Comparative Antioxidant Activity of Leucaena leucocephala Leaves Extracts Using CUPRAC and FRAP Methods

Abstract

Leucaena leucocephala is widely recognized as a medicinal and nutritional plant due to its abundance of bioactive phytochemicals, particularly phenolics and flavonoids, which play a major role in its antioxidant properties. This study aimed to compare the antioxidant activity of extracts and fractions of L. leucocephala leaves. The ethanol extraction was performed using the Ultrasound-Assisted Extraction (UAE) method, followed by fractionation with water, ethyl acetate, and n-hexane.  Total Phenolic Content (TPC) and Total Flavonoid Content (TFC) were determined using the Folin–Ciocalteu and aluminium chloride colorimetric methods, respectively. Their antioxidant activity was tested using CUPRAC and FRAP assays, along with method validation (accuracy, precision, linearity, limit of detection (LoD), and limit of quantification (LoQ). The ethyl acetate fraction exhibited the highest TPC (84.53 ± 0.11%) and TFC (87.26 ± 0.15%), followed by ethanol extract, aqueous, and n-hexane fractions. The ethyl acetate fraction showed the strongest antioxidant activity with IC₅₀ values of 31.76 ppm (CUPRAC) and 22.93 ppm (FRAP), while the n-hexane fraction demonstrated the weakest activity. Method validation of the analytical assays showed that the CUPRAC assay had a precision of 1.93%, accuracy of 100.12%, linearity (R²) of 0.99, LOD of 2.22 ppm, and LOQ of 7.39 ppm. The FRAP assay showed a precision of 0.81%, accuracy of 93.94%, linearity (R²) of 0.99, LOD of 2.30 ppm, and LOQ of 7.77 ppm. The CUPRAC assay met all validation criteria, while the FRAP assay did not meet the accuracy requirement. Overall, the CUPRAC assay demonstrated better performance than the FRAP assay. These results confirm the strong antioxidant potential of Leucaena leucocephala and emphasize the importance of solvent fractionation in enhancing the recovery of bioactive compounds. The findings support the potential use of Leucaena leucocephala as a natural antioxidant, with promising applications in pharmaceutical formulations and nutraceutical product development.