Echinacea and its alkylamides: Effects on the influenza A-induced secretion of cytokines,chemokines, and PGE2 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 PGE₂ 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 PGE₂ 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.     

GC-MS analysis of the lipophilic principles of Echinacea purpurea and evaluation of cucumber mosaic cucumovirus infection

An analytical GC–MS method based on nonpolar fused silica capillary column was developed to analyze the lipophilic constituents, mainly alkamides, from the root extracts of Echinacea purpurea (L.) Moench. In particular, the proposed method was applied to evaluate the phytochemical impacts of cucumber mosaic cucumovirus (CMV) infection on the plant's lipophilic marker phytochemicals. Methanolic (70% v/v) extracts, obtained from root materials by ultrasonic treatments, were subjected to liquid–liquid extraction with n-hexane-ethyl acetate (1:1 v/v) to recover the lipophilic, volatile to semivolatile, principles. Seventeen components, including the 11 alkamides known to E. purpurea roots, were identified in the GC–MS traces of the analyzed fractions and efficiently separated in a turnaround time of 25 min. CMV infection was found to be responsible for significant variations in the relative compositions of the major constituents, in particular germacrene D, Dodeca-2E, 4E, 8Z, 10Z(E)-tetraenoic acid isobutylamide cis/trans isomers, Undeca-2Z, 4E-diene-8, 10-diynoic acid isobutylamide and Dodeca-2E, 4Z-diene-8, 10-diynoic acid isobutylamide.     

Echinacea: anatomy, phytochemical pattern, and germination of the achene.

The achenes (fruits) of the therapeutically used Echinacea species E. purpurea, E. angustifolia, and E. pallida can be differentiated structurally (shape, anatomy) as well as phytochemically (essential oil components, alkamides). During germination in all three species dodeca-2E, 4E, 8Z, 10E(10Z)-tetraenoic acid isobutylamide (8/9) is mainly formed. Besides this a number of alkamides typical for the root of E. purpurea are synthesized in moderate amounts. Also, alkene derivatives of isovalerianic acid are produced. It is an interesting fact that neither 2-monoene alkalmides nor polyacetylenes could be detected during achene germination.     

GC–MS analysis of the lipophilic principles of Echinacea purpurea and evaluation of cucumber mosaic cucumovirus infection

An analytical GC–MS method based on nonpolar fused silica capillary column was developed to analyze the lipophilic constituents, mainly alkamides, from the root extracts of Echinacea purpurea (L.) Moench. In particular, the proposed method was applied to evaluate the phytochemical impacts of cucumber mosaic cucumovirus (CMV) infection on the plant's lipophilic marker phytochemicals. Methanolic (70% v/v) extracts, obtained from root materials by ultrasonic treatments, were subjected to liquid–liquid extraction with n-hexane-ethyl acetate (1:1 v/v) to recover the lipophilic, volatile to semivolatile, principles. Seventeen components, including the 11 alkamides known to E. purpurea roots, were identified in the GC–MS traces of the analyzed fractions and efficiently separated in a turnaround time of 25 min. CMV infection was found to be responsible for significant variations in the relative compositions of the major constituents, in particular germacrene D, Dodeca-2E, 4E, 8Z, 10Z(E)-tetraenoic acid isobutylamide cis/trans isomers, Undeca-2Z, 4E-diene-8, 10-diynoic acid isobutylamide and Dodeca-2E, 4Z-diene-8, 10-diynoic acid isobutylamide.     

Comparison of chemical components and antioxidants capacity of different Echinacea species.

Alcoholic extracts of the roots and leaves of three Echinacea species (E. purpurea, E. angustifolia and E. pallida) were analysed for the presence of characteristic chemicals by HPLC directly coupled to ultraviolet absorbance and electrospray mass spectrometric detectors. The method permitted rapid characterization and tentative identification of a large number of caffeoyl conjugates and alkamides in all the samples investigated. The roots of the three species differed markedly in their contents of characteristic compounds. Cichoric acid and verbascoside predominated in extracts of E. purpurea root whereas cynarine and dodeca-2E,4E,8Z,10Z/E-tetraenoic acid isobutylamide were the major chemicals characteristic of E. angustifolia root extracts. Echinacoside and 6-O-caffeoylechinacoside predominated in extracts of E. pallida roots. Characteristic alkamides were also examined by electrospray tandem mass spectrometry (MS/MS) and these compounds provided characteristic fragmentation patterns. Extracts of the roots and leaves of all three species were found to have antioxidant properties in a free radical scavenging assay and in a lipid peroxidation assay.     

Caffeoyl phenols and alkamides of cultivated Echinacea purpurea and Echinacea atrorubens var. paradoxa

Echinacea purpurea (L.) Moench was recently introduced into Taiwan. In the present study, the biomass, the contents of caffeoyl phenols, and the content of dodeca-2E,4E,8Z,10E-tetraenoic acid isobutylamide plus dodeca-2E,4E,8Z,10Z-tetraenoic acid isobutylamide (alkamides 8 and 9 respectively,) of locally selected line CLS-P2 and two introduced cultivars Magnus and White Swan of E. purpurea and an introduced E. atrorubens var. paradoxa were compared. The results indicated that both biomass and phytoactive constituents varied considerably among the introduced cultivars and selected line. Line CLS-P2 grew better and produced more aerial and ground parts than introduced cultivars Magnus and White Swan. It also produced more caffeoyl phenols, particularly cichoric acid and caftaric acid in its leaves than Magnus and White Swan. All the E. purpurea cultivars and line produced same amounts of alkamides 8 and 9 in their flower heads and leaves. But White Swan produced more alkamides 8 and 9 in its roots than CLS-P2 and Magnus. Line CLS-P2 was less homogenous in genetic background as compared to the introduced cultivars. E. atrorubens var. paradoxa also grew well in Taiwan, but it produced less aerial and ground dry mass than E. purpurea. E. atrorubens var. paradoxa produced more echinacoside in its flower heads, leaves, and root parts, while E. purpurea plants had more cichoric acid and caftaric acid in their flower heads and leaves. E. atrorubens var. paradoxa also produced more alkamides 8 and 9 in flower heads and leaves, while E. purpurea produced more alkamides 8 and 9 in roots.     

Comparative HPLC Analyses of Alkamides within the Achillea millefolium Group

Petrol/Et (2)O extractable alkamides from the subterranean parts of different members of the ACHILLEA MILLEFOLIUM group were separated and compared by reversed phase HPLC. Apart from different retention times, each peak could be characterized by its typical UV spectrum obtained by on-line photodiode array detection. Based on previously isolated and identified alkamides, a library search program of UV spectra and corresponding retention times was prepared which greatly facilitated a general comparison of the different HPLC profiles. Apart from the dominating deca-2 E,4 E,6Z-trienoic piperideide in the European representatives ( A. MILLEFOLIUM, A. PANNONICA, A. COLLINA, A. ASPLENIIFOLIA, A. SETACEA), the different cytotypes may be characterized by various accumulation tendencies, mainly towards two isomeric decatetraenoic piperideides and (+)-sesamin. The amide patterns of the Asian and North American members ( A. ASIATICA, A. LANULOSA) clearly deviate; the characteristic decatrienoic and decatetraenoic piperideides are replaced here by a preponderance of decadienoic acid-derived isobutylamide and piperideide. Since there are no diploid members known from North America, the striking chemical similarities with the diploid Asian members strongly suggest an Asian origin.     

Transport of alkamides from Echinacea species through Caco-2 monolayers

To gain more insights into the human intestinal absorption of alkamides from Echinacea species, transport studies were performed with the human adenocarcinoma colonic cell line Caco-2 (ATCC) as a model to assess the epithelial transport of dodeca-2 E,4 E,8 Z,10 E/ Z-tetraenoic acid isobutylamides (1/ 2). 30 minutes after apical loading of 25 microg/ml 1/ 2, about 15 % of these alkamides were detectable on the basolateral side. Close monitoring of the transport during 6 hours revealed a nearly complete transport to the basolateral side after 4 hours and no significant metabolism was observable. Transport experiments performed at 4 degrees C showed only a slight decrease in transport, which is a strong hint that dodeca-2 E,4 E,8 Z,10 E/ Z-tetraenoic acid isobutylamides (1/ 2) cross biological membranes by passive diffusion. Nearly the same results were obtained after preincubation of the Caco-2 cells with lipopolysaccharides (LPS) or phorbol 12-myristate-13-acetate (PMA) to mimic an inflammatory status. These results support the assumption that the alkamides can be easily transported from the intestinum and hence may contribute to the in vivo effects of Echinacea preparations.     

怀菊花茎叶中一个新聚炔类化合物

采用多种色谱技术从怀菊花茎叶中分离得到8个聚炔类化合物。通过UV、IR、ESI-MS、HR-ESI-MS及NMR等波谱技术对其进行结构鉴定,分别为菊花新炔C (2E,4E,12Z-十四碳三烯-1-吡咯烷基-1-酮-8,10-二炔)(1)、tetradeca-2E, 4E, 12E-trien-8, 10-diynoic acid pyrrolidide (2)、tetradeca-2E, 4E-dien-8, 10-diynoic acid pyrrolidide (3)、tetradeca-2E,4E,10Z-trien-8-ynoic acid pyrrolidide (4)、2E,4E,12E-tetradecatriene-8,10-diynoic acid isobutylamide (5)、2E,4E-undecyldiene-8,10-diynoic acid isobutylamide (6)、2E,4E,10E-N-isobutyl-2,4,10-tetradecatrien-8-ynoic acid amide(7)和十一-2E,4E-二烯-8,10-二炔酸苯乙胺(...     

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.     

Bioavailability and pharmacokinetics of Echinacea purpurea preparations and their interaction with the immune system

Echinacea is a widely used herbal remedy for the prevention and treatment of the common cold. Recently, many new insights concerning the molecular mode of action of the main lipophilic constituents, the alkamides, have renewed interest in this plant. In order to compare the bioavailability of alkamides from liquid and tablet preparations of E. purpurea (Echinaforce) in humans and to study the effects on ex vivo stimulated blood cells, a randomized, single-dose, crossover study with 10 (8 test, 2 placebo) volunteers has been performed. They received either 4 ml of the standardized E. purpurea (Echinaforce) tincture or 12 E. purpurea (Echinaforce) tablets or placebo. Both doses contained the same amount (0.07 mg) of the major alkamides, dodeca-2E,4E,8Z, 10E/Z-tetraenoic acid isobutylamides. Liquid chromatography electrospray ionization ion-trap mass spectrometry was used to determine the content of alkamides in serum. It was found that the arithmetic mean C(max) of dodeca-2E,4E, 8Z,10E/Z-tetraenoic acid isobutylamides absorbed after oral application of the Echinaforce tincture appeared after 30 min (0.40 ng/ml serum). In comparison, the t(max) of tablets was 45 min with a C(max) of 0.12 ng/ml. An ex vivo stimulation of blood by LPS was carried out to measure the influence of E. purpurea on the innate and adaptive immune system. Both E. purpurea preparations led to the same effects on the immune system according to the concentration of pro-inflammatory cytokines TNF-alpha and IL-8. 23 hours after oral application a significant down-regulation of TNF-alpha and IL-8 in LPS pre-stimulated whole blood was found. However, no significant changes in the concentration of IL-6 were observed. Although a quarter of the dodeca-2E,4E,8Z, 10E/Z-tetraenoic acid isobutylamides was absorbed from the tablets, the study shows that the formulations trigger the same effects on the measured immune parameters.     

Echinacea purpurea root extract enhances the adipocyte differentiation of 3T3-L1 cells

Echinacea purpurea has been shown to have anti-diabetic activities; for example, it activates peroxisome proliferator-activated receptor γ (PPARγ) and increases insulin-stimulated glucose uptake. Adipogenesis has been used to study the insulin signaling pathway and to screen anti-diabetic compounds. The present study was conducted to investigate the effects of an ethanol extract of E. purpurea (EEEP) and its constituents on the insulin-induced adipocyte differentiation of 3T3-L1 preadipocytes. When adipocyte differentiation was induced with insulin plus 3-isobutyl-1-methylxanthine and dexamethasone, the accumulation of lipid droplets and the cellular triglyceride content were significantly increased by EEEP. The expressions of PPARγ and C/EBPα in adipocytes treated with EEEP were gradually increased as compared with control cells. Fat accumulation and triglyceride content of adipocytes treated with dodeca-2(E),4(E)-dienoic acid isobutylamide were significantly increased as compared with control cells. The expressions of PPARγ and C/EBPα in adipocytes treated with dodeca-2(E),4(E)-dienoic acid isobutylamide were significantly higher than in control cells. These results suggest EEEP promotes the adipogenesis that is partially induced by insulin and that dodeca-2(E),4(E)-dienoic acid isobutylamide appears to be responsible for EEEP-enhanced adipocyte differentiation.     

Light-mediated antifungal activity of Echinacea extracts

This study demonstrated that plant extracts containing acetylenic isobutylamides and polyacetylenes, previously reported as occurring in Echinacea, have phototoxic antimicrobial activity against fungi, including clinically relevant pathogenic fungi. Results show that hexane extracts of Echinacea variably inhibit growth of yeast strains of Saccharomyces cerevisiae, Candida shehata, C. kefyr, C. albicans, C. steatulytica and C. tropicalis under near UV irradiation (phototoxicity) and to a lower extent without irradiation (conventional antifungal activity). The presence of polyacetylenes and alkylamides in extracts of different organs was confirmed in Echinacea purpurea by HPLC in agreement with previously reported data in the literature, and was related to phototoxic activity. Two representative pure compounds, undeca-2E,4Z-diene-8,10-diynoic acid isobutylamide and dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamide, were isolated from Echinacea purpurea root extracts, and compared in a disk assay (5 micrograms/disk) with the highly conjugated trideca-1-ene-3,5,7,9,10-pentayne (previously isolated in our laboratory and found here in E. purpurea). Significant phototoxicity was demonstrated by pure trideca-1-ene-3,5,7,9,10-pentayne, while only minor phototoxicity was induced by the other two acetylenic compounds. Phototoxic activity of Echinacea spp. is primarily attributed to the ketoalkenes and ketoalkynes abundantly present in the roots.     

Bioavailability and pharmacokinetics of alkamides from the roots of Echinacea angustifolia in humans

Alkamides are suspected to contribute to the activity of Echinacea preparations. They are mainly derived from undeca- and dodecanoic acid and differ in the degree of unsaturation and the configuration of the double bonds. In total, 6 alkamides have been isolated from the roots of Echinacea angustifolia as major lipophilic constituents and have been investigated regarding their pharmacokinetics. A sensitive and specific method has been developed for the identification and quantification of these alkamides in human plasma using liquid chromatography electrospray ionization ion-trap mass spectrometry. The method was applied to analyze plasma samples obtained from a randomized, open, single-dose, crossover study after oral administration of a 60% ethanolic extract from the roots of E. angustifolia to 11 healthy subjects. The maximum concentration of dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides, the main alkamides in the roots of E. angustifolia, appeared already after 30 minutes and was 10.88 ng/mL for the 2.5-mL dose.     

The Constituents of Echinacea atrorubens Roots and Aerial Parts

Eleven isobutylamides have been isolated from the n -hexane extract of the dried roots of Echinacea atrorubens Nutt. Undeca-2 E, 4 E -dien-8,10-diynoic acid isobutylamide represents a new compound. The lipophilic profiles of constituents of the roots and the herb were similar as the alkamide profiles previously found in E. purpurea and E. angustifolia. The polar components of E. atrorubens roots and the aerial parts, however, seem to be very different from E. purpurea and E. angustifolia, since neither cichoric acid nor echinacoside could be found.     

Absolute/relative bioavailability and metabolism of dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (tetraenes) after intravenous and oral single doses to rats

The present study assessed the absolute and relative bioavailabilities of dodeca-2 E,4 E,8 Z,10 E/ Z-tetraenoic acid isobutylamides (tetraenes), the main bioactive constituents in Echinacea, administered as pure compounds or in the form of an Echinacea purpurea root extract preparation. Tetraenes were administered orally by gavage or intravenously in a dose of 0.75?mg/kg. The extract was administered orally in a dose of 158.6?mg/kg which corresponds to the same amount of tetraenes. Pharmacokinetic parameters of tetraenes were calculated by non-compartmental analysis using WinNonlin? 5.2 software. Mean dodeca-2 E,4 E,8 Z,10 E/ Z-tetraenoic acid isobutylamide dose-normalized plasma area under the concentration-time curve (AUC?-∞/dose) was 3.24?±?0.32?min?·?ng/mL/μg and 0.95?±?0.16?min?·?ng/mL/μg after iv and oral administrations, respectively, and 1.53?±?0.18?min?·?ng/mL/μg after oral administration of the Echinacea root extract. The absolute oral bioavailability of dodeca-2 E,4 E,8 Z,10 E/ Z-tetraenoic acid isobutylamides was 29.2?±?2.3?%, which was increased to 47.1?±?7.2?% (1.6-fold) by administration of the Echinacea extract. Administration of an Echinacea extract increased blood exposure with no impact on C(max), but prolonged the elimination half-life to 123.3?±?15.7?min in comparison to 35.8?±?6.5?min after administration of the pure dodeca-2 E,4 E,8 Z,10 E/ Z-tetraenoic acid isobutylamides.     

Excitatory action of prostanoids on the ferret isolated vagus nerve preparation

We have investigated the actions of various prostanoid receptor agonists on an isolated preparation of the ferret cervical vagus using a grease-gap extracellular recording technique. The potency ranking for depolarization was BW245C (5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl) hydantoin; DP-selective, EC50=0.14 microM)>prostaglandin E2 (nonselective EP agonist)>U-46619 (11alpha, 9alpha-epoxymethano-15S-hydroxyprosta-5Z,13E-dienoic acid; TP agonist)>prostaglandin F2alpha (FP receptor agonist). Sulprostone (EP1/EP3-selective), fluprostenol (FP-selective) and cicaprost and iloprost (both IP-selective) had minimal effects. It is likely that DP, EP2/EP4 and TP receptors are present on the vagal fibres of the ferret.     

Alkamide levels in Echinacea purpurea: a rapid analytical method revealing differences among roots, rhizomes, stems, leaves and flowers

A rapid extraction, clean-up, and RPLC procedure suitable for routine quantitative analyses of alkamide levels in Echinacea purpurea extracts is described. The 13C-NMR spectra of the main diene-diyne alkamides in E. purpurea are reported. Alkamide levels differed significantly among roots, rhizomes, stems, leaves, and flowers of E. purpurea. Roots were distinguished from other plant parts by higher levels of the C12 diene-diyne alkamides, whereas levels of the C12 tetraene alkamides and C11 diene-diynes were highest in vegetative stems. The ratio of the two stereoisomeric C12 tetraene alkamides differed between flowers and all other E. purpurea parts. These results are important for standardisation of medicinal preparations of E. purpurea.     

Actions of prostanoids to induce emesis and defecation in the ferret

Several prostanoids were investigated for their ability to induce emesis and/or defecation and tenesmus in the ferret. The rank order of emetic potency (dose producing four episodes, D4) was: sulprostone (5 microg/kg)>11alpha,9alpha-epoxymethano-15S-hydroxyprosta-5Z,13E-dienoic acid (U46619; 8 microg/kg)>misoprostol (27 microg/kg)>17-phenyl-omega-trinor prostaglandin E2 (53 microg/kg)>prostaglandin E2 (94 microg/kg)>5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl) hydantoin (BW245C; 148 microg/kg)>prostaglandin F(2alpha) (13,500 microg/kg). Emesis was also induced by iloprost (D4 not determined) and prostaglandin E2 methyl ester (D4=350 microg/kg). Cicaprost and fluprostenol were virtually inactive; they also failed to modify copper sulphate (100 mg/kg, intragastric)-induced emesis (P>0.05), although cicaprost potentiated apomorphine (0.25 mg/kg, s.c.)-induced emesis (P<0.05). U46619-induced emesis was antagonised by vapiprost (P<0.05). The rank order of potency to produce defecation and tenesmus (dose producing three episodes) was: sulprostone (12 microg/kg)>misoprostol (15 microg/kg)>17-phenyl-omega-trinor prostaglandin E2 (94 microg/kg)>prostaglandin E2 (113 microg/kg)>fluprostenol (158 microg/kg)z.Gt;prostaglandin F(2alpha) (1759 microg/kg); prostaglandin E2 methyl ester also induced defecation (196 microg/kg). Data are discussed in relation to mechanisms involved in emesis and defecation.     

A novel (6S)-4,6-dimethyldodeca-2E,4E-dienoyl ester of phomalactone and related alpha-pyrone esters from a Phomopsis sp. with cytokine production inhibitory activity

A series of novel 6-substituted 5,6-dihydro-5-hydroxy-alpha-pyrone esters, 1 approximately 3, isolated from fermentations of a Phomopsis sp. (Xenova culture collection no. X22502) have been identified as inhibitors of lipopolysaccharide (LPS)-induced cytokine production. These include the (6S)-4,6-dimethyldodecadien-2E,4E-dienoyl ester of phomalactone, 1, and two analogues bearing a prop-2E-enoic acid moiety at the 6-position of the alpha-pyrone ring. (6S)-4,6-Dimethyl-2E,4E-dienoic acid, 4, and a hydroxylated analogue, 5, were also isolated and characterised. The most potent cytokine production inhibitor was 1, which inhibited LPS-induced tumour necrosis factor alpha (TNFalpha) production by U937 cells and LPS-induced interleukin 1beta (IL-1beta) production by peripheral blood mononuclear cells (PBMC) with IC50 values of 80 nM and 190 nM respectively. The effect of 1 in PBMC was selective for IL-1beta relative to TNFalpha. The inhibition of IL-1beta production by 1 involved a post-translational mechanism of action at the level of IL-1beta secretion as demonstrated by the lack of an effect on cell-associated IL-1beta production. 1 showed no effect on the activity of caspase 1 in cytosolic extracts from the THP1 monocytic cell line.