Betulin, a pentacyclic triterpenoid, abundantly accumulated in Inonotus obliquus (chaga mushroom), exhibits strong anti-inflammatory, antioxidant, anticancer, and wound-healing properties. However, its extraction remains challenging due to its poor solubility and thermal sensitivity. In this study, we optimized ultrasound-assisted extraction (UAE) using response surface methodology (RSM) to maximize simultaneous betulin and antioxidant compound recovery from I. obliquus. We evaluated three extraction variables (i.e., time, temperature, and ethanol concentration) using a three-factor, five-level design. All quadratic models were significant (p < 0.05), with R² values ranging from 0.83 to 0.93 and prediction errors remaining below 5 %, thereby confirming strong model reliability. Multi-response optimization using a superimposed response plot identified 92.08 % ethanol, 42.56 min, and 62.19 °C as a narrow optimal region, in which all responses simultaneously met the desired criteria. Under these conditions, extraction was predicted to yield high phenolic content (2.58 mg GAE/g DM), increased flavonoid levels (0.57 mg QE/ g DM), strong DPPH radical-scavenging activity (87.49 % DSA), and a betulin content of 2.00 mg/g DM. In contrast, low ethanol concentrations, excessive heating, or prolonged extraction times resulted in reduced yields due to the oxidative or thermal degradation of the bioactive constituents. Overall, the optimized extraction conditions emphasize the importance of controlling solvent polarity and balancing the temperature and time parameters to prevent thermolabile compound decomposition. These results provide a reproducible and eco-efficient framework for large-scale antioxidant constituent extraction from I. obliquus.

Edible mushrooms are recognized as a rich source of diverse metabolites, including polysaccharides, amino acid derivatives, terpenoids, polyphenols, sterols, and flavonoids. These compounds have been extensively reported to have antioxidant, anti-inflammatory, immunomodulatory, anticancer, and metabolic regulatory activities, thereby underscoring their scientific and industrial significance in nutraceutical and pharmaceutical applications. However, conventional extraction methods, which are simple and cost-effective, have limitations in large-scale processes, including long processing times, excessive use of solvents, low yields, and degradation of heat-sensitive compounds, which limit scalability. Therefore, eco-friendly extraction methodologies have emerged as sustainable alternatives. Advanced extraction strategies using food-grade solvents, such as deep eutectic solvents, in conjunction with ultrasound or microwave assistance, enable selective recovery of metabolites under mild conditions, enhancing yield and minimizing the environmental impact. In this review recent progress in advanced extraction methodologies, with respect to efficiency, selectivity, and sustainability, are critically assessed. The industrial implications, including case studies of advanced extraction methodologies applied to secondary metabolites, and the potential of edible mushrooms as renewable bioresources or the development of nextgeneration functional foods and natural therapeutics, are also discussed.

Phellinus linteus, a medicinal mushroom with potent antioxidant properties, contains bioactive compounds, such as polyphenols and flavonoids. To optimize the extraction of skin-whitening compounds, ultrasound-assisted extraction combined with statistically based optimization was used to simultaneously extract total polyphenol content (TPC), radical scavenging activity (RSA), and tyrosinase activity inhibition (TAI). Extraction variables, including extraction time (X1:4.8 ~ 55.2 min), extraction temperature (X2:26.4 ~ 93.6oC), and ethanol concentration (X3:13.0 ~ 97.0%), were varied in 17 experimental cycles based on a central composite design. Quadratic regression models for TPC, RSA, and TAI had coefficients of determination (R2) greater than 0.92, demonstrating well-fitted models and statistical significance. Analysis of variance revealed that all three variables significantly influenced extraction efficiency (p < 0.0041), with ethanol concentration (X3) having the most pronounced effect. The optimal extraction conditions were 80.0 min, 82.5oC, and 64.8% ethanol, yielding predicted values of 6.42 mg GAE/g DM for TPC, 73.71% for RSA, and 85.04% for TAI. These results suggest that a moderate ethanol concentration combined with adequate thermal input maximizes the extraction of antioxidant and tyrosinase inhibitory activities specifically associated with skin-whitening effects.