RESUMO
BACKGROUND: Schinus terebinthifolius is widely used in traditional medicine by Brazilian quilombola and indigenous communities for treatment of several diseases. Extracts from different tissues are being used to produce creams to treat cervicitis and cervicovaginitis. However, most studies are limited to the assessment of the essential oils and extracts obtained from the leaves. OBJECTIVE: The aim was to evaluate antioxidant and antibacterial activities, to assess the phytochemical profile and to quantify total phenolic compounds of various extracts prepared from S. terebinthifolius grown in the coast of Bahia, Brazil. MATERIALS AND METHODS: Extracts were obtained by hot continuous extraction (soxhlet) and by maceration. Quantification of phenolic compounds was performed using the Folin-Ciocalteu method and antioxidant properties were assessed by 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay. Phytochemical screening was performed as described by in the literature and antibacterial activity against Enterococcus faecalis (ATCC 29212) was determined by the microdilution broth assay. RESULTS: Extraction method greatly affected the metabolite profile of the extracts. Antioxidant activity varied between 21.92% and 85.76%, while total phenols ranged between 5.44 and 309.03 mg EAG/g of extract. Leaf extract obtained with soxhlet showed minimum inhibitory concentration (MIC) of 15.62 µg/mL, while stem extract obtained by maceration was able to inhibit the growth of E. faecalis at 62.5 µg/mL. Stem bark extracts showed a MIC of 500 µg/mL for both extraction methods, while no inhibition was observed for fruit extracts. CONCLUSION: In general, total phenolic content, antioxidant and antibacterial activities were higher in samples obtained by soxhlet. Our results provide important clues in order to identify alternative sources of bioactive compounds that can be used to develop new drugs.
RESUMO
Plant lectins, especially those purified from species of the Leguminosae family, represent the best-studied group of carbohydrate-binding proteins. Lectins purified from seeds of the Diocleinae subtribe exhibit a high degree of sequence identity notwithstanding that they show very distinct biological activities. Two main factors have been related to this feature: variance in key residues influencing the carbohydrate-binding site geometry and differences in the pH-dependent oligomeric state profile. In this work, we have isolated a lectin from Canavalia boliviana (Cbol) and solved its x-ray crystal structure in the unbound form and in complex with the carbohydrates Man(α1-3)Man(α1-O)Me, Man(α1-4)Man(α1-O)Me and 5-bromo-4-chloro-3-indolyl-α-D-mannose. We evaluated its oligomerization profile at different pH values using Small Angle X-ray Scattering and compared it to that of Concanavalin A. Based on predicted pKa-shifts of amino acids in the subunit interfaces we devised a model for the dimer-tetramer equilibrium phenomena of these proteins. Additionally, we demonstrated Cbol anti-inflammatory properties and further characterized them using in vivo and in vitro models.
Assuntos
Anti-Inflamatórios/farmacologia , Canavalia/química , Edema/tratamento farmacológico , Manosídeos/química , Peritonite/tratamento farmacológico , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Sementes/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Movimento Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Cristalografia por Raios X , Edema/induzido quimicamente , Manosídeos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Peritonite/induzido quimicamente , Conformação Proteica , Ratos , Ratos Wistar , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
Lectins are comprised of a large family of proteins capable of the specific and reversible recognition of carbohydrates. Legume lectins, the most studied plant lectins, show high structural similarity, but with modifications that imply a variation in the intensity of some biological activities. In this work, the primary and tertiary structures of Canavalia grandiflora (ConGF) were determined. ConGF, a lectin isolated from C. grandiflora seeds, is able to induce relaxant activity in rat aortic rings. The complete sequence of ConGF comprises 237 amino acids. This particular protein has primary sequence variations commonly found in lectins from Dioclea and Canavalia genera. The protein structure was solved at 2.3 Å resolution by X-ray crystallography. An X-Man molecule was modeled into the carbohydrate recognition domain. Still, ConGF (30 and 100 µg mL(-1)) elicited 25% of vasorelaxation (IC50=34.48 ± 5.07 µg mL(-1)) in endothelialized aortic rings. A nonselective inhibitor of nitric oxide blocked ConGF relaxant effect, showing mediation by nitric oxide. Key distances between ConGF carbohydrate recognition domain residues were determined in order to explain this effect, in turn revealing some structural aspects that could differentiate lectins from the Canavalia genera with respect to different efficacy in vasorelaxant effect.
Assuntos
Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Vasodilatadores/química , Vasodilatadores/farmacologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Técnicas In Vitro , Masculino , Manose/química , Manose/metabolismo , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Lectinas de Plantas/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Ratos , Ratos Wistar , Análise de Sequência , Relação Estrutura-Atividade , Vasodilatadores/metabolismoRESUMO
RATIONALE: Lectins are a family of proteins capable of deciphering the glycan code. Several authors have published works about crystallization and mass spectrometry analyses of ConA-like lectins. However, mass spectrometry has never been used to characterize lectin crystal content. In this study, Canavalia grandiflora lectin (ConGF), a ConA-like lectin, was crystallized, part of its primary structure sequenced and the pro-inflammatory activity evaluated. In addition, the crystal content was analyzed by mass spectrometry. METHODS: ConGF was crystallized in the presence of X-Man by hanging-drop vapor diffusion at 293 K and the protein crystal content was analyzed by electrospray ionization in a SYNAPT HDMS mass spectrometer. Partial sequence was obtained by protein digestion with several proteolytic enzymes and the peptides sequenced by liquid chromatography/tandem mass spectrometry (LC/MS/MS). The pro-inflammatory potential of ConGF was also evaluated in the model of rat paw edema. RESULTS: The protein crystals consist of mature α chain and ß and γ fragments measuring 25 612 ± 2 Da, 12 962 ± 2 Da and 12 667 ± 2 Da, respectively. The crystal belongs to the orthorhombic space group I222 (unit cell parameters: a = 67.70, b = 55.90, c = 107.46 Å), assuming a monomer in the asymmetric unit. The solvent content was calculated as 43.50% and the protein content as 2.5 µg. Furthermore, a significant part of the primary structure (65.8%) was determined by mass spectrometry. CONCLUSIONS: As far as we know this is the first report of lectin crystal content characterized by mass spectrometry. Like other ConA-like lectins, GonGF induced paw edema however differing in potency and duration. The observed pro-inflammatory activity suggests that ConGF might be a useful tool in the study of inflammation processes and structure/function relationships.