IFN-γ production.

Echinacea pallida suppressed production of all Cytokines; E. paradoxa and E. sanguinea behaved similarly, although to a lesser extent.

The results from these in vitro bioactivity assays indicate that dried Echinacea roots stored for sixteen months maintain Cytokine-modulating capacities.

Our data support and extend previous research and indicate that tinctures from different Echinacea species have different patterns of Immune modulation.

Further, they indicate that certain species may be efficacious in the Immune Response to Viral infection.

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.

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 mitogen-stimulated Splenic Cells.

The three herbal extracts significantly increased Interferon-alpha production, but inhibited the release of Tumor Necrosis Factor-gamma and InterLeukin-1beta (IL-1beta).

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.

The effects of Echinacea and several of its phytochemical components on NFkappaB expression by Jurkat Cells (a human T-Cell line) were investigated in vitro.

In the absence of stimulation, Echinacea and its components exerted no significant effect on basal NFkappaB expression levels. In the presence of EndoToxin (LPS), NFkappaB expression was decreased.

However, this decrease was significantly reversed by treatment with Cichoric Acid, an Echinacea root extract (prepared from both Echinacea angustifolia and Echinacea purpurea) and the Alkylamide fraction derived from this combination.

For the phorbol myristate acetate stimulation of Jurkat Cells, effects on NFkappaB expression were mixed.

Depending on the concentration, Cichoric Acid and a 2,4-diene Alkylamide significantly induced NFkappaB levels, whereas a 2-ene Alkylamide caused a significant inhibition.

In contrast, both the Echinacea and the mixed Alkylamide fraction exerted no effect.

The Alkylamide results indicate that the two basic forms of these compounds present in Echinacea may have opposing effects.

These opposing effects demonstrate the importance of a knowledge, not only of the phytochemical make-up of a herbal preparation.

But, also of the actions of each component and the consequences of differing relative amounts in the preparation being investigated.

Echinacea purpurea (L.) Moench, a top selling botanical medicine, is currently of considerable interest due to ImmunoModulatory, Anti-Inflammatory, AntiViral and Cannabinoid Receptor 2 (CB2) binding activities of its Alkylamide constituents.

The purpose of these studies was to comprehensively profile the Alkylamide (Alkamide) content of E. purpurea root, and to compare yields of Alkylamide constituents resulting from various ethanolic extraction procedures commonly employed by the dietary supplements industry.

To accomplish this goal, a high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) method was validated for quantitative analysis of several E. purpurea Alkylamides.

Using this method, at least 15 Alkylamides were identified.

And it was shown that fresh and dry E. purpurea extracts prepared from equivalent amounts (dry weight) of roots, with exceptions, exhibited similar yield of specific Alkylamides.

However, the amount of total dissolved solids in the dry extract was higher (by 38%) than the fresh extract.

Two extracts prepared from dried roots at different ratios of root:solvent (1:5, w:v and 1:11, w:v) were similar in yield of total dissolved solids.

But, there were differences in quantities of specific Alkylamides extracted using these two root:solvent ratios.

In addition, the important bioactive DoDecatetraenoic Acid IsoButylamides are fully extracted from dry E. purpurea root in 2 days.

Suggesting that the manufacturing practice of macerating Echinacea extracts for weeks may be unnecessary for optimal Alkylamide extraction. Finally, the identification of a new Alkylamide has been proposed.

These results demonstrate the differences of the described extractions and utility of the analytical methods used to determine the wide-ranging individual Alkylamide content of commonly consumed Echinacea extracts.

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 mMacrophage 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.