Influência da distribuição geográfica e dos aspectos fenológicos de Tithonia diversifolia Hemsl. A. Gray (Asteraceae) na sua composição química e atividade biológica

Abstract

Tithonia diversifolia Hemsl. A. Gray, popularly known as Mexican sunflower, is a species of the Asteraceae family with recognized pharmacological potential, including antioxidant, anti-inflammatory, and antiproliferative activities. The variability of the chemical composition of plant species can be influenced by environmental and phenological factors, directly impacting their bioactivity. This study aimed to analyze and correlate the chemical composition and antiproliferative activity of hydroalcoholic leaf extracts of Tithonia diversifolia from five locations in Espírito Santo (Colatina, Muniz Freire, Viana, UFES Vitória, and Santa Teresa), collected at vegetative (2014) and reproductive (2015) stages. Furthermore, to assess temporal stability, a new collection was performed after 10 years (2024) at the UFES and Santa Teresa locations. Chemical characterization of the extracts was performed using Fourier Transform Infrared Spectroscopy (FTIR), high-resolution Electrospray Ionization Mass Spectrometry (ESI-TOF), and chemometric analyses (Principal Component Analysis - PCA and Hierarchical Cluster Analysis - HCA). The results revealed a diversified chemical profile, with the annotation of secondary metabolites of interest, such as hydroxycinnamic acids (e.g., caffeoylquinic acid), flavonoids (e.g., apigenin, naringenin), sesquiterpene lactones (e.g., tagitinin A), and fatty acids. Multivariate analysis revealed a clustering of samples based on geographical origin, with samples from Santa Teresa standing out for presenting a distinct chemical profile and greater temporal variability. Biological activity was evaluated through in vitro assays using the MCF-7 (breast carcinoma) and MCF-10A (non-tumor mammary epithelium) cell lines. Cell viability assays (MTT) demonstrated that the extracts have concentration-dependent antiproliferative activity against MCF-7 cells. The MUF4 and MUF5 extracts, from Muniz Freire, showed the highest Selectivity Indices (SI > 2.0), indicating preferential action against tumor cells. Meanwhile, the proliferation assay (crystal violet) and clonogenicity assays corroborated the inhibition of growth and colony-forming capacity of MCF-7 cells treated with these extracts. Investigation of the mechanisms of action revealed that MUF4 and MUF5 induced a significant increase in the production of reactive oxygen species (ROS), caused cell cycle arrest, predominantly in the S and G2/M phases, and activated apoptosis, as confirmed by the Annexin V/PI assay. However, no significant induction of histone H2AX phosphorylation (γ-H2AX), a marker of double-strand DNA breaks, was observed, suggesting that cytotoxicity is not mediated by direct genotoxicity, but rather by redox imbalance and cellular stress. It is concluded that the chemical composition and antiproliferative activity of Tithonia diversifolia vary according to the geographical location and phenological stage of the plant. The extracts from Muniz Freire stood out for their selective and potent activity against breast cancer cells, acting through the induction of oxidative stress, disruption of the cell cycle, and activation of apoptosis. The results reinforce the therapeutic potential of the species and highlight the importance of considering botanical origin and growing conditions in the development of phytomedicines