Optimizations of Calcium‐Reinforced Nanophytosome of Pomegranate Extract Using Box–Behnken Design

This study developed reinforced nanophytosomes-innovative nanocarriers combining salts and lipids-using a thin layer hydration method to encapsulate bioactive compounds from pomegranate fruit extract (<i>Punica granatum L.</i>) from the red seed variety cultivated in Raver, Kerman, Iran. The extract was obtained via mechanical pressing and freeze-drying, preserving its bioactive constituents. Laboratory analysis revealed high phenolic content (approximately 371 mg/g dry weight with ethanol and 370 mg/g with water), significant anthocyanins (around 300.68 mg/g), flavonoids (194.97 mg/100 g), and strong antioxidant activity (~91%), indicating its suitability for phytosomal formulation. To optimize nanophytosome properties, three key factors were studied: the ratio of extract to phosphatidylcholine (1:1, 1:2, 1:3), calcium chloride concentrations (0, 1.35 and 2.70 mM), and the extract/PC to ethanol ratio (0.4, 0.7, and 1.0 w/w). These parameters were evaluated using response surface method (RSM) with the Box-Behnken design in Design Expert software. The optimal formula was identified at a ratio of 1:3 pomegranate extract to PC, calcium chloride concentration of 2.70 mM, and ethanol ratio of 0.82 (w/w). The resulting nanophytosomes demonstrated a particle size of 127.67 nm, a zeta potential of -39.7 mV, mobility of -3.145 μm.cm/Vs, dispersity index of 0.357, and an encapsulation efficiency of 100%, achieving a desirability score of 0.860.