Echinacea alkylamides modulate TNF-alpha gene expression via cannabinoid receptor CB2 and multiple signal transduction pathways

Echinacea plant preparations are widely used in the prevention and treatment of common cold. However, so far no molecular mechanism of action has been proposed. We analyzed the standardized tincture Echinaforce and found that it induced de novo synthesis of tumor necrosis factor alpha (TNF-alpha) mRNA in primary human monocytes/macrophages, but not TNF-alpha protein. Moreover, LPS-stimulated TNF-alpha protein was potently inhibited in the early phase but prolonged in the late phase. A study of the main constituents of the extract showed that the alkylamides dodeca-2E,4E,8Z,10E/Z-tetraenoic acid isobutylamides (1/2), trienoic (3) and dienoic acid (4) derivatives are responsible for this effect. The upregulation of TNF-alpha mRNA was found to be mediated by CB2 receptors, increased cAMP, p38/MAPK and JNK signaling, as well as NF-kappaB and ATF-2/CREB-1 activation. This study is the first to report a possible molecular mechanism of action of Echinacea, highlighting the role of alkylamides as potent immunomodulators and potential ligands for CB2 receptors.     

Cytochrome P450 enzyme-mediated degradation of Echinacea alkylamides in human liver microsomes

Echinacea preparations are widely used herbal remedies for the prevention and treatment of colds. In this study we have investigated the metabolism by human liver microsomes of the alkylamide components from an Echinacea preparation as well as that of pure synthetic alkylamides. No significant degradation of alkylamides was evident in cytosolic fractions. Time- and NADPH-dependent degradation of alkylamides was observed in microsomal fractions suggesting they are metabolised by cytochrome P450 (P450) enzymes in human liver. There was a difference in the susceptibility of 2-ene and 2,4-diene pure synthetic alkylamides to microsomal degradation with (2E)-N-isobutylundeca-2-ene-8,10-diynamide (1) metabolised to only a tenth the extent of (2E,4E,8Z,10Z)-N-isobutyldodeca-2,4,8,10-tetraenamide (3) under identical incubation conditions. Markedly less degradation of 3 was evident in the mixture of alkylamides present in an ethanolic Echinacea extract, suggesting that metabolism by liver P450s was dependent both on their chemistry and the combination present in the incubation. Co-incubation of 1 with 3 at equimolar concentrations resulted in a significant decrease in the metabolism of 3 by liver microsomes. This inhibition by 1, which has a terminal alkyne moiety, was found to be time- and concentration-dependent, and due to a mechanism-based inactivation of the P450s. Alkylamide metabolites were detected and found to be the predicted epoxidation, hydroxylation and dealkylation products. These findings suggest that Echinacea may effect the P450-mediated metabolism of other concurrently ingested pharmaceuticals.     

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.     

Sensitization of rat alveolar macrophages to enhanced TNF-alpha release by in vivo treatment with dexamethasone.

Treatment of rats with dexamethasone rapidly induced a marked weight loss which occurred within 3 days and persisted for several weeks. The cachectic state was paralleled by increased serum levels of triglycerides, albumin, and protein and a strong reduction of blood mononuclear leukocytes. In lung sections, an increased number of mononuclear giant cells was found but no bacteria, fungi, or Pneumocystis carinii organisms. Quite strikingly, alveolar macrophages from dexamethasone-treated rats, but not from control animals, were highly sensitive to LPS and released large amounts of TNF-alpha ex vivo. Also under in vivo conditions, high TNF-alpha serum concentrations were found in dexamethasone-treated but not control rats when examined 1 1/2 hr after an intravenous LPS injection. These data suggest that the glucocorticoid-induced cachexia of rats may be linked, at least in part, to readily inducible TNF-alpha release from primed macrophages.     

大孔吸附树脂对紫锥菊提取物中菊苣酸分离纯化的研究

采用大孔吸附树脂分离纯化紫锥菊提取物中免疫活性成分菊苣酸,7％(v／v)的甲醇-水溶液洗脱,HPLC法对产品中菊苣酸含量进行检测分析。该法能把紫锥菊提取物中菊苣酸含量(4％)提高9倍左右,回收率达到95％以上,且操作简单,可用于菊苣酸的分离纯化。     

Macrolide antibiotics protect against immune complex-induced lung injury in rats: role of nitric oxide from alveolar macrophages.

Macrolide antibiotics have unique immunomodulatory actions apart from antimicrobial properties. We studied the effects of macrolides on IgG immune complex (IgG-ICx)-induced lung injury in rats in vivo and in vitro. Intrapulmonary deposition of IgG-ICx produced a time-dependent increase in the concentration of NO in exhaled air. There were corresponding increases in the number of neutrophils accumulated into alveolar spaces, and lung wet-to-dry weight ratio. All of these changes were inhibited by pretreatment with erythromycin or josamycin, but not by amoxicillin or cephaclor. Incubation of cultured pulmonary alveolar macrophages caused up-regulation of NO production and expression of inducible NO synthase mRNA, an effect that was dose dependently inhibited by erythromycin, roxithromycin, or josamycin. The macrolides also reduced IgG-ICx-induced release of IL-1beta and TNF-alpha, but did not alter the release of NO induced by exogenously added IL-1beta and TNF-alpha. These results suggest that macrolide antibiotics specifically inhibit immune complex-induced lung injury presumably by inhibiting cytokine release and the resultant down-regulation of inducible NO synthase gene expression and NO production by rat pulmonary alveolar macrophages.     

Effect of temperature on stability of marker constituents in Echinacea purpurea root formulations.

Stability of an alkamide and a phenolic phytochemical marker in a hydro-alcoholic extract of Echinacea purpurea root and a dried powder prepared by evaporation of the extract was assessed in storage for 7 months at three temperature regimes: -20, 25 and 40 degrees Celsius. In the extract, the major alkamide, dodeca-2E, 4E, 8Z, 10E/Z-tetraenoic acid isobutyl amide, was not significantly affected by storage at any of the temperatures, but cichoric acid content declined as significantly (P = 0.05) at both 25 degrees C and 40 degrees C as compared to low-temperature storage. In the powder, the major alkamide showed a significantly reduced level at 25 degrees C and 40 degrees C while cichoric acid did not decline significantly. These results suggest that more attention should be given to the effect of formulation and temperature on storage of Echinacea products.     

Lung-specific delivery of cytokines induces sustained pulmonary and systemic immunomodulation in rats

The recombinant cytokines IFN-gamma and TNF-alpha stimulate several macrophage-mediated functions important in host defense. However, systemic administration of cytokines may be limited by significant host toxicity. We investigated whether aerosolized cytokines can stimulate alveolar macrophage and blood monocyte function, and whether they induce an inflammatory response in the lungs of normal rats. We found that aerosolized murine rIFN-gamma or recombinant human TNF-alpha increased IL-1 production by both alveolar macrophages and blood monocytes for at least 5 days after administration. Furthermore, murine rIFN-gamma increased the expression of Ia Ag on alveolar macrophages and human rTNF-alpha increased alveolar macrophage- and blood monocyte-mediated tumor lysis. Sequential aerosolization of IFN-gamma and TNF-alpha significantly increased both IL-1 release and Ia expression compared to either cytokine administered alone. Aerosolized human rTNF-alpha achieved lung levels comparable to those produced by an i.v. TNF-alpha dose reported to cause diffuse organ injury and death in rats. However, plasma TNF-alpha levels were several thousand-fold lower after aerosol administration. Aerosolized cytokines did not induce lung edema or an inflammatory cell infiltrate within the airways or alveoli. Aerosolized human rTNF-alpha alone, or murine rIFN-gamma and human rTNF-alpha, induced margination of leukocytes in pulmonary blood vessels 1 day after aerosolization, and a few small foci of pulmonary hemorrhage 5 days later. We conclude that aerosol administration of IFN-gamma or TNF-alpha enhances both pulmonary and systemic monocyte function, and that the combination of IFN-gamma and TNF-alpha produce additive or synergistic effects. Aerosolized cytokines induce only a minimal pulmonary inflammatory response. Aerosolized TNF-alpha produces high cytokine levels in the lung but very low uptake into the circulation.     

Isolated Pneumocystis carinii cell wall glucan provokes lower respiratory tract inflammatory responses

Macrophage-induced lung inflammation contributes substantially to respiratory failure during Pneumocystis carinii pneumonia. We isolated a P. carinii cell wall fraction rich in glucan carbohydrate, which potently induces TNF-alpha and macrophage-inflammatory protein-2 generation from alveolar macrophages. Instillation of this purified P. carinii carbohydrate cell wall fraction into healthy rodents is accompanied by substantial increases in whole lung TNF-alpha generation and is associated with neutrophilic infiltration of the lungs. Digestion of the P. carinii cell wall isolate with zymolyase, a preparation containing predominantly beta-1,3 glucanase, substantially reduces the ability of this P. carinii cell wall fraction to activate alveolar macrophages, thus suggesting that beta-glucan components of the P. carinii cell wall largely mediate TNF-alpha release. Furthermore, the soluble carbohydrate beta-glucan receptor antagonists laminariheptaose and laminarin also substantially reduce the ability of the P. carinii cell wall isolate to stimulate macrophage-inflammatory activation. In contrast, soluble alpha-mannan, a preparation that antagonizes macrophage mannose receptors, had minimal effect on TNF-alpha release induced by the P. carinii cell wall fraction. P. carinii beta-glucan-induced TNF-alpha release from alveolar macrophages was also inhibited by both dexamethasone and pentoxifylline, two pharmacological agents with potential activity in controlling P. carinii-induced lung inflammation. These data demonstrate that P. carinii beta-glucan cell wall components can directly stimulate alveolar macrophages to release proinflammatory cytokines mainly through interaction with cognate beta-glucan receptors on the phagocyte.     

Stimulation of TNF-alpha release by fungal cell wall polysaccharides

Carboxymethylated derivatives were prepared from the (1-->3)-beta-D-glucan isolated from the cell wall of baker's yeast Saccharomyces cerevisiae and from the chitin-glucan complex of the mycelium of the industrial filamentous fungus Aspergillus niger. The polysaccharides were applied to peritoneal mouse macrophages and after a 2-h incubation the release of TNF-alpha by the stimulated macrophages was measured using an enzyme-linked immunosorbent assay. As the third polysaccharide stimulant, a water-soluble derivative of chitin was assayed and the observed cytokine release was compared with the control experiment. In three concentrations of the polysaccharides applied, carboxymethyl glucan revealed a dramatic increase in the TNF-alpha release, while addition of carboxymethyl chitin-glucan resulted only in a moderate enhancement, and carboxymethyl chitin was inactive. The results indicate that fungal polysaccharides, especially (1-->3)-beta-D-glucan, are potent macrophage stimulators and activators of TNF-alpha release, which implies their potential application in antitumor therapy.     

Meconium enhances platelet-activating factor and tumor necrosis factor production by rat alveolar macrophages

Meconium aspiration syndrome (MAS) frequently results in inactivation of surfactant, persistent pulmonary hypertension (PPHN) and respiratory failure among newborn infants. Inflammation and inflammatory mediators play an important role in MAS. Since alveolar macrophages are thought to be very important cells in the pathogenesis of various inflammatory diseases, we evaluated whether meconium could stimulate rat alveolar macrophages to generate platelet-activating factor (PAF) and tumor necrosis factor (TNF)-alpha in vitro. We also examined the response to A23187 (calcium ionophore), 1-0-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (synthetic PAF) and dexamethasone on meconium-induced release of PAF and TNF-alpha. PAF and TNF-alpha concentrations from supernatant fluid were measured after high-performance liquid chromatography purification by specific radioimmunoassay, and TNF-alpha concentrations were determined by using an enzyme-linked immunosorbent assay. Our results showed that alveolar macrophages exposed to meconium could enhance PAF and TNF-alpha production in a dose (0.1, 1, 5 and 10%, P<0.01)-dependent way. In the presence of A23187, the capability of meconium to stimulate PAF production was further enhanced in the supernatant fluids. Furthermore, treatment with synthetic PAF significantly increased the generation of TNF-alpha in response to meconium. On the other hand, dexamethasone effectively inhibited both PAF and TNF-alpha production stimulated by 5% meconium (P<0.01, P<0.01; respectively). We suggest that alveolar macrophages and PAF, TNF-alpha play an important role in the pathogenesis of lung injury and severe complications in MAS. Furthermore, the protective effect of glucocorticoids in MAS could be due, at least in part, to a suppression of PAF and TNF-alpha generation.     

Chronic cigarette smoking enhances spontaneous release of tumour necrosis factor-alpha from alveolar macrophages of rats

Some biological effects of chronic cigarette smoking (two cigarettes for 2 h, daily for 4 months) in rats were evaluated. During the smoking period, body weight of smoker rats was always significantly lower than that of control rats. Immediately after the last smoking session the carboxyhaemoglobin concentration in the blood was about 8.5% and the polymorphonuclear cells in the bronchoalveolar fluid increased significantly. At the same time, enzymatic analyses on the supernatants of bronchoalveolar fluid revealed a significant increase of beta-glucuronidase in the smoker group. Alveolar macrophages, collected 0, 8 and 24 h after the last smoking session, significantly increased the generation of superoxide anion and, after incubation for 24 h at 37( degrees ) C in a humidified atmosphere, released significantly high amounts of TNF-alpha. When challenged with lipopolysaccharide, alveolar macrophages of smoker rats released much more TNF-alpha but, in such a case, TNF-alpha release was about one half of that observed in the control group. Peritoneal macrophages of both control and smoker rats were unable either to generate high levels of superoxide anion or to release significant amounts of TNF-alpha. The results clearly demonstrated the activated state of alveolar macrophages and the resting state of peritoneal macrophages.     

Application of an airlift bioreactor system for the production of adventitious root biomass and caffeic acid derivatives of Echinacea purpurea

In this study, we evaluated the feasibility of using mass cultivation of the adventitious roots of Echinacea purpurea in balloon type bubble (air-lift) bioreactors to produce caffeic acid derivatives, which have pharmaceutical and therapeutic values. An approximately 10 fold increase in biomass and secondary compounds was observed after 4 weeks of culture in balloon type bubble bioreactors (5 L capacity containing 4 L of half strength MS medium). In addition, a linear relationship was observed between the concentration of biomass and the sucrose and ion consumption rate. Furthermore, the concentration of biomass in the bioreactor culture was found to increase as the conductivity decreased. An inoculum density of 7 g/L FW and an aeration rate of 0.1 vvm were found to be suitable for inducing the accumulation of biomass and secondary metabolites. Of the three caffeic acid derivatives evaluated (caftaric acid, chlorogenic acid, and cichoric acid), the concentration of cichoric acid was the highest (26.64 mg/g DW).     

Enhanced production of cichoric acid in cell suspension culture of Echinacea purpurea by silver nanoparticle elicitation

Plant Cell, Tissue and Organ Culture (PCTOC) - This study was conducted to optimize callus induction, cell suspension culture, and enhance cichoric acid production in Echinacea purpurea. For this...     

Hepatic macrophage iron aggravates experimental alcoholic steatohepatitis

One prime feature of alcoholic liver disease (ALD) is iron accumulation in hepatic macrophages/Kupffer cells (KC) associated with enhanced NF-kappaB activation. Our recent work demonstrates a peroxynitrite-mediated transient rise in intracellular labile iron (ILI) as novel signaling for endotoxin-induced IKK and NF-kappaB activation in rodent KC. The present study investigated the mechanism of KC iron accumulation and its effects on ILI response in experimental ALD. We also tested ILI response in human blood monocytes. Chronic alcohol feeding in rats results in increased expression of transferrin (Tf) receptor-1 and hemochromatosis gene (HFE), enhanced iron uptake, an increase in nonheme iron content, and accentuated ILI response for NF-kappaB activation in KC. Ex vivo treatment of these KC with an iron chelator abrogates the increment of iron content, ILI response, and NF-kappaB activation. The ILI response is evident in macrophages derived from human blood monocytes by PMA treatment but not in vehicle-treated monocytes, and this differentiation-associated phenomenon is essential for maximal TNF-alpha release. PMA-induced macrophages load iron dextran and enhance ILI response and TNF-alpha release. These effects are reproduced in KC selectively loaded in vivo with iron dextran in mice and more importantly aggravate experimental ALD. Our results suggest enhanced iron uptake as a mechanism of KC iron loading in ALD and demonstrate the ILI response as a function acquired by differentiated macrophages in humans and as a priming mechanism for ALD.     

Clonal propagation and production of cichoric acid in three species of Echinaceae

In vitro tissue culture protocols were tested for propagation of Echinacea purpurea, Echinacea pallida and Echinacea angustifolia in order to obtain biomass for the production of cichoric acid, which is the major active compound in the Echinacea extracts. The in vitro culture process was initiated by seed germination on half-strength Murashige and Skoog (MS) medium. Multiplication was achieved on MS medium supplemented with naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2-iso-pentenyladenine (2iP), and N⁶-benzyladenine (BA) in different concentrations. Shoot explants produced the highest number of shootlets on MS medium, which was supplemented with 0.1 mg/l 2iP and 0.1 mg/l IBA. RAPD markers revealed genetic polymorphism in some instances between in vitro generated plantlets such as for E. purpurea plantlets analyzed with the OPO-8 primer. RAPD markers generated with the primer 4A-29 revealed low levels of genetic variation between in vitro plantlets for all three species of Echinacea, while remaining RAPD markers revealed no variation. Content of cichoric acid in leaves, shoots, and callus was analyzed by high-performance liquid chromatography/MS and was identified in all studied samples, independent of species or tissue type. Highest levels (0.39–0.73 mg/g dw) were observed in shoots and leaves.     

Alveolar sphingolipids generated in response to TNF-alpha modifies surfactant biophysical activity.

Sphingolipids represent a diverse group of bioactive lipid species that are generated intracellularly in response to tumor necrosis factor-alpha (TNF-alpha) and are implicated as potential mediators of acute lung injury. The purpose of these studies was to determine whether there was an extracellular, TNF-alpha-regulated pool of sphingolipids in the alveolus that modulates the surface tension lowering capacity of pulmonary surfactant. Intratracheal instillation of TNF-alpha in adult rats led to a twofold increase in the amount of surfactant-associated ceramide and tended to decrease levels of sphingomyelin without significantly altering sphingosine or sphinganine content. TNF-alpha induction of alveolar ceramide was associated with nearly an 80% increase in acid sphingomyelinase activity recovered in cell-free alveolar lavage. Ceramide administered in a dose-dependent manner potently antagonized the surface tension lowering effects of natural surfactant in vitro. Intratracheal TNF-alpha and ceramide treatment of rats significantly increased lung permeability, as was evidenced by extravasation of Evans blue dye into alveolar lavage and lung tissue. Thus these studies are the first to demonstrate the existence of a cytokine-regulated alveolar pool of sphingomyelin hydrolysis products that impairs the biophysical properties of the alveolar surfactant film. The results also suggest the presence of a secretory alveolar sphingomylinase that is TNF-alpha responsive and mediates effects of the cytokine on alveolar sphingolipid metabolism.     

TNF-alpha promoter, Nod2 and toll-like receptor-4 polymorphisms and the in vivo and ex vivo response to endotoxin

Humans exhibit substantial inter-individual differences in TNF-alpha production upon endotoxin stimulation. To determine to what extent the lipopolysaccharide-induced TNF-alpha production capacity in vivo and ex vivo is determined by polymorphisms in toll-like receptor-4 (TLR4), the TNF-alpha promoter region and Nod2, we screened for two TLR4 polymorphisms, a Nod2 polymorphism and the TNF-alpha promoter polymorphisms. We measured the perioperative endotoxemia and TNF-alpha production and the TNF-alpha production capacity of each patient in a whole-blood stimulation assay using blood drawn before anesthesia, using various LPS concentrations, in patients undergoing elective cardiac surgery. This operation represents a major surgical trauma associated with ischemia-reperfusion injury and triggers an endotoxemia and profound inflammatory response. In vivo TNF-alpha production was positively correlated with the level of endotoxemia after aortic declamping; thus TNF-alpha levels were higher in patients having endotoxemia compared to patients without endotoxemia. This correlation was observed in patients with any of the genotypes studied, and did not differ between the various genotypes. In vivo TNF-alpha levels correlated best with those ex vivo after stimulation with 1000 ng/mL LPS, and the estimated maximal TNF-alpha release capacity. Subjects with the wild-type TLR4 gene had similar levels of TNF-alpha upon LPS stimulation ex vivo as compared with patients carrying Asp299Gly and/or the Thr399Ile TLR4 polymorphism. Our results indicate that polymorphisms in the TLR4 receptor, Nod2 and TNF-alpha promoter region are not strongly associated with in vivo and ex vivo TNF-alpha production capacity upon endotoxin stimulation. This suggests that in this model of natural LPS release, the variation between individuals in TNF-alpha release can only modestly be determined by genetic background (TNF-alpha promoter, Nod2 and TLR4) of the individual.