Purificação, caracterização e avaliação de atividades antitumoral, antimicrobiana e sobre parâmetros hemostáticos das lectinas de Genipa americana L. (jenipapo), Rhizophora mangle L. (mangue vermelho) e da própolis vermelha de Alagoas

Abstract

The use of macromolecules obtained from plant tissues has great economic and pharmacological potential and this is due to the presence of bioactive compounds. Among these, lectins have been intensely explored due to the various biotechnological actions they may have, especially the antifungal, anticoagulant and antitumor activities. Thus, the present work aimed to purify, characterize and evaluate the antitumor, antimicrobial and hemostatic activity of lectins from Genipa americana L. (jenipapo), Rhizophora mangle L. (red mangrove) and red propolis from Alagoas. Lectins were isolated initially by extraction in 50mM Tris-Hcl pH 8.0 for G. americana seeds and bark, 0.15M NaCl for R. mangle seeds and 0.15M NaCl with 30% ethanol for red propolis. Then, salt fractions were made with ammonium sulfate and the fractions that showed better activity were submitted to molecular exclusion chromatography (Sephacryl S-100), for isolation of lectins from G. americana (GaBL); molecular exclusion chromatography (Sephacryl S-100) followed by a cation exchanger (DEAE Sepharose) for the lectin from G. americana seeds (GaSL); or chitin column chromatography to isolate lectins from R. mangle and red propolis. Through a single chromatographic step a lectin (GaBL) was isolated and by SDS-PAGE (10%) it was observed that GaBL has an approximate molecular mass of 242.5 kDa. The effect of lectin on blood clotting was evaluated through activated partial thromboplastin time (APTT), prothrombin time (PT). GaBL showed anticoagulant activity only in the intrinsic pathway, where the APTT was prolonged. In the cytotoxic test, GaBL was not toxic to 3T3 fibroblast cell lines at concentrations below 50μg/mL. The antitumor activity evaluated used human skin cancer (A431), melanoma (B16) and tongue squamous cell carcinoma (SCC9) cell lines. Cell proliferation and cell migration were decreased in all cell lines evaluated in contact with 10 µg/ml of GaBL decreased invasion of SCC9 cells. Apoptosis was higher in lectin treated B16 and SCC9 cells. GaBL's positive regulation of E-cadherin and suppression of Col1A1 in all strains tested indicated less cancer development. The lectin from G. americana seeds, GaSL, had its hemagglutinating activity (AH) strongly inhibited by rhamnose, but was not affected by divalent ions or EDTA. GaSL is classified as a thermostable lectin, with best activity at a temperature below 80°C and in the pH range 5.0 to 6.0. In the face of antifungal activity, GaSL, when tested with the fungi Candida albicans, Staphylococcus aureus and Cryptococcus neoformans, was shown to inhibit all fungal strains at concentrations below 12.5 μg/mL. The lectin from R. mangle seeds is also thermostable, but unlike the others, it was inhibited more strongly by arabinose and casein, had better activity at pH 8.0 and 9.0 and had its AH reduced in the presence of Mn2+, Mg2+ and Ca2+ ions, not altered by EDTA and stimulated in the presence of Zn2+. The lectin from propolis was also strongly inhibited by casein, is thermostable, showed better AH in the pH 5.0 to 6.0 range and was reduced in the presence of Mn2+, Mg2+, slightly stimulated by Zn2+ and not affected by EDTA and Ca2+. In view of the antifungal activity PVAL proved to be a promising biotechnological tool, when tested with the fungi Candida albicans, Staphylococcus aureus and Cryptococcus neoformans. PVAL at concentrations of 12.5 μg/mL was able to strongly inhibit Candida albicans and Staphylococcus aureus and 25 μg/mL Cryptococcus neoformans. The effect of PVAL on blood clotting was evaluated through activated partial thromboplastin time (APTT), prothrombin time (PT). Where PVLA promoted expressive prolongation of APTT, while for TP it promoted strong inhibition. In view of the above data, we can highlight that GaBL has high antitumor potential against cancer of the upper aerodigestive tract and has a notorious homeostatic activity. GaSL and PVAL have remarkable antifungal activity and can be a promising biotechnological tool, the latter having an important role in the homeostatic processes of coagulation, being a strong protractor of APTT and TP inhibitor. Finally, we emphasize that all the lectins studied have structural characteristics that favor the biotechnological study involving human health.