The Role of Alkamides as an Active Principle of Echinacea

Table of Contents

Abstract

Alkamides are the major lipophilic constituents of Echinacea preparations, which are widely used in some European countries and in North America for common colds. In earlier investigations they have been shown to possess stimulatory effects on phagocytosis. Recent experiments have demonstrated that alkamides are detectable in human blood in relevant concentrations after oral administration of Echinacea preparations. Alkamides show structural similarity with anandamide, an endogenous ligand of cannabinoid receptors. Consequently, it was found that alkamides bind significantly to CB₂ receptors, which is now considered as a possible molecular mode of action of Echinacea alkamides as immunomodulatory agents. It was also demonstrated recently in several studies that alkamide-containing Echinacea preparations trigger effects on the pro-inflammatory cytokines. They were therefore suggested as a new class of cannabinomimetics. However, the therapeutic relevance of these findings is still not clear as clinical studies on the common cold show contradictory results. Among the many pharmacological properties reported, investigations concerning herb-drug interactions have been neglected for a long time. Latest research concludes that prolonged use of Echinacea poses a minimal risk for co-medications metabolized by the P450 enzymes.

Key words

Alkamides - Echinacea purpurea - Echinacea angustifolia - Asteraceae - immunomodulation - pharmacokinetics - cannabinoid receptor binding - herb-drug interactions

Introduction

Echinacea preparations represent the most common herbal immunomodulators. They are marketed mainly for the treatment and prevention of the common cold and other upper respiratory tract infections (URTIs). An analysis reveals that completely different preparations are sold under the name Echinacea using different plant parts and different extraction solvents. The most abundantly used species are Echinacea angustifolia, Echinacea purpurea and Echinacea pallida [1]. Several reviews on the effectiveness of orally ingested Echinacea extracts in reducing the incidence, severity, or duration of acute URTIs have been published. The majority of trials investigated whether Echinacea preparations shorten the duration or decrease the severity of symptoms of the common cold (for reviews see [2], [3], [4]). A recent Cochrane review [3] concluded that especially preparations based on the aerial parts of E. purpurea might be effective for the early treatment of colds in adults but results are not fully consistent. Beneficial effects of other Echinacea preparations for preventative purposes might exist but have not been shown in independently replicated rigorously randomized trials.

So far, polysaccharides, glycoproteins, caffeic acid derivatives and alkamides have been considered as the constituents most relevant for activity [5]. Alkamides produce a strong tingling effect in the mouth, which has been considered as a marker for high quality Echinacea by the American Indians [6], [7], [8], [9]. In recent years, research has focused on these lipophilic constituents of Echinacea because of their bioavailability. This review summarizes the evidence for the contribution of alkamides to the immunomodulatory effect of Echinacea and their possible role as active principles in the use of Echinacea for common cold.

Distribution of Alkamides in Echinacea

Alkamides are the major lipophilic constituents and can be found in high concentrations in the roots of E. purpurea and E. angustifolia and with decreasing concentrations in the aerial parts of E. purpurea, E. pallida and E. angustifolia. They vary between species and between different parts of the plant [10], [11]. A distribution study indicated that the root bark and secondary roots of Echinacea angustifolia contained the highest concentrations, whereas the stems and leaves of E. angustifolia were devoid of alkamides [12]. High quality E. purpurea root material contains up to 6 mg/g alkamides [13]. The main alkamides are the isomeric dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (8a/8b) (Fig. [1]). Similar N-isobutylamides are also present in Spilanthes spp. and Xanthoxylum species [6], [7].

Fig. 1 Structures of the main Echinacea alkamides.

Pharmacology with Molecular Biological Aspects

A series of previous pharmacological experiments have shown that Echinacea extracts containing alkamides have significant anti-inflammatory and immunomodulatory properties. Among the many pharmacological effects reported, modulation of macrophages and PMN immune cells and effects on cytokine/chemokine expression in human cells have been demonstrated most convincingly [2], [4], [14], [15], [16], [17], [18], [19].

The lipophilic part of an ethanolic extract obtained from the roots of E. purpurea at a concentration of 10^-3 % and from the roots of E. angustifolia (10^-4 %), which contained the full spectrum of alkamides, stimulated phagocytosis of yeast particles by human polymorphonuclear neutrophils (PMN) in vitro by 37 % and 34 %, respectively. In mice, per os administration of lipophilic alkamide fractions from the roots of E. angustifolia and E. purpurea enhanced phagocytosis in the carbon-clearance assay by factors of 1.5 and 1.7, respectively [20], [21]. Other in vivo studies in rats have shown that administration of hydroalcoholic extracts (100 μL twice daily by oral gavage for 4 days) of E. purpurea roots and aerial parts containing defined concentrations of cichoric acid, polysaccharides and alkamides stimulated the phagocytic activity of macrophages dose-dependently [22]. An increase in lipopolysaccharide-stimulated nitric oxide release was observed in macrophages obtained from the spleens of rats previously treated with the standardized Echinacea purpurea extracts. A similar set of experiments demonstrated stimulation of alveolar macrophage function by alkamides administered to healthy rats [23]. In murine RAW264.7 macrophages, alkamides exerted an inhibitory effect on LPS-mediated activation, and a significant reduction in NO production was observed in comparison to cells treated with LPS alone. These data suggest that alkamides may have not only immunostimulatory but also anti-inflammatory activity [24]. An ”Echinacea Premium Liquid alkamide fraction”, which did not contain caffeic acid derivatives but only alkamides with undeca-2E-ene-8,10-diynoic acid isobutylamide as the major 2-ene alkamide and dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide as the major 2,4-diene alkamide, was found to significantly inhibit TNF-α production under LPS-stimulated conditions in the mouse macrophage cell line RAW 264. In these macrophages, only the alkamide mixture isolated from an ethanolic Echinacea extract (”Echinacea Premium Liquid”) of Echinacea purpurea (300 mg/mL) and Echinacea angustifolia roots (200 mg/mL) significantly decreased LPS-stimulated NO production at a concentration of 2.0 μg/mL. However, the individual alkamides, undeca-2E-ene-8,10-diynoic acid isobutylamide and dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide as found in the isolated alkamide mixture did not reduce the LPS-induced NO production in the same assay [25].

Clifford has reported in vitro inhibition of cyclooxygenase-1 (COX-1) and, to a lesser extent, of cyclooxygenase-2 (COX-2) by alkamides isolated from E. purpurea roots [26]. In vitro inhibition of 5-lipoxygenase and cyclooxygenase (from sheep seminal microsomes) has also been reported for polyunsaturated alkamides from E. angustifolia roots and Achillea species [27]. COX-1 inhibitory activity was highest for undeca-2Z,4E-diene-8,10-diynoic acid isobutylamide (2b), dodeca-2E,4Z-diene-8,10-diynoic acid 2-methylbutylamide (6) and undeca-2E,4Z-diene-8,10-diynoic acid 2-methylbutylamide (15), with inhibitions of 60, 48 and 55 %, respectively, at concentrations of 100 μg/mL. Undeca-2Z,4E-diene-8,10-diynoic acid isobutylamide (2b) possessed with 46 % the strongest COX-2 inhibitory activity. Compounds 6 and 15 showed 31 and 39 % inhibition of COX-2, respectively [26]. Inhibition of 5-lipoxygenase has also been described for extracts of roots of E. purpurea, E. pallida and E. angustifolia with IC₅₀ values of 0.642, 1.08 and 0.444 μg root/mL, respectively. The alkamide concentrations in the root of each species were 0.05 %, traces and 0.2 %, respectively [28]. Inhibition of cyclooxygenase is known as an effective strategy to suppress pain and inflammation. Alkamides isolated from the roots of E. angustifolia inhibited COX-2-dependent prostaglandin E₂ formation, but did not inhibit COX-2 expression at the transcriptional or translational level. An analysis of 8 different alkamides revealed a contribution of undeca-2Z-ene-8,10-diynoic acid isobutylamide (3b), dodeca-2E-ene-8,10-diynoic acid isobutylamide (5) and dodeca-2E,4Z-diene-8,10-diynoic acid 2-methylbutylamide (6) to this response [29]. Recently, Gertsch et al. [14] demonstrated the modulation of TNF-α gene expression and multiple signal transduction pathways by Echinacea alkamides and postulated a mechanism related to cannabinoid receptors. Parallel in vitro kinetic experiments measuring both mRNA and protein levels over a time-span of 39 h after a co-incubation with LPS and E. purpurea tincture (Echinaforce^TM, endotoxin < 0.5 EU/mL) have been performed. LPS-stimulated TNF-α protein expression was potently modulated by the Echinacea purpurea preparation, resulting in a significant inhibition (˜40 %) during the first 20 h, and subsequent stimulation of TNF-α protein expression (Fig. [2]).

To ascertain whether CB₂ was the receptor subtype involved in the observed effects, a CB₂ antagonist was used in combination with the dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides. The specific antagonist strongly abolished TNF-α transcription and thus indicated a strict peripheral cannabinoid-mediated process. In parallel, receptor binding studies to rodent CB₁ and CB₂ receptors were conducted by Woelkart et al. [30]. Most of the Echinacea alkamides (1, 2a, 2b, 6, 7, 8a, 8b, 9, 10, 11, 12) showed affinities to CB₂ receptors with Ki values lower than 20 μM, some only five times less active than anandamide, the endogenous ligand [30] (Fig. [3] and [4]). The most recent evidence of CB-receptor-binding has been demonstrated by Raduner et al. [16]. At concentrations below 100 nM, dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (8a) and dodeca-2E,4E-dienoic acid isobutylamide (12) potently displaced the radioligand from membrane recombinantly overexpressing CB₂ receptors with Ki values of 57 ± 14 nM and 60 ± 13 nM, respectively. In addition the interaction of alkamides with CB₂-receptors was explored in a homology model. In this binding arrangement, the importance of the aromatic ring of Tyr190 of the CB-receptor was highlighted [16] (Fig. [5], for a color version see Fig. 1S in the Supporting Information). Effects on CB₂ receptor-containing immune cells in humans were evaluated in a randomized, single-dose, crossover ex vivo study with LPS-stimulated blood cells after in vivo administration of two Echinacea purpurea preparations standardized on 0.07 mg dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (Echinaforce^TM tincture and tablets). Both forms of medication led to a significant decrease in production of pro-inflammatory cytokines (IL-8 and TNF-α), while changes in IL-6 concentration were not statistically significant [15]. IL-8 and TNF-α are pro-inflammatory immunomodulators. Therefore the effect of the Echinacea preparations can be considered as an anti-inflammatory action and corresponds with the observed in vitro effects of alkamides. This can explain why the symptoms of a common cold, like sore throat can be reduced. Also the direct effects of alkamides on T-lymphocytes, which are key mediators of antiviral immunity, have been investigated by testing inhibition of IL-2 production. The Echinacea purpurea extract (95 : 5 ethanol : water) and two Echinacea-derived alkamides, dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide and dodeca-2E,4E-dienoic acid isobutylamide at concentrations of 0.6 to 25 μg/mL significantly suppressed the ability of activated Jurkat T cells to produce IL-2, an important factor involved in response to infection, which consequently leads to a faster resolution of cold symptoms [31].

An ethanolic and an ethyl acetate fraction of alkamides obtained from the roots of Echinacea angustifolia exhibited significant activity against three viruses often involved in colds and influenza (herpes simplex virus, influenza virus, and rhinovirus). This activity correlated with the presence of alkamides [32]. In a previous paper, it was reported that Echinilin^TM (Factors R & D Technologies; Burnaby, Canada), a formulation prepared from freshly harvested E. purpurea plants and standardized on the basis of three known active components (alkamides, cichoric acid and polysaccharides) is effective for the treatment of a naturally acquired common cold [33]. However, the mechanism and the responsible constituents by which this effect was achieved remained unknown. In a recent clinical study, the effects of Echinacea were associated with a significant and sustained increase in the number of circulating total white blood cells, monocytes, neutrophils and NK cells. Furthermore, Echinacea treatment suppressed the cold-related increase in superoxide production by neutrophils [34], [35]. These data suggest that alkamides have the dual actions of anti-inflammatory and indirect antiviral effects, which together may influence the course of upper respiratory infection. In summary, a lot of recent pharmacological data suggest that Echinacea alkamides may not only have immunostimulatory but also anti-inflammatory and antiviral activity by a reduction in NO, TNF-α, IL-8, IL-2 and COX-dependent E₂ formation in different cell types and assays used.

Fig. 2 Kinetic study showing TNF-expression in primary human monocytes/macrophages-enriched PBMCs (3 × 10⁶ cells) from peripheral blood as protein levels, over a time course of 39 h. Echinaforce^TM (25 μg/mL) and LPS (1 μg/mL) were both tested alone and in combination. Echinaforce^TM was incubated for 1 h before addition of LPS. The protein concentrations were determined by ELISA. Data points were obtained every 3 h and are mean values ± S.E. from three independent experiments. Reprinted with permission by the publisher Elsevier, from reference Gertsch J, et al. FEBS Lett 2004; 577 : 563 - 9 [14].

Fig. 3 Selectivity of alkamides from E. angustifolia for the CB₁ receptor from rat membranes with and without PMSF, obtained by a standard receptor binding assay using a [³H]CP-55,940 as the radioligand and reported as mean Ki values [μM] with corresponding 95 % confidence intervals determined from at least three independent experiments (from Woelkart K, et al. [30]).

Fig. 4 Selectivity of alkamides from E. angustifolia for the CB₂ receptor from mouse membranes, obtained by a standard receptor binding assay using a [³H]CP-55,940 as the radioligand and reported as mean Ki values [μM] with corresponding 95 % confidence intervals determined from at least three independent experiments. (form Woelkart K, et al. [30]).

Fig. 5 Proposed binding conformation of dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (8a) and dodeca-2E,4E-dienoic acid isobutylamide (12) in the CB₂-receptor. A The putative binding site for CB₂ ligands is located adjacent to helices III, V, VI and VII at the near extracellular side of the 7TM bundles. B The putative interaction of alkamides with the CB₂-receptor is shown. The oxygen atom in the hydroxy group of Tyr190 forms a hydrogen bond with the amide hydrogen of the alkamide, and the aromatic ring of Tyr190 exhibits Π-Π interactions with the C-2/C-3 double bond in the alkamide. Reprinted with permission by publisher ASBMB, J Biol Chem from reference Raduner S et al. [16].

Biopharmaceutics and Herb-Drug Interactions

In 2001, Dietz et al. demonstrated for the first time the bioavailability of dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (8a/8b) in humans after oral administration of an ethanolic extract of E. purpurea [36]. Later Jager et al. could show the transport of dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides across Caco-2 monolayers, in an in vitro model for the intestinal epithelial barrier [37]. A similar study explored the transport of 12 alkamides from a proprietary preparation of Echinacea, which contained a 60 % ethanol-water extract of E. angustifolia root (200 mg/mL) and E. purpurea root (300 mg/mL). Both the 2,4-diene and the 2-ene alkamides readily penetrated the monolayers, although apparent permeability coefficient values varied (range 3 × 10^-6 to 3 × 10^-4 cm · s^-1) depending on the structure. Saturated compounds and those with N-terminal methylation had lower permeability coefficients [38]. More recently, a pharmacokinetic study in humans showed that the absorption maximum (C_max) of dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (”tetraenes”) is reached already 30 min after oral administration of a 60 % ethanolic extract from the roots of E. angustifolia (Fig. [6]). Initial concentrations of tetraenes could be detected in the blood as soon as 10 minutes after administration. Because of this fast absorption, the mucous membrane of the mouth is most likely already a major area of absorption [39]. In a subsequent study the absorption from tablets manufactured from ethanolic liquid extracts of E. angustifolia and E. purpurea which were taken immediately after a standard high fat breakfast was compared. Most alkamides were rapidly absorbed and were detected in plasma 20 min after tablet ingestion and remained detectable for up to 12 h. In contrast, caffeic acid derivatives could not be detected in any plasma sample at any time after tablet ingestion and therefore were reported not to be bioavailable. No obvious differences were observed in the pharmacokinetics of individual or total alkamides in two additional fasted subjects who took the same dose of the Echinacea preparation [40].

Based on the experience from these studies a cross-over study was performed to compare the pharmacokinetics and bioavailability of different formulations of an ethanolic extract from fresh E. purpurea prepared from 95 % aerial parts and 5 % roots (Echinaforce^TM tincture and tablets). With this study design two pharmacokinetic curves for each galenic form in every subject have been obtained in order to exclude variability from the individual metabolic systems. The mean C_max of dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (8a/8b) absorbed after oral administration of the tincture occurred at 30 min with a value of 0.40 ng/mL serum. In comparison the T_max of tablets was 45 min with a C_max of 0.12 ng/mL. (Fig. [7]) [15]. In all pharmacokinetic studies performed up-to-date, the time to reach the mean concentration maximum after administration of liquid Echinacea preparations (E. angustifolia or E. purpurea) was about 30 minutes (Table [1]). T_max after administration of Echinacea tablets, which has been investigated in two clinical studies, was varying within 45 minutes to 2.3 hours [40], [15]. As it has also been shown for Echinacea tinctures, the more concentrated preparations needed longer to attain T_max. T_max for the dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (8a/8b) was obtained 2.3 hours after administration of 17.16 mg and 45 minutes after administration of 0.07 mg.

One reason could be the reported micelle formation of the alkamides [16]. The solubility of the lipophilic compounds is limited in aqueous solutions as detected by a Tyndall effect at concentrations above 10 μM and even low alkamide concentrations (∼300 nM) resulted in the formation of detectable particles in buffer [16]. Although there are no investigations available concerning micellar structures of alkamides in blood, only crude estimates of (apparent) bioavailability can be calculated from the C_max for the dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (8a/8b) from all performed pharmacokinetic studies. In addition, all performed pharmacokinetic studies [15], [39], [40], [41] have been single-dose studies and provide evidence that alkamides are bioavailable and that their pharmacokinetics are in agreement with the one dose three times daily regimen already recommended for Echinacea. To accurately determine the inter-dosing interval, a study with a multiple-dose protocol would be needed to determine the steady-state plasma concentration for each of the active compounds.

Besides bioavailability, information concerning the metabolism and plant-drug interactions is an important issue in the evaluation of in vivo activity of alkamides. An interaction can be particularly important when the total drug absorption is altered. Most drug interaction studies which investigated the metabolism by human liver microsomes in vitro have been carried out with pure alkamides so far [42]. Time- and NADPH-dependent degradation of alkamides was observed in microsomal fractions suggesting that they are metabolized by cytochrome P450 enzymes in human liver. There was a difference in the susceptibility of 2-ene and 2,4-diene pure synthetic alkamides to microsomal degradation. Obviously less degradation of the dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides was evident in the mixture of alkamides present in an ethanolic Echinacea extract, suggesting that metabolism by liver P450 s was dependent both on their chemistry and the combination present in the incubation mixture. Matthias et al. [43] suggested that Echinacea may affect the P450-mediated metabolism of other concurrently ingested pharmaceuticals. However, in vivo assessments of Echinacea preparations are essential to predict possible CYP-mediated interactions. Twelve young adults participated in an open-label, randomized study at the University of Arkansas. Each participant took 800 mg E. purpurea (plant part and type of extract not defined) twice daily for 28 days and was followed by a 30-day washout period. Only cichoric acid was used as phytochemical marker compound. No significant effect on CYP activity in humans was observed [44]. Therefore, as in previous reports, Echinacea purpurea appeared to have a minor influence on CYP3A4 in vivo. On the other hand, Gorski et al. [45] demonstrated in an in vivo study with twelve healthy subjects the reduction of the oral clearance of substrates of CYP1A2 but not the oral clearance of substrates of CYP2C9 and CYP2D6 after taking 400 mg E. purpurea root extract four times a day for 8 days. Echinacea selectively modulates the catalytic activity of CYP3A at hepatic and intestinal sites. Therefore, care should be taken when Echinacea is coadministered with drugs dependent on CYP3A or CYP1A2 for their elimination [45]. More recently Yale et al. [46] investigated the inhibition of three of the most important drug metabolizing enzymes, cytochrome P450 3A4, 2D6, and 2C9 using high throughput CYP inhibition screening in vitro assays. Depending on the model substrate, a 70 % methanol extract of E. purpurea aerial parts exhibited mild inhibition of CYP3A4 activity or even mild inducing effects. Little effect on CYP2D6 and moderate inhibition of CYP2C9 were also observed for the E. purpurea preparation. The phytochemical content of the herbal preparation was not reanalyzed before analyses [46]. The few in vivo studies performed up to now, used preparations which were not standardized or fully characterized. Therefore no relevant statements can be made whether critical inhibitory levels of alkamides can be reached in the liver under normal therapeutic use. In future the level of achievable serum concentrations of Echinacea constituents should be considered in in vitro studies.

Fig. 6 Plasma concentration-time curve of tetraene (8a/8b) after a single oral dose of 2.5 mL 60 % EtOH extract of E. angustifolia roots. Each point represents the mean ± standard deviation of eleven volunteers. Reprinted with permission by publisher Sage Publications, Inc., J Clin Pharmacol from reference Woelkart K et al. [39].

Fig. 7 Serum concentration-time curves of tetraene (8a/8b) after a single oral dose of 4 mL Echinaforce^TM tincture and 12 Echinaforce^TM tablets. Each point represents the mean ± standard deviation (SD) of the eight volunteers. Reprinted with permission by publisher Dustri-Verlag Dr. Karl Feistle GmbH & Co. KG, Int J Clin Pharm Th from reference Woelkart K et al. [15].

Table 1 Summary of all pharmacokinetic studies of Echinacea preparations performed up to now

Reference	Plant species and preparation	Dose	Absorption	Detection
[41]	E. purpurea mother tincture, Weber & Weber for the alkamides.	10, 20, 30, 40 and 50 mL mother tincture with 0.25, 0.49, 0.74, 0.99 and 1.24 mg dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (tetraene) (8a/8b) (24,7 μg/mL)	Mean Cmax of 14.0, 21.6, 32.2, 45.2 and 37.7 ng/mL, respectively with a Tmax of 30, 30, 30, 27.5 and 50 minutes.	High performance liquid chromatography with PDA detection at 254 nm for the alkamides and 330 nm for cichoric acid.
	E. purpurea pressed juice, Schoenenberger for cichoric acid.	10, 30, 60, 90 and 120 mL of the pressed juice with 40, 120, 240, 360 and 480 mg cichoric acid (4 mg/mL)	Mean Cmax of 0, 31.5, 43.3, 82.6, 129.4 ng/mL, respectively with a Tmax of -, 2.0, 3.0, 3.6, 1.6 hours.
[36]	E. purpurea; mother tincture	65 mL concentrated mother tincture with 4.3 mg dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (tetraene) (8a/8b) (66.2 μg/mL)	44 ng/mL tetraene (8a/8b) one hour after application	High performance liquid chromatography with PDA detection (260 nm)
[39]	E. angustifolia; 60 % ethanolic extract from the roots	2.5 mL of the ethanolic extract with 2.0 mg tetraene (8a/8b) (0.8 mg/mL)	The mean Cmax reached after 30.3 (Tmax) minutes was 10.9 ng/mL with an area under the curve of 1 029.1 ng equivalent min/mL.	Liquid chromatography, electrospray ionization ion-trap mass spectrometry with MS/MS detection in positive SRM (selected reaction monitoring) mode.
[40]	Echinacea Premium tablets, MediHerb, Australia, each containing extract equivalent to 675 mg of E. purpurea root plus 600 mg of E. angustifolia root prepared from the dried ethanolic extracts of the two Echinacea species.	Four Echinacea Premium tablets with a total of 43.68 mg alkamides, especially a total of 17.16 mg dodeca-2E,4E,8Z, 10Z-tetraenoic acid isobutylamide (8a)	Cmax for total alkamides was 336 ng/mL, Tmax was 2.3 hours and the AUC was 714 μg equivalent h/L. Especially for (8a) Cmax was 221 ng/mL with a Tmax of 1.9 h and a AUC of 476 μg eq h/L.	Liquid chromatography-mass spectrometry equipped with an APCI interface operating in positive ion SIM mode.
[15]	An extract of fresh organically grown E. purpurea, 95 % herb and 5 % roots with 65 Vol. % alcohol. (EchinaforceTM, Bioforce AG, Switzerland)	4 mL EchinaforceTM tincture (0.018 mg/mL dodeca-2E,4E,8Z, 10E/Z-tetraenoic acid isobutylamides 8a/8b) or 12 EchinaforceTM tablets (0.006 mg/tablet 8a/8b), which both contained 0.07 mg of 8a/8b (tetraene).	The mean maximum concentration (Cmax) after administration of the EchinaforceTM tincture and EchinaforceTM tablets was reached after 30 minutes with 0.40 ng/mL and after 45 minutes with 0.12 ng/mL, respectively.	Liquid chromatography, electrospray ionization ion-trap mass spectrometry with MS/MS detection in positive SRM (selected reaction monitoring) mode.

Summary and Conclusion

It has been demonstrated in many animal, in vitro and ex vivo studies that an alkamide-enriched Echinacea fraction or isolated alkamides have significant anti-inflammatory and immunomodulatory properties. Modulation of macrophages, reduction of NO and TNF-α, and inhibition of the arachidonic acid metabolism could be directly related to alkamides. Dodeca-2E,4Z-diene-8,10-diynoic acid 2-methylbutylamide showed so far the best inhibition of COX-2-dependent prostaglandin E₂ formation. Although these animal and in vitro studies lend mechanistic credibility, the bioavailability and ex vivo studies give further information on therapeutic benefits in humans. Recent experiments have demonstrated that alkamides are detectable in human blood after oral ingestion of different Echinacea preparations. Effects on pro-inflammatory cytokines after LPS-stimulation could be shown in an ex vivo study. A single application of Echinacea led to a significant decrease in the production of IL-8 and TNF-α. There are also data showing that alkamides from Echinacea are a new class of CB₂-specific cannabinomimetics, which share the anti-inflammatory properties of anandamide and the cannabinoids from Cannabis sativa. However, as it has been shown in some studies that the anti-inflammatory effects exerted by cannabinomimetics are not strictly CB₂-dependent, this therefore raises the question about a possible common second target.

Furthermore, Echinacea-derived alkamides significantly suppressed T-lymphocytes, which are key mediators of antiviral immunity and exhibited activity against herpes simplex virus, influenza virus and rhinovirus, which are often implicated in colds and influenza. In a clinical study a standardized E. purpurea preparation was effective for the treatment of a naturally acquired common cold [33], while an alkamide-enriched E. angustifolia root extract failed in a recent experimental rhinovirus inoculation study [47]. It has be argued whether the dose of 300 mg of Echinacea root, given three times each day, containing 1.5 mg or less dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides was too low to obtain a significant effect on symptoms associated with rhinovirus infection [48]. Therefore, further clinical studies are needed to evaluate the therapeutic role of alkamides. Also the influence of alkamides on cytochrome P450 enzymes needs further in vivo evaluation. Most of the in vivo studies performed so far used phytochemically insufficiently characterized Echinacea preparations. Thus, with the present knowledge it is not possible to make a final statement on the therapeutic role of alkamides, but it is quite likely that they are relevant constituents of Echinacea.

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