title: Evaluation of anti-inflammatory and mechanism of action of extract of Macrosiphonia longiflora (Desf.) Müll. Arg.
abstract: BACKGROUND
  Macrosiphonia longiflora (Desf.) Müll. Arg (Apocynaceae), popularly known as 'velame' and 'velame branco', is a native subshrub that grows in the Brazilian Cerrado. This plant is widely used in traditional medicine in the form of decoction and infusion, particularly as anti-inflammatory, depurative, anti-rheumatic, antisyphilitic and antiulcer remedy. There is no available information in the literature that has addressed its pharmacological activity and phytochemical analysis.
  
  
  OBJECTIVE
  This study aimed to evaluate the anti-inflammatory pharmacological profile of the hydroethanolic extract of Macrosiphonia longiflora, using in vivo and in vitro acute inflammation experimental models, as well as investigate the roles of cytokines and nitric oxide in its mechanism of action, and including phytochemical analysis constitution of its hydroethanolic extract.
  
  
  METHODS
  Hydroethanolic (70%) extract of Macrosiphonia longiflora (HEMl) was prepared by maceration. The preliminary phytochemical analysis was performed according to procedures described in the literature. Selected secondary metabolites detected were quantified by spectrophotometry and high performance liquid chromatography (HPLC). Its cytotoxic potential in Chinese hamster ovary (CHO-k1) epithelial cell lines was evaluated using Alamar Blue. in vivo anti-inflammatory activity was evaluated with carrageenan- and dextran-induced paw edemas, carrageenan-induced pleurisy in rats and lipopolysaccharide (LPS)-induced peritonitis in mice. The in vitro anti-inflammatory activity was evaluated using RAW 264.7 cells stimulated with LPS and interferon (INF)-γ. Effects of HEMl on the inflammatory cytokines (IL-1β, IL-10, IL-17, INF-γ and TNF-α) concentrations in the peritoneal lavage were evaluated using commercial ELISA kits, while the Griess method was employed to determine nitric oxide (NO) concentrations in the peritoneal lavage, as well as in the supernatants of RAW 264.7 cells.
  
  
  RESULTS
  Preliminary phytochemical analysis, revealed the presence of phenolics compounds, terpenoids, alkaloids and flavonoids. Spectrophotometric analysis revealed the presence of relatively high content of phenolics and flavonoids in HEMl. HPLC analysis confirmed the presence of the quantified compounds and demonstrated the presence of ellagic acid in the detected matrix of compounds. HEMl appeared to be non-cytotoxic. It effectively inhibited (p<0.05) paw edema induced by carrageenan and dextran. Furthermore, HEMl also significantly reduced exudates volume and leukocyte migration in the carrageenan-induced pleurisy and LPS-induced peritonitis, neutrophils counts in LPS-induced peritonitis. HEMl also acts by effectively inhibiting the following inflammatory cytokines: IL-1β and IL-10 levels in the peritoneal lavage, but had no effect on IL-17 level in the peritonitis model. In addition, HEMl had no effect on the levels of tumor necrosis factor alpha (TNF-α) present in the peritoneal lavage and cells supernatants. The concentration of NO, as assessed by measurement of nitrite (NO2(-)), showed that pretreatment with HEMl reduced NO significantly in the peritoneal lavage and in RAW 264.7 cells co-stimulated with LPS and INF-γ.
  
  
  CONCLUSIONS
  The results obtained in this study indicate that HEMl possesses very low cytotoxic potential. In addition, it demonstrated a potent anti-inflammatory activity in both the in vivo and in vitro models of acute inflammation. The anti-inflammatory effect is partly related to the inhibition of IL-1β, IL-10, and nitric oxide releases, but independent of TNF-α and IL-17 modulation. Phytochemical analysis revealed the predominant presence of the flavonoids (naringin, rutin, myricetin, morin, quercetin, (±)-naringenin, and luteolin) and phenols (ellagic acid), which are possibly involved in the anti-inflammatory effect of HEMl. The current study provided supportive evidence for the popular use of HEMl in the treatment of inflammatory conditions, and shed more light on the possible roles of the inflammatory cytokines in its mechanisms of action as anti-inflammatory agent.
authors.affiliation: Department of Basic Sciences in Health, Faculty of Medicine, Federal University of Mato Grosso, Av. Fernando Correa da Costa, n. 2367, Cuiabá 78060-900, Mato Grosso, Brazil.
authors.forename: Anísio Onório
authors.initials: AO
authors.lastname: da Silva
authors.affiliation: Department of Basic Sciences in Health, Faculty of Medicine, Federal University of Mato Grosso, Av. Fernando Correa da Costa, n. 2367, Cuiabá 78060-900, Mato Grosso, Brazil.
authors.forename: Aurea
authors.initials: A
authors.lastname: Damaceno Alves
authors.affiliation: Department of Basic Sciences in Health, Faculty of Medicine, Federal University of Mato Grosso, Av. Fernando Correa da Costa, n. 2367, Cuiabá 78060-900, Mato Grosso, Brazil.
authors.forename: Danielle Ayr Tavares de
authors.initials: DA
authors.lastname: Almeida
authors.affiliation: Department of Basic Sciences in Health, Faculty of Medicine, Federal University of Mato Grosso, Av. Fernando Correa da Costa, n. 2367, Cuiabá 78060-900, Mato Grosso, Brazil.
authors.forename: Sikiru Olaitan
authors.initials: SO
authors.lastname: Balogun
authors.affiliation: Department of Basic Sciences in Health, Faculty of Medicine, Federal University of Mato Grosso, Av. Fernando Correa da Costa, n. 2367, Cuiabá 78060-900, Mato Grosso, Brazil.
authors.forename: Ruberlei Godinho
authors.initials: RG
authors.lastname: de Oliveira
authors.affiliation: Research Laboratory of Natural Products (LPPN), Federal University of Tocantins, Av. NS15, Palmas, Tocantins 77020-210, Brazil.
authors.forename: Aline
authors.initials: A
authors.lastname: Aires Aguiar
authors.affiliation: Research Laboratory of Natural Products (LPPN), Federal University of Tocantins, Av. NS15, Palmas, Tocantins 77020-210, Brazil.
authors.forename: Ilsamar Mendes
authors.initials: IM
authors.lastname: Soares
authors.affiliation: Research Laboratory of Natural Products (LPPN), Federal University of Tocantins, Av. NS15, Palmas, Tocantins 77020-210, Brazil.
authors.forename: Poliana Guerino
authors.initials: PG
authors.lastname: Marson-Ascêncio
authors.affiliation: Research Laboratory of Natural Products (LPPN), Federal University of Tocantins, Av. NS15, Palmas, Tocantins 77020-210, Brazil.
authors.forename: Sérgio Donizeti
authors.initials: SD
authors.lastname: Ascêncio
authors.affiliation: Department of Basic Sciences in Health, Faculty of Medicine, Federal University of Mato Grosso, Av. Fernando Correa da Costa, n. 2367, Cuiabá 78060-900, Mato Grosso, Brazil. Electronic address: taba@terra.com.br.
authors.forename: Domingos Tabajara
authors.initials: DT
authors.lastname: de Oliveira Martins
mesh_terms: D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000302:isolation & purification / Q000494:pharmacology* / Q000627:therapeutic use; D029062:Apocynaceae / Q000737:chemistry* / Q000254:growth & development; D001938:Brazil; D016466:CHO Cells; D002470:Cell Survival / Q000187:drug effects; D003412:Cricetulus; D016207:Cytokines / Q000032:analysis / Q000276:immunology; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D004487:Edema / Q000188:drug therapy / Q000276:immunology; D020834:Ethnopharmacology; D008264:Macrophages / Q000187:drug effects* / Q000276:immunology; D051379:Mice; D010538:Peritonitis / Q000188:drug therapy / Q000276:immunology; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology* / Q000627:therapeutic use; D017208:Rats, Wistar
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D016207:Cytokines; D010936:Plant Extracts
keywords: Cytokines; Cytotoxicity; Inflammation; Macrosiphonia longiflora; Nitric oxide