OPTIMIZATION OF POLYPHENOL RECOVERY AND ANTIOXIDANT ACTIVITY FROM Nelumbo nucifera LEAF USING BOX-BEHNKEN DESIGN AND LC-HRMS PROFILING

The leaves of Nelumbo nucifera are rich in polyphenols, known for their antioxidant and therapeutic properties. This study aimed to optimize extraction conditions to maximize total phenolic content (TPC) and total flavonoid content (TFC) using the Box-Behnken Design (BBD). The relationship between polyphenol content and antioxidant activity was also evaluated. Solid-liquid extraction was employed under various conditions designed using BBD, varying extraction time (X1), ethanol concentration (X2), and solid-to-liquid ratio (X3). The TPC and TFC were quantified using colorimetric methods, while antioxidant activity was assessed through DPPH radical scavenging assays. Polyphenolic compounds were identified using LC-HRMS. Data analysis included ANOVA to validate the model and its predictions. Optimal extraction conditions were determined as 53.87 X1, 69.03% X2, and an X3 of 10.48:1. Under these conditions, the predicted TPC and TFC were 25.894 mg GAE/g and 12.158 mg QE/g, respectively, with a high desirability score of 0.931. Experimental validation confirmed these values with no significant differences (p = 0.253). The optimized extract (PRN) exhibited stronger antioxidant activity compared to the non-optimized extract (NCR), achieving up to 84.23% DPPH inhibition at 500 µg/mL. LC-HRMS analysis identified key polyphenolic compounds, including quercetin-3-β-D-glucoside, rutin, kaempferol, and myricetin, which contribute to the extract's antioxidant potential. The findings support SDG 3 (Good Health and Well-being) and highlight the potential of N. nucifera as a natural antioxidant source for pharmaceutical, nutraceutical, and cosmetic applications. Future studies should further explore its bioactivity and applications in vivo.