Avaliação in vitro dos efeitos antiparasitário, antitumoral e antimicrobiano de proteínas extraídas e purificadas de semente de Cycas revoluta

Abstract

Cycas revoluta (Thunb.), belonging to the Cycadaceae family, is a gymnosperm native to China, known for its significant ornamental appeal. Recently, the scientific community has been investigating its therapeutic potential against a variety of pathogens. This study focuses on analyzing the proteins present in its seeds, aiming to provide insights into their biological activities, particularly their antiparasitic, antitumor, and antimicrobial properties. Firstly, protein extraction from C. revoluta seeds was performed by maceration and homogenization with NaCl, followed by protein purification using a Sephadex G-75 gel filtration column. The proteins were then visualized via SDS-PAGE gel electrophoresis, quantified using the BCA method, and analyzed by mass spectrometry. Before assessing biological activities, cytotoxicity tests were conducted to ensure that safe dosages were used for non-tumor cells, thereby avoiding potential biases in the study. The study includes tests to evaluate the antiparasitic effect by analyzing the invasion and proliferation of Toxoplasma gondii tachyzoites. Additionally, it assesses the antitumor effect on lung and prostate cancer, with experiments focused on both direct treatment of cells (MTT) and prevention (tumor colony formation). Antimicrobial activity tests were also conducted to determine the minimum inhibitory concentration (MIC) required to inhibit the growth of Escherichia coli, Staphylococcus aureus, and Salmonella enterica. The crude extract from Cycas revoluta seeds and the four purified protein fractions, designated as Peak I (Multifunctional fusion protein - Glycine-tRNA synthetase – 113,7 kDa; Prostaglandin G/H synthase – 63,5 kDa; Chitinase A – 38,7 kDa), Peak II (Chitinase A – 38,7 kDa), Associated Peak (Chitinase A – 38,7 kDa; Hydrolase – 15,4 kDa), and Peak III (Hydrolase – 15,4 kDa), were investigated. Each protein fraction exhibited distinct results in biological analysis. In the evaluation of anti-Toxoplasma gondii activity, Peak II was particularly notable for its efficacy in treating the parasite and infected cells. In the antitumor assay, the MTT analysis revealed that only the crude extract reduced the viability of prostate cancer cells. In the clonogenic assay, both the crude extract and the Associated Peak effectively reduced colony formation in prostate (PC3) and lung (A549) cancer cells. In the antibacterial evaluation, the fractions with the lowest MIC values were Peak III for Escherichia coli and Staphylococcus aureus, and Peak II for Salmonella enterica. In conclusion, the purified protein fractions of C. revoluta exhibit promising therapeutic potential. Further scientific studies on this species are encouraged in the search for new therapeutic molecules with significant application potential.