Systematic Optimization of Multi-Mode Extraction Process and Accurate Evaluation of In Vitro Antioxidant Activity of Tannins from Punica granatum L. Peel

Abstract

Punica granatum L. peel is an abundant agro-industrial by-product rich in tannins with notable antioxidant potential, yet conventional single-mode extraction methods often suffer from limited efficiency and unclear quantitative benchmarks. This study aimed to systematically optimize a multi-mode extraction process and precisely evaluate the in vitro antioxidant activity of the obtained tannins. A cavitation-permeation synergistic mechanism was innovatively introduced, and orthogonal experiments were conducted to compare water bath heating, ultrasonic-assisted, and microwave-assisted extraction using various solvents. Among these, ultrasonic-assisted acetone extraction under optimized conditions (acetone concentration 40%, solid-to-liquid ratio 1:40, extraction time 45 min, temperature 35°C) yielded the highest tannin extraction rate of 61.6%, significantly outperforming the other three methods (water bath heating: 56.97%; ultrasonic ethanol: 44.19%; microwave ethanol: 55.2%; p < 0.05). For purification, gradient elution with D101 macroporous resin using 40% ethanol produced tannins with a purity of 95.24%. Antioxidant evaluation via the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺) radical scavenging assay showed that the purified tannins exhibited a scavenging rate of 93.7 ± 0.9%, which was significantly higher than that of ascorbic acid (87.2 ± 0.6%) under identical conditions (p < 0.01). The superior activity at low concentrations highlights the dominant role of the electron transfer mechanism facilitated by the polyphenolic hydroxyl structure. These findings provide both process optimization and activity validation for the high-value utilization of Punica granatum L. peel tannins, suggesting promising applications in functional foods and pharmaceutical formulations.