Ultrasonic-assisted extraction of flavonoids from Amomum villosum Lour. Using natural deep eutectic solvent: Process optimization, comparison, identification, and bioactivity

Abstract

Amomum villosum Lour. (A. villosum) is widely utilized in culinary and medicinal contexts due to its rich diversity of bioactive compounds. In this study, an ultrasonic-assisted extraction (UAE) method using a natural deep eutectic solvent (NaDES) was employed to extract flavonoids from A. villosum. The NaDES composed of betaine and glycerol (Bet-Gly) exhibited effective performance as an extraction solvent, as evidenced by its high efficiency. Optimal extraction conditions (water content: 30 %, solid-liquid ratio: 1:20 g/mL, temperature: 70 °C, ultrasonic power: 85 % P, time: 30 mins) were established via single-factor experiments. The maximum total flavonoid content (TFC) of 82.22 ± 0.39 mg rutin equivalent per gram dry weight (mg Rut/g DW) was achieved under optimized conditions. Comparative studies revealed that the UAE-NaDES method afforded higher TFC and DPPH radical scavenging activity values, broader flavonoid diversity, and demonstrated a better capacity to stabilize them compared to conventional solutions. Furthermore, NaDES exhibited favorable biocompatibility properties, and the resulting extract displayed remarkable anti-inflammatory and anti-cancer effects. These findings demonstrate that NaDES is an efficient solvent for flavonoids extraction from A. villosum, with potential applications in food and pharmaceutical industries, thereby minimizing solvent removal steps.

Keywords: Bioactivity; Composition identification; Flavonoids; Natural deep eutectic solvent; Stability; Ultrasonic-assisted extraction.

Fig. 1

Photos captured after a 60-day storage period of the prepared NaDESs (A: Betaine-based NaDESs, B: L-proline-based NaDESs).

Fig. 2

Characterization of the optimal NaDES and its constituents (A:FT-IR spectra, B: TGA curves); the influence of different factors on TFC (C: water content, D: solid–liquid ratio, E: temperature, F: ultrasonic power, G: extraction time); the correlation coefficient among the physical properties of Bet-Gly, including viscosity, Kamlet-Taft parameters, density, and pH, with varying water contents (10–50%, w/w) and the corresponding extraction yield (H).

Fig. 3

Comparative analysis of TFC and antioxidant activity of extracts obtained using NaDES and conventional solvents (A); the microstructure of A. villosum before and after extraction with various solvents (B: raw powder, C: Bet-Gly, D: EtOH, E: ME, F: W). Note: Different letters indicate significant differences (p < 0.05). Letters are assigned in descending order of mean values, with group 'a' representing the highest value, followed by 'b', 'c', etc.

Fig. 4

Stability of flavonoids at different temperatures for 28 days (A: 25 °C, B: 4 °C, C: −20 °C); viscosity-temperature curves of different solvents (D).

Fig. 5

Toxicity of NaDES (A: cell viability of RAW264.7, B: cell viability of L929). Note: The dashed horizontal line indicates the 80% cell viability threshold.

Fig. 6

Anti-inflammatory effect of Bet-Gly extract (A: cell viability of RAW264.7, B: the fold change in NO release relative to control, C: concentration of IL-6, D: concentration of TNF-α). Note:Each value is expressed as mean ± SD (n = 3), ns > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (compared with LPS).

Fig. 7

Anti-cancer effect of Bet-Gly extract (A: cell viability of GES-1, B: cell viability of MGC803, C: numbers of colonies calculated, D: wound healing rate calculated, E: optical microscopy images of wound healing at 0 and 48 h, F: representative images of colony formation). Note:Each value is expressed as mean ± SD (n = 3), ns > 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (compared with the control).