Identificação, purificação e caracterização de uma lectina de folhas de Jatropha multifida L. (MALPIGHIALES: EUPHORBIACEAE)

Abstract

Jatropha multifida, popularly known by the names merthiolate, coral flower, coral, garden coral, blood flower, is a plant grown in South America, southern China and Africa, and is also found in Alagoas. Various tissues of the plant are used in folk medicine as a healing agent for the treatment of oral candidiasis, gonorrhea, fever, arthritis, wounds and infections. Secondary and primary metabolites may be related to the medicinal properties of this plant. Through thin layer chromatography, the presence of alkaloids, flavonoids, steroid tannins and phenols was observed in the crude extracts in methanol and ethyl acetate. Among the primary metabolites, lectins are proteins capable of binding to carbohydrates and exerting various biological activities such as antimicrobial, insecticide, antitumor and immunomodulatory. The present study aimed to identify, isolate and characterize a lectin extracted from the dry leaves of J. multifida. Proteins were extracted from the leaf powder through homogenization (16 h, 4 ºC) in four different solutions: NaCl 0.15 M and in Tris-HCl 50 mM pH 8.0, sodium phosphate 50 mM pH 7.2 and the sodium acetate pH 5.0. The extraction performed with Tris-HCl 50 mM pH 8.0 showed a higher specific haemagglutinating activity (AHE: 162.44) as well as extracting a larger amount of proteins (3.15 mg / mL). The obtained extract was treated with ammonium sulfate in different concentrations (0-20%, 20-40%, 40-60% and 60-80%) for protein fractionation and the fraction 0-20% was the only one that presented AHE ( 556.98). The 0-20% fraction was subjected to chitin column chromatography balanced with 50 mM Tris - HCl pH 8.0. 2 mL fractions were collected and evaluated for absorbance at 280 nm and HA. The active protein peak obtained with the eluent 0.5 M acetic acid was collected and dialyzed against 50 mM Tris HCl pH 8.0 for 6 hours. To check the purity of the lectin and determine the apparent molecular mass, the sample was analyzed by electrophoretic method on 10% polyacrylamide gel under denaturing conditions, using sodium dodecyl sulfate, and in the presence and absence of reducing agent, where it was possible the visualization of a protein band of apparent molecular mass of 56 kDa in a non-reducing and reducing condition. Therefore, a J. multifida leaf lectin (JamuLL) was isolated using a single chromatographic step in milligram quantities (2.5 mg; AHE: 10.240). The measurement of proteins and phenols was carried out at all stages of the purification, showing that the purification procedure removed phenols found in the extract and fraction. The characterization of lectin showed that it has hemagglutinating activity partially inhibited by casein, but that it is not affected by divalent ions (Ca2 + and Mg2 +) or by the presence of EDTA. JamuLL is classified as a thermostable lectin, as it remained active between temperatures of 30 ° C to 100 ° C for 60 minutes, as well as between the pH range 5.0 to 8.0, showing better activity at acidic pH values . In this work it was possible to isolate a lectin from a medicinal plant with possible biotechnological potential.