RESUMO

Plant cell suspension culture of T. peruviana is a feasible biotechnological platform for the production of secondary metabolites with anti-proliferative/cytotoxic activity, as phenolic compounds (PC); however, different in in vitro growth conditions may affect the production, demanding strategies to increase the metabolite biosynthesis, as well as the development of sensitive and rapid analytical methods for metabolite monitoring. The Fourier transform near-infrared (FT-NIR) spectroscopy and Reversed-phase high-performance liquid chromatography (RP-HPLC) combined with Multivariate analysis (MVA) were used to detect significant differences in the PC production in cultures treated with two elicitors. The results suggest that the FT-NIR-MVA is useful for discriminating samples according to the treatment, showed significant influence of the PC signal. RP-HPLC-MVA showed that the elicitor effect occurs at 72 h post-elicitation. Detection of dihydroquercetin (maximum concentration = 12.59 mg/L), a flavonoid with anti-cancer properties, is highlighted. Future studies will be aimed at scaling this culture to increase the productivity of dihydroquercetin.

RESUMO

Abstract Thevetia peruviana is an ornamental shrub grown-up in many tropical region of the world. This plant produces secondary metabolites with biological properties of interest for the pharmaceutical industry. The objective was to determine the secondary metabolites profile of callus and cell suspension cultures of T. peruviana and compare them with those from explant (fruit pulp). Extracts in 50% aqueous ethanol and ethyl acetate were prepared. The phytochemical analysis was performed using standard chemical tests and thin layer chromatography. In addition, total phenolic and flavonoids compounds (TPC and TFC), total cardiac glycosides (TCG) and total antioxidant activity (TAA) was determined during the cell suspension growth. Phenolic chemical profile was also analyzed by high performance liquid chromatography (HPLC). Common metabolites (alkaloids, amino acids, antioxidants, cardiac glycosides, leucoanthocyanidins, flavonoids, phenols, sugars and triterpenes) were detected in all samples. The maximum production of extracellular TCG, TPC, TFC and TAA in cells suspensions were at 6-12 days; in contrast, intracellular content was relatively constant during the exponential grown phase (0 to 12-days). HPLC analysis detected one compound with retention time at 11.6 min; this compound was tentatively identified as dihydroquercetin, a flavonoid with anti-cancer properties. These results provide evidence on the utility of the in vitro cell cultures of T. peruviana for valuable pharmaceutical compounds production.

Assuntos

Células Cultivadas , Thevetia/citologia , Compostos Fitoquímicos/biossíntese , Triterpenos , Flavonoides , Cromatografia Líquida de Alta Pressão , Anticarcinógenos , Thevetia/química , Técnicas de Cultura , Compostos Fitoquímicos/análise , Antioxidantes

RESUMO

The objective was to enhance the production of the phenolic compounds in plant cell suspension cultures of T. peruviana at shake flask scale. The effects of salicylic acid (SA), methyl-jasmonate (MeJA) and the combination of both (SA/MeJA) were studied. Elicitor concentration, elicitation time and harvest time of cells were optimized. Phenolic compound content (PCC), flavonoid content (FC) and antioxidant activity (AA) were determined by the folin-ciocalteu method, flavonoid-aluminum complexation method and the ABTS assay, respectively. Differences between intracellular metabolite profiles due to the mentioned treatments were analyzed by Thin-layer chromatography and High-performance liquid chromatography. Highest PCC, FC and AA were obtained under the following treatments: 3 µM MeJA > 3 µM MeJA/300 µM SA > 300 µM SA > control, when elicited on the 4th day and harvested 96-h post-elicitation. It was demonstrated that exposure to 3 µM MeJA increase 1.49-fold of PCC, 1.66-fold of AA and 2.55-fold of FC compared to the control culture.