Echinacea Angustifolia Roots Research Articles

Anxiofit-1 and reduction of subthreshold and mild anxiety: evaluation of a health claim pursuant to Article 14 of Regulation (EC) No 1924/2006.

Following an application from Anxiofit Ltd., submitted for authorisation of a health claim pursuant to Article 14 of Regulation (EC) No 1924/2006 via the Competent Authority of Hungary, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on the scientific substantiation of a health claim related to Anxiofit‐1 and reduction of subthreshold and mild anxiety. The food, Anxiofit‐1, Echinacea angustifolia root extract, standardised for the content of echinacoside (at least 3%) and the profile of alkamides, which is the subject of the health claim, is sufficiently characterised. The Panel considers that reduction of subthreshold and mild anxiety is a beneficial physiological effect. Subthreshold and mild anxiety are risk factors for anxiety and depressive disorders. One human intervention study showed an effect of Anxiofit‐1 (80 mg/day given for 7 days) on the state anxiety and not on the trait subscale in subjects with subthreshold or mild anxiety. These results are supported by two human intervention studies conducted with Anxiofit‐1 at 40 mg/day for 7 days and 6 weeks, respectively, which, on their own, cannot be used for the substantiation of the claim either because of methodological limitations or because the results cannot be extrapolated to the target population for the claim. All the human intervention studies submitted have been conducted in a similar setting, the results of the study with Anxiofit‐1 given at 80 mg/day have not been confirmed by other research groups. The information submitted by the applicant does not provide evidence for a plausible mechanism by which Anxiofit‐1 could exert the claimed effect. The Panel concludes that the scientific evidence is insufficient to establish a cause and effect relationship between the consumption of Anxiofit‐1 and reduction of subthreshold and mild anxiety.

Anxiofit‐1 and reduction of subthreshold and mild anxiety: evaluation of a health claim pursuant to Article 14 of Regulation (EC) No 1924/2006

Following an application from Anxiofit Ltd. and ExtractumPharma Co Ltd., submitted for authorisation of a health claim pursuant to Article 14 of Regulation (EC) No 1924/2006 via the Competent Authority of Hungary, the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA) was asked to deliver an opinion on the scientific substantiation of a health claim related to Anxiofit-1 and reduction of subthreshold and mild anxiety. The food, Anxiofit-1, Echinacea angustifolia root extract, standardised for the content of echinacoside (at least 3%) and the alkamide profile, which is the subject of the health claim, is sufficiently characterised. The Panel considers that reduction of subthreshold and mild anxiety is a beneficial physiological effect. Subthreshold and mild anxiety is a risk factor for anxiety and depressive disorders. One human intervention study with a small number of participants showed a decrease in anxiety scores under the conditions of use proposed by the applicant. Another small study, performed by the same research group with patients with diagnosed generalised anxiety disorder, also showed a decrease in anxiety in this population. However, the Panel notes that the results have not been replicated in other studies. The information supplied by the applicant did not provide evidence for a plausible mechanism by which Anxiofit-1 could exert the claimed effect. The Panel concludes that the scientific evidence is insufficient to establish a cause and effect relationship between the consumption of Anxiofit-1 and reduction of subthreshold and mild anxiety.

Harvest in different years of growth influences chemical composition of Echinacea angustifolia roots

Echinacea angustifolia DC., native from North America, is one of the three Echinacea species that are widely used as herbal remedy, mainly for their immunostimulant properties. Echinacoside, cynarin and alkylamides are the main constituents of the species.Five plants of two accessions (JA2 and JA9) of narrow-leaved coneflower were harvested from the second to the fifth year of cultivation and secondary metabolites content were determined in the whole root as well as in different samples such as the crown, cortex, internal tissue and thin roots.The highest content of marker compounds was found in the thin roots (0.20% for cynarin and 1.11% for echinacoside and from 0.29 to 0.44% for dodeca-2E,4E,8Z,10E/Z-tetraenoic isobutylamide) and in the root cortex (0.16% for cynarin and 1.01% for echinacoside and from 0.31 to 0.48% for dodeca-2E,4E,8Z,10E/Z-tetraenoic isobutylamide). Our results demonstrated that root cortex tissue accumulates the marker compounds, explaining why the thin roots present the higher amount of secondary metabolite.Regarding the entire roots, differences between the accessions (JA9 versus JA2, except for cynarin) and among the years were observed. The lowest and the highest echinacoside content was measured in the JA9 accession, in the second (0.39%) and in the fifth (1.47%) year of growing, respectively. The lowest and the highest dodeca-2E,4E,8Z,10E/Z-tetraenoic isobutylamide content was found in the third year, 0.43% in JA9 and 0.16% in JA2, respectively. The lowest and the highest cynarin content was obtained in the fourth year (0.11% in JA9) and in the fifth (0.22% in JA2), respectively.These results may be useful for Echinacea growers and processors to improve their agronomical and post-harvesting choices.

Proliferative activity of a blend of Echinacea angustifolia and Echinacea purpurea root extracts in human vein epithelial, HeLa, and QBC-939 cell lines, but not in Beas-2b cell lines

Echinacea is used for its immunostimulating properties and may have a role in modulating adverse immune effects of chemotherapy (i.e., use of 5-fluorouracil (5-FU); fluorouracil and its immunosuppressive effect). Patients may seek herbal remedies such as Echinacea (Echinacea angustifolia and Echinacea purpurea) for immune stimulation. Echinacea extracts have been prescribed to supplement cancer chemotherapy for their immune-supportive effects; however, the extracts may also influence tumourgenesis. Our study aimed to determine the proliferative effect of the ethanolic blend of E. angustifolia and E. purpurea on various cancer cervical and bile duct cell lines, including HELA and QBC-939. Various cancer cells (HeLa and QBC-939) and human vein epithelial cells (HUVEC) were treated with the Echinacea blend sample that was evaporated and reconstituted in Dimethyl sulfoxide (DMSO). As the extract concentration of Echinacea was increased from 12.5 μg/mL to 25 μg/mL, there was an increase in cell inhibition up to 100%, which then reduced to 90% over the next three concentrations, 50 μg/mL, 100 μg/mL, and 200 μg/mL, in HeLa cells; further inhibitory effects were observed in QBC-939 cells, from 9% inhibition at a concentration of 25 μg/mL up to 37.96% inhibition at 100 μg/mL concentration. Moreover, this is the first study to report the growth-promoting effects of this Echinacea blend in HUVEC, up to 800% at a dose concentration of 200 μg/mL. Previous studies have suggested that chicoric acid of Echinacea spp. is responsible for the increased cell growth. The results of this study show that the hydroethanolic extract of Echinacea herbal medicine promotes the growth of HeLa cells and QBC-939 cancer cell proliferation, and may interfere with cancer treatment (i.e., chemotherapy drugs such as 5-fluorouracil and Cisplatin (DDP)). However, the Echinacea blend shows potential in neurodegenerative diseases with growth-promoting effects in HUVEC. Further animal trials (in vivo effect) measuring dose toxicology are necessary to demonstrate the interaction of this blend with body and tumor growth, and also any positive synergistic or adverse interaction with chemotherapeutic drugs listed, so as to confirm the current observation and epithelial tissue growth or regeneration in a neurodegenerative disease model.

Possible Role of Fat Tissue in the Pharmacokinetics of Dodeca-2E,4E,8Z,10E/Z-tetraenoic Acid Isobutylamides after Oral Administration of Echinacea angustifolia Extract in Rats

Alkamides are one of the most important constituents of lipophilic extracts of Echinacea angustifolia roots. These compounds play an important role in the versatile pharmacological actions of this plant. The present study aimed to compare the concentrations of isomeric dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (DTAI) in brain and periepididymal fat tissues and blood plasma of rats. Thirty, 60, 240 and 720 min after the oral administration of E. angustifolia root extract, tissue and plasma concentrations were determined by reversed-phase HPLC with ESI-MS/MS detection. The calculated terminal t1/2 of the mixture of DTAI was 8.28 h, which indicates a relatively slow elimination. In the 0.5-4 h period the brain/plasma and fat/plasma concentration ratios were continuously above 3 and 18, respectively, followed by equilibrium at 12 h. Our results indicate substantial accumulation of alkamides in lipid-rich tissues, which presumably contributes to a maintained pharmacological action.

Immunomodulation mediated by a herbal syrup containing a standardized Echinacea root extract: A pilot study in healthy human subjects on cytokine gene expression

In this study, the immunomodulatory effect of a triply standardized Echinacea angustifolia root extract (Polinacea®) was evaluated in 10 healthy subjects. Ten ml of syrup containing one hundred mg of extract (corresponding to 4.7mg of Echinacoside and 8.0mg of a high molecular weight-20,000Da- polysaccharide) were administered as a herbal syrup once a day for one month. The immunomodulatory effect was evaluated before and after herbal syrup administration evaluating the expression levels of the cytokines IL-2, IL-8, IL-6 and TNF-α. Cytokine expression was studied in lympho-monocytes and in plasma samples measuring the mRNA and protein levels, respectively. The results were analysed by ANOVA and non-parametric Friedman rank sum tests; when possible it was adopted a pair-wise comparisons at different post-treatment times, using the paired t-tests with Holm correction. The correlation between the variations of cytokine plasma levels and the respective mRNA was carried out using a linear regression model.In lympho-monocytes our data indicate the up-regulation of the mRNA levels of IL-2 and IL-8 and the down regulation of the mRNA levels of the pro-inflammatory cytokines TNF-α and IL6. The differential regulation was maximal after 14 days of treatment. IL-2 up-regulation and IL-6 down-regulation were also confirmed at the protein level in plasma. Finally, the up-regulation of the mRNA of IL-2/IL-8 and the down-regulation of IL-6 positively correlated with the protein levels detected in the plasma.In conclusion, this pilot study suggests a relevant role for the standardized Echinacea angustifolia root extract in the control of cytokine expression. This first demonstration of the immuno-modulating activity of Echinacea angustifolia root extract in the healthy subject, supports at least in part the common use of such products as health promoting supplement.

Management of Gastrointestinal Mucositis Due to Cancer Therapies in Pediatric Patients: Results of a Case Series with SAMITAL ®

Gastrointestinal mucositis is a common debilitating complication of chemotherapy and one for which there is currently no effective long-term treatment. To report our experience with the use of SAMITAL(®), a new oral suspension formulation based on the combination of three standardized extracts from Vaccinium myrtillus, Macleaya cordata fruits and Echinacea angustifolia roots in the prevention and treatment of chemotherapy-induced gastrointestinal mucositis in pediatric patients. 20 pediatric patients undergoing chemotherapy for a range of oncological conditions were followed. Patients initially received oral SAMITAL(®) to treat gastrointestinal mucositis and were then given SAMITAL(®) prophylactically to prevent recurrences with successive cycles of chemotherapy. SAMITAL(®) significantly decreased gastrointestinal mucositis grade after the first episode with a reduction of mean scores from 3.2 ± 0.7 at baseline to 0.4 ± 0.6 at the end of treatment (p < 0.001). SAMITAL(®) reduced pain, mucosal erosions, bleeding and dysphagia/feeding impairment. SAMITAL(®) improved patients' overall condition and quality of life after the first administration and lowered the need for parenteral nutrition. Importantly, it allowed chemotherapy cycles to be continued without complications. Results from this case series suggest that SAMITAL(®) may play an important role in the prevention and treatment of chemotherapy-induced gastrointestinal mucositis in children and adolescents and as such warrants investigation in controlled studies.

Administration of botanicals with the diet regulates gene expression in peripheral blood cells of Sarda sheep during ACTH challenge

The aim of the present research was to investigate the regulation of gene expression in ovine blood leukocytes during ACTH-induced cortisol release and the effect of dietary administration of botanicals to counteract the evoked response in polymorphonucleate cells (PMNCs). Thirty-six homogeneous Sarda sheep (age, 18 ± 4.1 mo; BW, 38.7 ± 1.3 kg) were allotted to six groups of six sheep each. One group was used as a negative control (Saline) and five groups were treated, every 12 h for 48 h, with 0.5 mL of ACTH agonist (250 μg/mL of tetracosactrin). Before ACTH treatment, four of the five ACTH-treated groups were separated and fed for 22 d with a basal diet supplemented with extracts from Echinacea angustifolia roots (PO + ACTH), Echinacea angustifolia flowers (EA + ACTH), Andrographis paniculata (AP + ACTH), and the bark of Larix decidua milled (LB + ACTH). Control groups (Saline and ACTH) were fed with the same basal diet without botanicals. Total RNA was extracted from blood samples collected before (T0) and after 3 h (T3) and 51 h (T51) from the first ACTH injection, and transcriptome analysis was performed using a custom oligoarray, designed from 12,194 Ovis aries UniGenes on a CombiMatrix platform. At T3, treatment with ACTH caused down-regulation of transcripts (P < 0.001) involved in “response to stress” (GADD45A, GADD45B, WRNIP1, and XRCC6) and in “innate immune response” (MAPK3 and NFkBIB). At T51, treatment with ACTH caused down-regulation (P < 0.001) of genes involved in “immune response” (IFNG and IL2) and up-regulation (P < 0.001) of NF-κB1 and TP53. Each botanical produced a different (P < 0.001) molecular signature for these genes at T3 and T51. The most active botanical in modulating transcriptome modifications in PMNCs after ACTH-induced cortisol release was Larix decidua Mill bark followed by Polinacea roots. These botanicals can be viewed as promising feed supplements in ruminants to cope with conditions associated with increased concentrations of plasma cortisol.

Antifungal activity of extracts by supercritical carbon dioxide extraction from roots of Echinacea angustifolia and analysis of their constituents using gas chromatography-mass spectrometry (GC-MS)

In this work the supercritical fluid extraction with carbon dioxide was applied to obtain the extracts from roots of Echinacea angustifolia DC. The extracts were evaluated through the antifungal in vitro assay against fungal strain Botrytis cinerea. Furthermore, the active compounds from the extracts were separated by macroporous resin and analyzed using gas chromatography-mass spectrometry (GC-MS). The results show that the extracts, obtained using SC-CO2 extraction technique, had strong antifungal activity against B. cinerea with EC50 and EC90 values of 948 and 1869 μg/ml, minimum inhibitory concentration (MIC) of 250 μg/ml, and minimum fungicidal concentration (MFC) of 2000 μg/ml, respectively. After separation with macroporous resin chromatography, nine fractions were collected from theSC-CO2 extracts. At concentration of 2000 μg/ml, fractions 3, 7, and 8 presented highly antifungal activity against B. cinerea, with inhibition ratio of 40.59, 73.04 and 53.37%, respectively. Determined by the GC-MS analysis, the composition of fraction 7 consisted of different kinds of volatile oils, most of which were ketones (82.71%) and unsaturated hydrocarbons (9.59%). The present study has provided the new evidence that the Echinaceaextracts are involved in the antifungal activity. Key words: Antifungal activity, Echinacea angustifolia DC, gas chromatography-mass spectrometry (GC-MS), SC–CO2 extraction.

Analysis of Alkylamides in Echinacea Plant Materials and Dietary Supplements by Ultrafast Liquid Chromatography with Diode Array and Mass Spectrometric Detection

Alkylamides are a class of compounds present in plants of the genus Echinacea (Asteraceae), which have been shown to have high bioavailability and immunomodulatory effects. Fast analysis to identify these components in a variety of products is essential to profile products used in clinical trials and for quality control of these products. A method based on ultrafast liquid chromatography (UFLC) coupled with diode array detection and electrospray ionization mass spectrometry was developed for the analysis of alkylamides from the roots of Echinacea angustifolia (DC.) Hell., Echinacea purpurea (L.) Moench, and commercial dietary supplements. A total of 24 alkylamides were identified by LC-MS. The analysis time for these components is 15 min. Compared to the alkylamide profiles determined in the Echinacea root materials, the commercial products showed a more complex profile due to the blending of root and aerial parts of E. purpurea. This versatile method allows for the identification of alkylamides in a variety of Echinacea products and presents the most extensive characterization of alkylamides in E. angustifolia roots so far.

Anti-inflammatory activity of a polysaccharidic fraction of Echinacea angustifolia.

The anti-inflammatory activity of a polysaccharidic fraction (EPF) obtained from Echinacea angustifolia roots has been examined using the carrageenan paw oedema and the croton oil ear test. EPF (0.5 mg kg-1 i.v.) almost inhibited the carrageenan-induced oedema over 8 h and furthermore, EPF, topically applied, inhibited mouse ear oedema induced by croton oil. EPF also reduced the leukocytic infiltration of the croton oil dermatitis, evaluated both as peroxidase activity and histologically. After topical application EPF appears to be slightly inferior in potency to indomethacin. The results suggest that the anti-inflammatory activity of E. angustifolia resides in its polysaccharidic content.

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

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

F.20. A c-jun N-terminal Kinase (JNK)/Apoptosis/Inflammation-based Gene Card for Multiple Sclerosis Diagnosis and Patient Stratification

The aim of the present research was to investigate the regulation of gene expression in ovine blood leukocytes during ACTH-induced cortisol release and the effect of dietary administration of botanicals to counteract the evoked response in polymorphonucleate cells (PMNCs). Thirty-six homogeneous Sarda sheep (age, 18 ± 4.1 mo; BW, 38.7 ± 1.3 kg) were allotted to six groups of six sheep each. One group was used as a negative control (Saline) and five groups were treated, every 12 h for 48 h, with 0.5 mL of ACTH agonist (250 μg/mL of tetracosactrin). Before ACTH treatment, four of the five ACTH-treated groups were separated and fed for 22 d with a basal diet supplemented with extracts from Echinacea angustifolia roots (PO + ACTH), Echinacea angustifolia flowers (EA + ACTH), Andrographis paniculata (AP + ACTH), and the bark of Larix decidua milled (LB + ACTH). Control groups (Saline and ACTH) were fed with the same basal diet without botanicals. Total RNA was extracted from blood samples collected before (T0) and after 3 h (T3) and 51 h (T51) from the first ACTH injection, and transcriptome analysis was performed using a custom oligoarray, designed from 12,194 Ovis aries UniGenes on a CombiMatrix platform. At T3, treatment with ACTH caused down-regulation of transcripts (P < 0.001) involved in “response to stress” (GADD45A, GADD45B, WRNIP1, and XRCC6) and in “innate immune response” (MAPK3 and NFkBIB). At T51, treatment with ACTH caused down-regulation (P < 0.001) of genes involved in “immune response” (IFNG and IL2) and up-regulation (P < 0.001) of NF-κB1 and TP53. Each botanical produced a different (P < 0.001) molecular signature for these genes at T3 and T51. The most active botanical in modulating transcriptome modifications in PMNCs after ACTH-induced cortisol release was Larix decidua Mill bark followed by Polinacea roots. These botanicals can be viewed as promising feed supplements in ruminants to cope with conditions associated with increased concentrations of plasma cortisol.

OPTIMIZATION OF THE CULTURE CONDITIONS FOR THE BIOMASS AND PHENOLICS PRODUCTION FROM ADVENTITIOUS ROOTS OF ECHINACEA ANGUSTIFOLIA

In this study, we have investigated the effect of different concentrations of auxins (IAA, IBA, NAA), MS medium strength, sucrose, ammonium/nitrate, initial medium pH and inoculum size on bioniass increase and total phenols and flavonoids accumulation from adventitious roots of Echinacea angustifolia. Adventitious roots were cultured in shake flask for 4 weeks under dark conditions. Among different auxins tested, IBA was best auxin for adventitious root proliferation compare to IAA and NAA. Root growth was inhibited when the initial pH was maintained below 5.0 or above 6.0. Nitrate rather than ammonium was necessary for root growth and phenolics accumulation. Biomass increase and total phenols and flavonoids contents were maximized in half strength MS media supplemented with 2 mg L -1 IBA, 5% (w/v), sucrose, 5:25 (mM) ammonium/nitrate, adjusted medium pH to 6.0 before autoclaving, and inoculum size of 10 g L -1 FW. The results indicated that in vitro environments strongly affect the growth and accumulation of phenolics from Echinacea adventitious root culture. Culture optimization is beneficial to large-scale production of biomass and secondary metabolites in Echinacea angustifolia.

Comparison of Echinacea alkylamide pharmacokinetics between liquid and tablet preparations

The relative oral bioavailability of alkylamides from two different Echinacea dosage forms (liquid and tablet) were compared in a small two-way crossover study in humans (n=3). The liquid preparation investigated contained a mixture of Echinacea purpurea root (300 mg/ml) and Echinacea angustifolia root (200 mg/ml) extracted in 60% ethanol. The tablet preparation investigated was also a mixture of E. purpurea root (675 mg/tablet) and E. angustifolia root (600 mg/tablet), but was prepared from the dried 60% ethanolic extracts of these two Echinacea species. Alkylamides were found to be rapidly absorbed and measurable in plasma from both preparations. No significant differences in the tetraene alkylamide pharmacokinetic parameters for T(1/2), AUC(t-lin) and C(max) in the two different preparations were found. T(max) increased from 20 min for the liquid to 30 min for the tablet, which is not unexpected as the tablet required time for disintegration before absorption could occur. These results suggested that there was no significant difference in the bioavailability of alkylamides from the liquid and tablet Echinacea formulations. Furthermore, the results also indicated that the absorption site and any alkylamide loss due to digestive processes were similar in both preparations.

Comparison of alkamide pharmacokinetics between equivalent liquid and tablet echinacea preparations

The traditional liquid preparations of echinacea suffer poor compliance due to the strong, characteristic tingling sensation associated with the alkamides. Tablet preparations are a dose form which offers much better compliance, but there have been concerns that alkamides from these are not as well absorbed. To investigate this concern, the relative oral bioavailability of alkamides from two different echinacea formulations (liquid and tablet) were compared in a small two way crossover study in humans (n=3) and their pharmacokinetics parameters compared. The liquid preparation was a mixture of Echinacea purpurea L. (Moench) root (300mg/mL) and Echinacea angustifolia DC. root (200mg/mL) extracted in 60% ethanol. The tablet preparation was also a mixture of Echinacea purpurea root (675mg/tablet) and Echinacea angustifolia root (600mg/tablet) but was prepared from the dried ethanolic extracts of the two Echinacea species. 9 mL of the liquid, containing 9.1mg of the tetraene alkamide, was diluted to 25 mL with water and then swallowed immediately. 3 tablets, containing 7.8mg of the tetraene alkamide were swallowed with 25 mL of water. Alkamides were rapidly absorbed and measurable in plasma from both preparations. No significant differences in the tetraene alkamide pharmacokinetic parameters for T1/2, AUCt-lin and Cmax in the two different preparations were found. Tmax increased from 20 minutes with the liquid to 30 minutes with the tablet which is not unexpected as the tablet required time for disintegration before absorption could occur. By swallowing the liquid product immediately, the normal method of dosing for this product, any buccal absorption should have been minimised. These results suggest that there is no significant difference in the bioavailability of alkamides from liquid and tablet echinacea formulations and that both any alkamide loss due to digestive processes and the absorption site is similar in both preparations.

Alkamides from Echinacea angustifolia roots inhibit Cyclooxygenase-2-dependent Prostaglandin synthesis in Human Neuroglioma Cells

During past years inhibition of the cyclooxygenase-2 (COX-2) enzyme has been proven as an effective strategy to suppress pain and inflammation. Based on this and other mechanistic findings, interest has also renewed in the molecular pathways underlying the anti-inflammatory effects of herbal drugs. The present study addressed this issue and investigated the impact of several polyunsaturated alkamides isolated from a CO2 extract of the roots of Echinacea angustifolia DC. with both activity and expression of COX-2. Experiments were performed using the human neuroglioma cell line H4, which has been established as a suitable model for studying molecular mechanisms and pathways involved in COX-2 expression [1, 2]. A 48-h treatment of H4 human neuroglioma cells with the CO2 extract led to a significant downregulation of prostaglandin E2 formation. Analysis of 8 different alkamides revealed a contribution of undeca 2Z-ene-8,10-diynoic acid isobutylamide (A5), dodeca-2E-ene-8,10-diynoic acid isobutylamide (A7) and dodeca-2E,4Z-diene-8,10-diynoic acid 2-methylbutylamide (A8) to this response. Using an established short-term COX-2 activity assay all three alkamides were shown to interfere with COX-2 activity. In contrast, none of the COX-2-suppressing nor any other tested alkamide was found to inhibit COX-2 expression at the transcriptional and translational level. Overall, our results suggest that certain alkamides derived from Echinacea angustifolia roots may contribute to the pharmacological action of the herbal extract by inhibiting COX-2-dependent prostaglandin E2 formation at sites of inflammation.

Optimization of culturing conditions for the production of biomass and phenolics from adventitious roots ofEchinacea angustifolia

We investigated different concentrations of auxins (1AA, IBA, NAA), the strength of the MS medium, sucrose and ammonium/nitrate contents, initial medium pH, and inoculum size to determine their effects on biomass increase and the accumulation of total phenols and flavonoids in adventitious roots ofEchinacea angustifolia. These roots were cultured under darkness in shake flasks for 4 weeks. IBA proved the best auxin for inducing root proliferation. Root growth was inhibited when the initial pH was maintained below 5.0 or above 6.0. Nitrate, rather than ammonium, was more necessary for root growth and phenolics accumulations. Overall, biomass increased and total phenol and flavonoid contents were maximized under the following conditions: half-strength MS medium supplemented with 2 mg L-1 IBA, 5% (w/v) sucrose, 5:25 (mM) ammonium/nitrate ratio, pH adjusted to 6.0 before autoclaving, and an inoculum size of 10 g L-1 FW. These results indicate that the type ofin vitro environment strongly affects growth and the accumulation of phenolics from adventitious root cultures ofE. angustifolia. Such optimization is beneficial to large-scale production of biomass and secondary metabolites in that species.

Proliferative Activity ofEchinacea angustifolia Root Extracts on Cancer Cells: Interference with Doxorubicin Cytotoxicity

Doxorubicin is an anticancer drug that causes apoptosis in cells, but cardiotoxicity limits the cumulative dose that can remain in the blood. Echinacea extracts have been prescribed to supplement cancer chemotherapy. In a recent study, it was reported that Echinacea purpurea extracts protected noncancerous cells from apoptosis. Our study aimed to determine interference with doxorubicin chemotherapy, and if fractions and compounds from Echinacea angustifolia roots protected the cells. Cervical and breast cancer cells were treated with the Echinacea samples and doxorubicin. At 0.05 and 0.5 microM doxorubicin concentration, cynarine increased HeLa cell growth by 48-125% and 29-101%, respectively (p<0.01). At 0.05 microM doxorubicin concentration, chicoric acid increased cell growth by 23-100% (p<0.01). When MCF-7 cells were treated with Echinacea and doxorubicin, the ethyl acetate fraction increased cell growth by 20-25%, and chicoric acid increased cell growth by 10-15%. Cynarine showed proliferative activity on HeLa cells, but showed antiproliferative activity on MCF-7 cells. Results indicate that phenolic compounds are responsible for proliferative activity. Studies with individual compounds show that chicoric acid and cynarine interfered with cells treated with 0.5 microM doxorubicin. The results of this study show that Echinacea herbal medicines affect cell proliferation despite cancer treatment, and that herbal medicines require further study with respect to anticancer drugs.

Bioavailability of Echinacea root powder phenolics in humans

We hypothesized that Echinacea angustifolia, purpurea, and pallida root powder phenolics were metabolized and found in the body as caffeic acid (the main subunit of most of these phenolics). Healthy adult subjects, 6 female and 6 male received 4 g of each Echinacea root powder in a single dose, each dose separated by a one-week washout period in a randomized crossover design. Subjects were instructed to consume diets of minimal phenolic content for 2 days before dosing, and foods provided for all meals during the day of each feeding were low in phenolics. Phenolic contents of E. angustifolia, E. purpurea, and E. pallida were 49.6 μmol (mainly echinacoside), 41.2 μmol (mainly cichoric acid), and 41.0 μmol (mainly echinacoside and cichoric acid)/4 g, respectively. In subjects who consumed E. purpurea and E. pallida, cichoric acid and a smaller peak tentatively identified as caffeic acid, were detected in urine. The 24h urinary recovery of cichoric acid in subjects who consumed E. purpurea and E. pallida were 0.36 ± 0.25 and 0.34 ± 0.31 % of ingested dose, respectively. These results indicate a lack of any significant conversion of Echinacea phenolic acids into caffeic acid, and very low bioavailability of cichoric acid, such that is unlikely that this compound contributes much to the bioactivities of Echinacea. Funded by NIEHS Grant No. PO1 ES12020, Center for Research on Dietary Botanical Supplements, Iowa State University.