OPTIMIZATION OF THE FOLIN-CIOCALTEU METHOD SPECIFICALLY FOR PROPOLIS ANALYSIS

Propolis is a complex bee-derived matrix rich in structurally diverse phenolic compounds, whose quantification is commonly performed to characterize its composition and potential biological properties (Felício et al., 2025). Chromatographic methods, such as HPLC, provide detailed information on individual compounds but require greater analytical resources, whereas the Folin-Ciocalteu spectrophotometric assay is a more accessible alternative for total phenolic content (TPC) determination (Pavlovic et al., 2020). However, methodological variations reported in the literature may affect assay performance, making standardization particularly important for a complex matrix such as propolis (Escriche & Juan-Borrás, 2018). This study aimed to optimize and standardize the Folin-Ciocalteu method for TPC determination in propolis extract, emphasizing analytical reproducibility, based on an extensive literature review of the main analytical variables reported for propolis analysis. A propolis extract and two blind standards were evaluated by systematically varying the analytical format (microplate versus spectrophotometer), wavelength (740 versus 760 nm), Folin-Ciocalteu reagent source (FC1, FC2, and FC3; one from an international manufacturer and two from Brazilian manufacturers), reagent dilution (10, 20 and 30-fold), Na₂CO₃ concentration (2.5-20%), alkaline reagent type (Na₂CO₃ versus NaOH), reaction time (30-120 min), and operator, and the results were compared based on TPC, variability and the ability to obtain consistent responses for the blind standards. The analytical format and wavelength did not significantly affect TPC, indicating that the microplate format can be used as a higher-throughput alternative to spectrophotometric analysis while reducing material consumption. On the other hand, the Folin-Ciocalteu reagent source significantly influenced the results, with FC1 exhibiting the lowest variability and the lowest coefficient of variation and consequently being selected for subsequent analyses. Reagent concentration also significantly affected the results, as greater reagent dilution significantly reduced TPC. For Na₂CO₃, 5% and 7.5% produced similar TPC values, whereas 2.5% resulted in lower values, and concentrations of 10% and 20% were associated with instability and precipitate formation. NaOH produced higher TPC values, greater variability, and did not adequately discriminate between the blind standards. Regarding reaction time, 30 min showed greater variability, whereas 60 and 120 min provided stable and comparable responses; therefore, 60 min was selected as the shortest stable reaction time. Finally, analyses performed by three operators under the same conditions yielded similar results, indicating good operator-to-operator consistency. Overall, TPC determination in propolis by the Folin-Ciocalteu method is sensitive to analytical conditions, making it essential to standardize analytical format, reagent source and dilution, alkaline conditions, reaction time, and operational procedures to improve comparability and reproducibility of results.

Acknowledgment: The authors acknowledge the São Paulo Research Foundation (FAPESP, Grant No. 2025/24420-6) for financial support.