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 (Punica granatum L.) 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.