Optimization of antioxidant extraction from Citrullus colocynthis seed using response surface methodology

The growing demand for natural antioxidants has spurred interest in underutilized plant sources such as Citrullus colocynthis seeds, which are rich in bioactive compounds like phenolics and flavonoids. This study successfully optimized the extraction of these valuable antioxidants using the Soxhlet method with ethanol solvent and Response Surface Methodology (RSM) to overcome inefficiencies in traditional protocols. A Box-Behnken design investigated the effects of three critical variables: ethanol concentration (50-90% v/v), temperature (40-70 °C), and solid-to-solvent ratio (1:10-1:30 g/mL). The models revealed that ethanol concentration and temperature significantly influenced the extraction yield, total phenolic content (TPC), total flavonoid content (TFC), and antioxidant activities (DPPH and FRAP). These properties were quantified using the Folin-Ciocalteu assay for TPC, a colorimetric method for TFC, and the DPPH radical scavenging assay and FRAP (Ferric Reducing Antioxidant Power) assay for antioxidant capacity. The optimal extraction conditions for maximizing antioxidant activity were precisely determined as 76.31% ethanol (v/v), 55.00 °C, and a 49.19 mL/g liquid-to-solid ratio. Under these optimal parameters, the extract yielded a maximum TPC of 9.94 mg CAE/g (Catechin Equivalent per gram dry matter), TFC of 5.42 mg CAE/g, and strong antioxidant values of 10.52 mg AAE/g (DPPH) and 6.54 mg AAE/g (FRAP). Strong correlations (r > 0.85) between TPC and antioxidant activities confirmed that phenolic compounds were the primary drivers of the extract's efficacy. The strong correlations observed between TPC and antioxidant activity (r > 0.85) substantiate that phenolic compounds are the primary drivers of the extract's efficacy. In conclusion, this research provides a validated, optimized protocol, establishing Citrullus colocynthis seeds as an efficiently extractable source of potent natural antioxidants.