Ultrasonic-Assisted Extraction of Astaxanthin Using Hydrophobic Deep Eutectic Solvent: Process Optimization and...

Deep eutectic solvent (DES) extraction is a green and efficient technology. As a substitute for organic reagents, DESs are widely used to extract active ingredients from traditional Chinese medicine. This study established an environmentally friendly and efficient method for extracting astaxanthin (AST) from Phaffia rhodozyma (PR) using ultrasound-assisted deep eutectic solvents (DESs-UAE). The astaxanthin content was determined by high-performance liquid chromatography (HPLC). Six types of deep eutectic solvents composed of DL-menthol and selected hydrogen bond donors were prepared and evaluated, among which the DL-menthol-acetic acid system showed superior extraction performance. Response surface methodology (RSM) was employed to optimize extraction parameters (ultrasonic power, time, and temperature), and the optimal conditions were determined as follows: ultrasonic power 420 W, ultrasonic time 20 min, and ultrasonic temperature 60 °C, achieving an AST extraction rate of 62% (2.49 mg/g). Compared with conventional organic solvent extraction, DESs exhibited a significantly higher AST extraction rate from PR, except for dimethyl sulfoxide (DMSO). Scanning electron microscopy (SEM) analysis demonstrated that DES-UAE treatment disrupted the cellular structure of PR, resulting in numerous surface pores; this facilitated the release of intracellular bioactive components and significantly improved AST extraction efficiency. The PR extract showed no significant cytotoxicity and could effectively promote L929 cell proliferation. It concentration-dependently increased superoxide dismutase (SOD) activity and decreased malondialdehyde (MDA) content in H₂O₂-induced oxidative stress L929 cells, thereby alleviating oxidative damage. Additionally, it concentration-dependently upregulated type I collagen expression in these cells, ameliorated the decline in collagen synthesis function, and exerted a protective effect against cellular oxidative damage. This study provides a green alternative to toxic solvents and offers important theoretical and chemical support for the extraction of natural products and the high-value utilization of Phaffia rhodozyma (PR). Deep eutectic solvents have emerged as promising green alternatives to hazardous organic solvents, yet hydrophobic DESs tailored for lipophilic astaxanthin extraction from Phaffia rhodozyma and the linkage between extraction performance and anti-aging bioactivity remain insufficiently explored. Here, an ultrasound-assisted hydrophobic deep eutectic solvent extraction strategy was constructed to acquire astaxanthin, aiming to overcome low efficiency and environmental risks of conventional organic extraction techniques. Six DL-menthol-based DESs were prepared and screened, and DL-menthol-acetic acid possessed the optimal extraction capacity. Key extraction parameters were optimized via response surface methodology, and the maximum astaxanthin extraction recovery reached 62% (2.49 mg/g) under 420 W ultrasonic power, 20 min treatment and 60 °C. This yield was markedly higher than that of most common organic solvents; though comparable extraction effect was obtained with DMSO, the adopted DES possessed outstanding low-toxic and biodegradable superiorities that DMSO cannot match. SEM characterization verified that the combined treatment destroyed yeast cell structure and formed porous morphology, which accelerated intracellular astaxanthin release and accounted for improved extraction efficiency. Biological assays proved the extract possessed good biosafety and proliferation-promoting effect on L929 cells. It effectively relieved cellular oxidative injury by elevating the SOD level and reducing MDA accumulation in oxidative damaged cells, and upregulated type I collagen expression to mitigate aging-related collagen loss. This work develops an eco-friendly and high-efficiency extraction route for lipophilic active substance, confirms the practical value of hydrophobic DES, and provides experimental basis for high-value utilization of Phaffia rhodozyma resources.