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Abstract Achyrocline satureioides Lam. Here, the effects of the in vivo actions of the hydroalcoholic extract obtained from inflorescences of A. Male Wistar rats were orally treated with A. The number of leukocytes and the amount of chemotactic mediators were quantified in the inflammatory exudate, and adhesion molecule and toll-like receptor 4 TLR-4 expressions and phorbol-myristate-acetate- PMA- stimulated oxidative burst were quantified in circulating neutrophils.
Leukocyte-endothelial interactions were quantified in the mesentery tissue. Enzymes and tissue morphology of the liver and kidney were evaluated. Treatment with A. Together, the data show that A. Introduction Achyrocline satureioides Lam. In Brazil, it predominantly occurs in southern regions, where it is popularly known as Marcela or Macela and is largely employed in folk medicine [ 1 ]. The ethnopharmacological use of infusions prepared from inflorescences of A.
Investigations on its chemical composition showed that the extract obtained from inflorescences is rich in flavonoids, mainly quercetin and luteolin [ 1 , 8 , 9 ]. Experimental assays in vivo and in vitro have confirmed the ethnopharmacological employment of the extracts obtained from inflorescences of A.
In this context, the anti-inflammatory activity of aqueous and ethanolic extracts from A. Puhlmann and coauthors [ 11 ] showed enhanced in vivo phagocytic activity of carbon particles by macrophages obtained from rats treated with A. In addition, A. Although the anti-inflammatory effects of A. Neutrophils express a wide range of membrane receptors, such as adhesion molecules and chemoattractant receptors, prompting them to react to exogenous stimuli and endogenous chemical mediators.
In this context, neutrophils express the toll-like receptor 4 TLR-4 , which is responsible for the recognition of lipopolysaccharides LPSs from Gram-negative bacteria. L-selectin mediates the rolling of the neutrophils along endothelial cells, as it is rapidly expressed by activated neutrophils and interacts with constitutive carbohydrates or even with P- or E-selectin expressed on endothelial cells [ 14 ]. L-selectin is cleaved, by action of metalloproteases, pointing to the leukocytes become arrested to the endothelium [ 15 ].
Ultimately, neutrophils transmigrate between interendothelial junctions, via heterophilic and homophilic interactions of immunoglobulin superfamily molecules, such as platelet endothelial cell adhesion molecule-1 PECAM-1 , to subsequently migrate to the inflammatory focus [ 16 ]. In the extravascular matrix, neutrophils directly move to the site of the lesion in response to chemotactic chemical mediators [ 17 ].
At the lesion site, they phagocyte the lesion-causing agent and release preformed chemical substances that contribute to the destruction of the damaging agent. However, in the case of noncontrolled inflammation, blockage of phases of neutrophil mobilization into focus of the inflammatory reaction is an important therapeutic strategy.
This study investigated the in vivo actions of hydroalcoholic extract obtained from inflorescences of A. Data confirmed quercetin and luteolin to be the main constituents in the hydroalcoholic extract and showed the absence of systemic toxicity. However, results clearly showed that A. Additional anti-inflammatory mechanisms might exist, as the extract also inhibited neutrophil activation caused by a direct intracellular stimulation of protein kinases.
Materials and Methods 2. Plant Material Inflorescences of A. The macerate was filtered and the solvent removed by rotary evaporation under reduced pressure. A reverse-phase C18 column 25 cm, 4. Chromatographic separation was performed at room temperature with a flow rate 0. UV-Vis spectra were recorded at wavelengths — nm detection at nm. Solvents used were of HPLC grade, filtered 0.
The samples of hydroalcoholic extract 0. Hydroalcoholic extract of A. The mass spectrum of the individual unknown compounds was compared with that of known compounds stored in the software database Library. Treatments The doses used in this study were based on data previously published by Santin et al.
Assays were carried out 1 or 2 hours after treatments. After six days, the pouch was refilled with 10 mL air. After 1 h, LPS from E. At 1 h or 4 h after the LPS injection, the animals were reanesthetized and sacrificed. The pouches were washed with 2 mL ice-cold PBS, and the total leukocyte number was determined using a Neubauer chamber. Subsequently, the whole blood was hemolyzed hypotonic lysis with NaCl solution at 0. The total number of leukocytes was quantified using a Neubauer chamber.
Data were obtained from 10, cells, and only the morphologically viable leukocytes were considered for analysis. The neutrophil population was characterized by different size and complexity parameters of different cell types detected by flow cytometry. The optical signals emitted were converted into electronic signals and were analyzed by FlowJo software Tree Star, Inc. Results of TLR-4 expression are presented as fluorescence units, and apoptosis and necrosis are shown as the percentage of Annexin V- or PI-positive cells.
Subsequently, the leukocyte suspension was washed with 1 mL ice-cold PBS. The supernatant was discarded, and leukocytes were incubated with monoclonal anti-CD62L antibodies conjugated to PE 1 : and anti-CD18 conjugated to FITC 1 : , for 20 minutes at room temperature.
Immediately after incubations, the samples were centrifuged 5 min, g and were resuspended in PBS for quantification by flow cytometry. The neutrophil population was characterized as described in Section 2.
Results of oxidative metabolism are expressed as units of fluorescence, and data of adhesion molecules are expressed as the percentage of positive cells.
The rats were anesthetized 1 h after the treatment, and 30 min later, the mesentery was surgically exteriorized. The rate of solution outflow onto the exposed tissue was controlled to keep the preparation in continuous contact with a film of the solution. Images obtained on the TV monitor were recorded on software. Digitized images on the computer monitor were subsequently analyzed by image analyzing software AxioVision. Leukocytes moving in the periphery of the axial stream, in contact with the endothelium, were considered to be rolling, and their number was determined in 10 min periods.
The number of leukocytes that adhered to the endothelium stopped at the vessel wall was determined in the same vascular segment after 10 min. Three fields were evaluated per animal after application. The results were then averaged for each animal. Inflammatory Mediators The air pouch lavage fluid was collected 1 h after LPS injection to evaluate the concentration of inflammatory mediators.
Biochemical Parameters The whole blood was collected 2 hour after treatments without anticoagulant to serum separation after centrifugation 10 min, g. The concentration of kidney and liver markers was analyzed using commercial biochemical kits for urea, creatinine, aspartate aminotransferase AST , alanine aminotransferase ALT , and gamma-GT.
Tissue samples were dehydrated in graded ethanol solutions, cleared in xylene, and embedded in paraffin wax. Statistical Analysis Means and the standard error of the mean S. GraphPad Prism 4. Results 3. Chromatographic Analysis The phytochemical profile of the A.
The major components of the extract were compounds 1 and 2, which were identified by direct comparison with authentic samples and area peaks as luteolin and quercetin, respectively. Although these compounds occurred in other tissues of A. The GC-MS analysis showed seven distinct peaks, identified via the NIST08 library software database as ethyl ester derivatives of oleic, palmitic and stearic acids.
In addition, the steroids stigmasterol, gamma-sitosterol, and sitostenone were also identified in the A.
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