Alkamide production from hairy root cultures of <Emphasis Type="Italic">Echinacea</Emphasis>

Hairy root cultures of Echinacea, one of the most important medicinal plants in the US, represent a valuable alternative to field cultivation for the production of bioactive secondary metabolites. In this study, the three most economically important species of Echinacea (Echinacea purpurea, Echinacea pallida, and Echinacea angustifolia) were readily transformed with two strains of Agrobacterium that produce the hairy root phenotype. Transformed roots of all three species exhibited consistent accelerated growth and increased levels of alkamide production. Optimization of the culture of Echinacea hairy roots was implemented to enhance both growth and alkamide production concomitantly. The use of half-strength Gamborg’s B5 medium supplemented with 3.0% sucrose was twice as effective in maintaining hairy root production than any other media tested. The addition of indolebutyric acid increased the growth rate of roots by as much as 14-fold. Alkamide production increased severalfold in response to the addition of the elicitor, jasmonic acid, but did not respond to the addition of indolebutyric acid. Induced accumulation of the important bioactive compounds, alkamides 2 and 8, was observed both in transformed roots and in response to jasmonic acid treatments. The results of this study demonstrate the efficacy of hairy root cultures of Echinacea for the in vitro production of alkamides and establish guidelines for optimum yield.     

Bauer ketones 23 and 24 from Echinacea paradoxa var. paradoxa inhibit lipopolysaccharide-induced nitric oxide,prostaglandin E2 and cytokines in RAW264.7 mouse macrophages

Among the nine Echinacea species, E. purpurea, E. angustifolia and E. pallida, have been widely used to treat the common cold, flu and other infections. In this study, ethanol extracts of these three Echinacea species and E. paradoxa, including its typical variety, E. paradoxa var. paradoxa, were screened in lipopolysaccharide (LPS)-stimulated macrophage cells to assess potential anti-inflammatory activity. E. paradoxa var. paradoxa, rich in polyenes/polyacetylenes, was an especially efficient inhibitor of LPS-induced production of nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) by 46%, 32%, 53% and 26%, respectively, when tested at 20 μg/ml in comparison to DMSO control. By bioactivity-guided fractionation, pentadeca-8Z-ene-11, 13-diyn-2-one (Bauer ketone 23) and pentadeca-8Z, 13Z-dien-11-yn-2-one (Bauer ketone 24) from E. paradoxa var. paradoxa were found primarily responsible for inhibitory effects on NO and PGE2 production. Moreover, Bauer ketone 24 was the major contributor to inhibition of inflammatory cytokine production in LPS-induced mouse macrophage cells. These results provide a rationale for exploring the medicinal effects of the Bauer ketone-rich taxon, E. paradoxa var. paradoxa, and confirm the anti-inflammatory properties of Bauer ketones 23 and 24.     

Alkamides and a neolignan from Echinacea purpurea roots and the interaction of alkamides with G-protein-coupled cannabinoid receptors

Multiple chromatographic separations of the CHCl₃-soluble extract of the roots of Echinacea purpurea led to the isolation of 19 compounds. Four natural products, three alkamides and nitidanin diisovalerianate, were identified, and five further compounds were detected for the first time in this species. Additionally, 10 known E. purpurea metabolites were isolated. The structures were determined by mass spectrometry and advanced 1D and 2D NMR techniques. The bioactivity of the isolated compounds was studied in [³⁵S]GTPγS-binding experiments performed on rat brain membrane preparations. Both partial and inverse agonist compounds for cannabinoid (CB1) receptors were identified among the metabolites, characterized by weak to moderate interactions with the G-protein signaling mechanisms. The G-protein-modulating activities of the Echinacea compounds are rather far from the full agonist effects seen with the CB1 receptor agonist reference compound arachidonyl-2′-chloroethylamide (ACEA). However, upon coadministration with ACEA, a number of them proved capable of inhibiting the stimulation of the pure agonist, thereby demonstrating cannabinoid receptor antagonist properties.     

Biotechnological production of caffeic acid derivatives from cell and organ cultures of Echinacea species

Caffeic acid derivatives (CADs) are a group of bioactive compounds which are produced in Echinacea species especially Echinacea purpurea, Echinacea angustifolia, and Echinacea pallida. Echinacea is a popular herbal medicine used in the treatment of common cold and it is also a prominent dietary supplement used throughout the world. Caffeic acid, chlorogenic acid (5-O-caffeoylquinic acid), caftaric acid (2-O-caffeoyltartaric acid), cichoric acid (2, 3-O-dicaffeoyltartaric acid), cynarin, and echinacoside are some of the important CADs which have varied pharmacological activities. The concentrations of these bioactive compounds are species specific and also they vary considerably with the cultivated Echinacea species due to geographical location, stage of development, time of harvest, and growth conditions. Due to these reasons, plant cell and organ cultures have become attractive alternative for the production of biomass and caffeic acid derivatives. Adventitious and hairy roots have been induced in E. pupurea and E. angustifolia, and suspension cultures have been established from flask to bioreactor scale for the production of biomass and CADs. Tremendous progress has been made in this area; various bioprocess methods and strategies have been developed for constant high-quality productivity of biomass and secondary products. This review is aimed to discuss biotechnological methods and approaches employed for the sustainable production of CADs.     

Subtracted Diversity Array Identifies Novel Molecular Markers Including Retrotransposons for Fingerprinting Echinacea Species

Echinacea, native to the Canadian prairies and the prairie states of the United States, has a long tradition as a folk medicine for the Native Americans. Currently, Echinacea are among the top 10 selling herbal medicines in the U.S. and Europe, due to increasing popularity for the treatment of common cold and ability to stimulate the immune system. However, the genetic relationship within the species of this genus is unclear, making the authentication of the species used for the medicinal industry more difficult. We report the construction of a novel Subtracted Diversity Array (SDA) for Echinacea species and demonstrate the potential of this array for isolating highly polymorphic sequences. In order to selectively isolate Echinacea-specific sequences, a Suppression Subtractive Hybridization (SSH) was performed between a pool of twenty-four Echinacea genotypes and a pool of other angiosperms and non-angiosperms. A total of 283 subtracted genomic DNA (gDNA) fragments were amplified and arrayed. Twenty-seven Echinacea genotypes including four that were not used in the array construction could be successfully discriminated. Interestingly, unknown samples of E. paradoxa and E. purpurea could be unambiguously identified from the cluster analysis. Furthermore, this Echinacea-specific SDA was also able to isolate highly polymorphic retrotransposon sequences. Five out of the eleven most discriminatory features matched to known retrotransposons. This is the first time retrotransposon sequences have been used to fingerprint Echinacea, highlighting the potential of retrotransposons as based molecular markers useful for fingerprinting and studying diversity patterns in Echinacea.     

Alkamides from Echinacea disrupt the fungal cell wall-membrane complex

We tested the hypothesis that alkamides from Echinacea exert antifungal activity by disrupting the fungal cell wall/membrane complex. Saccharomyces cerevisiae cells were treated separately with each of seven synthetic alkamides found in Echinacea extracts. The resulting cell wall damage and cell viability were assessed by fluorescence microscopy after mild sonication. Membrane disrupting properties of test compounds were studied using liposomes encapsulating carboxyfluorescein. Negative controls included hygromycin and nourseothricin (aminoglycosides that inhibit protein synthesis), and the positive control used was caspofungin (an echinocandin that disrupts fungal cell walls). The results show that yeast cells exposed to sub-inhibitory concentrations of each of the seven alkamides and Echinacea extract exhibit increased frequencies of cell wall damage and death that were comparable to caspofungin and significantly greater than negative controls. Consistent with effects of cell wall damaging agents, the growth inhibition by three representative alkamides tested and caspofungin, but not hygromycin B, were partially reversed in sorbitol protection assays. Membrane disruption assays showed that the Echinacea extract and alkamides have pronounced membrane disruption activity, in contrast to caspofungin and other controls that all had little effect on membrane stability. A Quantitative Structure-Activity Relationship (QSAR) analysis was performed to study the effect of structural substituents on the antifungal activity of the alkamides. Among the set studied, diynoic alkamides showed the greatest antifungal and cell wall disruption activities while an opposite trend was observed in the membrane disruption assay where the dienoic group was more effective. We propose that alkamides found in Echinacea act synergistically to disrupt the fungal cell wall/membrane complex, an excellent target for specific inhibition of fungal pathogens. Structure-function relationships provide opportunities for synthesis of alkamide analogs with improved antifungal activities.     

Clonal propagation and production of cichoric acid in three species of Echinaceae

In vitro tissue culture protocols were tested for propagation of Echinacea purpurea, Echinacea pallida and Echinacea angustifolia in order to obtain biomass for the production of cichoric acid, which is the major active compound in the Echinacea extracts. The in vitro culture process was initiated by seed germination on half-strength Murashige and Skoog (MS) medium. Multiplication was achieved on MS medium supplemented with naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2-iso-pentenyladenine (2iP), and N⁶-benzyladenine (BA) in different concentrations. Shoot explants produced the highest number of shootlets on MS medium, which was supplemented with 0.1 mg/l 2iP and 0.1 mg/l IBA. RAPD markers revealed genetic polymorphism in some instances between in vitro generated plantlets such as for E. purpurea plantlets analyzed with the OPO-8 primer. RAPD markers generated with the primer 4A-29 revealed low levels of genetic variation between in vitro plantlets for all three species of Echinacea, while remaining RAPD markers revealed no variation. Content of cichoric acid in leaves, shoots, and callus was analyzed by high-performance liquid chromatography/MS and was identified in all studied samples, independent of species or tissue type. Highest levels (0.39–0.73 mg/g dw) were observed in shoots and leaves.     

A comparative study of bioactive secondary metabolite production in diploid and tetraploid Echinacea purpurea (L.) Moench

The impact of the ploidy level on biomass accumulation and the production of high-value secondary metabolites was studied in Echinacea purpurea (L.) Moench. Tetraploid E. purpurea was obtained by treating diploid explants with colchicine. The morphology, biomass yield, the contents of caffeic acid derivatives and alkamides, and the activities of phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) were compared between diploid plants and tetraploid plants of E. purpurea. The total fresh root weight and total dry root weight of the tetraploid plants were 39.32 and 40.48 % higher than those of the diploid plants, respectively. The chemical profiles of the diploid and tetraploid E. purpurea plants were similar, as determined through a comparison of their FTIR spectra and second derivative spectra. The caffeic acid derivatives and alkamides in the diploid and tetraploid plants were determined by HPLC. The tetraploid plants had higher contents of both of these types of molecules. In addition, the tetraploid plants had higher PAL and C4H activities compared with the diploid plants. The enhancement in the PAL and C4H activities was accompanied with an increase in the cichoric acid content, which indicates that the induction of polyploidy in E. purpurea resulted in higher PAL and C4H expression and promoted the biosynthesis of cichoric acid. Therefore, the induction of polyploidy may be a valid strategy to achieve a higher yield of biomass and bioactive compounds in E. purpurea.     

Active polyphenolic compounds,nutrient contents and antioxidant capacity of extruded fish feed containing purple coneflower (Echinacea purpurea (L.) Moench.)

The growth of fish is directly dependent on feed composition and quality. Medicinal plants can be added to fish feed as adjuvant therapy for the prevention of fish diseases. The purple coneflower (Echinacea purpurea (L.) Moench.) has been reported to have multiple biological effects, including immunomodulatory and antioxidant activity. The most active compounds of E. purpurea are polyphenols - caffeic acid derivatives: caftaric acid, chlorogenic acid, cynarin, echinacoside and cichoric acid.Due to a relatively limited number of studies on the use of the purple coneflower as a nutritional supplement for fish feeding, extruded fish feed with addition of Echinacea roots was produced. In the feed total phenolic content, selected polyphenol contents, the energetic value, nutrient contents and antioxidant capacity were examined.The results indicate that fish feed with addition of the Echinacea has a great potential to be a good source of natural radical scavengers, for example polyphenols, and nutritive ingredients. Antioxidant properties of feed were well correlated with the coneflower content. The study findings confirmed that high-temperature extrusion-cooking process does not deactivate phenolic antioxidant compounds, which are present both in the Echinacea roots and in the final product. Fish feed with addition of E. purpurea can be used as a nutritional supplement in the prevention of fish diseases caused by oxidative stress.     

Ethanolic Echinacea purpurea Extracts Contain a Mixture of Cytokine-Suppressive and Cytokine-Inducing Compounds,Including Some That Originate from Endophytic Bacteria

Echinacea preparations, which are used for the prevention and treatment of upper respiratory infections, account for 10% of the dietary supplement market in the U.S., with sales totaling more than $100 million annually. In an attempt to shed light on Echinacea''s mechanism of action, we evaluated the effects of a 75% ethanolic root extract of Echinacea purpurea, prepared in accord with industry methods, on cytokine and chemokine production from RAW 264.7 macrophage-like cells. We found that the extract displayed dual activities; the extract could itself stimulate production of the cytokine TNF-α, and also suppress production of TNF-α in response to stimulation with exogenous LPS. Liquid:liquid partitioning followed by normal-phase flash chromatography resulted in separation of the stimulatory and inhibitory activities into different fractions, confirming the complex nature of this extract. We also studied the role of alkylamides in the suppressive activity of this E. purpurea extract. Our fractionation method concentrated the alkylamides into a single fraction, which suppressed production of TNF-α, CCL3, and CCL5; however fractions that did not contain detectable alkylamides also displayed similar suppressive effects. Alkylamides, therefore, likely contribute to the suppressive activity of the extract but are not solely responsible for that activity. From the fractions without detectable alkylamides, we purified xanthienopyran, a compound not previously known to be a constituent of the Echinacea genus. Xanthienopyran suppressed production of TNF-α suggesting that it may contribute to the suppressive activity of the crude ethanolic extract. Finally, we show that ethanolic extracts prepared from E. purpurea plants grown under sterile conditions and from sterilized seeds, do not contain LPS and do not stimulate macrophage production of TNF-α, supporting the hypothesis that the macrophage-stimulating activity in E. purpurea extracts can originate from endophytic bacteria. Together, our findings indicate that ethanolic E. purpurea extracts contain multiple constituents that differentially regulate cytokine production by macrophages.     

Ethnobotany of purple coneflower (Echinacea angustifolia,Asteraceae) and OtherEchinacea Species

The purple coneflower, Echinacea angustifolia, was the most widely used medicinal plant of the Plains Indians. It was used for a variety of ailments, including toothache, coughs, colds, sore throats, snakebite, and as a painkiller. H. C. F. Meyer used it as a patent medicine in the 1870s and introduced it to the medical profession. Recent scientific research (mostly German) onEchinacea species has shown that they possess immunostimulatory activity. Increased cultivation ofE. purpurea andE. angustifolia may be needed to meet the increased demand for its roots and to alleviate the effects of overharvesting of wild stands.     

New C9-polyacetylenes from the essential oil of the highly endangered species Baccharis palustris Heering (Asteraceae)

The essential oil composition of the aerial parts from Baccharis palustris Heering (Asteraceae), a highly endangered species, was analyzed by GC/qMS, GC/HRMS-TOF, FT-IR, UV–vis and NMR spectroscopy using 1D and 2D techniques. To our knowledge, this is the first report on the essential oil composition of B. palustris, as well as the first phytochemical study on this plant species. The volatile extracts were obtained from different phenologicals stages of plant, at flowering stage (FS) and vegetative stage (VS), where 51 components were identified accounting for 96.3 % and 99.0 % of the oil, respectively. The oil was rich in polyacetylene compounds (> 75 %), being the main components identified the new natural C₉-polyacetylenes 1-nonene-3,5-diyne (1, here named as baccharisdiyne) (52.7–65.0 %), 1,7(Z)-nonadiene-3,5-diyne [2, 7(Z)-dehydro-baccharisdiyne] (14.4–17.8 %), and 1,7(E)-nonadiene-3,5-diyne [3, 7(E)-dehydro-baccharisdiyne] (1.5–2.4 %). In addition, the known polyacetylenic compounds (Z)-lachnophyllum acid methyl ester (4) (4.3–5.3 %) and (E)-lachnophyllum acid methyl ester (5) (0.2 %) were also identified. Moreover, GC/qMS analysis allowed the identification of other 46 components in the essential oil samples, mainly mono- and sesquiterpenoids. In B. palustris, C₉-polyacetylenes probably derive biogenetically from a C₁₀ precursor: baccharisdiyne (1) would be derived from either or both lachnophyllum methyl ester geometric isomers (4 or 5), by enzymatic hydrolysis followed by decarboxylation. Dehydro-baccharisdiynes (2 and 3) could be produced by a similar pathway starting from the corresponding matricaria acid methyl ester isomers, one of which was tentatively identified at trace-level in B. palustris essential oil.     

Minor alkamides from Heliopsis longipes S.F. Blake (Asteraceae) fresh roots

From the fresh roots of Heliopsis longipes three new minor alkamides: longipinamide A (N-isobutyl-8,10-diynoic-3Z-undecenamide), longipenamide A (N-isobutyl-syn-8,9-dihydroxy-2E,6Z-decadienamide) and longipenamide B (N-isobutyl-syn-6,9-dihydroxy-2E,7E-decadienamide); three known alkamides: affinin (spilanthol, N-isobutyl-2E,6Z,8E-decatrienamide), N-isobutyl-2E,6Z-decadienamide and N-isobutyl-2E-decenamide; and 11 other known compounds were isolated. The structures of the three new minor alkamides were established by 1D and 2D NMR spectroscopy including ¹H, ¹³C, DEPT, COSY, HSQC, and HMBC experiments, as well as by EI and FAB⁺ mass spectrometry. To our knowledge, this is the first report of the isolation of linear dihydroxyalkamides as natural products.     

Unprecedented NES non-antagonistic inhibitor for nuclear export of Rev from Sida cordifolia

Bioassay-guided separation from the MeOH extract of the South American medicinal plant Sida cordifolia resulted in isolation of (10E,12Z)-9-hydroxyoctadeca-10,12-dienoic acid (1) as an unprecedented NES non-antagonistic inhibitor for nuclear export of Rev. This mechanism of action was established by competitive experiment by the biotinylated probe derived from leptomycin B, the known NES antagonistic inhibitor. Additionally, structure–activity relationship analysis by use of the synthesized analogs clarified cooperation of several functionalities in the Rev-export inhibitory activity of 1.     

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-methylbutylamide¹, dodeca-2E,4E-dienoic acid isobutylamide¹, dodeca-2E-ene-8,10-diynoic acid isobutylamide⁷, hexadeca-2E,9Z-diene-12,14-diynoic acid isobutylamide¹, cichoric acid⁷, caftaric acid¹, and echinacoside⁷ (¹p<0.0001, ⁷p<0.05). Five of those compounds were also significantly associated with latitudinal variation by regression analyses (p<0.05).     

How well do herbarium data predict the locationof present populations? A test using <Emphasis Type="Italic">Echinacea</Emphasis> species in Missouri

The four native Missouri taxa of Echinacea were used as a model to test the predictive value of herbarium data with regard to present-day distribution. Specimens with label data considered potentially adequate to relocate the population were databased and the sites in question were visited. Most of the historical populations were not relocated, although a greater percentage of those collected post-1980 were found to still exist. Time since collection significantly affected the probability of relocation of E. purpurea and E. pallida var. pallida, the two native taxa commonly harvested for medicinal products. The collection rate for E. pallida var. pallida remained high over time. However, the collection rate for E. purpurea has been much reduced in recent decades. Few of the historical populations were relocated, and located populations on unprotected public land were very small, perhaps indicating that habitat loss or human activity is causing a decline in Missouri populations of E. purpurea.     

Leishmania donovani pteridine reductase 1: comparative protein modeling and protein–ligand interaction studies of the leishmanicidal constituents isolated from the fruits of Piper longum

Visceral leishmaniasis or kala-azar is caused by the dimorphic parasite Leishmania donovani in the Indian subcontinent. Treatment options for kala-azar are currently inadequate due to various limitations. Currently, drug discovery for leishmaniases is oriented towards rational drug design; the aim is to identify specific inhibitors that target particular metabolic activities as a possible means of controlling the parasites without affecting the host. Leishmania salvages pteridin from its host and reduces it using pteridine reductase 1 (PTR1, EC 1.5.1.33), which makes this reductase an excellent drug target. Recently, we identified six alkamides and one benzenoid compound from the n-hexane fraction of the fruit of Piper longum that possess potent leishmanicidal activity against promastigotes as well as axenic amastigotes. Based on a homology model derived for recombinant pteridine reductase isolated from a clinical isolate of L. donovani, we carried out molecular modeling and docking studies with these compounds to evaluate their binding affinity. A fairly good agreement between experimental data and the results of molecular modeling investigation of the bioactive and inactive compounds was observed. The amide group in the conjugated alkamides and the 3,4-methylenedioxystyrene moiety in the benzenoid compound acts as heads and the long aliphatic chain acts as a tail, thus playing important roles in the binding of the inhibitor to the appropriate position at the active site. The remarkably high activity of a component containing piperine and piperine isomers (3.36:1) as observed by our group prompted us to study the activities of all four isomers of piperine—piperine (2E,4E), isopiperine (2Z,4E), isochavicine (2E,4Z), and chavicine (2Z,4Z)—against LdPTR1. The maximum inhibitory effect was demonstrated by isochavicine. The identification of these predicted inhibitors of LdPTR1 allowed us to build up a stereoview of the structure of the binding site in relation to activity, affording significant information that should prove useful during the structure-based design of leishmanicidal drugs.     

A novel co-culture system of adventitious roots of <Emphasis Type="Italic">Echinacea</Emphasis> species in bioreactors for high production of bioactive compounds

To efficiently produce bioactive compounds of Echinacea via adventitious root (AR) culture, we established an AR co-culture system of Echinacea species. ARs of different combination of Echinacea species [E. pallida (Epa), E. purpurea (Epu), and E. angustifolia (Ean)] were inoculated into 5 L balloon-type airlift bioreactors to select a suitable combination group. The biomass of ARs increased in the Epa+Epu group but decreased in co-culture groups of Epa+Ean and Epa+Epu+Ean. In the Epa+Epu group, the content and productivity of total phenolics, flavonoids, and caffeic acid derivatives increased, a monomer of caffeic acid derivatives (caffeic acid) that is absent in single-species cultures (Epa or Epu) were synthesized; moreover, Epa+Epu also showed the highest antioxidant activity. The inoculum proportions in Epa+Epu significantly influenced the co-culture effect; among the proportion groups (Epa:Epu?=?1:6, 2:5, 3:4, 1:1, 4:4, 5:2, and 6:1), the Epa:Epu proportion of 4:3 was the most favorable for AR biomass and bioactive compound accumulation, and the antioxidant activity also peaked at 4:3 proportion. In addition, the co-culture system is suitable for large bioreactors (10 and 20 L), wherein the AR biomass increased without a decrease in the amount of bioactive compounds. A co-culture system was thus successfully established in this study, and AR cultures are expected to be used as an alternative raw material for the production of Echinacea-derived products.     

Full characterization of PDX, a neuroprotectin/protectin D1 isomer, which inhibits blood platelet aggregation

Our study aimed to establish the complete structure of the main dihydroxy conjugated triene issued from the lipoxygenation (soybean enzyme) of docosahexaenoic acid, named PDX, an isomer of protectin/neuroprotectin D1 (PD1/NPD1) described by Bazan and Serhan. NMR approaches and other chemical characterization (e.g. GC-MS, HPLC and LC-MS/MS) indicated that PDX is 10(S),17(S)-dihydroxy-docosahexa-4Z,7Z,11E,13Z,15E,19Z-enoic acid. The use of ¹⁸O₂ and mass spectrometry showed that PDX is a double lipoxygenation product. Its structure differs from PD1, with E,Z,E geometry (PDX) instead of E,E,Z (PD1) and S configuration at carbon 10 instead of R. PDX inhibits human blood platelet aggregation at sub-micromolar concentrations.