Optimization of compound K extraction using natural deep eutectic solvents and evaluation of mitochondrial protective...

Compound K (CK), a deglycosylated ginsenoside metabolite, exhibits high bioavailability and diverse pharmacological activities but has limited solubility in conventional solvents. Natural Deep Eutectic Solvents (NADES) composed of Generally Recognized as Safe (GRAS) compounds provide an eco-friendly alternative for efficient CK extraction.

A full factorial design was applied to optimize CK dissolution. Three variables-hydrogen bond donor (HBD; glycerol, 1,3-propanediol, butylene glycol), solvent ratio (betaine: HBD = 1:1 or 1:2), and water content (0-20%)-were evaluated. CK concentration was quantified by HPLC, and the optimized NADES was validated through triplicate experiments. Mitochondrial membrane potential (Δψm) was assessed in zebrafish embryos using JC-1 fluorescence to compare the bioactivity of CK extracts obtained with NADES, 50% ethanol, and water.

Water content significantly affected CK extraction (p < 0.05). The optimized NADES (betaine:1,3-propanediol = 1:2, 20% water) yielded 339.5 μg/mL CK at 3 mg/mL loading concentration. Confirmation experiments showed excellent reproducibility (339.2 ± 0.9 μg/mL). Comparative validation demonstrated that NADES achieved 1.30-1.40-fold higher extraction efficiency than water and 1.09-1.27-fold higher than 50% ethanol across all tested concentrations (3, 5, and 10 mg/mL). CK-NADES extracts exhibited superior mitochondrial protection (EC₅₀ = 1.10 μg/mL) compared with 50% ethanol (EC₅₀ not reached) and water (not detectable).

Betaine-1,3-propanediol NADES significantly enhanced CK extraction efficiency and mitochondrial protective activity. This study establishes NADES as a sustainable and effective green-chemistry platform for developing CK-enriched cosmeceutical and functional food formulations.