Optimization of Extraction Process for Total Flavonoids and Crude Polysaccharides from Nephrolepis cordifolia and Assessment of Their In Vitro Antioxidant Properties

This study systematically optimized the extraction processes of two bioactive constituents, total flavonoids and crude polysaccharides, from the dried underground tubers of Nephrolepis cordifolia (L.) C. Presl, and further comprehensively characterized their in vitro antioxidant activities. For total flavonoid extraction, an ethanol–ammonium sulfate aqueous two-phase system (ATPS) was integrated with ultrasonic-assisted aqueous two-phase extraction (UATPE), and the process parameters were optimized via Box–Behnken design-based response surface methodology (RSM) using total flavonoid content (TFC) as the primary response indicator. The validated optimal conditions were determined as follows: extraction time of 40.67 min, solid-to-liquid ratio of 1:30.71 (g/mL), ammonium sulfate mass fraction of 24.03%, and ethanol concentration of 35% (v/v). Under these optimized conditions, the TFC reached (54.83 ± 1.58) mg rutin equivalents per gram dry weight (mg RE/g DW), which was significantly higher than the values obtained from conventional ATPS [(49.05 ± 0.41) (mg RE/g DW)] and traditional ethanol extraction [(28.68 ± 1.54) (mg RE/g DW)] (p < 0.05). For crude polysaccharide production, an ultrasonic-assisted water extraction followed by ethanol precipitation protocol was adopted, and parameters were screened through an L9 (33) orthogonal experimental design. The obtained optimal extraction parameters were an extraction time of 10 min, three extraction cycles, and solid-to-liquid ratio of 1:15 (g/mL). A maximum crude polysaccharide yield of (14.89 ± 0.18) % was achieved under these conditions, with a relative standard deviation of 1.21% (n = 3) across parallel validation tests. Subsequent antioxidant assays demonstrated that the total flavonoid fraction from Nephrolepis cordifolia exhibited potent scavenging capacities against both 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH•) and 2,2′-Azinobis-(3-ethylbenzthiazoline-6-sulphonate) radical cation (ABTS•+) radicals. Within the tested concentration range of 0.1–0.5 m g/mL, the crude polysaccharide presented a significant positive concentration-dependent enhancement of activity in four classical in vitro antioxidant systems, including DPPH radical scavenging, ABTS cation radical scavenging, hydroxyl radical scavenging, and total reducing power. Collectively, these findings confirm that the introduced ultrasonic-assisted extraction strategy remarkably improves the recovery efficiency of target bioactive components from Nephrolepis cordifolia. Both total flavonoids and crude polysaccharides display excellent in vitro free radical scavenging potential. This work provides a solid methodological basis for the efficient preparation of the two bioactive compounds, and highlights that Nephrolepis cordifolia is a promising candidate for further exploration as a natural antioxidant source.