Produção in vitro, análise fitoquímica e avaliação da atividade antineoplásica de metabólitos de Hovenia dulcis Thunb. (Rhamnaceae) obtidos por diferentes estratégias biotecnológicas

Abstract

Hovenia dulcis Thunberg, naturally occurring in East Asia, is cultivated in Brazil and known as uva-do-japão. In folk medicine, the species is used on the treatment of several diseases and some studies point to its activity as antineoplasic, tripanocidal and hepatoprotective agent. Compounds known as secondary metabolites are not essential to plant cell survival, but provide adaptative advantages and important biological activities are related to these compounds. Natural products have been obtained through tissue culture techniques, such as callus cultures and cell suspension cultures, which provide the continuous and homogenous production of raw materials, regardless of seasonal and environmental factors. The present work aimed the establishment of H. dulcis in vitro cultures on the purpose of secondary metabolites production, intending the evaluation of its antineoplasic potential on K562 cells. Protocols to establish different systems such as callus cultures, cell suspension cultures (CSC) and compact callus clusters (CCC) were tested and the use of elicitors on the improvement of metabolites production was also evaluated. The addition of KIN and TDZ in replacement of BAP was not suitable to produce friable calli, and the same was observed for dark conditions. The addition of silver nitrate promoted callus friability in all tested concentrations and 2.0 mg.L-1 was considered the best condition. Rates of 100% were obtained on CSC formation with or without the AgNO3. Highest biomass production was observed on the lowest PIC concentration tested (0.625 mg.L-1). NMR spectral analysis suggested the presence of (+)-dihydromirycetin, (+)-gallocatechin, hovenitin II, hovenoside G, hoduloside III, hoduloside IV, hoduloside I and hovenidulcioside B1 in friable callus., All NAA tested concentrations induced green compact callus formation on CCC cultures. The elevation of rotation speed to 135 rpm increased cell dispersion with formation of the desired cell clusters. Cell line selection proved to be a suitable method for clusters size homogeneity, which was maintained over five subcultures in 100% of the cultures. A saponin rich fraction was obtained from the cell clusters, corresponding to 1.4% of dry mass. NMR spectral analysis suggested the presence of Hovenoside G and hovenidulciosides A2 and B2 as saponins. Elicitation was considered and efficient approach to secondary metabolites production having no effect on callus morphology. The elicitors were capable of altering chromatographic profiles obtained on HPLC. The use of 5.0 mg.L-1 of yeast extract as elicitor increased the total flavonoid production almost 3 folds (12.280 ± 3.396 quercetin equivalents/mg of extract). Finally, the in vitro antitumoral studies showed cytotoxicity of H. dulcis callus extract on chronic myeloid leukemia cell line (IC50 of 74.05 μg.mL-1) and growth inhibition, suggesting the antineoplasic potential for a biotechnological product (callus) of this specie.