Optimized formulation of a three-component extract mixture from Moroccan Crocus sativus L. (Stigmas, leaves, and Tepals) for enhanced antioxidant activity.

Abstract

This study evaluated the antioxidant potential of a three-component mixture derived from Crocus sativus L. stigmas, leaves, and tepals, aiming to valorize these underutilized by-products. Hydroethanolic extracts were chemically profiled using High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD), identifying major bioactive compounds including crocin (11.04%), rutin (22.32%), and ellagic acid (62.47%). Antioxidant capacity was assessed using DPPH and ABTS assays, yielding IC₅₀ values ranging from 88.67 ± 0.83 µg/mL to 291.41 ± 0.91 µg/mL for DPPH, and from 116.76 ± 1.31 µg/mL to 286.87 ± 0.25 µg/mL for ABTS. A simplex-centroid mixture design was employed to determine optimal component ratios. The best-performing DPPH mixture (43% stigmas, 35% leaves, 22% tepals) achieved an IC₅₀ of 97.36 ± 1.23 µg/mL, closely matching the predicted 95.96 µg/mL. Similarly, the optimal ABTS blend (45% stigmas, 35% leaves, 20% tepals) reached an IC₅₀ of 110.59 ± 6.82 µg/mL, aligning with the predicted 106.31 µg/mL. Both combinations outperformed standard antioxidants like BHT and ascorbic acid. These findings support the synergistic use of saffron by-products in food preservation, nutraceuticals, and biopharmaceuticals, offering an eco-friendly, cost-effective solution aligned with circular economy principles.