Baccharis dracunculifolia DC (Asteraceae) selectively modulates the effector functions of human neutrophils.

OBJECTIVES: To examine whether the hydroalcoholic extract from Baccharis dracunculifolia leaves (BdE) modulates the human neutrophil oxidative metabolism, degranulation, phagocytosis and microbial killing capacity. METHODS: In-vitro assays based on chemiluminescence, spectrophotometry, flow cytometry and polarimetry were used, as well as docking calculations. KEY FINDINGS: At concentrations that effectively suppressed the neutrophil oxidative metabolism elicited by soluble and particulate stimuli (<10 µg/ml), without clear signs of cytotoxicity, BdE (1) inhibited NADPH oxidase and myeloperoxidase activity; (2) scavenged H2 O2 and HOCl; (3) weakly inhibited phagocytosis; and (4) did not affect neutrophil degranulation and microbial killing capacity, the expression levels of TLR2, TLR4, FcγRIIa, FcγRIIIb and CR3 and the activity of elastase and lysozyme. Caffeic acid, one of the major B. dracunculifolia secondary metabolites, did not inhibit phagocytosis but interfered in the myeloperoxidase-H2 O2 -HOCl system by scavenging H2 O2 and HOCl, and interacting with the catalytic residues His-95, Arg-239 and Gln-91. CONCLUSIONS: BdE selectively modulates the effector functions of human neutrophils, inhibits the activity of key enzymes and scavenges physiological oxidant species. Caffeic acid contributes to lower the levels of oxidant species. Our findings help to unravel the mechanisms by which these natural products exert immunomodulatory action towards neutrophils.

Inhibition of the human neutrophil oxidative metabolism by Baccharis dracunculifolia DC (Asteraceae) is influenced by seasonality and the ratio of caffeic acid to other phenolic compounds.

ETHNOPHARMACOLOGICAL RELEVANCE: The great potential of phytotherapic drugs for treating and preventing inflammatory diseases mediated by increased neutrophil reactive oxygen species (ROS) generation has guided the search for new natural products with antioxidant and immunomodulatory properties. Baccharis dracunculifolia D.C. (Asteraceae), the main botanical source of Brazilian green propolis, is a native plant from Brazil widely used in folk medicine as anti-inflammatory. This study aims: (a) to determine the influence of seasonality on the chemical profile and biological activity of Baccharis dracunculifolia (Asteraceae) leaf extracts (BdE); (b) to analyze the correlation between the major compounds and the ability of BdE to modulate the superoxide anion and total ROS generation by human neutrophils. MATERIALS AND METHODS: The extracts were obtained from leaf samples collected monthly during one year. The superoxide anion and total ROS generation were assessed by the lucigenin (CL-luc)- and luminol (CL-lum)-enhanced chemiluminescence assays. RESULTS: Seasonality influenced more the quantitative than the qualitative chemical profile of B. dracunculifolia, and affected its biological activity. The major compounds identified were caffeic acid, p-coumaric acid, aromadendrin-4'-methyl ether (AME), isosakuranetin and artepillin C. The IC50 values obtained for CL-lum and CL-luc inhibition by BdE ranged from 8.1-15.8 and 5.8-13.3µgmL(-1), respectively, and correlated positively with caffeic acid concentration. CL-luc inhibition correlated negatively with the concentration of artepillin C, AME, isosakuranetin and total flavonoids. The BdE sample from May/07 inhibited CL-lum and CL-luc the most strongly (IC50=8.1 ± 1.6 and 5.8 ± 1.0 µg mL(-1), respectively), and contained the highest ratio of caffeic acid to the other isolated compounds; so, this ratio could be employed as chemical marker for this biological activity of B. dracunculifolia. CONCLUSION: The ability of B. dracunculifolia to inhibit the neutrophil ROS generation depends more on the type and ratio of phenolic compounds and flavonoids than on their high absolute concentrations. Together, our results help select the most appropriate plant material for the production of phytotherapic drugs to be used in the treatment of inflammatory diseases mediated by increased neutrophil activation.

7-Hydroxycoumarin modulates the oxidative metabolism, degranulation and microbial killing of human neutrophils.

In the present study, we assessed whether 7-hydroxycoumarin (umbelliferone), 7-hydroxy-4-methylcoumarin, and their acetylated analogs modulate some of the effector functions of human neutrophils and display antioxidant activity. These compounds decreased the ability of neutrophils to generate superoxide anion, release primary granule enzymes, and kill Candida albicans. Cytotoxicity did not mediate their inhibitory effect, at least under the assessed conditions. These coumarins scavenged hypochlorous acid and protected ascorbic acid from electrochemical oxidation in cell-free systems. On the other hand, the four coumarins increased the luminol-enhanced chemiluminescence of human neutrophils stimulated with phorbol-12-myristate-13-acetate and serum-opsonized zymosan. Oxidation of the hydroxylated coumarins by the neutrophil myeloperoxidase produced highly reactive coumarin radical intermediates, which mediated the prooxidant effect observed in the luminol-enhanced chemiluminescence assay. These species also oxidized ascorbic acid and the spin traps α-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 5-dimethyl-1-pyrroline-N-oxide. Therefore, 7-hydroxycoumarin and the derivatives investigated here were able to modulate the effector functions of human neutrophils and scavenge reactive oxidizing species; they also generated reactive coumarin derivatives in the presence of myeloperoxidase. Acetylation of the free hydroxyl group, but not addition of the 4-methyl group, suppressed the biological effects of 7-hydroxycoumarin. These findings help clarify how 7-hydroxycoumarin acts on neutrophils to produce relevant anti-inflammatory effects.

4-methylcoumarin derivatives inhibit human neutrophil oxidative metabolism and elastase activity.

Increased neutrophil activation significantly contributes to the tissue damage in inflammatory illnesses; this phenomenon has motivated the search for new compounds to modulate their effector functions. Coumarins are natural products that are widely consumed in the human diet. We have evaluated the antioxidant and immunomodulator potential of five 4-methylcoumarin derivatives. We found that the 4-methylcoumarin derivatives inhibited the generation of reactive oxygen species by human neutrophils triggered by serum-opsonized zymosan or phorbol-12-myristate-13-acetate; this inhibition occurred in a concentration-dependent manner, as revealed by lucigenin- and luminol-enhanced chemiluminescence assays. Cytotoxicity did not mediate this inhibitory effect. The 7,8-dihydroxy-4-methylcoumarin suppressed the neutrophil oxidative metabolism more effectively than the 6,7- and 5,7-dihydroxy-4-methylcoumarins, but the 5,7- and 7,8-diacetoxy-4-methylcoumarins were less effective than their hydroxylated counterparts. An analysis of the biochemical pathways suggested that the 6,7- and 7,8-dihydroxy-4-methylcoumarins inhibit the protein kinase C-mediated signaling pathway, but 5,7-dihydroxy-4-methylcoumarin, as well as 5,7- and 7,8-diacetoxy-4-methylcoumarins do not significantly interfere in this pathway of the activation of the human neutrophil oxidative metabolism. The 4-methylcoumarin derivatives bearing the catechol group suppressed the elastase and myeloperoxidase activity and reduced the 1,1-diphenyl-2-picrylhydrazyl free radical the most strongly. Interestingly, the 5,7-dihydroxy-4-methylcoumarin scavenged hypochlorous acid more effectively than the o-dihydroxy-substituted 4-methylcoumarin derivatives, and the diacetoxylated 4-methylcoumarin derivatives scavenged hypochlorous acid as effectively as the 7,8-dihydroxy-4-methylcoumarin. The significant influence of small structural modifications in the inhibitory potential of 4-methylcoumarin derivatives on the effector functions of neutrophil makes them interesting candidates to develop new drugs for the treatment of inflammatory diseases mediated by increased neutrophil activation.

Modulation of human neutrophil oxidative metabolism and degranulation by extract of Tamarindus indica L. fruit pulp.

The tamarind (Tamarindus indica L.) is indigenous to Asian countries and widely cultivated in the American continents. The tamarind fruit pulp extract (ExT), traditionally used in spices, food components and juices, is rich in polyphenols that have demonstrated anti-atherosclerotic, antioxidant and immunomodulatory activities. This study evaluated the modulator effect of a crude hydroalcoholic ExT on some peripheral human neutrophil functions. The neutrophil reactive oxygen species generation, triggered by opsonized zymosan (OZ), n-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), and assessed by luminol- and lucigenin-enhanced chemiluminescence (LumCL and LucCL, respectively), was inhibited by ExT in a concentration-dependent manner. ExT was a more effective inhibitor of the PMA-stimulated neutrophil function [IC50 (in microg/10(6)cells)=115.7+/-9.7 (LumCL) and 174.5+/-25.9 (LucCL)], than the OZ- [IC50=248.5+/-23.1 (LumCL) and 324.1+/-34.6 (LucCL)] or fMLP-stimulated cells [IC50=178.5+/-12.2 (LumCL)]. The ExT also inhibited neutrophil NADPH oxidase activity (evaluated by O2 consumption), degranulation and elastase activity (evaluated by spectrophotometric methods) at concentrations higher than 200 microg/10(6)cells, without being toxic to the cells, under the conditions assessed. Together, these results indicate the potential of ExT as a source of compounds that can modulate the neutrophil-mediated inflammatory diseases.

Ethanolic crude extract and flavonoids isolated from Alternanthera maritima: neutrophil chemiluminescence inhibition and free radical scavenging activity.

Extracts from Alternanthera maritima are used in Brazilian folk medicine for the treatment of infectious and inflammatory diseases. Bioassay-guided fractionation of A. maritima aerial parts yielded an ethanolic crude extract, its butanolic fraction and seven isolated flavonoids (two aglycones, two O-glycosides and three C-glycosides) with antioxidative activity. The ability of these samples to scavenge enzymatically generated free radicals (luminol-horseradish peroxidase-H2O2 reaction) and inhibit reactive oxygen species (ROS) production by opsonized zymosan-stimulated human neutrophils (PMNLs) was evaluated by chemiluminescence methods. In both assays, the butanolic fraction was significantly more active than the ethanolic crude extract, the flavonoid aglycones had high inhibitory activities and the C-glycosylated flavonoids had no significant effect even at the highest concentration tested (50 micromol/L). However, the O-glycosylated flavonoids inhibitory effects on chemiluminescence were strongly dependent on the chemical structure and assay type (cellular or cell-free system). Under the conditions tested, active samples were not toxic to human PMNLs.

Effect of the extract of the tamarind (Tamarindus indica) fruit on the complement system: studies in vitro and in hamsters submitted to a cholesterol-enriched diet.

This work evaluated a crude hydroalcoholic extract (ExT) from the pulp of the tamarind (Tamarindus indica) fruit as a source of compounds active on the complement system (CS) in vitro. ExT, previously characterized by other authors, had time and concentration dependent effects on the lytic activity of the CS. The activity of 0.8 mg/mL of the extract on the classical/lectin pathways (CP/LP) increased after 30 min of pre-incubation, while that of the alternative pathway (AP) decreased after 15 min at 1mg/mL. Since the CS is a mediator of inflammation, studies were also made in vivo, taking advantage of a model of hypercholesterolemia in hamsters to investigate the role of CS in the phase preceding the inflammatory process of atherosclerosis. Hamsters submitted to a diet rich in cholesterol showed increased lytic activity of the CP/LP and AP after 45 days. The activity levels of C2 and factor B components on respectively, classical/lectin and alternative pathways of the CS also increased. Early events cooperating to trigger the process of atherosclerotic lesions are not completely understood, and these alterations of complement may participate in these events. When treatment with a diet rich in cholesterol was associated to the furnishing of ExT, evaluation of complement components and complement lytic activity showed values similar to those of the controls, showing that treatment with ExT blocked the increase of complement activity caused by the cholesterol-rich diet. By itself, ExT had no effect on the complement system in vivo. ExT activity on the CS may be of interest for therapy and research purposes.

Sesquiterpene lactones from Lychnophora pohlii: neutrophil chemiluminescence inhibition and free radical scavenger activity.

Excessive production of reactive oxygen species (ROS) by polymorphonuclear leucocytes (PMNLs) is thought to contribute to the pathology of many inflammatory diseases. Sesquiterpene lactones (STLs) seem to be important contributors to the anti-inflammatory activity of many species of Lychnophora (Asteraceae), which have been widely used in Brazilian folk medicine because of this pharmacological property. In this study, the inhibitory effects of three STLs isolated from Lychnophora pohlii (lychnopholide, centratherin and goyazensolide) on rabbit PMNL oxidative burst were evaluated by the luminol-enhanced chemiluminescence (CL-lum) assay. All STLs tested showed concentration-dependent inhibitory activity on CL-lum but were not cytotoxic to PMNLs (evaluated by lactate dehydrogenase release) under the assessed conditions. Moreover, goyazensolide, the most active STL, had no free radical scavenger property, as assessed by 1,1-diphenyl-2picrylhydrazyl radical assay, and had no inhibitory effect on the luminol-horseradish peroxidase-hydrogen peroxide chemiluminescence. Taken together, the results of this investigation suggest that the concomitant presence of methacrylate ester and hydroxyl groups contributes to a high inhibitory effect on PMNL oxidative metabolism. This effect was not mediated by free radical scavenger or cytotoxic effects, but probably by inhibition of enzymes involved in the signal transduction pathways of the ROS generation process.