Screening Factorial Design for Extraction of Bioactive Metabolites from Tithonia diversifolia (Hmsl.) A. Gray/Asteraceae Leaf Extract: Phytochemical Assays and Chemical Characterization by Synchronous Fluorescence and Phosphorescence Spectroscopy

Recent studies highlight the rich medicinal flora of São Tomé and Príncipe (STP) islands due to their therapeutic potential against various diseases. To maximize bioactive compounds’ extraction from Tithonia diversifolia leaves, we have used a two-level, three-factor (23) full factorial design. The screening evaluated water and hexane solvents at 25 °C and 40 °C, across incubation periods of 0 and 5 days, maintaining a constant agitation of 150 rpm. The highest TPC among the tested conditions was 72.16 µmole gallic acid equivalent/g of leaves using water at 40 °C with a 5-day incubation period. The lea extracts were characterized through several phytochemical assays, including TFC, reducing power, ABTS, DPPH, and SOD activity. This report introduces a novel, non-destructive analytical fingerprinting approach using intrinsic synchronous fluorescence and phosphorescence. Intrinsic, non-destructive synchronous fluorescence spectroscopy was performed across a wavelength range of 250 to 750 nm, utilizing a Δ λ interval of 5 to 30 nm. The hexane leaf extract displayed distinct peaks at 290, 320, 345, 400, 490, and 675 nm. Conversely, the aqueous extracts only showed peaks at 490, 560, and 675 nm. Intrinsic synchronous phosphorescence testing revealed peaks at 325, 400, 490, 550, and 675 nm for the hexane extract, and at 500 and 560 nm for the aqueous extract. Additionally, 3D fluorescence fingerprinting spectra successfully validated the emission peaks at 290, 320, 345, 400, 490, and 675 nm. Therefore, the present work describes novel SFS, SPS and 3D-SFS as fingerprinting/comparative profiling techniques for leaf extracts which require definitive compound identification methods.