High performance liquid chromatography determined alkamide levels in Australian-grown Echinacea spp.

Extracts of Echinacea spp. are widely used as therapeutic immunostimulants with such activity being attributed in part to the alkamide fractions of these plants. Using high performance liquid chromatography, the levels of 8 alkamides, including 2 tetraene alkamides (dodeca-2E, 4E, 8Z, 10E/Z-tetraenoic acid isobutylamide), were quantitatively determined in 2 Australian-grown Echinacea spp. Overall, the levels of alkamides in Australian-grown E. angustifolia were found to be comparable with levels obtained in this study and other studies for USA and European Echinacea spp. However, results obtained for one sample of E. angustifolia suggested that it may have been mislabelled and that it was most likely a sample of E. pallida. Levels of tetraene alkamides in Australian-grown E. purpurea were also similar to, if not higher, than levels which have been reported for the same species grown in Germany and the USA. Preliminary studies on the stability of alkamide compounds in E. angustifolia indicated that they are susceptible to degradation, with a 13% loss of alkamide level over 2 months. Overall, results indicate that there is considerable potential to develop Echinacea as a viable crop in Australia.

Bisresorcinol Derivatives from Grevillea glauca

Seven new and three known bisresorcinols, grevirobstol A(=5,5'-((6Z,9Z)-hexadeca-6,9-diene-1,16-diyl)bisresorcinol; 8), 5,5'-[(8Z)-hexadec-8-ene-1,16-diyl]bisresorcinol (9), and 2-methyl-5,5'-[8Z)-hexadec-8-ene-1,16-diyl] bisresorcinol (10) were isolated from the stems of Grevillea glauca. The new compounds were identified on the basis of spectroscopic data as (Z)-6,7-didehydroglaucone A (1), glaucones A and B (2 and 3, resp.), 2-(3-hydroxyisopentyl)bisnorstriatol (4), 2-(3-methylbut-2-en-1-yl)bisnorstriatol (5), 2'-methylgrebustol A (6), and glaucane (7).

Solvent-induced 7R-dioxygenase activity of soybean 15-lipoxygenase-1 in the formation of omega-3 DPA-derived resolvin analogs

The resolvin family contains important anti-inflammatory and pro-resolution compounds enzymatically derived in vivo from the polyunsaturated omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). More recently, docosapentaenoic acid (DPA) has emerged as another potentially important precursor in the biological production of resolvin compounds. In this work we have used medium engineering to develop a simple method for the controlled synthesis of two di-hydroxylated diastereomers of DPAn-3 catalyzed by soybean 15-lipoxygenase-1 (15-sLOX-1) in the presence of short chain n-alcohols, including methanol, ethanol and propan-1-ol. The complete structures of the two major products, 7S,17S-dihydroxydocosapenta-8Z,10E,13Z,15E,19Z-enoic acid (7S,17S-diHDPAn-3) and 7R,17S-dihydroxydocosapenta-8Z,10E,13Z,15E,19Z- enoic acid (7R,17S-diHDPAn-3), have been elucidated using spectroscopic analysis. The alcohol-dependent R-dioxygenase activity of soybean 15-lipoxygenase with mono-hydroperoxide intermediate substrates has also been demonstrated with other biologically relevant PUFAs, including DHA, EPA and ARA. The developed method has applications in the production of closely related isomers of naturally occurring resolvins and protectins, demonstrating the versatility of 15-sLOX-1 as a biocatalyst. © 2014 Elsevier B.V.

Self-assembling cannabinomimetics: supramolecular structures of N-alkyl amides

Certain fatty acid N-alkyl amides from the medicinal plant Echinacea activate cannabinoid type-2 (CB2) receptors. In this study we show that the CB2-binding Echinacea constituents dodeca-2E,4E-dienoic acid isobutylamide (1) and dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (2) form micelles in aqueous medium. In contrast, micelle formation is not observed for undeca-2E-ene-8,10-diynoic acid isobutylamide (3), which does not bind to CB2, or structurally related endogenous cannabinoids, such as arachidonoyl ethanolamine (anandamide). The critical micelle concentration (CMC) range of 1 and 2 was determined by fluorescence spectroscopy as 200-300 and 7400-10000 nM, respectively. The size of premicelle aggregates, micelles, and supermicelles was studied by dynamic light scattering. Microscopy images show that compound 1, but not 2, forms globular and rod-like supermicelles with radii of approximately 75 nm. The self-assembling N-alkyl amides partition between themselves and the CB2 receptor, and aggregation of N-alkyl amides thus determines their in vitro pharmacological effects. Molecular mechanics by Monte Carlo simulations of the aggregation process support the experimental data, suggesting that both 1 and 2 can readily aggregate into premicelles, but only 1 spontaneously assembles into larger aggregates. These findings have important implications for biological studies with this class of compounds.

Cytochrome P450 inhibitory action of Echinacea preparations differs widely and co-varies with alkylamide content

Echinacea preparations are one of the best selling herbal medicinal products with a well established therapeutic use in the prophylaxis of upper respiratory tract infections. Their consumption is increasing, but information about their ability to inhibit cytochrome P450 enzymes (CYP) is fragmentary. The picture is further complicated by a lack of phytochemical characterization of previously tested preparations. Due to its well characterized immunomodulatory activity, the standardized Swiss registered Echinacea purpurea (L.) Moench Echinaforce extract was selected for detailed study. With the single baculovirus-expressed CYP isoforms 1A2, 2C19, 2D9 and 3A4, inhibitory actions were measured by monitoring fluorescent metabolites derived from enzyme substrates (supersome assay). The Echinaforce extract induced mild inhibition of all these isoforms, with CYP 3A4 being the most, and CYP 2D6 the least sensitive enzyme. To assess whether CYP inhibition might be a general feature of Echinacea preparations, an additional nine commercially available preparations were screened using CYP 3A4. All tested preparations were able to inhibit CYP 3A4, but inhibitory potencies (expressed as median inhibitory concentration, IC50) varied by a factor of 150. The alkylamides are thought to be responsible for the immunomodulatory activity of Echinacea, and so the concentration of 2E,4E,8Z,10E/Z-tetranoic acid isobutylamide (1) and total alkylamide content were determined in all preparations, and the latter was found to be associated with their CYP 3A4 inhibitory potency. The chemically pure alkylamides dodeca-2E,4E,8Z,10E/Z-tetranoic acid isobutylamide (1) and dodeca-2E,4E-dieonoic acid isobutylamide (2) showed inhibitory activity on CYP 2C19, 2D6 and 3A4. However, unlike the Echinaforce extract, the alkylamides did not induce CYP 1A2 inhibition. Thus, other, as yet unidentified constituents also contribute to the overall weak inhibitory effects seen with Echinacea preparations in-vitro.

Alkylamides from Echinacea are a new class of cannabinomimetics. Cannabinoid type 2 receptor-dependent and -independent immunomodulatory effects

Alkylamides (alkamides) from Echinacea modulate tumor necrosis factor alpha mRNA expression in human monocytes/macrophages via the cannabinoid type 2 (CB2) receptor (Gertsch, J., Schoop, R., Kuenzle, U., and Suter, A. (2004) FEBS Lett. 577, 563-569). Here we show that the alkylamides dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (A1) and dodeca-2E,4E-dienoic acid isobutylamide (A2) bind to the CB2 receptor more strongly than the endogenous cannabinoids. The Ki values of A1 and A2 (CB2 approximately 60 nM; CB1 >1500 nM) were determined by displacement of the synthetic high affinity cannabinoid ligand [3H]CP-55,940. Molecular modeling suggests that alkylamides bind in the solvent-accessible cavity in CB2, directed by H-bonding and pi-pi interactions. In a screen with 49 other pharmacologically relevant receptors, it could be shown that A1 and A2 specifically bind to CB2 and CB1. A1 and A2 elevated total intracellular Ca2+ in CB2-positive but not in CB2-negative promyelocytic HL60 cells, an effect that was inhibited by the CB2 antagonist SR144528. At 50 nM, A1, A2, and the endogenous cannabinoid anandamide (CB2 Ki >200 nM) up-regulated constitutive interleukin (IL)-6 expression in human whole blood in a seemingly CB2-dependent manner. A1, A2, anandamide, the CB2 antagonist SR144528 (Ki <10 nM), and also the non-CB2-binding alkylamide undeca-2E-ene,8,10-diynoic acid isobutylamide all significantly inhibited lipopolysaccharide-induced tumor necrosis factor alpha, IL-1beta, and IL-12p70 expression (5-500 nM) in a CB2-independent manner. Alkylamides and anandamide also showed weak differential effects on anti-CD3-versus anti-CD28-stimulated cytokine expression in human whole blood. Overall, alkylamides, anandamide, and SR144528 potently inhibited lipopolysaccharide-induced inflammation in human whole blood and exerted modulatory effects on cytokine expression, but these effects are not exclusively related to CB2 binding.

Quantitative analysis of arachidonic acid, endocannabinoids, N-acylethanolamines and steroids in biological samples by LCMS/MS: Fit to purpose.

Arachidonic acid (5Z,8Z,11Z,14Z-eicosatetraenoic acid; C20:4) (arachidonate, AA) is a vital polyunsaturated omega-6 fatty acid (PUFA) without its presence the mammalian brain, muscles, and possibly other organs cannot develop or function [1] and [2]. AA fulfils numerous known and possibly yet unknown functions as integral part of mammalian phospholipid membranes and as free AA which also acts as a precursor of a variety of biologically active lipid mediators generally referred to as eicosanoids (e.g., prostaglandins, leukotrienes). A more recent class of eicosanoids is composed of the endogenous cannabinoids (endocannabinoids) 2-arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamide (anandamide, AEA), which act on cannabinoid CB1 and CB2 receptors but also modulate ion channels and nuclear receptors [3] and [4]. In recent years, the role of endocannabinoids as prominent anti-inflammatory and neuromodulatory eicosanoids has been shown by numerous studies [5].

Cytochrome P450 enzyme-mediated degradation of Echinacea alkylamides in human liver microsomes

Echinacea preparations are widely used herbal remedies for the prevention and treatment of colds. In this study we have investigated the metabolism by human liver microsomes of the alkylamide components from an Echinacea preparation as well as that of pure synthetic alkylamides. No significant degradation of alkylamides was evident in cytosolic fractions. Time and NADPH-dependent degradation of alkylamides was observed in microsomal fractions suggesting they are metabolised by cytochrome P450 (P450) enzymes in human liver. There was a difference in the susceptibility of 2-ene and 2,4-diene pure synthetic alkylamides to microsomal degradation with (2E)-N-isobutylundeca-2-ene-8,10-diynamide (1) metabolised to only a tenth the extent of (2E,4E,8Z,IOZ)-N-isobutyldodeca-2,4,8,10-tetracnamide (3) under identical incubation conditions. Markedly less degradation of 3 was evident in the mixture of alkylamides present in an ethanolic Echinacea extract, suggesting that metabolism by liver P450s was dependent both on their chemistry and the combination present in the incubation. Co-incubation of 1 with 3 at equimolar concentrations resulted in a significant decrease in the metabolism of 3 by liver microsomes. This inhibition by 1, which has a terminal alkyne moiety, was found to be time- and concentration-dependent, and due to a mechanism-based inactivation of the P450s. Alkylamide metabolites were detected and found to be the predicted epoxidation, hydroxylation and dealkylation products. These findings suggest that Echinacea may effect the P450-mediated metabolism of other concurrently ingested pharmaceuticals. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Effect of Melatonin and Analogues on Corneal Wound Healing: Involvement of Mt2 Melatonin Receptor

Purpose: We have investigated the effect of melatonin and its analogues on rabbit corneal epithelial wound healing. Methods: New Zealand rabbits were anaesthetised and wounds were made by placing Whatman paper discs soaked in n-heptanol on the cornea. Melatonin and analogues (all 10 nmol) were instilled. Wound diameter was measured every 2 hours by means of fluorescein application with a Topcon SL-8Z slit lamp. Melatonin antagonists (all 10 nmol) were applied 2 hours before the application of the n-heptanol-soaked disc and then every 6 hours together with melatonin. To confirm the presence of MT2 receptors in corneal epithelial cells immunohistochemistry, Western blot and RT-PCR assays in native tissue and in rabbit corneal epithelial cells were performed. The tear components were extracted then processed by HPLC to quantify melatonin in tears. Results: Migration assays revealed that melatonin and particularly the treatment with the MT2 agonist IIK7, accelerated the rate of healing (p < 0.001). The application of the non-selective melatonin receptor antagonist luzindole and the MT2 antagonist DH97 (but not prazosin), prevented the effect of melatonin on wound healing (both p < 0.001). Immunohistochemistry, Western blot and RT-PCR assays showed the presence of MT2 melatonin receptor in corneal epithelial cells. In addition, we have identified melatonin in tears and determined its daily variations. Conclusions: These data suggest that MT2 receptors are implicated in the effect of melatonin on corneal wound healing regulating migration rate. This suggests the potential use of melatonin and its analogues to enhance epithelial wound healing in ocular surface disease.