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.     

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.     

Effect of drying temperature on alkylamide and cichoric acid concentrations of Echinacea purpurea

Root and aerial sections (flower, stem, and leaf) of Echinacea purpurea were dried with hot air at temperatures in the range of 40-70 degrees C, and the concentrations of alkylamides and cichoric acid were determined after drying. Increasing drying temperature decreased from 48 h at 40 degrees C to 9 h at 70 degrees C but resulted in a decreased concentration of cichoric acid in all plant sections with a greater loss from aerial plant parts than from the root. There was, however, no significant difference in the concentration of the alkylamides at any drying temperature. Establishment of operational parameters for the drying of echinacea must therefore be structured around the more labile cichoric acid.     

Studies on the antioxidant activity of Echinacea root extract

Methanol extracts of freeze-dried Echinacea (E. angustifolia, E. pallida, and E. purpurea) roots were examined for free radical scavenging capacities and antioxidant activities. Root extracts of E. angustifolia, E. pallida, and E. purpurea were capable of scavenging hydroxyl radical. Similar scavenging activities for each variety were found for both 1,1-diphenyl-2-picrylhydrazyl radical and ABTS radical. Meanwhile, antioxidant activities of all three varieties of Echinacea were found to delay the formation of conjugated diene hydroperoxide induced by the thermal decomposition of 2, 2'-azobis(2-amidinopropane) dihydrochloride and extend the lag phase of peroxidation of soybean liposomes. Echinacea root extracts suppressed the oxidation of human low-density lipoprotein, as evaluated by reduced agarose electrophoretic mobility following oxidative modification by Cu(2+). The mechanisms of antioxidant activity of extracts derived from Echinacea roots included free radical scavenging and transition metal chelating.     

Apple peels as a value-added food ingredient

There is some evidence that chronic diseases, such as cancer and cardiovascular disease, may occur as a result of oxidative stress. Apple peels have high concentrations of phenolic compounds and may assist in the prevention of chronic diseases. Millions of pounds of waste apple peels are generated in the production of applesauce and canned apples in New York State each year. We proposed that a valuable food ingredient could be made using the peels of these apples if they could be dried and ground to a powder without large losses of phytochemicals. Rome Beauty apple peels were treated with citric acid dips, ascorbic acid dips, and blanches before being oven-dried at 60 degrees C. Only blanching treatments greatly preserved the phenolic compounds, and peels blanched for 10 s had the highest total phenolic content. Rome Beauty apple peels were then blanched for 10 s and dried under various conditions (oven-dried at 40, 60, or 80 degrees C, air-dried, or freeze-dried). The air-dried and freeze-dried apple peels had the highest total phenolic, flavonoid, and anthocyanin contents. On a fresh weight basis, the total phenolic and flavonoid contents of these samples were similar to those of the fresh apple peels. Freeze-dried peels had a lower water activity than air-dried peels on a fresh weight basis. The optimal processing conditions for the ingredient were blanching for 10s and freeze-drying. The process was scaled up, and the apple peel powder ingredient was characterized. The total phenolic content was 3342 +/- 12 mg gallic acid equivalents/100 g dried peels, the flavonoid content was 2299 +/- 52 mg catechin equivalents/100 g dried peels, and the anthocyanin content was 169.7 +/- 1.6 mg cyanidin 3-glucoside equivalents/100 g dried peels. These phytochemical contents were a significantly higher than those of the fresh apple peels if calculated on a fresh weight basis (p < 0.05). The apple peel powder had a total antioxidant activity of 1251 +/- 56 micromol vitamin C equivalents/g, similar to fresh Rome Beauty peels on a fresh weight basis (p > 0.05). One gram of powder had an antioxidant activity equivalent to 220 mg of vitamin C. The freeze-dried apple peels also had a strong antiproliferative effect on HepG(2) liver cancer cells with a median effective dose (EC(50)) of 1.88 +/- 0.01 mg/mL. This was lower than the EC(50) exhibited by the fresh apple peels (p < 0.05). Apple peel powder may be used in a various food products to add phytochemicals and promote good health.     

Effects of mixing and various temperature regimes on the respiration of fresh and air-dried coniferous raw humus materials

O₂ uptake of fresh and air-dried L. F and H layer materials from a coniferous-forest floor was studied in the laboratory under regimes of progressively-increasing temperature and also at constant temperature. Experiments were carried out on separate materials from each layer and on mixtures of these materials. A marked apparent “priming” of microbial activity was found when previously air-dried materials were mixed and then rewetted. Some evidence of “priming” was also found when fresh L. F and H layer materials which had not been air dried were mixed. There were large differences between the O₂ uptake patterns of the fresh and air-dried materials when incubated in the same manner. Microbial activity in fresh materials was found to be stimulated when the same number of accumulated heat units were provided under a regime of increasing temperatures as compared to a regime of constant temperature. Results of the respiration studies are discussed in the light of possible differences in species diversity of the soil microbial population resulting from treatment of these organic horizon materials.     

Variability in in vitro macrophage activation by commercially diverse bulk echinacea plant material is predominantly due to bacterial lipoproteins and lipopolysaccharides

We previously reported that the majority of in vitro monocyte/macrophage activation exhibited by extracts of Echinacea and other botanicals depends upon bacterial lipopolysaccharides and Braun-type bacterial lipoproteins. We determined the contribution made by these bacterial components to the overall immune-enhancing activity detected in E. purpurea and E. angustifolia bulk root and aerial material obtained from six major growers/suppliers in North America. Substantial variation in activity (up to 200-fold) was observed in extracts of these materials when tested in two monocyte/macrophage cell lines. The majority of activity was negated by treatment with agents that target bacterial lipoproteins (lipoprotein lipase) and lipopolysaccharides (polymyxin B). Experiments comparing the activity of freeze-dried, freshly harvested Echinacea plants to those harvested and dried using various commercially relevant conditions suggest that postharvesting procedures do not substantially contribute to the variation observed in the commercial material.     

Evaluation of radical scavenging activity of fresh and air-dried tomatoes by three model reactions.

The radical scavenging activity and the antioxidant content of fresh and air-dried tomatoes were investigated. Tomato halves were dried in a pilot-scale dryer under the following conditions: air temperature, 80 degrees C; air flow rate, 1.5 m/s; drying time, 400 min; final moisture, 25%. Carotenoid (lycopene, beta-carotene, lutein) and ascorbic acid were analyzed by HPLC with a spectrophotometric and an electrochemical detector, respectively. Total phenolics were determined by using the Folin-Ciocalteu reagent. The radical scavenging activity was studied in three model systems: (a) the xanthine oxidase and xanthine system, which generates superoxide radical and hydrogen peroxide; (b) the 3-morpholinosydnonimine system, which releases spontaneously superoxide radical and nitrogen monoxide, forming peroxynitrite; (c) the linoleic acid and CuSO(4) system, which promotes lipid peroxidation. These model systems allow the simulation of key reactions involved in the pathogenesis of certain chronic diseases and may be related to the in vivo activity of tomato antioxidants. Hence, these measurements can be used for optimizing tomato processing and storage. The drying process resulted in a decrease of ascorbic acid content, whereas phenol reagent reducing compounds increased. Carotenoid levels were substantially unchanged upon drying. Fresh and air-dried tomato extracts could act as radical scavengers both in the reactive oxygen species-mediated reactions and in lipid peroxidation. Drying affected the antioxidant effectiveness as measured in the xanthine/xanthine oxidase system, which was found to be the most sensitive method for the measurement of tomato antioxidant activity (lower I(50)) but retained the antioxidant effectiveness in the other two systems.     

Isolation and partial characterization of rennet-like proteases from Australian cardoon (Cynara cardunculus L.)

The yield, protein content, proteolytic activity, and substrate specificity of crude and partially purified extracts from dried and fresh Australian cardoon (Cynara cardunculus L.) flowers were determined. Crude water extracts had high yield but low protein content and proteolytic activity, whereas citric acid extracts had low yield but high protein content and proteolytic activity. Fresh flower extracts gave higher yield and proteolytic activity but lower protein content in comparison with dried flower extracts. Purification with ammonium sulfate resulted in significantly increased proteolytic activity for water extracts from both fresh and dried cardoon flowers, whereas the proteolytic activity of citric acid extracts did not change significantly after purification. Irrespective of extraction method, all extracts had higher proteolytic activity against ovine whole and kappa-caseins compared to their bovine counterparts, showing optimal activity at 37 degrees C and pH 6.0. Separation of purified extracts by ion-exchange liquid chromatography yielded three active fractions, each of which when assayed with sodium dodecyl sulfate capillary electrophoresis revealed two subunits with molecular masses of 15.5 and 33.1 kDa, respectively.     

Volatile components of roots, stems, leaves, and flowers of Echinacea species.

The headspace volatile components of roots, stems, leaves, and flowers of Echinacea angustifolia,E. pallida, and E. purpurea were analyzed by capillary gas chromatography/mass spectrometry (GC/MS). Over 70 compounds were identified in the samples. All plant tissues, irrespective of the species, contain acetaldehyde, dimethyl sulfide, camphene, hexanal, beta-pinene, and limonene. The main headspace constituents of the aerial parts of the plant are beta-myrcene, alpha-pinene, limonene, camphene, beta-pinene, trans-ocimene, 3-hexen-1-ol, and 2-methyl-4-pentenal. The major headspace components of root tissue are alpha-phellandrene (present only in the roots of E. purpurea and E.angustifolia), dimethyl sulfide, 2-methylbutanal, 3-methylbutanal, 2-methylpropanal, acetaldehyde, camphene, 2-propanal, and limonene. Aldehydes, particularly butanals and propanals, make up 41-57% of the headspace of root tissue, 19-29% of the headspace of the leaf tissue, and only 6-14% of the headspace of flower and stem tissues. Terpenoids including alpha- and beta-pinene, beta-myrcene, ocimene, limonene, camphene, and terpinene make up 81-91% of the headspace of flowers and stems, 46-58% of the headspace of the leaf tissue, and only 6-21% of the roots. Of the 70 compounds identified, >50 are reported in Echinacea for the first time.     

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.     

不同地面覆盖对土壤性状和秋播大蒜产量及品质的影响

为探究秋播大蒜适宜的地面覆盖类型及其对大蒜产量的影响机制,从而为地面覆盖在大蒜高产高效栽培中的应用提供理论依据,以'麻江红蒜'为试验材料,以不覆盖处理为对照,分别设置白色地膜、黑色地膜、银灰色地膜、1～2cm和3～4cm稻草、1～2cm和3～4 cm稻壳共7种地面覆盖处理,分析不同处理对土壤含水量、温度和酶活性以及大蒜...     

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.     

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.     

Antioxidant capacity changes and phenolic profile of Echinacea purpurea, nettle (Urtica dioica L.), and dandelion (Taraxacum officinale) after application of polyamine and phenolic biosynthesis regulators

The changes of the antioxidant (AOA) and antiradical activities (ARA) and the total contents of phenolics, anthocyanins, flavonols, and hydroxybenzoic acid in roots and different aerial sections of Echinacea purpurea, nettle, and dandelion, after treatment with ornithine decarboxylase inhibitor, a polyamine inhibitor (O-phosphoethanolamine, KF), and a phenol biosynthesis stimulator (carboxymethyl chitin glucan, CCHG) were analyzed spectrophotometrically; hydroxycinnamic acids content was analyzed by RP-HPLC with UV detection. Both regulators increased the AOA measured as inhibition of peroxidation (IP) in all herb sections, with the exception of Echinacea stems after treatment with KF. In root tissues IP was dramatically elevated mainly after CCHG application: 8.5-fold in Echinacea, 4.14-fold in nettle, and 2.08-fold in dandelion. ARA decrease of Echinacea leaves treated with regulators was in direct relation only with cichoric acid and caftaric acid contents. Both regulators uphold the formation of cinnamic acid conjugates, the most expressive being that of cichoric acid after treatment with CCHG in Echinacea roots from 2.71 to 20.92 mg g(-1). There was a strong relationship between increase of the total phenolics in all sections of Echinacea, as well as in the studied sections of dandelion, and the anthocyanin content.     

Compaction of Corn Distillers Dried Grains

Corn distillers dried grains (DDGS) were compacted into cylindrical pellets (3.5 cm in length, 1.5 cm in diameter) utilizing a closed‐end die under axial stress from a vertical piston applied by an Instron universal testing machine. The effects of independent variables, including the raw material moisture content (25–35% db), processing temperature (100–120°C), pressure (12.5–37.5 MPa), and dwell time (5–15 sec) on pellet density, durability, and stability were determined using response surface methodology. Moisture content, temperature, and pressure significantly affected (P < 0.05) the properties of DDGS pellets, while the influence of dwell time was negligible (P > 0.05). Increasing temperature initially increased and then decreased unit density. High moisture and pressure had favorable effects on unit density and durability rating. The density ratio increased with increasing pressure and moisture content. The results suggested technical feasibility of compacting DDGS. For the range of variables tested, optimum levels were identified as 34.6% moisture content, 107°C press temperature, and 36.8 MPa pressure to obtain maximum durability and density and acceptable dimensional stability.     

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.     

Diurnal changes in moisture content and isothermal and thermally induced moisture fluxes under n-tillage and c-tillage in Nigeria

A study on the diurnal changes of soil moisture content and on the isothermal and thermally induced moisture fluxes was conducted on an Alfisol at the International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria, on no-tillage and conventional-tillage plots. These studies were conducted during the 1980 dry season, 9 years after initiating the tillage treatments in 1971. Three bare 5 × 5 m² plots per treatment were used to study diurnal changes in moisture content as soil dried from the initial moisture status of field capacity. The latter was attained by excessive and deep irrigation. Moisture content, moisture potential, and soil temperature were monitored three times a day (08:00, 14:00 and 18:00 h) at the depths of 0–7, 7–14 and 14–21 cm for four 7 day periods at weekly intervals. These results, along with physical characterization of the soil profile and changes in air temperature, were used to calculate isothermal, thermally induced liquid and vapor fluxes.

Results showed that there was a general increase in soil moisture content with sampling depth during the night (18:00 to 08:00 h), and a general decrease with depth during the day (08:00 to 18:00 h). The amplitude of the diurnal cycle of water content changes decreased with depth, and was superimposed on a progressive depletion in water content in the layers studied. The first layer of the conventional-tillage treatment dried to a lower water content than that of the no-tillage treatment. Partition of moisture fluxes, induced by isothermal and thermal conditions, showed that isothermal liquid flux was dominant in no-tillage, and that thermal vapor flux was very important as soil dried in conventional-tillage.

The direction of the fluxes observed (i.e. isothermal liquid flux always being positive upwards and thermal vapor flux positive during the night and negative downwards during the day) was of critical importance as the soil dried. The liquid fluxes became less important and thermal vapor and probably isothermal vapor fluxes became more important with soil moisture depletion. Vapor movement under these circumstances may have played an important role in supplying water to roots both during the day (deep roots) and night (shallow roots) depending on the magnitude of the fluxes. Vapor fluxes were higher and started earlier in conventional-tillage than in no-tillage.     

Relating Rice Milling Quality Changes During Adsorption to Individual Kernel Moisture Content Distribution

Several varieties of rough rice that were either stored for an extended period of time or freshly harvested were conditioned to initial moisture contents ranging from 10 to 17%. After the individual kernel moisture content distributions were measured, the samples were soaked in water at temperatures ranging from 10 to 40°C. The samples were then dried and milled. The bulk critical moisture content, at which head rice yield began to decline due to moisture adsorption, ranged from 12.5 to 14.9%, depending on the variety, harvest moisture content, and storage conditions. The kernel critical moisture content, determined from each sample from the cumulative kernel moisture content frequency distribution, increased with increasing sample initial moisture content.     

Effects of trenching on soil processes and properties in a New Mexico mixed-conifer forest

Summary Trenching was used to reduce root activity in treeless plots in a New Mexico mixed-conifer forest to examine the effects of plant roots on soil processes. Trenching led to increases in moisture content (104%), inorganic N concentration (115%), and mass loss from cellulose (196%). In laboratory incubations, trenched soils collected in the 1st and 2nd year after trenching evolved 52% and 115% more CO₂, respectively, than control soils. Amending incubated trenched and control soils with moisture and inorganic N indicated that increased soil moisture content in trenched plots could explain the increased microbial activity. Trenching also had statistically significant but inconsistent effects on net N mineralization in incubated soils. The greatest effect of trenching was to increase net N mineralization under favorable temperature and moisture conditions. Irrigation of field plots increased both CO₂ evolution and net N mineralization. Overall, these data are consistent with the hypothesis that plant roots reduced microbial activity by moisture uptake during the time of the study.