Optimization of Chondroitin Sulfate Extraction From Skate ( <i>Raja</i> spp.) Cartilage Using Response Surface Methodology: Anti‐Inflammatory and Chondroprotective Activities

The enzymatic hydrolysis of skate cartilage utilizing Alcalase and Protamex was optimized by response surface methodology (RSM). The quadratic model was adequate, confirming its appropriateness for optimization. The optimal parameters for the extraction of total uronic acids were a 0.98% enzyme concentration, 264 min of hydrolysis, and a hydrolysate-to-ethanol ratio of 1:2.14. High-performance liquid chromatography analysis revealed ∆Di-6S as the principal disaccharide. To validate the optimized extract, biological assays were performed in lipopolysaccharide-stimulated RAW264.7 macrophages and interleukin (IL)-1β-induced SW1353 chondrocytes. Skate cartilage extract (SCE) significantly reduced nitric oxide levels (p < 0.001) and cytokine levels of IL-1β (p = 0.028), IL-6 (p = 0.004), and tumor necrosis factor-α (p < 0.001) in a dose-dependent manner in RAW264.7 macrophages, indicating the suppression of inflammatory signaling pathways. The high chondroitin-6-sulfate may contribute to these anti-inflammatory effects. In IL-1β-induced SW1353 chondrocytes, SCE dose-dependently increased the mRNA expression of aggrecan (p = 0.022) and type II collagen (p = 0.033), indicating that SCE may counteract IL-1β-induced suppression of gene expression. This study indicates that RSM is a useful approach for optimizing extraction conditions from skate cartilage. In vitro assays showed that the optimized extract attenuated inflammatory markers in macrophages and upregulated aggrecan and type II collagen mRNA in chondrocytes. Therefore, the SCE produced under RSM-optimized conditions demonstrates modulatory effects on inflammatory responses and cartilage matrix gene expression in vitro. PRACTICAL APPLICATIONS: This paper describes the RSM-based optimization of enzymatic extraction conditions to maximize CS-rich extract yield from skate (Raja spp.) cartilage, a sustainable marine by-product of fish processing. The optimized extract was rich in chondroitin sulfate, particularly chondroitin-6-sulfate, and demonstrated in vitro anti-inflammatory and chondroprotective effects.