Bioguided purification and characterization of an antibacterial component of bark tincture from Caesalpinia paraguariensis Burk

Abstract

In previous works, antibacterial activity and toxicity of partially purified fractions of bark tincture from Caesalpinia paraguariensis Burkart. (Fabaceae) were determined. The fraction with the highest antibacterial activity (ethyl acetate), also exhibited high toxicity. Therefore, this work focused on purifying and separating the antibacterial component/s from the toxic/s present in the acetate-ethylic fraction (AEF), to characterize it/them chemically, determine their Minimum Inhibitory Concentration (MIC) and cytotoxicity. AEF was fractionated by CC-RP (C18) with watermethanol gradient (0–100%), sub-fractions obtained were analyzed by TLC silica gel and NP-PEG reagents under UV365nm light, whose composition profiles allowed to separate eight different groups (G1-G8). Antibacterial activity of sub-fractions was assessed against Staphilococcus aureus ATCC 25923 (106 cfu/mL) by direct bioautography (3 mL ssMH inoculated medium, incubation at 37°C during 24 h), which was revealed by spray of MTT solution (2.5 mg/mL). MIC/MBC were determined against S. aureus ATCC 25923 and Enterococcus faecalis ATCC 29212, by microdilution in MH broth, and next subculture on agarized MH medium, according to CLSI protocols. The toxicity was tested determining viability of A. salina exposed to AEF, G6 or reference compounds between 1 and 1000 μg/mL (LC50) for 24 h (25°C). Survival percent human lymphocytes isolated from peripheral blood (HLPBs) cultured in RPMI 1640, exposed to G6 between 1–200 µg/mL and incubated (37°C, 5% CO2, 24 h), was determined by metabolic activity assay measurable through MTT redox reagent, by ELISA multiplate reader at 550 nm. Chemical characterization of the most active sub-fraction was done by analyzing HPLC(DAD)-EM(ESI/Q-TOF), UV-Vis spectroscopy, TLC with specific revealers to phenolic compounds, searching in data base and specialized bibliography. Through bio-guided sub-fractionation a component was purified (G6) with bacteriostatic activity against assayed strains (MICs: 125-500 µg/mL), not toxic (CL50 > 1000 µg/mL), and not cytotoxic on HLPBs (cell survival > 75 percent at 200 µg/mL). TLC analysis revealed at Rf 0.7 a fluorescent yellow-orange spot, consistent with flavonoid-type phenolic compounds. A peak at RT 13.7 min was detected by HPLC-MS, compatible with the presence of flavonoid compound according to its UV-DAD spectrum [λmax (MeOH) 248, 366 nm], whose mass was 286 a.m.u., and C15H10O6 its most probable molecular formula. The database and bibliography searching yielded on least six structures of same kind. The UV-Vis spectrum and characteristic color on TLC allowed to define the identity of the compound as fisetin. This flavonol was reported previously on species of Fabaceae and other families, in colorful fruits and their juices, but it is the first time that it has been identified for C. paraguariensis.