Brazilian Conference on Natural Products
7th Brazilian Conference on Natural Product/ XXXIII RESEM Proceedings

Several species of Baccharis are popularly used to treat stomach pain, ulcers, cramps, fever, wounds, and rheumatism. Rheumatoid and gouty arthritis are chronic diseases that affect multiple organ systems and have in common an exacerbated inflammatory response. The current anti-arthritis therapy is limited to the emergence of adverse effects and the high cost of biological drugs. Natural products comprise a promising source of compounds for the development of novel anti-inflammatory drugs. The objective of this work was to evaluate the in vitro activity of Baccharis species on the release of pro-inflammatory mediators and to analyze the chemical composition of the most active extracts. Vegetal materials of 19 Baccharis species were collected in Parque Nacional do Caparaó (Espírito Santo and Minas Gerais states) and Serra da Calçada (Minas Gerais state). The dried plant materials (leaf, stalk or aerial parts) were subjected to static maceration with 96°GL ethanol to afford 31 extracts after solvent removal in a rotary evaporator. The extracts had their chromatographic profiles analyzed by UPLC-ESI-MS/MS on an ODS column eluted with a gradient of acidified ACN/H2O (0.1% v/v HCOOH). Based on the differences of the chemical composition evidenced by UPLC-ESI-MS/MS analyses, 18 extracts were selected for biological assays. The cytotoxicity of the selected extracts against THP-1 cells was evaluated by the sulforhodamine B method. Non-cytotoxic extracts (cell viability > 80%) were then evaluated in LPS-stimulated THP-1 cells at 20 µg/mL for the inhibition of IL-1β and TNF-α production. The quantification of the pro-inflammatory mediators was performed by enzyme-linked immunosorbent assay (ELISA). Among the 18 extracts tested, those from B. calvescens (65.0% inhibition of IL-1β and 21.1% of TNF-α) and B. imbricata (82.6% inhibition of IL-1β and 42.6% of TNF-α) were the most active ones. For these extracts, concentration-response curves were obtained. The extract of B. calvescens showed EC50 values of 14.8 µg/mL (IL-1β) and > 20 µg/mL (TNF-α), whereas EC50 values of 5.6 µg/mL (IL-1β) and 14.5 µg/mL (TNF-α) were obtained for B. imbricata extract. Additional amounts of extracts were prepared by percolation of B. calvescens and B. imbricata leaves with ethanol 96ºGL. Portions of these extracts were suspended in water and fractionated by partition between immiscible solvents, sequentially with n-hexane, dichloromethane and ethyl acetate. The obtained fractions were tested in vitro in LPS-stimulated THP-1 cells, as described above. The dichloromethane fractions from B. calvescens and B. imbricata were the most active ones, with inhibition values of 72.2% (IL-1β) and 59.7% (TNF-α) for the first, and 84.4% (IL-1β) and 67.7% (TNF-α) for the second. Dereplication of the extracts and fractions by UPLC-ESI-MS/MS demonstrated the putative presence of flavonoids and phenolic compounds such as chlorogenic acid, naringenin, apigenin, and hispidulin in B. calvescens, whereas luteolin, kaempferol and isokaempferol were identified in B. imbricata. In conclusion, B. imbricata and B. calvescens extracts inhibited in vitro the production of proinflammatory cytokines and this activity is probably due to the presence of flavonoids and phenolic compounds.