Prediction-Guided Biotransformation of p -Hydroxyphenethyl Anisate into a Novel 3',4'-Dihydroxyphenethyl Anisate with Potent Anti-Inflammatory, Anti-Melanoma, and Antioxidant Activities

-dihydroxyphenyl compound, 3',4'-dihydroxyphenethyl anisate. This biotransformation yielded a product with significantly enhanced therapeutic potential. Notably, the product showed a potent 11-fold increase in anti-inflammatory activity compared to the precursor. Mechanistically, 3',4'-dihydroxyphenethyl anisate effectively mitigated the hyperimmune response in lipopolysaccharide-stimulated RAW 264.7 macrophages by suppressing the gene expression of pro-inflammatory mediators, including tumor necrosis factor-alpha, interleukin-1 beta, interleukin 6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2. Notably, Western blot analysis confirmed that this inhibitory effect was sustained at the protein level, where 3',4'-dihydroxyphenethyl anisate induced a significant downregulation of iNOS expression. Furthermore, the hydroxylated product showed preliminary antioxidant capacity (absent in the parent compound) and observable anti-melanoma activity. This study validates PDMA as an effective strategy for generating novel and high-value bioactive molecules via biotransformation. Our newly produced catechol is a promising candidate for future pharmacological applications.