Cultura de calos e suspensão celular de Duroia saccifera: estudo fitoquímico, cinética de crescimento e avaliação das atividades biológicas

Abstract

Tissue and plant cell culture is an efficient strategy for the safe and continuous production of secondary metabolites under controlled physical and chemical conditions. Duroia saccifera (Roem. & Schult.) K. Schum, as well as other species of the genus and the Rubiaceae family, is a potential target for phytochemical and pharmacological studies due to the occurrence of a wide variety of classes of biologically active molecules. Based on this, the objective of this work was to evaluate the growth potential, from the determination of the growth curves of the culture of calli and cell suspensions of the D. saccifera and to perform the phytochemical study of the extracts obtained from the callus and suspensions, and evaluate their chemical and biological activities. For this, calli previously established in vitro in Murashige and Skoog medium supplemented with 4 mg.L-1 of 2,4-dichlorophenoxyacetic acid (2,4D) and 2 mg.L-1 kinetin (KIN) were multiplied in successive subcultures every 30 days and kept in a growth room with controlled temperature of 26 ± 2 ºC, under photoperiod of 16/8 hours (light/dark), provided by fluorescent lamps. The material was extracted with the organic solvents hexane, ethyl acetate (EtOAc) and methanol. The extracts were tested in antioxidant, antimicrobial and antimycobacterial assays and fractionated. Suspension cell culture was established, when 1 g of callus was inoculated into 100 mL of the liquid culture medium. The growth curves of both forms of cultivation were determined during 40 days, in order to perform a comparative study through the calculation of growth kinetic parameters. In the phytochemical study of the extracts it was possible to verify the presence of terpenoids, steroids, aromatic substances, fatty acids, among others secondary metabolites. In fraction 2 (6.3 mg) of the hexane extract the evidence of the presence of the lupen-3-one steroid was observed. Regarding the biological and chemical assays, the EtOAc extract presented high antimycobacterial activity against Mycobacterium tuberculosis, with minimal inhibitory concentration (MIC), 90% inhibitory concentration (IC90) and minimum bactericidal concentration (MBC) of 25, 19.5 and 200 μg.L-1, respectively. The extracts were not active in the antimicrobial assays against the tested bacteria, as the extracts had no antioxidant potential against the DPPH free radical and the Fe3+/phenanthroline complex. The results showed that the callus followed a slow growth pattern, reaching a higher fresh (FM) and dry mass (DM) on the 40th day, respectively, 72.6 and 2.808 g.L-1. The accumulation of biomass and the growth of suspension cell culture was significantly higher, reaching higher MF and MS, on the 36th day of cultivation, respectively of 413.7 and 12.995 g.L-1. It was not possible to determine the stationary stages in the growth, however, it was inferred that the maximum production of secondary metabolites occurred on the 40th day of the culture of callus and from the 24th to the 36th day in the culture of cell suspension. The calculated kinetic parameters demonstrate that the cell suspension has a high potential for the supply of plant biomass of the species, becoming a promising source for the in vitro secondary metabolites production.