A. Matthias

Permeability studies of alkylamides and caffeic acid conjugates from echinacea using a Caco-2 cell monolayer model

Background:  Echinacea is composed of three major groups of compounds that are thought to be responsible for stimulation of the immune system – the caffeic acid conjugates, alkylamides and polysaccharides. This study has focussed on the former two classes, as these are the constituents found in ethanolic liquid extracts.

Alkylamides from echinacea modulate induced immune responses in macrophages.

The ability of Echinacea and its components to alter the immune response was examined in vitro in a macrophage cell line under either basal or immunostimulated conditions. Potential immunostimulatory and inflammatory activity was determined using a nuclear transcription factor (NFκB) expression, tumour necrosis factor α (TNFα) and nitric oxide (NO) production as biomarkers. In the absence of alternate stimulation, the only significant effects seen were a decrease in NFκB expression by a 2-ene alkylamide ((2E)-N-isobutylundeca-2-ene-8,10-diynamide (1)) and a decrease in TNFα levels by cichoric acid and an Echinacea alkylamide fraction (EPL AA). When the cells were stimulated by lipopolysaccharide (LPS), inhibition of the increased NFκB expression levels was caused by cichoric acid, an Echinacea preparation (EPL), EPL AA and a 2,4-diene ((2E,4E,8Z,10Z)-N-isobutyldodeca-2,4,8,10-tetraenamide (2)). Increases in TNFα levels were inhibited by cichoric acid, EPL and EPL AA but enhanced by 1 in the presence of LPS, while only EPL AA was able to inhibit the stimulated increases in NO. When using phorbol myristate acetate to stimulate the cells, NFκB and NO levels were unaffected by Echinacea or its components while only cichoric acid and 2 inhibited TNFα levels. Although cichoric acid was found to have an effect, it is probably not an important contributor to the Echinacea modulation of the immune response in vivo, as it is not bioavailable. Echinacea appears to attenuate the response of macrophages to an immune stimulus and its combination of phytochemicals exhibits different pharmacological properties to one or more of the isolated major individual components.

Cytotoxic activity of polyacetylenes and polyenes isolated from roots of Echinacea pallida

The n‐hexane extracts of the roots of three medicinally used Echinacea species exhibited cytotoxic activity on human cancer cell lines, with Echinacea pallida found to be the most cytotoxic. Acetylenes are present in E. pallida lipophilic extracts but essentially absent in extracts from the other two species. In the present study, the cytotoxic effects of five compounds, two polyacetylenes (namely, 8‐hydroxy‐pentadeca‐(9E)‐ene‐11,13‐diyn‐2‐one (1) and pentadeca‐(9E)‐ene‐11,13‐diyne‐2,8‐dione (3)) and three polyenes (namely, 8‐hydroxy‐pentadeca‐(9E,13Z)‐dien‐11‐yn‐2‐one (2), pentadeca‐(9E,13Z)‐dien‐11‐yne‐2,8‐dione (4) and pentadeca‐(8Z,13Z)‐dien‐11‐yn‐2‐one (5)), isolated from the n‐hexane extract of E. pallida roots by bioassay‐guided fractionation, were investigated and the potential bioavailability of these compounds in the extract was studied.

Investigations into the antibacterial activities of phytotherapeutics against Helicobacter pylori and Campylobacter jejuni.

The prevalence of gastric diseases is increasing with H. pylori, the causative agent of acute and chronic gastritis, being a major predisposing factor for peptic ulcer disease and gastric carcinoma. C. jejuni is the most common cause of enteric infections, particularly among children, resulting in severe diarrhoea. Increasing drug resistance of these bacteria against standard antibiotics, and the more widespread use of herbal medicines, favours investigations into additional anti‐Helicobacter and anti‐Campylobacter effects of phytotherapeutics that are already used for their beneficial effects on bowel and digestive functions.

Pharmacokinetic and pharmacodynamic interactions of echinacea and policosanol with warfarin in healthy subjects

AIMS This study investigated the pharmacokinetic and pharmacodynamic interactions of echinacea and policosanol with warfarin in healthy subjects. METHODS This was an open-label, randomized, three-treatment, cross-over, clinical trial in healthy male subjects (n= 12) of known CYP2C9 and VKORC1 genotype who received a single oral dose of warfarin alone or after 2 weeks of pre-treatment with each herbal medicine at recommended doses. Pharmacodynamic (INR, platelet activity) and pharmacokinetic (warfarin enantiomer concentrations) end points were evaluated. RESULTS The apparent clearance of (S)-warfarin (90% CI of ratio; 1.01, 1.18) was significantly higher during concomitant treatment with echinacea but this did not lead to a clinically significant change in INR (90% CI of AUC of INR; 0.91, 1.31). Policosanol did not significantly affect warfarin enantiomer pharmacokinetics or warfarin response. Neither echinacea nor policosanol had a significant effect on platelet aggregation after 2 weeks of pre-treatment with the respective herbal medicines. CONCLUSION Echinacea significantly reduced plasma concentrations of S-warfarin. However, neither echinacea nor policosanol significantly affected warfarin pharmacodynamics, platelet aggregation or baseline clotting status in healthy subjects.

Echinacea metabolism and drug interactions: The case for standardization of a complementary medicine

The herbal medicine, Echinacea, is used for treatment and prevention of upper respiratory tract infections. Among the phytochemicals found in Echinacea, the bioavailable alkylamides are thought to be the compounds responsible for its effects on the human immune system. Cytochrome P450 enzymes (P450s) appear to be the principal system responsible for the metabolism of Echinacea components and most of the main hepatic and some extrahepatic isoforms appear to be involved. Epoxide formation, N-dealkylation and hydroxylation are the main metabolic pathways mediated by P450s. Interactions with P450s determine the circulating concentrations and duration of action of these phytochemicals as well as any potential interactions with other chemicals. Most research to date has focused on the potential of Echinacea to interact with other drugs. Literature reports are equivocal and comparisons between studies are difficult as the phytochemical composition of the preparations examined is rarely assessed. Certain alkylamides containing a terminal acetylene appear to exert a time- and NADPH-dependent inhibition on the metabolism of other compounds. However as there are no industry standardization requirements, differences in the relative concentrations of individual alkylamides between preparations could alter the potential for interactions. A thorough phytochemical analysis of samples investigated is necessary in further studies so that sound conclusions can be drawn regarding the potential for inter-individual variation in pharmacokinetics and therapeutic effects and interactions with other chemicals. Moreover standardization of alkylamide content may allow the exploitation of beneficial interactions between alkylamide components to enhance the therapeutic effect of this widely used complementary medicine.

Modulation of macrophage immune responses by Echinacea

Echinacea preparations are widely used herbal medicines for the prevention and treatment of colds and minor infections. There is little evidence for the individual components in Echinacea that contribute to immune regulatory activity. Activity of an ethanolic Echinacea extract and several constituents, including cichoric acid, have been examined using three in vitro measures of macrophage immune function – NF-κB, TNF- α and nitric oxide (NO). In cultured macrophages, all components except the monoene alkylamide (AA1) decreased lipopolysaccharide (LPS) stimulated NF-κB levels. 0.2 µg/ml cichoric acid and 2.0µg/mL Echinacea Premium Liquid (EPL) and EPL alkylamide fraction (EPL AA) were found to significantly decrease TNF-α production under LPS stimulated conditions in macrophages. In macrophages, only the alkylamide mixture isolated from the ethanolic Echinacea extract decreased LPS stimulated NO production. In this study, the mixture of alkylamides in the Echinacea ethanolic liquid extract did not respond in the same manner in the assays as the individual alkylamides investigated. While cichoric acid has been shown to affect NF-κB, TNF-α and NO levels, it is unlikely to be relevant in the Echinacea alterations of the immune response in vivo due to its non- bioavailability – i.e. no demonstrated absorption across the intestinal barrier and no detectable levels in plasma. These results demonstrate that Echinacea is an effective modulator of macrophage immune responses in vitro.

Echinacea intake induces an immune response through altered expression of leucocyte hsp70, increased white cell counts and improved erythrocyte antioxidant defences

Objective:  To study the effect of Echinacea tablets on the expression of leucocyte heat shock protein 70 (hsp70), erythrocyte haemolysis, plasma antioxidant status, serum chemistry, haematological values and plasma alkylamide concentrations.

Bioavailability of Echinacea constituents: Caco-2 monolayers and pharmacokinetics of the alkylamides and caffeic acid conjugates.

Many studies have been done over the years to assess the effectiveness of Echinacea as an immunomodulator. We have assessed the potential bioavailability of alkylamides and caffeic acid conjugates using Caco-2 monolayers and compared it to their actual bioavailability in a Phase I clinical trial. The caffeic acid conjugates permeated poorly through the Caco-2 monolayers. Alkylamides were found to diffuse rapidly through Caco-2 monolayers. Differences in diffusion rates for each alkylamide correlated to structural variations, with saturation and N-terminal methylation contributing to decreases in diffusion rates. Alkylamide diffusion is not affected by the presence of other constituents and the results for a synthetic alkylamide were in line with those for alkylamides found in an ethanolic Echinacea preparation. We examined plasma from healthy volunteers for 12 hours after ingestion of Echinacea tablets manufactured from an ethanolic liquid extract. Caffeic acid conjugates could not be identified in any plasma sample at any time after tablet ingestion. Alkylamides were detected in plasma 20 minutes after tablet ingestion and for each alkylamide, pharmacokinetic profiles were devised. The data are consistent with the dosing regimen of one tablet three times daily and supports their usage as the primary markers for quality Echinacea preparations.

Stereoselective synthesis, natural occurrence and CB2 receptor binding affinities of alkylamides from herbal medicines such as Echinacea sp.

A divergent synthesis of (2E,4E,8E,10E)- and (2E,4E,8E,10Z)-N-isobutyldodeca-2,4,8,10-tetraenamides from pent-4-yn-1-ol allowed identification of the (2E,4E,8E,10Z)-isomer for the first time in Echinacea species. A short, stereoselective synthesis of the (2E,4E,8E,10Z)-isomer is also described which allowed further biological evaluation of this material, and the demonstration that this isomer does not occur in Spilanthes mauritiana as previously reported.