E-dienoic Acid Isobutylamide Research Articles

Mast cell degranulation and calcium influx are inhibited by an Echinacea purpurea extract and the alkylamide dodeca-2E,4E-dienoic acid isobutylamide

Ethnopharmacological relevanceNative Americans used plants from the genus Echinacea to treat a variety of different inflammatory conditions including swollen gums, sore throats, skin inflammation, and gastrointestinal disorders. Today, various Echinacea spp. preparations are used primarily to treat upper respiratory infections. Aim of the studyThe goal of this study was to evaluate the effects of an ethanolic E. purpurea (L) Moench root extract and the alkylamide dodeca-2E,4E-dienoic acid isobutylamide (A15) on mast cells, which are important mediators of allergic and inflammatory responses. Inhibition of mast cell activation may help explain the traditional use of Echinacea. Materials and methodsA15 was evaluated for its effects on degranulation, calcium influx, cytokine and lipid mediator production using bone marrow derived mast cells (BMMCs) and the transformed rat basophilic leukemia mast cell line RBL-2H3. Methods included enzymatic assays, fluorimetry, ELISAs, and microscopy. A root extract of E. purpurea, and low and high alkylamide-containing fractions prepared from this extract, were also tested for effects on mast cell function. Finally, we tested A15 for effects on calcium responses in RAW 264.7 macrophage and Jurkat T cell lines. ResultsA15 inhibited ß-hexosaminidase release from BMMCs and RBL-2H3 cells after treatment with the calcium ionophore A23187 by 83.5% and 48.4% at 100µM, respectively. Inhibition also occurred following stimulation with IgE anti-DNP/DNP-HSA. In addition, A15 inhibited 47% of histamine release from A23187-treated RBL-2H3 cells. A15 prevented the rapid rise in intracellular calcium following FcεRI crosslinking and A23187 treatment suggesting it acts on the signals controlling granule release. An E. purpurea root extract and a fraction with high alkylamide content derived from this extract also displayed these activities while fractions with little to no detectable amounts of alkylamide did not. A15 mediated inhibition of calcium influx was not limited to mast cells as A23187-stimulated calcium influx was blocked in both RAW 264.7 and Jurkat cell lines with 60.2% and 43.6% inhibition at 1min post-stimulation, respectively. A15 also inhibited the release of TNF-α, and PGE2 to a lesser degree, following A23187 stimulation indicating its broad activity on mast cell mediator production. ConclusionsThese findings suggest that Echinacea extracts and alkylamides may be useful for treating allergic and inflammatory responses mediated by mast cells. More broadly, since calcium is a critical second messenger, the inhibitory effects of alkylamides on calcium uptake would be predicted to dampen a variety of pathological responses, suggesting new uses for this plant and its constituents.

Study on screening antitumor active fractions and chemical components in active fractions from root of Anaycclus pyrethrum

Components that systematic separated from the root of Anaycclus pyrethrum were identified, in order to lay a foundation for future study of the root of A. pyrethrum. The CCK-8 assay showed that dichloromethane fraction exhibited the highest degree of cytotoxicity than others. Ten monomeric components were obtained from dichloromethane fraction and ethyl acetate fraction extracted from the root of A. pyrethrum, including 7 N-alkylamides, one coumarin and two flavonoid glycosides. They were identified as tetradeca-2E,4E,8E-trienoic acid 4-hydroxyphenylethylamide(1), deca-2E,4E-dienoicacid isobutylamide(2), undeca-2E,4E-diene-8,10-diynoic acid phenylethylamide(3), tetradeca-2E,4E-dienoic acid 4-hydroxyphenylethylamide(4), tetradeca-2E,4E-diene-8,10-diynoic acid isobutylamide(5), deca-2E,4E- dienoic acid 4-hydroxyphenylethylamide(6), dodeca-2E,4E-dienoic acid 4-hydroxy -phenyl-ethylamide(7), isoscopoletin(8), quercetin-7-O-β-D-glucopyranoside(9), isorhamnetin-7-O-β-D-glucopyranoside(10). Among them, compound 1 was identified as a new compound, Compounds 2-4, 8-10 were isolated from this herb for the first time.

Alkamides from Anacyclus pyrethrum L. and Their in Vitro Antiprotozoal Activity.

In our ongoing study to evaluate the antiprotozoal activity of alkamides from Asteraceae, a dichloromethane extract from the roots of Anacyclus pyrethrum L. showed a moderate in vitro activity against the NF54 strain of Plasmodium falciparum and against Leishmania donovani (amastigotes, MHOM/ET/67/L82 strain). Seven pure alkamides and a mixture of two further alkamides were isolated by column chromatography followed by preparative high performance liquid chromatography. The alkamides were identified by mass- and NMR-spectroscopic methods as tetradeca-2E,4E-dien-8,10-diynoic acid isobutylamide (anacycline, 1), deca-2E,4E-dienoic acid isobutylamide (pellitorine, 2), deca-2E,4E,9-trienoic acid isobutylamide (3), deca-2E,4E-dienoic acid 2-phenylethylamide (4), undeca-2E,4E-dien-8,10-diynoic acid isopentylamide (5), tetradeca-2E,4E,12Z-trien-8,10-diynoic acid isobutylamide (6), and dodeca-2E,4E-dien acid 4-hydroxy-2-phenylethylamide (7). Two compounds—undeca-2E,4E-dien-8,10-diynoic acid 2-phenylethylamide (8) and deca-2E,4E-dienoic acid 4-hydroxy-2-phenylethylamide (9)—were isolated as an inseparable mixture (1:4). Compounds 3, 4, and 5 were isolated from Anacyclus pyrethrum L. for the first time. While compounds 4 and 5 were previously known from the genus Achillea, compound 3 is a new natural product, to the best of our knowledge. All isolated alkamides were tested in vitro for antiprotozoal activity against Plasmodium falciparum, Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani and for cytotoxicity against L6 rat skeletal myoblasts.

Pharmacokinetics and immunomodulatory effect of lipophilic Echinacea extract formulated in softgel capsules

An attractive herbal product, softgel capsules containing 10mg of Echinacea angustifolia lipophilic extract, was given in a single oral administration to 10 human volunteers to perform a pharmacokinetic and immunological study.The plasma concentration of the major constituent was monitored, quantifying at predetermined time points the dodeca-2E,4E,8Z,10E/Z-tetraenoic isobutylamides (tetraene). The plasmatic levels of IL-2, IL-6, IL-8, IL-10 and TNF-a in samples collected before and 24h after drug administration were analyzed by cytokine assay. The total RNA was extracted from limpho-monocyte isolated from the same blood samples and the same cytokines in terms of gene expression were evaluated. With the help of proper statistical tests the differences between the values obtained at 0 and 24h were evaluated.Results of pharmacokinetic studies attest an approximately 3.5-fold improvement of tetraene oral bioavailability compared with previously published studies. Dodeca-2E,4E-dienoic acid isobutylamide exerts immunomodulatory effects down-regulating the gene expression and reducing the protein plasmatic levels of pro-inflammatory cytokines such as IL-6, TNF-α and IL-8, and up-regulating the expression of anti-inflammatory molecules as IL-10. Student’s two-sided paired t-test and non-parametric Wilcoxon–Mann–Whitney signed rank test agree in the conclusions about the differences between the ln values at 24h and corresponding ln values at 0h.

A New Geranylbenzofuranone from Zanthoxylum armatum

A new geranylbenzofuranone, zantholide (1), and eight known compounds, dodeca-2E,4E-dienoic acid isobutylamide (2), dodeca-2E,4E,8Z,11-tetraenoic acid isobutyl amide (3), zanthoxylin (4), sesamin (5), kobusin (6), asarinin (7), fargesin (8) and armatamide (9), have been isolated from the bark of Zanthoxylum armatum. The structures of the isolated compounds were determined on the basis of spectroscopic (1D, 2D NMR) and HR-ESI-MS data. This is the first report on the isolation of 2 and 3 from the Rutaceae family and 4 from Z. armatum.

Purification of Alkylamides from Echinacea angustifolia (DC.) Hell. Roots by High-Speed Countercurrent Chromatography

High-speed countercurrent chromatography (HSCCC) was used for the separation of alkylamides from the roots of Echinacea angustifolia (DC.) Hell. For this purpose, the alkylamides were extracted with hexane and subjected to semipreparative HSCCC using a two-phase solvent system consisting of n-hexane, ethyl acetate, methanol, and water (4:1:2:1). The lower aqueous phase was used as the mobile phase at a flow rate of 3 mL/min and a rotary speed of 1000 rpm. This procedure led to the isolation of four pure alkylamides, that is, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide (38.9 mg, 97% purity), dodeca-2E,4E,8Z-trienoic acid isobutylamide (4.4 mg, 92% purity), dodeca-2E,4E-dienoic acid isobutylamide (3.2 mg, 99% purity), and dodeca-2E,4E-dienoic acid 2-methylbutylamide (0.3 mg, 92% purity). The identity and purity of the isolated alkylamides were confirmed by LC-ESI-MS and (1)H NMR and (13)C NMR data. To the best of the authors' knowledge, this is the first report of dodeca-2E,4E-dienoic acid 2-methylbutylamide in E. angustifolia roots.

Echinacea and its alkylamides: Effects on the influenza A-induced secretion of cytokines, chemokines, and PGE 2 from RAW 264.7 macrophage-like cells

The goal of this study was to determine whether extracts and isolated alkylamides from Echinacea purpurea would be useful for prevention of the inflammatory response that accompanies infections with H1N1 influenza A. Seventeen extracts and 4 alkylamides were tested for the ability to inhibit production of cytokines, chemokines, and PGE 2 from RAW 264.7 macrophage-like cells infected with the H1N1 influenza A strain PR/8/34. The alkylamides undeca-2Z,4E-diene-8,10-diynic acid isobutylamide, dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide, dodeca-2E,4E-dienoic acid isobutylamide, and undeca-2E-ene-8,10-diynoic acid isobutylamide suppressed production of TNF-α and PGE 2 from infected cells. Dodeca-2E,4E-dienoic acid isobutylamide was especially effective at inhibiting production of these mediators and also strongly inhibited production of G-CSF, CCL2/MCP-1, CCL3/MIP-1α and CCL5/RANTES. In contrast, the ethanol extracts (75%), which were prepared from dormant roots of E. purpurea grown in different locations throughout North Carolina, displayed a range of effects from suppression to stimulation of mediator production. Precipitation of the extracts with ethanol removed the stimulatory activity, however, even after precipitation; many of the extracts did not display any suppressive activity. Analysis of the extracts revealed slight variations in concentration of alkylamides, caftaric acid, and cichoric acid, but the activity of the extracts did not strongly correlate with concentrations of these compounds. Our in vitro experiments suggest that E. purpurea extracts have the potential for use in alleviating the symptoms and pathology associated with infections with influenza A; however, further study will be necessary to define procedures necessary to unmask the alkylamide activity in crude extracts.

Establishment of recombinant cannabinoid receptor assays and characterization of several natural and synthetic ligands

Cannabinoid receptors (CBR) are important drug targets for the treatment of various inflammatory, metabolic and neurological diseases. Therefore, sensitive test systems for the assessment of ligands are needed. In this study, a steady-state GTPase assay for human CBR subtypes 1 and 2 was developed to characterize the pharmacological property of ligands at a very proximal point of the signal transduction cascade. Establishing these in vitro test sytems, we studied cell or tissue membranes heterogenously or endogenously expressing CBR, such as CBR-infected Human Embryonic Kidney (HEK) 293 cells, rat cerebellum and spleen cells. The lack of effects in the GTPase assay and in [(35)S]GTPgammaS binding experiments in these expression system, directed us to use Spodoptera frugiperda (Sf9) cells. Co-expressing CBR, different Galpha-subunits, Gbetagamma heterodimer, and RGS (Regulator of G-protein signaling)-proteins in Sf9 cell membranes greatly improved the sensitivity of the assay, with highest GTPase activation in the CBR + Galpha(i2) + Gbeta(1)gamma(2) + RGS4 system. We examined exogenous and endogenous standard ligands as well as secondary metabolites as Delta(9)-tetrahydrocannabinol (Delta(9)-THC), dodeca-2E,4E-dienoic acid isobutylamide, an alkylamide from Echinacea purpurea, and an E. purpurea hexane extract according their agonistic and antagonistic properties. The suitability of the assay for screening procedures was also proven by detecting the activity of Delta(9)-THC in a matrix of other less active compounds (Delta(9)-THC-free Cannabis sativa extract). In conclusion, we have developed highly sensitive test systems for the analysis of CBR ligands.

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.

Exploration of natural alkylamides and synthetic analogs as source for new ligands for the cannabinoid type-2 receptor

The cannabinoid type-2 (CB2) receptor is an attractive target for the development of drugs against inflammatory disease, atherosclerosis, and osteoporosis. Based on the discovery that alkylamides (alkamides) from Echinacea constitute a new class of CB2-specific cannabinomimetics1, we have screened a series of synthetic analogs of Echinacea alkylamides as well as other plant-derived natural N-alkyl amides for competitive binding to the CB2-receptor. Because dodeca-2E,4E-dienoic acid isobutylamide from Echinacea has a high affinity to the human CB2-receptor we synthesized analogs with modified head groups, as reported for the endogenous cannabinoid anandamide2. The resulting preliminary structure-activity relationship clearly shows that the CB2 receptor binding mode of Echinacea alkylamides is different from anandamide. To further explore the potential of natural N-alkyl amides as a new general class of CB2 ligands we have initiated a screening of plant extracts. The hexane extracts of the medicinal plants Spilanthes oleracea L. and Lepidium meyenii Walper. which are known to contain distinct types of alkylamides, were tested in receptor binding assays. The alkylamide-fraction of the L. meyenii extract showed significant receptor binding and five isolated benzylated alkylamides (macamides) were assessed for both their CB2-receptor affinity as well as CB2-mediated functional effects. Our data show that natural alkylamides from Echinacea and Lepidium are promising candidates for the development of novel CB2-receptor ligands.

Alkylamides from Echinacea Are a New Class of Cannabinomimetics

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.

Phytochemical variation within populations of Echinacea angustifolia (Asteraceae)

Quantitative evaluation of phytochemical diversity in Echinacea angustifolia DC. populations from different natural geographic areas supports the existence of distinct natural chemotypes within the species. Consumers, growers and manufacturers of phytomedicines are interested in chemotype identification for prediction of phytochemical content in cultivar development. Six month old E. angustifolia roots, grown from nine different wild seed sources in a controlled environment, were extracted into 70% ethanol and 28 reported phytochemicals were measured by HPLC separation. Two-way ANOVA between the nine populations revealed quantitative differences ( p <0.05) in the caffeic acid derivatives 2,3- O -dicaffeoyl tartaric acid (cichoric acid), 2- O -caffeoyl tartaric acid (caftaric acid), 1,3-dicaffeoyl-quinic acid (cynarin), echinacoside and ten reported alkamides. Canonical discriminant analysis determined the phytochemical variables which contributed the most towards chemotype distinction for five of the nine populations: undeca-2E,4Z-diene-8,10-diynoic acid-2-methylbutylamide ∗ , dodeca-2E,4E-dienoic acid isobutylamide ∗ , dodeca-2E-ene-8,10-diynoic acid isobutylamide ∗∗ , hexadeca-2E,9Z-diene-12,14-diynoic acid isobutylamide ∗ , cichoric acid ∗∗ , caftaric acid ∗ , and echinacoside ∗∗ ( ∗ p <0.0001, ∗∗ p <0.05). Five of those compounds were also significantly associated with latitudinal variation by regression analyses ( p <0.05).

Alkamides fromCissampelos glaberrima

Two alkamides, deca-2E,4E-dienoic acid isobutylamide (1) and octa-2E,4E-dienoic acid isobutylamide (2), were isolated from the roots of Cissampelos glaberrima (Menispermaceae). Their structures were established by spectral and physical methods. Traces of two new alkamides, decen-2-oic acid isobutylamide (3) and decanoic acid isobutylamide (4), were identified by mass spectrometry.