Alkamide stability in Echinacea purpurea extracts with and without phenolic acids in dry films and in solution

Degradation of the major alkamides in E. purpurea extracts was monitored under four different accelerated storage conditions, phenolic-depleted and phenolic-rich dry E. purpurea extracts and phenolic-depleted and phenolic-rich DMSO E. purpurea extracts at 70, 80, and 90 degrees C. Degradation of alkamides followed apparent first-order reaction rate kinetics. Alkamides degraded faster in dry films than in DMSO solution. The phenolic acids acted as antioxidants by limiting the loss of the alkamides in dry E. purpurea extracts. In contrast, E. purpurea alkamides in DMSO degraded faster when the phenolic fraction was absent. The overall order of degradation rate constants was alkamides 1 approximately 2 approximately 6 > 9 approximately 8 > 3 approximately 5 approximately 7. The energy of activation (Ea) predicted for alkamide degradation averaged 101 +/- 12 kJ/mol in dry films +/- phenolic acids, suggesting the oxidation mechanism was the same under both conditions. In DMSO solutions, Ea values were about one-half of those in dry films (61 +/- 14 kJ/mol), suggesting a different mechanism for alkamide oxidation in solution compared to dry. Predicted half-lives for alkamides in extracts suggested very good stability.     

Retention of alkamides in dried Echinacea purpurea

Different drying methods, such as freeze-drying (FD), vacuum microwave drying (VMD), and air-drying (AD), were applied to fresh roots and leaves of Canadian-grown Echinacea purpurea to determine the optimal method for preserving alkamide levels. Using HPLC, six alkamide fractions (alkamides 1, 2, 3, 6a/6, 7, 8/9) were quantitated in dried roots, whereas four alkamide fractions (alkamides 1, 2, 3, 8/9) were measured in dried leaves. Different elution conditions used in HPLC for alkamide analysis did not affect the eluted fractions nor the quantitation of different alkamides. Individual alkamide concentrations in roots and leaves were affected by the drying methods used. To preserve higher levels of total alkamides, FD was found to be the best method, VMD was a superior method for drying roots than AD at 70 degrees C, while AD at 50 degrees C was the preferred method for drying leaves of E. purpurea.     

Synergistic antioxidative effects of alkamides, caffeic acid derivatives, and polysaccharide fractions from Echinacea purpurea on in vitro oxidation of human low-density lipoproteins

Preparations of Echinacea are widely used as alternative remedies to prevent the common cold and infections in the upper respiratory tract. After extraction, fractionation, and isolation, the antioxidant activity of three extracts, one alkamide fraction, four polysaccharide-containing fractions, and three caffeic acid derivatives from Echinacea purpurea root was evaluated by measuring their inhibition of in vitro Cu(II)-catalyzed oxidation of human low-density lipoprotein (LDL). The antioxidant activities of the isolated caffeic acid derivatives were compared to those of echinacoside, caffeic acid, and rosmarinic acid for reference. The order of antioxidant activity of the tested substances was cichoric acid > echinacoside > or = derivative II > or = caffeic acid > or = rosmarinic acid > derivative I. Among the extracts the 80% aqueous ethanolic extract exhibited a 10 times longer lag phase prolongation (LPP) than the 50% ethanolic extract, which in turn exhibited a longer LPP than the water extract. Following ion-exchange chromatography of the water extract, the majority of its antioxidant activity was found in the latest eluted fraction (H2O-acidic 3). The antioxidant activity of the tested Echinacea extracts, fractions, and isolated compounds was dose dependent. Synergistic antioxidant effects of Echinacea constituents were found when cichoric acid (major caffeic acid derivative in E. purpurea) or echinacoside (major caffeic acid derivative in Echinacea pallida and Echinacea angustifolia) were combined with a natural mixture of alkamides and/or a water extract containing the high molecular weight compounds. This contributes to the hypothesis that the physiologically beneficial effects of Echinacea are exerted by the multitude of constituents present in the preparations.     

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.     

HPLC method validated for the simultaneous analysis of cichoric acid and alkamides in Echinacea purpurea plants and products

A reversed-phase high-performance liquid chromatography (HPLC) method has been developed to determine caffeic acid derivatives, for example, cichoric acid, and alkamides in plant parts and herbal products of Echinacea purpurea. The method consists of an extraction procedure whereby the hydrophilic phenolics as well as the lipophilic alkamides are released from the samples, followed by the analytical HPLC procedure for quantitative determination of these compounds. The method is the first one validated for the determination of these two groups of compounds in the same procedure. Naringenin has been used as an internal standard, as no other flavanones are present in the extract and it does not interfere with any of the compounds under investigation. Analysis of Danish-grown plant material shows that it is possible to raise plants of a very high chemical quality in Denmark. A selection of international herbal products available on the Danish market show surprisingly variable quality, not necessarily reflecting the product information given on the labels.     

Phytochemical variation in echinacea from roots and flowerheads of wild and cultivated populations

Quantitative phytochemical variation was determined from roots and inflorescences of native plant populations in the genus Echinacea. Specimens were collected in situ throughout the natural range of each putative taxon and transplanted to greenhouse cultivation. Ethanolic extracts from individual plants were separated by reversed-phase HPLC to quantify the alkamides, polyenes/ynes, and phenolics, and then grouped by age and taxonomically, according to a recent morphometric taxonomic revision of the genus. Canonical discriminant analysis revealed that cichoric acid, the diene alkamides 1-3 and 7, and ketoalkene 24 were the best taxonomic markers. Mean content for each of 26 phytochemicals revealed useful agronomic information, such as those varieties and organs with the highest accumulations, as well as the optimal age and growth conditions for each variety. The highest amounts of cichoric acid were measured from the older, wild inflorescences of E. pallida var.sanguinea, whereas the highest quantities of the alkamides 1-3 and 7 were present in roots of wild and transplanted E. purpurea. Baseline phytochemical data and chromatographic profiles for all types of wild Echinacea may be used for protection of wild stands, germplasm identification, and crop improvement.     

Stabilization of caffeic acid derivatives in Echinacea purpurea L. glycerin extract

Recent work has shown that enzymatic degradation and oxidation of cichoric acid and other caffeic derivatives occurs in Echinacea preparations. However, very little is known as to the means of stabilizing these phytopreparations. To stabilize the glycerin extract of Echinacea purpurea, we have evaluated the effects of 3 natural antioxidants (citric acid, malic acid, and hibiscus extract) on the stability of the major caffeic acid derivatives (caftaric acid, caffeic acid, cichoric acid, and 2-O-feruloyl-tartaric acid). Chlorogenic acid, which normally occurs in an ethanol extract of E. purpurea, was not present in the glycerin extract. The caffeic acid derivatives, with the exception of 2-O-feruloyl-tartaric acid, were subject to degradation in the control sample. 2-O-Feruloyl-tartaric acid was stable during the whole testing period. All antioxidant treatments greatly improved the stability of caffeic acid derivatives. Stability was dependent upon the concentration of antioxidant added.     

Inhibition of diacylglycerol acyltransferase by alkamides isolated from the fruits of Piper longum and Piper nigrum

Pharmacological inhibition of acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) has emerged as a potential therapy for the treatment of obesity and type 2 diabetes. Bioassay-guided isolation of CHCl3 extracts of the fruits of Piper longum and Piper nigum (Piperaceae), using an in vitro DGAT inhibitory assay, lead to isolation of a new alkamide named (2E,4Z,8E)-N-[9-(3,4-methylenedioxyphenyl)-2,4,8-nonatrienoyl]piperidine (2), together with four known alkamides: retrofractamide C (1), pipernonaline (3), piperrolein B (4), and dehydropipernonaline (5). Compounds 2-5 inhibited DGAT with IC50 values of 29.8 (2), 37.2 (3), 20.1 (4), and 21.2 (5) microM, respectively, but the IC50 value for 1 was more than 900 microM. This finding indicates that compounds possessing piperidine groups (2-5) can be potential DGAT inhibitors.     

Fungistatic and bacteriostatic activities of alkamides from Heliopsis longipes roots: affinin and reduced amides

This work demonstrates the fungistatic and bacteriostatic activities of affinin, the main alkamide of Heliopsis longipes (Gray) Blake (Asteraceae) roots and two alkamides obtained by catalytic reduction of affinin: N-isobutyl-2E-decenamide and N-isobutyl-decanamide. The bioactivity was tested against Rhizoctonia solani groups AG3 and AG5, Sclerotium rolfsii, Sclerotium cepivorum, Fusarium sp., Vertcillium sp., phytopathogenic fungi; Phytophthora infestans, a phytopathogenic Chromista; Saccharomyces cerevisiae, a nonphytopathogenic ascomycete; and Escherichia coli, Erwinia carotovora, and Bacillus subtilis, bacteria. Affinin, being the primary component of the lipidic fraction, is expected to be responsible for the fungitoxic activity observed in roots of this plant species. Four of the assayed fungi showed an important sensitivity to the presence of affinin: S. rolfsii, S. cepivorum, P. infestans, and R. solani AG-3 and AG-5, displaying a growth inhibition of 100%. S. cerevisiaeshowed a similar growth inhibition with affinin. None of the alkamides obtained by catalytic reduction of affinin showed a fungitoxic activity. Affinin had a definite negative effect on the growth of E. coli and B. subtilis, but E. carotovora carotovora was not sensitive to the highest dose of affinin assayed. N-Isobutyl-2E-decenamide displayed a higher bacteriostatic activity against E. coli and E. carotovora carotovora. In both cases, this alkamide was more potent than affinin. On the other hand, only N-isobutyl-decanamide displayed a significant activity on the growth of B. subtilis.     

Retention of caffeic acid derivatives in dried Echinacea purpurea

Different drying methods were applied to fresh Canadian-grown Echinacea purpurea flowers to determine optimal drying procedures for preserving caffeic acid derivatives. Fresh flowers of E. purpurea were dried by freeze-drying (FD), vacuum microwave drying with full vacuum (VMD), and air-drying (AD) at 25, 40, and 70 degrees C. Using HPLC, chicoric acid and caftaric acid levels were quantitated in dried flowers. These acids were significantly affected by the drying method conditions used. Although significant (p < 0.05) loss of chicoric acid was observed when flowers were stored at high moisture, VMD flowers with a low moisture content retained the highest levels of chicoric acid and caftaric acid similar to FD flowers. Flowers that were AD at 25 degrees C retained about 50%, while those dried by AD at 70 degrees C resulted in the lowest retention of these acids. Although flowers dried by AD at 40 degrees C retained relatively high amounts of chicoric acid and caftaric acid, the time (55 h) required to reach optimal drying was considerably longer than that (47 min) for VMD.     

How does tomato quality (sugar, acid, and nutritional quality) vary with ripening stage, temperature, and irradiance?

The objective of this study was to understand the respective impact of ripening stage, temperature, and irradiance on seasonal variations of tomato fruit quality. During ripening, concentrations in reducing sugars, carotenes, ascorbate, rutin, and caffeic acid derivates increased, whereas those in titratable acidity, chlorophylls, and chlorogenic acid content decreased. Fruit temperature and irradiance affected final fruit composition. Sugars and acids (linked to fruit gustative quality) were not considerably modified, but secondary metabolites with antioxidant properties were very sensitive to fruit environment. Increased fruit irradiance enhanced ascorbate, lycopene, beta-carotene, rutin, and caffeic acid derivate concentrations and the disappearance of oxidized ascorbate and chlorophylls. Increasing the temperature from 21 to 26 degrees C reduced total carotene content without affecting lycopene content. A further temperature increase from 27 to 32 degrees C reduced ascorbate, lycopene, and its precursor's content, but enhanced rutin, caffeic acid derivates, and glucoside contents. The regulation by light and temperature of the biosynthesis pathways of secondary metabolites is discussed.     

Transepithelial transport of chlorogenic acid, caffeic acid, and their colonic metabolites in intestinal caco-2 cell monolayers

Both chlorogenic and caffeic acids exhibited nonsaturable transport in Caco-2 cells, whereas caffeic acid also showed proton-coupled polarized absorption. Thus, the absorption efficiency of caffeic acid was greater than that of chlorogenic acid. Polarized transport of caffeic acid was inhibited by substrates of MCT such as benzoic and acetic acids. Almost all of the apically loaded chlorogenic and caffeic acid was retained on the apical side, and the transepithelial flux was inversely correlated with the paracellular permeability of Caco-2 cells. These results indicate that transport was mainly via paracellular diffusion, although caffeic acid was absorbed to a lesser extent by the monocarboxylic acid transporter (MCT). Furthermore, m-coumaric acid and 3-(m-hydroxyphenyl)propionic acid, the main metabolites of chlorogenic and caffeic acid by colonic microflora, competitively inhibited the transport of fluorescein, a known substrate of MCT. This suggests that their absorption could also be mediated by MCT. These findings have exemplified the physiological importance of MCT-mediated absorption in both phenolic acids per se and their colonic metabolites.     

Echinacea standardization: analytical methods for phenolic compounds and typical levels in medicinal species

A proposed standard extraction and HPLC analysis method has been used to measure typical levels of various phenolic compounds in the medicinally used Echinacea species. Chicoric acid was the main phenolic in E. purpurea roots (mean 2.27% summer, 1.68% autumn) and tops (2.02% summer, 0.52% autumn), and echinacoside was the main phenolic in E. angustifolia (1.04%) and E. pallida roots (0.34%). Caftaric acid was the other main phenolic compound in E. purpurea roots (0.40% summer, 0.35% autumn) and tops (0.82% summer, 0.18% autumn), and cynarin was a characteristic component of E. angustifolia roots (0.12%). Enzymatic browning during extraction could reduce the measured levels of phenolic compounds by >50%. Colorimetric analyses for total phenolics correlated well with the HPLC results for E. purpurea and E. angustifolia, but the colorimetric method gave higher values.     

Caffeic acid derivatives production by hairy root cultures of Echinacea purpurea

Inoculation of leaf explants of Echinacea purpurea (Moench) with Agrobacterium rhizogenes induced hairy roots with the capacity to produce biologically active caffeic acid derivatives (CADs), especially cichoric acid. The kinetics of growth, the uptake of macronutrients, and the accumulation of CADs were investigated in heterotrophically cultured hairy roots for a 50 day period. A maximum of 12.2 g L(-1) dry biomass was achieved in MS nutrients supplemented with 30 g L(-1) sucrose on day 40. The mathematical relationship between hairy root growth and conductivity was established during the exponential phase in Erlenmeyer flasks. HPLC analyses of methanolic (0.1% phosphoric acid; 70:30, v/v) extracts from hairy roots revealed the presence of important CADs: cichoric acid (19.21 mg g(-1) dry biomass), caftaric acid (3.56 mg g(-1) dry biomass), and chlorogenic acid (0.93 mg g(-1) dry biomass). These results demonstrate that biotechnological production of CADs in hairy roots of E. purpurea is possible. Furthermore, these hairy root cultures offer, for the very first time, an excellent biological model to study the biosynthetic pathway of medicinally important CADs.     

Absorption, disposition, metabolism, and excretion of [3-(14)C]caffeic acid in rats

Male Sprague-Dawley rats ingested 140 × 10(6) dpm of [3-(14)C]trans-caffeic acid, and over the ensuing 72 h period, body tissues, plasma, urine, and feces were collected and the overall levels of radioactivity determined. Where sufficient radioactivity had accumulated, samples were analyzed by HPLC with online radioactivity and tandem mass spectrometric detection. Nine labeled compounds were identified, the substrate and its cis isomer, 3'-O- and 4'-O-sulfates and glucuronides of caffeic acid, 4'-O-sulfates and glucuronides of ferulic acid, and isoferulic acid-4'-O-sulfate. Four unidentified metabolites were also detected. After passing down the gastrointestinal tract, the majority of the radiolabeled metabolites were excreted in urine with minimal accumulation in plasma. Only relatively small amounts of an unidentified (14)C-labeled metabolite were expelled in feces. There was little or no accumulation of radioactivity in body tissues, including the brain. The overall recovery of radioactivity 72 h after ingestion of [3-(14)C]caffeic acid was ～80% of intake.     

Content of polyphenolic compounds in the Nigerian stimulants Cola nitida ssp. alba, Cola nitida ssp. rubra A. Chev, and Cola acuminata Schott & Endl and their antioxidant capacity

Varieties of kola nuts (Cola nitida alba, Cola nitida rubra A. Chev, and Cola acuminata Schott & Endl), a group of popular Nigerian and West African stimulants, were analyzed for their content of secondary plant metabolites. The three varieties of the kola nuts contained appreciable levels of (+)-catechin (27-37 g/kg), caffeine (18-24 g/kg), (-)-epicatechin (20-21 g/kg), procyanidin B 1 [epicatechin-(4beta-->8)-catechin] (15-19 g/kg), and procyanidin B2 [epicatechin-(4beta-->8)-epicatechin] (7-10 g/kg). Antioxidant capacity of the extracts and purified metabolites was assessed by two HPLC-based and two colorimetric in vitro assays. Extracts of all varieties exhibited antioxidant capacity with IC 50 values in the range 1.70-2.83 and 2.74-4.08 mg/mL in the hypoxanthine/xanthine oxidase and 2-deoxyguanosine HPLC-based assays, respectively. Utilization of HPLC-based assays designed to reflect in situ generation of free radicals (e.g., HO(*)), as opposed to general assays (DPPH, FRAP) in common use which do not, indicate that, of the major secondary plant metabolites present in kola nut extracts, caffeine is potentially the more effective cancer chemopreventive metabolite in terms of its antioxidant capacity.     

Absorption of chlorogenic acid and caffeic acid in rats after oral administration

Absorption of orally administered chlorogenic acid (5-caffeoylquinic acid) and caffeic acid in rats was studied to obtain plasma pharmacokinetic profiles of their metabolites. Rats were administered 700 micromol/kg body weight of chlorogenic or caffeic acid, and blood was collected from the tail for 6 h after administration. Ingested caffeic acid was absorbed from the alimentary tract and was present in the rat blood circulation in the form of various metabolites. On the other hand, only traces of metabolites, supposedly caffeic and ferulic acids conjugates, were detected in rat plasma for 6 h after chlorogenic acid administration. Chlorogenic acid and small amounts of caffeic acid were found in the small intestine for 6 h after chlorogenic acid administration. These results suggest that chlorogenic acid is not well absorbed from the digestive tract, unlike caffeic acid, and subject to almost no structural changes to the easily absorbed forms.     

Antioxidative activity of propolis extract in yeast cells

The antioxidative activities of propolis and its main phenolic compounds, caffeic acid, p-coumaric acid, ferulic acid, and caffeic acid phenethyl ester, were investigated in the yeast Saccharomyces cerevisiae. After 1 h of exposure of the yeast cells, their intracellular oxidation was measured using 2',7'-dichlorofluorescein. Yeast cells exposed to 96% ethanolic extracts of propolis in DMSO (EEP) showed decreased intracellular oxidation, with no significant differences seen for the individual phenolic compounds. However, cellular uptake was seen only for a moderately polar fraction of EEP (E2) and caffeic acid phenethyl ester. The EEP antioxidative activity thus resulted from this E2 fraction of EEP. The influence of EEP was also investigated at the mitochondrial proteome level, by analyzing its profile after 1 h of exposure of the yeast cells to EEP and E2. Changes in the levels of antioxidative proteins and proteins involved in ATP synthesis were seen.     

Chemical and biological characterization of cinnamic acid derivatives from cell cultures of lavender (Lavandula officinalis) induced by stress and jasmonic acid

Cell cultures of lavender (Lavandula officinalis) were analyzed for the metabolite profile under normal growth conditions and under stress as well as after jasmonic acid treatment. The main compound synthesized was rosmarinic acid, which was also secreted into the culture medium. Different solvent extraction methods at different pH values altered the profile slightly. Anoxic stress induced the synthesis of a cinnamic acid derivative, which was identified as caffeic acid by gas chromatography-mass spectrometry. Caffeic acid was also induced after treatment of the cell cultures with jasmonic acid. Although the antioxidative activity of both compounds, rosmarinic acid and caffeic acid, was confirmed in an assay using 2,2-diphenyl-1-picrylhydrazyl (DPPH), it was demonstrated that both substances have a low cytotoxic potential in vitro using acute myeloid leukemia (HL-60) cells. The potential of the system for finding new bioactive compounds is discussed.