Phytochemical and biotechnological study of Sinningia magnifica (Otto & A. Dietr.) Wiehler and Sinningia schiffneri Fritsch (Gesneriaceae) and evaluation of the photoinduced biological effect for antitumoral PDT application

The aim of this work was the phytochemical and biotechnological study of Sinningia schiffneri Fritsch and Sinningia magnifica (Otto & A. Dietr.) species, members of Gesneriaceae family, and the evaluation of the photoinduced biological effect for application in antitumor photodynamic therapy (PDT). To this, we sought to analyze the chromatographic profile of these plant species (plant in natura and plant obtained in vitro) employing to guide the study the evaluation of the absorption in the region of 400 to 800 nm, the analysis of photochemical efficiency and the bioassays with the evaluation of photoinduced biological effect in tumor cell lines, as prostate cancer, melanoma and ovarian cancer. Thus, a phytochemical approach was performed with the identification of 19 substances, 4 of which are new to Sinningia (Digiferrol, Digiferrugineol e 2,6,7-tri-epi-cedrol, from S. schiffneri and a 2,4’,6-trihydroxy-4-methoxydiidrochalcone derivative, from S. magnifica), most of them being molecules individually identified to the class of anthraquinones and naphthoquinones. This study was performed using several chromatographic procedures (UHPLC-ESI-MS/MS, using standard substances previously isolated from Sinningia species in a dereplication approach and classic column, analytical and preparative thin layer chromatography for fractionation and isolation). For isolated substances the structural identification was performed using spectroscopic methods of analysis (1D and 2D NMR) and mass spectrometry (ESI-MS/MS). In the biotechnological study, was done the development of procedures for in vitro plantlets culture and callus culture of S. schiffineri and S. magnifica, aiming to the application or establishment of plant tissue culture for the production of secondary metabolites, focusing on quinones and their derivatives documented in literature from Sinningia species and for presenting potential to act as natural photosensitizers for application in PDT. The in vitro culture establishment was only possible for S. magnifica, and allowed the development of in vitro procedures for plantlets culture and callus culture, involving steps such as the choice of the most suitable antioxidant for callus growth, a determination of the type and amount of growth regulator with better results for the formation of callogenesis, better conditions for the development of the callus secondary culture, as well as its growth curve, in which was stablished the use of charcoal as antioxidant and hytormones in the concentration of 8 mg L-1 BAP and 8 mg L-1 2,4-D, in MS medium, stored in the dark, with stationary growth phase at 42 days. From the dereplication study, photophysical and photochemistry evaluation, in vitro biological studies were performed in tumor cell culture (PC-3 and SKMEL-103), applying PDT, in which the statistics that affect between the presence and absence of LEDs in the wavelength of 420nm to 5,5 J/cm2 at low concentrations, with better results for Betulinic acid, Dunniol, 7,8-dimethoxy-?-dunnione, 7-hydroxy-?-dunnione, 7-hydroxy-6-methoxy-alpha-dunnione and Pustulin (7-hydroxy-6-methoxy-tectoquinone). Also, a study with betulinic acid was performed, as it is a commercialized product, and with promising results in terms of bioactivity, proceeding with the ovarian cancer investigation, but its solubility is a limiting factor in terms of kinetic supply. For this, an betulinic acid in HP-beta-cyclodextrin complex (BAC) was prepared and quantification methods of the complex in plasma and tumors were performed using HPLC-MS and MALDI-MSI. The photoinduced biological effect of betulinic acid free and complexed with HP-beta-CD was evaluated in vitro and the results showed decreased cell viability of ovarian carcinoma culture (A2780) in the presence of LED irradiation (lambda = 420nm) after 5 min of contact with the cells, with similar results in fluorescence microscopy analysis and compared to the experimental controls. In the in vivo experiments, the inclusion complex of betulinic acid with HP-beta-CD was effective in doubling the bioavailability of plasma betulinic acid at the administered concentration of 100 mg kg-1. However, it was not possible to detect this molecule in tumor tissue by HPLC-MS and MSI under the experimental conditions used (AU)