QQS orphan gene and its interactor NF-YC4 reduce susceptibility to pathogens and pests.

Enhancing the nutritional quality and disease resistance of crops without sacrificing productivity is a key issue for developing varieties that are valuable to farmers and for simultaneously improving food security and sustainability. Expression of the Arabidopsis thaliana species-specific AtQQS (Qua-Quine Starch) orphan gene or its interactor, NF-YC4 (Nuclear Factor Y, subunit C4), has been shown to increase levels of leaf/seed protein without affecting the growth and yield of agronomic species. Here, we demonstrate that overexpression of AtQQS and NF-YC4 in Arabidopsis and soybean enhances resistance/reduces susceptibility to viruses, bacteria, fungi, aphids and soybean cyst nematodes. A series of Arabidopsis mutants in starch metabolism were used to explore the relationships between QQS expression, carbon and nitrogen partitioning, and defense. The enhanced basal defenses mediated by QQS were independent of changes in protein/carbohydrate composition of the plants. We demonstrate that either AtQQS or NF-YC4 overexpression in Arabidopsis and in soybean reduces susceptibility of these plants to pathogens/pests. Transgenic soybean lines overexpressing NF-YC4 produce seeds with increased protein while maintaining healthy growth. Pull-down studies reveal that QQS interacts with human NF-YC, as well as with Arabidopsis NF-YC4, and indicate two QQS binding sites near the NF-YC-histone-binding domain. A new model for QQS interaction with NF-YC is speculated. Our findings illustrate the potential of QQS and NF-YC4 to increase protein and improve defensive traits in crops, overcoming the normal growth-defense trade-offs.

Integrating metabolomics and transcriptomics data to discover a biocatalyst that can generate the amine precursors for alkamide biosynthesis.

The Echinacea genus is exemplary of over 30 plant families that produce a set of bioactive amides, called alkamides. The Echinacea alkamides may be assembled from two distinct moieties, a branched-chain amine that is acylated with a novel polyunsaturated fatty acid. In this study we identified the potential enzymological source of the amine moiety as a pyridoxal phosphate-dependent decarboxylating enzyme that uses branched-chain amino acids as substrate. This identification was based on a correlative analysis of the transcriptomes and metabolomes of 36 different E. purpurea tissues and organs, which expressed distinct alkamide profiles. Although no correlation was found between the accumulation patterns of the alkamides and their putative metabolic precursors (i.e., fatty acids and branched-chain amino acids), isotope labeling analyses supported the transformation of valine and isoleucine to isobutylamine and 2-methylbutylamine as reactions of alkamide biosynthesis. Sequence homology identified the pyridoxal phosphate-dependent decarboxylase-like proteins in the translated proteome of E. purpurea. These sequences were prioritized for direct characterization by correlating their transcript levels with alkamide accumulation patterns in different organs and tissues, and this multi-pronged approach led to the identification and characterization of a branched-chain amino acid decarboxylase, which would appear to be responsible for generating the amine moieties of naturally occurring alkamides.

A Clade-Specific Arabidopsis Gene Connects Primary Metabolism and Senescence.

Nearly immobile, plants have evolved new components to be able to respond to changing environments. One example is Qua Quine Starch (QQS, AT3G30720), an Arabidopsis thaliana-specific orphan gene that integrates primary metabolism with adaptation to environment changes. SAQR (Senescence-Associated and QQS-Related, AT1G64360), is unique to a clade within the family Brassicaceae; as such, the gene may have arisen about 20 million years ago. SAQR is up-regulated in QQS RNAi mutant and in the apx1 mutant under light-induced oxidative stress. SAQR plays a role in carbon allocation: overexpression lines of SAQR have significantly decreased starch content; conversely, in a saqr T-DNA knockout (KO) line, starch accumulation is increased. Meta-analysis of public microarray data indicates that SAQR expression is correlated with expression of a subset of genes involved in senescence, defense, and stress responses. SAQR promoter::GUS expression analysis reveals that SAQR expression increases after leaf expansion and photosynthetic capacity have peaked, just prior to visible natural senescence. SAQR is expressed predominantly within leaf and cotyledon vasculature, increasing in intensity as natural senescence continues, and then decreasing prior to death. In contrast, under experimentally induced senescence, SAQR expression increases in vasculature of cotyledons but not in true leaves. In SAQR KO line, the transcript level of the dirigent-like disease resistance gene (AT1G22900) is increased, while that of the Early Light Induced Protein 1 gene (ELIP1, AT3G22840) is decreased. Taken together, these data indicate that SAQR may function in the QQS network, playing a role in integration of primary metabolism with adaptation to internal and environmental changes, specifically those that affect the process of senescence.

Microbial Community and Chemical Characteristics of Swine Manure during Maturation.

Swine diet formulations have the potential to lower animal emissions, including odor and ammonia (NH). The purpose of this study was to determine the impact of manure storage duration on manure chemical and microbial properties in swine feeding trials. Three groups of 12 pigs were fed a standard corn-soybean meal diet over a 13-wk period. Urine and feces were collected at each feeding and transferred to 12 manure storage tanks. Manure chemical characteristics and headspace gas concentrations were monitored for NH, hydrogen sulfide (HS), volatile fatty acids, phenols, and indoles. Microbial analysis of the stored manure included plate counts, community structure (denaturing gradient gel electrophoresis), and metabolic function (Biolog). All odorants in manure and headspace gas concentrations were significantly ( < 0.01) correlated for length of storage using quadratic equations, peaking after Week 5 for all headspace gases and most manure chemical characteristics. Microbial community structure and metabolic utilization patterns showed continued change throughout the 13-wk trial. Denaturing gradient gel electrophoresis species diversity patterns declined significantly ( < 0.01) with time as substrate utilization declined for sugars and certain amino acids, but functionality increased in the utilization of short chain fatty acids as levels of these compounds increased in manure. Studies to assess the effect of swine diet formulations on manure emissions for odor need to be conducted for a minimum of 5 wk. Efforts to determine the impact of diets on greenhouse gas emissions will require longer periods of study (>13 wk).

Patterns of metabolite changes identified from large-scale gene perturbations in Arabidopsis using a genome-scale metabolic network.

Metabolomics enables quantitative evaluation of metabolic changes caused by genetic or environmental perturbations. However, little is known about how perturbing a single gene changes the metabolic system as a whole and which network and functional properties are involved in this response. To answer this question, we investigated the metabolite profiles from 136 mutants with single gene perturbations of functionally diverse Arabidopsis (Arabidopsis thaliana) genes. Fewer than 10 metabolites were changed significantly relative to the wild type in most of the mutants, indicating that the metabolic network was robust to perturbations of single metabolic genes. These changed metabolites were closer to each other in a genome-scale metabolic network than expected by chance, supporting the notion that the genetic perturbations changed the network more locally than globally. Surprisingly, the changed metabolites were close to the perturbed reactions in only 30% of the mutants of the well-characterized genes. To determine the factors that contributed to the distance between the observed metabolic changes and the perturbation site in the network, we examined nine network and functional properties of the perturbed genes. Only the isozyme number affected the distance between the perturbed reactions and changed metabolites. This study revealed patterns of metabolic changes from large-scale gene perturbations and relationships between characteristics of the perturbed genes and metabolic changes.

Metabolomics as a Hypothesis-Generating Functional Genomics Tool for the Annotation of Arabidopsis thaliana Genes of "Unknown Function".

Metabolomics is the methodology that identifies and measures global pools of small molecules (of less than about 1,000 Da) of a biological sample, which are collectively called the metabolome. Metabolomics can therefore reveal the metabolic outcome of a genetic or environmental perturbation of a metabolic regulatory network, and thus provide insights into the structure and regulation of that network. Because of the chemical complexity of the metabolome and limitations associated with individual analytical platforms for determining the metabolome, it is currently difficult to capture the complete metabolome of an organism or tissue, which is in contrast to genomics and transcriptomics. This paper describes the analysis of Arabidopsis metabolomics data sets acquired by a consortium that includes five analytical laboratories, bioinformaticists, and biostatisticians, which aims to develop and validate metabolomics as a hypothesis-generating functional genomics tool. The consortium is determining the metabolomes of Arabidopsis T-DNA mutant stocks, grown in standardized controlled environment optimized to minimize environmental impacts on the metabolomes. Metabolomics data were generated with seven analytical platforms, and the combined data is being provided to the research community to formulate initial hypotheses about genes of unknown function (GUFs). A public database (www.PlantMetabolomics.org) has been developed to provide the scientific community with access to the data along with tools to allow for its interactive analysis. Exemplary datasets are discussed to validate the approach, which illustrate how initial hypotheses can be generated from the consortium-produced metabolomics data, integrated with prior knowledge to provide a testable hypothesis concerning the functionality of GUFs.

Genetic dissection of methylcrotonyl CoA carboxylase indicates a complex role for mitochondrial leucine catabolism during seed development and germination.

3-methylcrotonyl CoA carboxylase (MCCase) is a nuclear-encoded, mitochondrial-localized biotin-containing enzyme. The reaction catalyzed by this enzyme is required for leucine (Leu) catabolism, and it may also play a role in the catabolism of isoprenoids and the mevalonate shunt. In Arabidopsis, two MCCase subunits (the biotinylated MCCA subunit and the non-biotinylated MCCB subunit) are each encoded by single genes (At1g03090 and At4g34030, respectively). A reverse genetic approach was used to assess the physiological role of MCCase in plants. We recovered and characterized T-DNA and transposon-tagged knockout alleles of the MCCA and MCCB genes. Metabolite profiling studies indicate that mutations in either MCCA or MCCB block mitochondrial Leu catabolism, as inferred from the increased accumulation of Leu. Under light deprivation conditions, the hyper-accumulation of Leu, 3-methylcrotonyl CoA and isovaleryl CoA indicates that mitochondrial and peroxisomal Leu catabolism pathways are independently regulated. This biochemical block in mitochondrial Leu catabolism is associated with an impaired reproductive growth phenotype, which includes aberrant flower and silique development and decreased seed germination. The decreased seed germination phenotype is only observed for homozygous mutant seeds collected from a parent plant that is itself homozygous, but not from a parent plant that is heterozygous. These characterizations may shed light on the role of catabolic processes in growth and development, an area of plant biology that is poorly understood.

MetNetGE: interactive views of biological networks and ontologies.

BACKGROUND: Linking high-throughput experimental data with biological networks is a key step for understanding complex biological systems. Currently, visualization tools for large metabolic networks often result in a dense web of connections that is difficult to interpret biologically. The MetNetGE application organizes and visualizes biological networks in a meaningful way to improve performance and biological interpretability. RESULTS: MetNetGE is an interactive visualization tool based on the Google Earth platform. MetNetGE features novel visualization techniques for pathway and ontology information display. Instead of simply showing hundreds of pathways in a complex graph, MetNetGE gives an overview of the network using the hierarchical pathway ontology using a novel layout, called the Enhanced Radial Space-Filling (ERSF) approach that allows the network to be summarized compactly. The non-tree edges in the pathway or gene ontology, which represent pathways or genes that belong to multiple categories, are linked using orbital connections in a third dimension. Biologists can easily identify highly activated pathways or gene ontology categories by mapping of summary experiment statistics such as coefficient of variation and overrepresentation values onto the visualization. After identifying such pathways, biologists can focus on the corresponding region to explore detailed pathway structure and experimental data in an aligned 3D tiered layout. In this paper, the use of MetNetGE is illustrated with pathway diagrams and data from E. coli and Arabidopsis. CONCLUSIONS: MetNetGE is a visualization tool that organizes biological networks according to a hierarchical ontology structure. The ERSF technique assigns attributes in 3D space, such as color, height, and transparency, to any ontological structure. For hierarchical data, the novel ERSF layout enables the user to identify pathways or categories that are differentially regulated in particular experiments. MetNetGE also displays complex biological pathway in an aligned 3D tiered layout for exploration.

Enrichment of Echinacea angustifolia with Bauer alkylamide 11 and Bauer ketone 23 increased anti-inflammatory potential through interference with cox-2 enzyme activity.

Bauer alkylamide 11 and Bauer ketone 23 were previously found to be partially responsible for Echinacea angustifolia anti-inflammatory properties. This study further tested their importance using the inhibition of prostaglandin E(2) (PGE(2)) and nitric oxide (NO) production by RAW264.7 mouse macrophages in the absence and presence of lipopolysaccharide (LPS) and E. angustifolia extracts, phytochemical enriched fractions, or pure synthesized standards. Molecular targets were probed using microarray, qRT-PCR, Western blot, and enzyme assays. Fractions with these phytochemicals were more potent inhibitors of LPS-induced PGE(2) production than E. angustifolia extracts. Microarray did not detect changes in transcripts with phytochemical treatments; however, qRT-PCR showed a decrease in TNF-alpha and an increase of iNOS transcripts. LPS-induced COX-2 protein was increased by an E. angustifolia fraction containing Bauer ketone 23 and by pure phytochemical. COX-2 activity was decreased with all treatments. The phytochemical inhibition of PGE(2) production by Echinacea may be due to the direct targeting of COX-2 enzyme.

Hypericum in infection: Identification of anti-viral and anti-inflammatory constituents.

The Iowa Center for Research on Botanical Dietary Supplements seeks to optimize Echinacea, Hypericum, and Prunella botanical supplements for human-health benefit, emphasizing antiviral, anti-inflammatory and anti-pain activities. This mini-review reports on ongoing studies on Hypericum. The Center uses the genetically diverse, well-documented Hypericum populations collected and maintained at the USDA-ARS North Central Regional Plant Introduction Station (NCRPIS), and the strength of research in synthetic chemistry at Iowa State University to tap natural diversity, to help discover key constituents and interactions among constituents that impact bioactivity and toxicity. The NCRPIS has acquired more than 180 distinct populations of Hypericum, with a focus on Hypericum perforatum L. (Hypericaceae), representing about 13% of currently recognized taxa. Center chemists have developed novel synthetic pathways for key flavones, acyl phloroglucinols, hyperolactones and a tetralin that have been found in Hypericum, and these compounds are used as standards and for bioactivity studies. Both light-dependent and light-independent anti-viral activities have been identified by using bioactivity-guided fractionation of H. perforatum and a HIV-1 infection test system. Our Center has focused on light-independent activity, potentially due to novel chemicals, and polar fractions are undergoing further fractionation. Anti-inflammatory activity has been found to be light-independent, and fractionation of a flavonoid-rich extract revealed four compounds (amentoflavone, chlorogenic acid, pseudohypericin and quercetin) that interacted in the light to inhibit lipopolysaccharide-induced prostaglandin E(2) activity. The Center continues to explore novel populations of H. perforatum and related species to identify constituents and interactions of constituents that contribute to potential health benefits related to infection.

Endogenous levels of Echinacea alkylamides and ketones are important contributors to the inhibition of prostaglandin E2 and nitric oxide production in cultured macrophages.

Because of the popularity of Echinacea as a dietary supplement, researchers have been actively investigating which Echinacea constituent or groups of constituents are necessary for immune-modulating bioactivities. Our prior studies indicate that alkylamides may play an important role in the inhibition of prostaglandin E2 (PGE(2)) production. High-performance liquid chromatography fractionation, employed to elucidate interacting anti-inflammatory constituents from ethanol extracts of Echinacea purpurea, Echinacea angustifolia, Echinacea pallida, and Echinacea tennesseensis, identified fractions containing alkylamides and ketones as key anti-inflammatory contributors using lipopolysaccharide-induced PGE(2) production in RAW264.7 mouse macrophage cells. Nitric oxide (NO) production and parallel cytotoxicity screens were also employed to substantiate an anti-inflammatory response. E. pallida showed significant inhibition of PGE(2) with a first round fraction, containing gas chromatography-mass spectrometry (GC-MS) peaks for Bauer ketones 20, 21, 22, 23, and 24, with 23 and 24 identified as significant contributors to this PGE(2) inhibition. Chemically synthesized Bauer ketones 21 and 23 at 1 microM each significantly inhibited both PGE(2) and NO production. Three rounds of fractionation were produced from an E. angustifolia extract. GC-MS analysis identified the presence of Bauer ketone 23 in third round fraction 3D32 and Bauer alkylamide 11 making up 96% of third round fraction 3E40. Synthetic Bauer ketone 23 inhibited PGE(2) production to 83% of control, and synthetic Bauer alkylamide 11 significantly inhibited PGE(2) and NO production at the endogenous concentrations determined to be present in their respective fraction; thus, each constituent partially explained the in vitro anti-inflammatory activity of their respective fraction. From this study, two key contributors to the anti-inflammatory properties of E. angustifolia were identified as Bauer alkylamide 11 and Bauer ketone 23.

Echinacea tennesseensis ethanol tinctures harbor cytokine- and proliferation-enhancing capacities.

BACKGROUND: Members of the genus Echinacea are used medicinally to treat upper respiratory infections such as colds and influenza. The aim of the present investigation was to characterize the phytomedicinal properties of the American federally endangered species Echinacea tennesseensis. METHODS: Fifty-percent ethanol tinctures were prepared from roots, stems, leaves, and flowers and tested separately for their ability to influence production of IL-1beta, IL-2, IL-10, and TNF-alpha as well as proliferation by young human adult peripheral blood mononuclear cells (PMBC) in vitro. Tincture aliquots were stored at three different temperatures (4, -20, and -80 degrees C) for 21h before testing. At 1-month post-extraction, tinctures stored at -20 degrees C were tested again for cytokine modulation. Phytochemical analyses were performed using HPLC. RESULTS: Fresh root, leaf, and flower tinctures stimulated PBMC proliferation. Fresh root tinctures alone stimulated IL-1beta, IL-10, and TNF-alpha production. No tinctures modulated IL-2 production. Stem tinctures showed no activity. Storage temperature did not influence any outcomes. Root tinctures maintained their ability to modulate IL-1beta, IL-10, and TNF-alpha production after 1month of storage at -20 degrees C. CONCLUSIONS: These results suggest E. tennesseensis harbors phytomedicinal properties that vary by plant organ, with roots demonstrating the strongest activities.

Alcohol extract of Echinacea pallida reverses stress-delayed wound healing in mice.

Healing of open skin wounds begins with an inflammatory response. Restraint stress has been well documented to delay wound closure, partially via glucocorticoid (GC)-mediated immunosuppression of inflammation. Echinacea, a popular herbal immunomodulator, is purported to be beneficial for wound healing. To test the hypothesis, an alcohol extract of E. pallida was administrated orally to mice for 3 days prior to, and 4 days post wounding with a dermal biopsy on the dorsum. Concomitantly, mice were exposed to 3 cycles of daily restraint stress prior to, and 4 cycles post wounding. Echinacea accelerated wound closure in the stressed mice, but had no apparent wound healing effect for the non-stressed mice when compared to their respective controls. To test if the positive healing effect is through modulation of GC release, plasma corticosterone concentrations were measured in unwounded mice treated with restraint stress and the herbal extract for 4 days. Plasma GC in restraint stressed mice gavaged with Echinacea was not different from mice treated with restraint only, but was increased compared to the vehicle control. This data suggests that the improved wound healing effect of Echinacea in stressed mice is not mediated through modulation of GC signaling.

Echinacea increases arginase activity and has anti-inflammatory properties in RAW 264.7 macrophage cells, indicative of alternative macrophage activation.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Echinacea is a popular herbal immunomodulator. Recent reports indicate that Echinacea products inhibit nitric oxide (NO) production in activated macrophages. AIM OF THE STUDY: In the present study we determined the inhibitory effects of alcohol extracts and individual fractions of alcohol extracts of Echinacea on NO production, and explored the mechanism underlying the pharmacological anti-inflammatory activity. MATERIALS AND METHODS: Alcohol extracts of three medicinal Echinacea species, Echinacea angustifolia, Echinacea pallida and Echinacea purpurea, were prepared using Soxhlet apparatus and fractionated using HPLC. NO production by LPS activated RAW 264.7 macrophage cells was measured using a Griess reagent and iNOS detected using immunoblotting. In addition, effects on arginase activity were measured in RAW 264.7 cells stimulated with 8-bromo-cAMP +/- LPS. RESULTS: Alcohol extracts of all three Echinacea species significantly inhibited NO production by lipopolysaccharide (LPS)-activated the RAW 264.7 macrophage cell line; among them Echinacea pallida was the most active. The Echinacea-mediated decrease in NO production was unlikely due to a direct scavenging of NO because the extracts did not directly inhibit NO released from an NO donor, sodium nitroprusside. An immunoblotting assay demonstrated that the extract of Echinacea pallida inhibited inducible nitric oxide synthase (iNOS) protein expression in LPS-treated macrophages. The enzymes iNOS and arginase metabolize a common substrate, l-arginine, but produce distinct biological effects. While iNOS is involved in inflammatory response and host defense, arginase participates actively in anti-inflammatory activation. Arginase activity of RAW 264.7 cells stimulated with 8-bromo-cAMP was significantly increased by alcohol extracts of all three Echinacea species. The polar fraction containing caffeic acid derivatives enhanced arginase activity, while the lipophilic fraction containing alkamides exhibited a potential of inhibiting NO production and iNOS expression. CONCLUSIONS: These results suggest that the anti-inflammatory activity of Echinacea might be due to multiple active metabolites, which work together to switch macrophage activation from classical activation towards alternative activation.

Pseudohypericin is necessary for the light-activated inhibition of prostaglandin E2 pathways by a 4 component system mimicking an Hypericum perforatum fraction.

Hypericum perforatum (Hp) has been used medicinally to treat a variety of conditions including mild-to-moderate depression. Recently, several anti-inflammatory activities of Hp have been reported. An ethanol extract of Hp was fractionated with the guidance of an anti-inflammatory bioassay (lipopolysaccharide (LPS)-induced prostaglandin E2 production (PGE2)), and four constituents were identified. When combined together at concentrations detected in the Hp fraction to make a 4 component system, these constituents (0.1microM chlorogenic acid (compound 1), 0.08microM amentoflavone (compound 2), 0.07microM quercetin (compound 3), and 0.03microM pseudohypericin (compound 4)) explained the majority of the activity of the fraction when activated by light, but only partially explained the activity of this Hp fraction in dark conditions. One of the constituents, light-activated pseudohypericin, was necessary, but not sufficient to explain the reduction in LPS-induced PGE2 of the 4 component system. The Hp fraction and the 4 component system inhibited lipoxygenase and cytosolic phospholipase A2, two enzymes in the PGE2-mediated inflammatory response. The 4 component system inhibited the production of the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha), and the Hp fraction inhibited the anti-inflammatory cytokine interleukin-10 (IL-10). Thus, the Hp fraction and selected constituents from this fraction showed evidence of blocking pro-inflammatory mediators but not enhancing inflammation-suppressing mediators.

Echinacea in infection.

Ongoing studies have developed strategies for identifying key bioactive compounds and chemical profiles in Echinacea with the goal of improving its human health benefits. Antiviral and antiinflammatory-antipain assays have targeted various classes of chemicals responsible for these activities. Analysis of polar fractions of E. purpurea extracts showed the presence of antiviral activity, with evidence suggesting that polyphenolic compounds other than the known HIV inhibitor, cichoric acid, may be involved. Antiinflammatory activity differed by species, with E. sanguinea having the greatest activity and E. angustifolia, E. pallida, and E. simulata having somewhat less. Fractionation and studies with pure compounds indicate that this activity is explained, at least in part, by the alkamide constituents. Ethanol extracts from Echinacea roots had potent activity as novel agonists of TRPV1, a mammalian pain receptor reported as an integrator of inflammatory pain and hyperalgesia and a prime therapeutic target for analgesic and antiinflammatory drugs. One fraction from E. purpurea ethanol extract was bioactive in this system. Interestingly, the antiinflammatory compounds identified to inhibit prostaglandin E(2) production differed from those involved in TRPV1 receptor activation.

Enhancement of innate and adaptive immune functions by multiple Echinacea species.

Echinacea preparations are commonly used as nonspecific immunomodulatory agents. Alcohol extracts from three widely used Echinacea species, Echinacea angustifolia, Echinacea pallida, and Echinacea purpurea, were investigated for immunomodulating properties. The three Echinacea species demonstrated a broad difference in concentrations of individual lipophilic amides and hydrophilic caffeic acid derivatives. Mice were gavaged once a day (for 7 days) with one of the Echinacea extracts (130 mg/kg) or vehicle and immunized with sheep red blood cells (sRBC) 4 days prior to collection of immune cells for multiple immunological assays. The three herb extracts induced similar, but differential, changes in the percentage of immune cell populations and their biological functions, including increased percentages of CD49+ and CD19+ lymphocytes in spleen and natural killer cell cytotoxicity. Antibody response to sRBC was significantly increased equally by extracts of all three Echinacea species. Concanavalin A-stimulated splenocytes from E. angustifolia- and E. pallida-treated mice demonstrated significantly higher T cell proliferation. In addition, the Echinacea treatment significantly altered the cytokine production by mitogen-stimulated splenic cells. The three herbal extracts significantly increased interferon-alpha production, but inhibited the release of tumor necrosis factor-gamma and interleukin (IL)-1beta. Only E. angustifolia- and E. pallida-treated mice demonstrated significantly higher production of IL-4 and increased IL-10 production. Taken together, these findings demonstrated that Echinacea is a wide-spectrum immunomodulator that modulates both innate and adaptive immune responses. In particular, E. angustifolia or E. pallida may have more anti-inflammatory potential.

Inhibition of prostaglandin E(2) production by anti-inflammatory hypericum perforatum extracts and constituents in RAW264.7 Mouse Macrophage Cells.

Hypericum perforatum (Hp) is commonly known for its antiviral, antidepressant, and cytotoxic properties, but traditionally Hp was also used to treat inflammation. In this study, the anti-inflammatory activity and cytotoxicity of different Hp extractions and accessions and constituents present within Hp extracts were characterized. In contrast to the antiviral activity of Hp, the anti-inflammatory activity observed with all Hp extracts was light-independent. When pure constituents were tested, the flavonoids, amentoflavone, hyperforin, and light-activated pseudohypericin, displayed anti-inflammatory activity, albeit at concentrations generally higher than the amount present in the Hp extracts. Constituents that were present in the Hp extracts at concentrations that inhibited the production of prostaglandin E(2) (PGE(2)) were pseudohypericin and hyperforin, suggesting that they are the primary anti-inflammatory constituents along with the flavonoids, and perhaps the interactions of these constituents and other unidentified compounds are important for the anti-inflammatory activity of the Hp extracts.

Echinacea species and alkamides inhibit prostaglandin E(2) production in RAW264.7 mouse macrophage cells.

Inhibition of prostaglandin E(2) (PGE(2)) production in lipopolysaccharide-stimulated RAW264.7 mouse macrophage cells was assessed with an enzyme immunoassay following treatments with Echinacea extracts or synthesized alkamides. Results indicated that ethanol extracts diluted in media to a concentration of 15 microg/mL from E. angustifolia, E. pallida, E. simulata, and E. sanguinea significantly inhibited PGE2 production. In further studies, PGE2 production was significantly reduced by all synthesized alkamides assayed at 50 microM, by Bauer alkamides 8, 12A analogue, and 14, Chen alkamide 2, and Chen alkamide 2 analogue at 25 microM and by Bauer alkamide 14 at 10 microM. Cytotoxicity did not play a role in the noted reduction of PGE2 production in either the Echinacea extracts or synthesized alkamides. High-performance liquid chromatography analysis identified individual alkamides present at concentrations below 2.8 microM in the extracts from the six Echinacea species (15 microg/mL crude extract). Because active extracts contained <2.8 microM of specific alkamide and the results showed that synthetic alkamides must have a minimum concentration of 10 microM to inhibit PGE2, it is likely that alkamides may contribute toward the anti-inflammatory activity of Echinacea in a synergistic or additive manner.

Alcohol extracts of Echinacea inhibit production of nitric oxide and tumor necrosis factor-alpha by macrophages in vitro.

It has been suggested that Echinacea has anti-inflammatory activity in vivo. Nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), and interleukin-1beta are important mediators in the inflammatory response. The effect of alcohol extracts of E. angustifolia (EA), E. pallida (EPA) and E. purpurea (EP) on the production of these inflammatory mediators in both LPS-stimulated RAW 264.7 macrophages in vitro and murine peritoneal exudate cells (PECs) in vivo were investigated. As macrophages produce these inflammatory mediators in response to pathogenic infection, parallel cultures of macrophages were studied for phagocytosis and intracellular killing of Salmonella enterica. EPA and EP in vitro inhibited NO production and TNF-α release in a dose-dependent manner. RAW 264.7 cells treated with EA or EP showed decreased killing over 24 h, although EA enhanced bacterial phagocytosis. Upon bacterial infection, RAW 264.7 cells produce high levels of NO; however, an Echinacea-mediated decrease in NO production was observed. Echinacea alcohol extracts administered orally at 130 mg/kg per day for seven days had a weak effect on NO production and phagocytosis by LPS-stimulated PECs. The results indicated that all Echinacea species significantly decreased inflammatory mediators in vitro, however, only EA and EP reduced bacterial killing. Oral administration of Echinacea alcohol extracts did not adversely affect the development and anti-bacterial function of inflammatory PECs in vivo, however, NO production was decreased during bacterial infection of PECs.