Sequential Supercritical CO2 and Subcritical Water Extraction for the Valorisation of Pomegranate (Punica granatum L.) By-Products: A Response Surface Methodology Approach

Pomegranate marc is a major, underutilized juice industry by-product rich in lipophilic polyunsaturated fatty acids-notably conjugated α-linolenic acids (CLnAs)-and hydrophilic polyphenols with potent antioxidant and anti-inflammatory properties. Despite its potential for nutraceutical, cosmetic, and pharmaceutical applications, this matrix remains largely unexploited. This study presents a novel, sequential in-line extraction strategy combining supercritical CO2 (ScCO2) and subcritical water (scW) to recover complementary bioactive fractions. Both extraction steps were optimized via Response Surface Methodology (RSM). Box-Behnken optimization of ScCO2 (43 MPa, 76 °C, 6.4 L min-1, 124 min) yielded 30 g kg-1 dry weight (dw) of oleoresin, achieving a 68% recovery of total oil. Subsequent scW extraction was optimized at 149 °C, with a 40 L kg-1 water-to-solute ratio and 73 min extraction time, yielding 47 g kg-1 dw of total phenolics (58% recovery). Strong agreement between experimental and predicted values confirmed the robustness of the models. Comprehensive profiling revealed a diverse phytocomplex including fatty acids, tocopherols, flavonoids, soluble sugars, and polysaccharides. Antioxidant assays confirmed that both γ-tocopherol and polyphenols significantly contribute to the extracts' bioactivity. To improve physical handling, the aqueous fractions were converted into solid dispersions via spray drying with maltodextrin. Preliminary in vitro biological assessments on HEK-293 (human embryonic kidney) and MCF-7 (Michigan Cancer Foundation-7) cell lines suggested that the maltodextrin-based formulations may modulate the cytotoxic profile compared to the free extract, with exploratory results showing dosage-dependent variations in cell viability across the two lines. This work suggests a potentially scalable and sustainable biorefinery approach for the integral valorisation of pomegranate marc, offering a basis for a pathway to produce solvent-free bioactives.