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Immuno-suppressive effect of blocking the CD28 signaling pathway in T-cells by an active component of Echinacea found by a novel pharmaceutical screening method.

AFTIR (after flowing through immobilized receptor) is a novel method for screening herbal extracts for pharmaceutical properties. Using AFTIR, we identified Cynarin in Echinacea purpurea by its selective binding to chip immobilized CD28, a receptor of T-cells, which is instrumental to immune functioning. The results of surface plasma resonance show that binding between immobilized CD28 and Cynarin is stronger than the binding between CD28 and CD80, a co-stimulated receptor of antigen presenting cells. Cynarin's function was verified by its ability to downregulate CD28-dependent interleukin-2 (IL-2) expression in a T-cell culture line. AFTIR offers promise as an efficient screening method for herbal medicines.

Animals↗

Macrophage activating effects of new alkamides from the roots of Echinacea species.

Chemical investigation of the roots of Echinacea angustifolia, E. purpurea, and E. pallida yielded two new alkamides, identified by analysis of spectroscopic data and comparison with reported alkamides. The new compounds were dodeca-2Z,4E,10Z-trien-8-ynoic acid isobutylamide (1) from E. angustifolia and dodeca-2Z,4E-diene-8,10-diynoic acid isobutylamide (2) from E. purpurea and E. pallida. These two components, as well as previously identified alkamides, exerted inhibition on LPS-mediated activation of a murine macrophage line, RAW264.7. These data suggest that these alkamides may have anti-inflammatory activity. The cytotoxicity of these alkamides using MTT assays was also investigated.

Amides↗

Stereoselective synthesis, natural occurrence and CB(2) receptor binding affinities of alkylamides from herbal medicines such as Echinacea sp.

A divergent synthesis of (2E,4E,8E,10E)- and (2E,4E,8E,10Z)-N-isobutyldodeca-2,4,8,10-tetraenamides from pent-4-yn-1-ol allowed identification of the (2E,4E,8E,10Z)-isomer for the first time in Echinacea species. A short, stereoselective synthesis of the (2E,4E,8E,10Z)-isomer is also described which allowed further biological evaluation of this material, and the demonstration that this isomer does not occur in Spilanthes mauritiana as previously reported.

Echinacea↗

[New knowledge regarding the effect and effectiveness of Echinacea purpurea extracts].

Preparations made from the pressed juice of the flowering aerial parts of the Purple Coneflower, Echinacea purpurea (L.) Moench represent the most commonly used herbal immunnomodulatory agents. They are frequently administered for the supportive treatment of common viral infections of the upper respiratory tract. New clinical studies are reviewed, which demonstrated that corresponding preparations can diminish the severity and the length of common colds significantly, and that they can also be used efficiently for the treatment of children. New investigations have also shown, that stimulation of makrophages and induction of cytokins are major parts of the mode of action, and that glycoproteins/polysaccharides and alkamides are part of the activity relevant constituents.

Adjuvants, Immunologic↗

Efficacy of Echinacea purpurea in patients with a common cold. A placebo-controlled, randomised, double-blind clinical trial.

Common colds are one of the most frequent acute illnesses with major economical impact. Echinaceae purpureae herba (Echinacin, EC31J0) has shown promising results in the relief of common cold symptoms and the time taken to improvement compared to placebo. This study was aimed to confirm these findings by performing a randomised, double-blind, placebo-controlled clinical trial. A total of 80 adult male or female patients with first signs of a cold were recruited. The number of days of illness with a complete picture of the common cold (defined by the modified Jackson score of at least 5 points and experience of rhinorrhea and/or a subjective sensation of having a cold) was the primary end-point. In the verum group the median time of illness was 6.0 days compared to 9.0 days in the placebo group, assigning zero time for patients without a complete picture (one-sided p = 0.0112). EC31J0 was well tolerated and clinically effective in alleviating symptoms more rapidly than placebo in patients with a common cold.

Adult↗

Alkamide levels in Echinacea purpurea: effects of processing, drying and storage.

The effects of processing, drying, and storage time and temperature on alkamide levels in Echinacea purpurea roots are described. Chopping altered the levels of some alkamides slightly, whereas drying had no effect. Levels of all alkamides fell by over 80% during storage at 24 degrees C for 64 weeks. Alkamide levels also dropped significantly during storage at -18 degrees C.

Amides↗

Genetic characterization of the three medicinal Echinacea species using RAPD analysis.

The three medicinal species of the Echinacea genus, E. angustifolia DC., E. pallida (Nutt.) Nutt. and E. purpurea (L.) Moench were distinguished using the RAPD (random amplified polymorphic DNA) technique. Species-specific markers were identified from amplicons obtained with four of the twenty 10-mer primers contained in the Operon RAPD kit A. In particular, one marker was identified for E. angustifolia (OPA 20, 1800 pb) and E. pallida (OPA 10, 600 pb) and three markers for E. purpurea (OPA 11 : 1250 pb; OPA 17 : 750, 1800 pb). Genetic distance analysis indicated a high degree of difference among the three species with a relative lower difference between E. angustifolia and E. pallida.

Echinacea↗

The arabinogalactan-proteins from pressed juice of Echinacea purpurea belong to the hybrid class of hydroxyproline-rich glycoproteins.

Pressed juices from Echinacea purpurea are used as non-specific immunostimulants, and arabinogalactan-proteins (AGPs) are part of the active principle. An AGP fraction was isolated from pressed juice of E. purpurea by precipitation with ss-glucosyl Yariv reagent, followed by gel-permeation chromatography. Polyclonal antibodies directed against the carbohydrate moiety of this AGP fraction showed a preferential specificity for E. purpurea AGPs from pressed juice over those extracted from E. purpurea suspension culture and other plant species. Native AGPs purified from this AGP fraction by RP-HPLC were then deglycosylated for N-terminal protein sequencing resulting in the identification of three major polypeptides. They show characteristic motifs of classical AGPs but also some features of extensins, suggesting these may be "hybrid" hydroxyproline-rich glycoproteins (HRGPs).

Chromatography, High Pressure Liquid↗

Structure and accumulation of phenolics in elicited Echinacea purpurea cell cultures.

Echinacea purpurea cell cultures have been established for the production of secondary and possibly bioactive compounds using elicitation techniques. Different elicitors including yeast elicitor, methyl jasmonate, glutathione, and manganese ions were used and compared for their effects on the formation of phenolics in the cell cultures. The accumulation of phenolics in the medium after elicitation with glutathione and manganese ions showed similar patterns as detected by HPLC in control medium. However, in yeast elicitor- and methyl jasmonate-treated cell suspension cultures, other phenolics were formed and accumulated in the medium. In contrast, both control and elicited cells contained the same pattern of phenolics. The main phenolics both in the medium and cells, 19 in total, were isolated and identified on the basis of chromatographic, chemical (derivatisation), enzymatic and spectroscopic techniques. The medium contained lignans, neolignans and acetophenone derivatives as main elicitor-enhanced products. The cells mainly contained phenolic glycosides including a new compound, alpha- O-beta-D-glucopyranosylacetovanillone. Detailed collision-induced dissociation electrospray ionisation mass spectrometric fragmentation pathways for 8,4'-oxyneolignan glycosides are discussed.

Cell Culture Techniques↗

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

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

Animals↗

Bioactivity of alkamides isolated from Echinacea purpurea (L.) Moench.

Alkamides from the roots of Echinacea purpurea (L.) Moench were examined for anti-inflammatory activity in an in vitro model system. Cyclooxygenase-I (COX-I) and cyclooxygenase-II (COX-II) inhibitory activities were assessed at pH 7 for alkamides isolated from E. purpurea roots to compare inhibitory activities between the two cyclooxygenase isozymes. At 100 microg/ml, several E. purpurea alkamides inhibited COX-I and COX-II enzymes in the range of 36-60% and 15-46%, respectively, as compared to controls. Mosquitocidal activity was assessed at 100 and 10 microg/ml, with 100% mortality against Aedes aegyptii L. larvae noted for several E. purpurea alkamides at 100 microg/ml.

Aedes↗

Enhancement of the humoral immune response by Echinacea purpurea in female Swiss mice.

Various preparations of the plant Echinacea purpurea have been investigated for their potential to enhance immune function, primarily through activation of innate immune responses. Few studies have examined the potential for enhancement of humoral immunity. Using female Swiss mice we administered a volumetric dose of a glycerine extract of E. purpurea by oral gavage, to evaluate effects on the IgM specific antibody forming cell (AFC) response. Four days of treatment following immunization with sheep red blood cells (SRBC) produced a significant enhancement over naive controls at doses of 0.4 and 0.8 mL/kg/day. A few clinical trials and anecdotal reports have suggested that the greatest efficacy for E. purpurea occurs in acute use following onset of illness. A time course study, using the time of SRBC immunization to mimic the onset of illness, examined the effects of 8 and 4 days of E. purpurea treatment at 0.6 mL/kg/day. Only in the 4-day administration, with dosing beginning 1 hour after SRBC immunization, was there an observed enhancement of the antibody forming cell response. This supports the acute use of E. purpurea as suggested by anecdotal reports, and demonstrates the potential for enhancement of humoral immune responses as well as innate immune responses.

Adjuvants, Immunologic↗

Echinacea purpurea and melatonin augment natural-killer cells in leukemic mice and prolong life span.

OBJECTIVE: We recently showed that daily dietary administration of Echinacea purpurea root extract to normal mice for as little as 1 week resulted in significant elevations of natural-killer (NK) cells (immune cells that are cytolytic to virus-containing cells and many tumor cells). Such boosting of this fundamental immune cell population suggests a prophylactic role for this herb in normal animals. Based on this evidence, our goal in the present work was to assess the role of dietary administration of this herbal extract to mice bearing leukemia, a type of tumor well known to be a target for NK cells. DESIGN: A commercially available root extract of E. purpurea, which we have already shown to be highly effective in mice, was administered daily for 50 days from the onset of leukemia (day 0). Control leukemic mice received no extract. Other leukemic mice received the NK-enhancing neurohormone, melatonin, administered precisely as above. In all treatment and control categories, some mice were sampled at 9 days after tumor onset, others were sampled at 3 months, and still others were left to assess treatment effect on life span. RESULTS: At 9 days (intermediate stage leukemia; death beginning by day 17-18), E. purpurea-treated mice had a 2.5-fold increase in the absolute numbers of NK cells in their spleens. By 3 months after leukemia onset, E. purpurea-treated mice still had 2-3 times the normal numbers of NK cells in their spleens. No leukemic, untreated (control) mice remained alive at 3 months, hence the comparison with normal animals. Moreover, at 3 months post-tumor onset, all the major hemopoietic and immune cell lineages in their bone marrow birth site, were recorded at normal numbers, in E. purpurea-consuming, leukemic mice. The survival advantage provided by administering these leukemic mice with E. purpurea was highly significant versus untreated, leukemic mice when analyzed by Kaplan-Meier survival statistics. CONCLUSION: The present study has provided the first systematic analysis, under controlled laboratory conditions, of the effect(s) of the botanical, E. purpurea, in vivo, in leukemic hosts. The profoundly positive effects of this herb in disease abatement observed in this study suggest the therapeutic potential of E. purpurea, at least with respect to leukemia, if not other tumors as well.

Animals↗

The effect of immunization with killed tumor cells, with/without feeding of Echinacea purpurea in an erythroleukemic mouse model.

OBJECTIVE: Tumor amelioration via vaccination/immunization is a practice for which considerable experimental and clinical support is growing. Combination therapies have proven to be more beneficial than treatment with single agents. We hypothesized that immunization of mice with killed erythroleukemia cells prior to the induction of erythroleukemia via injection of viable tumor cells, plus dietary administration of a known immuno-enhancing phytocompound, Echinacea purpurea, would be more effective than immunization alone. DESIGN: A commercially available extract of E. purpurea root, already proven as a natural killer (NK) cell stimulant, was administered via the chow, for periods of 9 days or 3 months after the onset of leukemia to mice which had been injected (immunized) 5 weeks earlier with killed leukemia cells. RESULTS: Immunized mice (+/- E. purpurea) had significantly prolonged life spans versus non-immunized mice, with an even greater proportion of hosts surviving long-term in the E. purpurea-fed group. NK cells, the mediators of nonspecific immunity and well-demonstrated mediators of tumor cytolysis, were very significantly elevated in immunized, leukemic mice receiving E. purpurea in their diet versus those receiving untreated chow. Early in tumor development (9 days), cells mediating specific immunity (T, B lymphocytes) were 10-12 times higher in absolute numbers in the spleens in all immunized, leukemic mice vs unimmunized, leukemic mice at the same stage of tumor progression. CONCLUSIONS: The results demonstrate that combination therapy, involving specific tumor cell immunization, followed by daily phytotherapy (dietary E. purpurea), sensitized the immune cells and led to life span prolongation greater than that provided by immunization alone.

Animals↗

Analysis of the inhibitory potential of Ginkgo biloba, Echinacea purpurea, and Serenoa repens on the metabolic activity of cytochrome P450 3A4, 2D6, and 2C9.

OBJECTIVE: To study the potential of three top-selling herbal products, Ginkgo biloba, Echinacea purpurea, and Serenoa repens to inhibit the in vitro enzymatic activity of three of the most important drug metabolizing enzymes, cytochrome P450 (CYP) 3A4, 2D6, and 2C9. METHODS: High throughput CYP inhibition screening was used to test the inhibitory capacity of extracts of commercially available herbal medications on the metabolism of CYP enzyme substrates. RESULTS: S. repens showed potent inhibition of the metabolic activity of all three CYPs tested. The effects of G. biloba and E. purpurea varied. E. purpurea demonstrated mild inhibition of CYP3A4 activity with 7- benzyloxy-4-trifluoromethylcoumarin (BFC) as the model substrate, but mild inducing effects in the presence of the model substrate resorufin benzyl ether (BzRes). Little effect on CYP2D6 and moderate inhibition of CYP2C9 was seen with both E. purpurea and G. biloba. G. biloba also showed mild-to-moderate inhibition of CYP3A4 depending on the model substrate. CONCLUSIONS: The inhibitory capacity of herbal products varies depending on the concentrations of the model substrate and the herbal extract, as well as the identity of the model substrate, as demonstrated by the varied effects of CYP3A4 enzymatic activity with different model substrates. The potential for strong adverse interactions exists for Serenoa repens, which was a potent inhibitor of all three CYPs examined. Physicians are encouraged to advise patients of the risks of combining herbal products with prescription medications.

Aryl Hydrocarbon Hydroxylases↗

Floral nectar production and nectary anatomy and ultrastructure of Echinacea purpurea (Asteraceae).

BACKGROUND AND AIMS: In spite of the impressive species diversity in the Asteraceae and their widespread appeal to many generalist pollinators, floral-nectary ultrastructure in the family has rarely been investigated. To redress this, a study using Echinacea purpurea, a plant of horticultural and nutraceutical value, was undertaken. Nectar secretion of disc florets was compared with floral nectary ultrastructure taking into account nectar's potential impact upon the reproductive success of this outcrossing species. METHODS: Micropipette collections of nectar in conjunction with refractometry were used to determine the volume and nectar-sugar quantities of disc florets throughout their phenology, from commencement of its production to cessation of secretion. Light, scanning-electron and transmission-electron microscopy were utilized to examine morphology, anatomy and ultrastructure of nectaries of the disc florets. KEY RESULTS: Florets were protandrous with nectar being secreted from anthesis until the third day of the pistillate phase. Nectar production per floret peaked on the first day of stigma receptivity, making the two innermost whorls of open florets most attractive to foraging visitors. Modified stomata were situated along the apical rim of the collar-like nectary, which surrounds the style base and sits on top of the inferior ovary. The floral nectary was supplied by phloem only, and both sieve elements and companion cells were found adjacent to the epidermis; the latter participated in the origin of some of the precursor cells that yielded these specialized cells of phloem. Companion cells possessed wall ingrowths (transfer cells). Lobed nuclei were a key feature of secretory parenchyma cells. CONCLUSIONS: The abundance of mitochondria suggests an eccrine mechanism of secretion, although dictyosomal vesicles may contribute to a granulocrine process. Phloem sap evidently is the main contributor of nectar carbohydrates. From the sieve elements and companion cells, an apoplastic route via intercellular spaces and cell walls, leading to the pores of modified stomata, is available. A symplastic pathway, via plasmodesmata connecting sieve elements to companion, parenchyma and epidermal cells, is also feasible. Uncollected nectar was reabsorbed, and the direct innervation of the nectary by sieve tubes potentially serves a second important route for nectar-sugar reclamation. Microchannels in the outer cuticle may facilitate both secretion and reabsorption.

Carbohydrates↗

Sequence assessment of comigrating AFLPTM bands in Echinacea -- implications for comparative biological studies.

The extent of sequence identity among clones derived from monomorphic and polymorphic AFLPTM polymorphism bands was quantified. A total of 79 fragments from a monomorphic band of 273 bp and 48 fragments from a polymorphic band of 159 bp, isolated from individuals belonging to different populations, varieties, and species of Echinacea, were cloned and sequenced. The monomorphic fragments exhibited above 90% sequence identity among clones within samples. Sequence identity within variety ranged from 82.78% to 94.87% and within species from 75.82% to 98.9% and was 57.97% in the genus. The polymorphic fragments exhibited much less sequence identity. In some instances, even two clones from the same fragment were different in their size and sequence. Within sample, clone sequence identity ranged from 100% to 51.57%, within variety from 33.33% to 100% in one variety, and from 23.66% to 45% within species and was as low as 1.25% within the genus. In addition, sequences of the same size were aligned to verify the nature of their sequence dissimilarity/similarity. Within each size group, identical sequences were found across species and varieties. In general, comigrating bands cannot be considered homologous. Thus, the use of AFLPTM band data for comparative studies is appropriate only if the results emanating from such analyses are considered as approximations and are interpreted as phenotypic but not genotypic.

Base Sequence↗

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.

Antioxidants↗