| Summary: | As a new and green solvent, ionic liquids (ILs) have received more attention during the green extraction and separation process for natural medicines. In this paper, IL-ultrasound-assisted extraction (IL-UAE) of total biflavonoids (TBFs) from <i>Selaginella helvetica</i> was firstly developed, and different ILs were employed and compared. Based on single-factor experiment, solid⁻liquid ratio (1:10⁻1:14 g/mL), IL concentration (0.6⁻1.0 mmol/mL), and extract temperature (40⁻60 °C) were further explored, according to response surface methodology (RSM), with TBF yields as the index. Moreover, antioxidant activity of TBF extract was analyzed by four methods, i.e., 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) and 2,2′-azinobis-(3-ethylbenzth-iazoline-6-sulphonate (ABTS) free radical scavenging assay, ferric ion reducing power assay, and chelation of ferrous ions assay. The results indicated that [C<sub>6</sub>mim]PF<sub>6</sub> had a high selectivity and efficiency. Moreover, important parameters for the extraction process were investigated and optimized. Through parameter optimization (0.8 mmol/L, 250 W, 40 min, 1:12.7 g/mL, and 47 °C), a yield of 18.69 mg/g biflavonoids was obtained from the extract of <i>S. helvetica</i>. Compared with ethanol-UAE, heat-reflux extraction, Soxhlet extraction, and percolation extraction, IL-UAE could not only obtain higher yield in a shorter time, but also reduce the solvent consumption. In addition, TBF extract showed potential antioxidant activity based on the above four antioxidant methods. In short, IL-UAE was first employed to develop a novel and green extraction method for TBF content, and this experiment provides valuable references for further utilization of <i>S. helvetica</i>.
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