Optimization of the rapid effective extraction, antioxidant, antiproliferative and alpha-amylase activities in Plantago ovata seed non-adherent and adherent mucilage by RSM

Plantago ovata seed is comprises two distinct layers: a non-adherent outer layer that can easily extracted by gentle agitation, and an adherent inner layer, which is extremely difficult to separate from the seed coat. This study aims to develop and optimize an efficient method for extracting adherent and non-adherent mucilage from P. ovata seeds and to evaluate its biological activities. The extraction process variables were optimized using the response surface methodology (RSM) based on Box-behnken Design with three factors at three levels, to test different experimental conditions and achieve the highest yield. Our results demonstrate that ultrasound treatment was an efficient method for the rapid extraction of both mucilage layers. The aqueous treatment involved a seed-to-water ratio of 1:60, an extraction time of 1.4 h with constant stirring at 45 °C and pH 8, followed by sonication at 100% amplitude for 60 min with pulses of 10 s ON and 20 s OFF for non-adherent mucilage extraction. For the adherent mucilage, the extraction time was adjusted to 1 h with constant stirring at 45 °C and a seed-to-water ratio of 1:50. This process facilitated the recovery of nearly all P. ovata seed mucilage, estimated at approximately 40 mg/g of seeds (39.85% ~ 40%). Evaluation of antioxidant activity in vitro showed that aqueous extracts of P.ovata mucilage had antiradical activity (IC50 = 202 µg/ml), ferric reducing power (IC50 = 310 µg/ml), important total antioxidant activity (16.84 mg EAG/g MS), inhibitory effect on alpha amylase (IC50 = 2.13 mg/ml), and exhibits strong antiproliferative effects on Caco2 cells (IC₅₀ = 90 µg/ml).