RESUMO
INTRODUCTION: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest. OBJECTIVES: This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds (BpuTI), describing its kinetic mechanism and anticoagulant effect. METHODS: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. In vitro assays were performed to verify its ability to prolong blood clotting time. RESULTS: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. BpuTI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and BpuTI showed similarity to several Kunitz-type trypsin inhibitors. BpuTI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10-6 M) and Ki (1.05 x 10-6 M). Additionally, BpuTI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. BpuTI showed an anticoagulant effect in vitro at a concentration of 33 µM, prolonging clotting time by 2.6 times. CONCLUSION: Our results suggest that BpuTI can be a biological tool to be used in blood clotting studies.
Assuntos
Bauhinia , Inibidores da Tripsina , Animais , Bovinos , Inibidores da Tripsina/farmacologia , Inibidores da Tripsina/química , Bauhinia/metabolismo , Tripsina/análise , Tripsina/química , Tripsina/metabolismo , Espectrometria de Massas em Tandem , Sementes/química , Anticoagulantes/farmacologia , Anticoagulantes/análise , Anticoagulantes/químicaRESUMO
The present work aimed to investigate the antioxidant, anti-inflammatory and wound healing potential of ethyl acetate fraction from Bauhinia ungulata L. (FABU) on in vitro and in vivo models. Wound healing assay using human lung adenocarcinoma A549 cell line was employed to evaluate the ability of FABU in modulating cell migration. In addition, a surgical wound model in C57BL/6 mice was used to study the healing potential of FABU incorporated into gel carbomer 940 (Carbopol®). Evaluation of lipid peroxidation, inflammatory and anti-inflammatory mediator gene expression, rate of wound closure, and histological analysis were done. FABU significantly reduced the gap area in in vitro wound healing assay, 24 h after treatment. In the animal model, FABU at 0.5% topically applied once-daily for 5 days to the surgical wounds significantly reduced the lesion area. Moreover, it significantly decreased the levels of lipid peroxidation in the lesions and decreased the relative gene expression levels of IL-1ß and TNF-α in the injured region. In conclusion, our study suggests that Bauhinia ungulata can effectively promote the wound healing, probably by regulating the inflammatory environment during the early stages of the process.
Assuntos
Bauhinia/metabolismo , Extratos Vegetais/farmacologia , Cicatrização/efeitos dos fármacos , Células A549 , Acetatos/química , Acetatos/farmacologia , Resinas Acrílicas/farmacologia , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Modelos Animais de Doenças , Fabaceae/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fitoterapia , Pele/metabolismo , Cicatrização/fisiologiaRESUMO
Several plants have been studied for their medicinal properties, especially concerning the management of chronic diseases, such as diabetes, aiming at a more accessible form of treatment. In this context, the aim of this study was to characterize plant proteins used in folk medicine as hypoglycemic agents for the treatment of diabetes, namely "abajerú" (Chrysobalanus icaco) and "cow's paw" (Bauhinia forficata and Bauhinia variegata). The species were differentiated by proteome characterization. Proteins were in-solution digested using trypsin by the filter-assisted sample preparation (FASP) method. Peptides were then analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) for protein characterization. In total, 131 proteins were identified. The main biological functions of these proteins were cellular respiration, transport, metabolism and photosynthesis. Insulin-like proteins were not detected, but proteins involved in controlling glucose levels were. The results are of value in the proteomic characterization of phytotherapeutic plants, and may serve as baseline for the assessed species in Brazil, where a lack of knowledge in this regard is observed.
Assuntos
Bauhinia/metabolismo , Chrysobalanaceae/metabolismo , Diabetes Mellitus/tratamento farmacológico , Hipoglicemiantes/farmacologia , Extratos Vegetais/farmacologia , Proteoma , Brasil , Cromatografia Líquida , Geografia , Hipoglicemiantes/química , Medicina Tradicional , Extratos Vegetais/química , Folhas de Planta/metabolismo , Plantas Medicinais , Proteômica , Espectrometria de Massas em TandemRESUMO
A chromatographic fingerprint is a comprehensive method that reveals the distinctive pattern of peaks across the chromatogram for a given sample. It is considered an effective strategy to assess the identity and quality of herbal materials, as well as for the control of the quality of their derived products. HPLC is the most employed technique for these purposes and it is used routinely for quality control in industry. Hence, its impact on the environment should not be neglected. This work provides a rational and generic procedure to qualitatively fingerprint complex matrices. Resource- and time-saving experimental designs were selected; an alternative safer organic solvent was tested and a time-saving and innovative response entitled the green chromatographic fingerprinting response was developed and employed. This procedure was applied in the development of chromatographic fingerprints for extracts of Bauhinia forficata and Casearia sylvestris. Moreover, the response proposed here can be combined with a complementary metric available in the literature to compare methods using different solvents. According to this, the chromatographic fingerprints developed here using ethanol as the organic solvent provided a performance better than that of reference methods in which more harmful acetonitrile or methanol were employed.
Assuntos
Bauhinia/química , Casearia/química , Cromatografia Líquida de Alta Pressão/métodos , Química Verde/métodos , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extração em Fase Sólida/métodos , Bauhinia/metabolismo , Casearia/metabolismo , Metaboloma , Extratos Vegetais/metabolismoRESUMO
Three plant proteinase inhibitors BbKI (kallikrein inhibitor) and BbCI (cruzipain inhibitor) from Bauhinia bauhinioides, and a BrTI (trypsin inhibitor) from B. rufa, were examined for other effects in Callosobruchus maculatus development; of these only BrTI affected bruchid emergence. BrTI and BbKI share 81% identities in their primary sequences and the major differences between them are the regions comprising the RGD and RGE motifs in BrTI. These sequences were shown to be essential for BrTI insecticidal activity, since a modified BbKI [that is a recombinant form (BbKIm) with some amino acid residues replaced by those found in BrTI sequence] also strongly inhibited insect development. By using synthetic peptides related to the BrTI sequence, YLEAPVARGDGGLA-NH2 (RGE) and IVYYPDRGETGL-NH2 (RGE), it was found that the peptide with an RGE sequence was able to block normal development of C. maculatus larvae (ED(50) 0.16% and LD(50) 0.09%), this being even more effective than the native protein.
Assuntos
Bauhinia/metabolismo , Besouros/crescimento & desenvolvimento , Inibidores Enzimáticos/metabolismo , Inseticidas/metabolismo , Peptídeo Hidrolases/metabolismo , Doenças das Plantas/genética , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Animais , Bauhinia/química , Bauhinia/genética , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/genética , Inibidores de Cisteína Proteinase/metabolismo , Inibidores Enzimáticos/química , Genes de Plantas , Inseticidas/química , Calicreínas/antagonistas & inibidores , Larva/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Dados de Sequência Molecular , Estrutura Molecular , Peptídeos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Protozoários , Proteínas Recombinantes , Alinhamento de Sequência , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Inibidores da Tripsina/química , Inibidores da Tripsina/genética , Inibidores da Tripsina/metabolismoRESUMO
The aqueous extract from aerial parts of Bauhinia forficata was able to neutralize the clotting activity induced by Bothrops and Crotalus crude venoms. The clotting time, upon human plasma, induced by B. moojeni venom was significantly prolonged. Clotting and fibrinogenolytic activities induced by isolated thrombin-like enzyme from Bothrops jararacussu were totally inhibited after incubation at different ratios. The extract was not able to neutralize the hemorrhagic activity induced by an Bothrops venoms, but it efficiently inhibited the edema induced by Crotalus durissus terrificus venom and isolated PLA2s. In addition, it did not inhibited the phospholipase A2 activity of Bothrops snake venoms. Interaction studies between Bauhinia forficata extract and snake venoms, when analyzed by SDS-PAGE, did not reveal any apparent degradation of the venom proteins. This extract is a promising source of natural inhibitors of serine-proteases involved in blood clotting disturbances induced by snake venoms.
Assuntos
Anticoagulantes/farmacologia , Antifibrinolíticos/farmacologia , Bauhinia/metabolismo , Venenos de Serpentes/antagonistas & inibidores , Animais , Anticoagulantes/química , Antifibrinolíticos/química , Bauhinia/química , Venenos de Crotalídeos/efeitos adversos , Venenos de Crotalídeos/antagonistas & inibidores , Avaliação Pré-Clínica de Medicamentos/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Componentes Aéreos da Planta/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Venenos de Serpentes/efeitos adversos , Venenos de Serpentes/química , Água/química , Água/farmacologiaRESUMO
A Kunitz-type protease inhibitor (BbKI) found in Bauhinia bauhinioides seeds has been overexpressed in Escherichia coli and crystallized at 293 K using PEG 4000 as the precipitant. X-ray diffraction data have been collected to 1.87 A resolution using an in-house X-ray generator. The crystals of the recombinant protein (rBbKI) belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 46.70, b = 64.14, c = 59.24 A. Calculation of the Matthews coefficient suggests the presence of one monomer of rBbKI in the asymmetric unit, with a corresponding solvent content of 51% (VM = 2.5 A3 Da(-1)). Iodinated crystals were prepared and a derivative data set was also collected at 2.1 A resolution. Crystals soaked for a few seconds in a cryogenic solution containing 0.5 M NaI were found to be reasonably isomorphous to the native crystals. Furthermore, the presence of iodide anions could be confirmed in the NaI-derivatized crystal. Data sets from native and derivative crystals are being evaluated for use in crystal structure determination by means of the SIRAS (single isomorphous replacement with anomalous scattering) method.