Natural ternary deep eutectic solvent-based green extraction of flavonoids from Trollius chinensis flowers: process optimization, mechanistic insights and in vitro activities

Trollius chinensis flowers are acclaimed as “Longjing Tea from Beyond the Great Wall” and always imperial tribute. Flavonoids are one of the main active ingredients in T. chinensis flowers. Trollius chinensis flowers are acclaimed as “Longjing Tea from Beyond the Great Wall” and always imperial tribute. Flavonoids are one of the main active ingredients in T. chinensis flowers. This study aimed to explore natural ternary deep eutectic solvents-ultrasound assisted extraction (NATDES-UAE) for total flavonoids from T. chinensis flowers. L-proline-acetamide-levulinic acid (Pro-AM-LA, 1:3:1) was selected as the optimal NATDES, and artificial neural network (ANN) was employed to optimize extraction parameters. Ultra-high performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS/MS) was used to characterize flavonoid compositions. Scanning electron microscopy (SEM), Fourier-transform infrared (FT-IR) spectroscopy, density functional theory calculations and molecular dynamics simulations were performed to clarify the extraction mechanism. Recyclability, greenness metrics (AGREE, AGREEprep, BAGI), and in vitro bioactivities of extracts were also assessed. Under ANN optimized conditions (55 °C extraction temperature, 58 min extraction time, 17 mg/mL solidtoliquid ratio, 38 % water content), the rutin equivalent total flavonoid yield reached 14.312 mg RE/g DW, and 16 flavonoid compounds including orientin and vitexin were identified. SEM results demonstrated that Pro-AM-LA-UAE disrupted the surface structure of flower materials. Hydrogen bonding interactions between Pro-AM-LA and flavonoids were the key driver for satisfactory extraction performance. Pro-AM-LA retained 65 % extraction efficiency after four recycling cycles. The NATDES-UAE extract exhibited antioxidant activity, antibacterial activity against Serratia marcescens , and cytotoxic effects on HCT116, HT29 and HepG2 cell lines. These findings indicate that NATDES-UAE is a promising green and efficient strategy for obtaining total flavonoids from T. chinensis flowers, with prospective applications in cosmetics and pharmaceutical industries.