Comparative Ultrasonic Bath and Probe Extraction of Piperine from Piper nigrum L. Using Natural Deep Eutectic Solvents: RSM Optimization, Characterization, and In Vitro Bioactivity

Background: Piper nigrum L. (PNL) is a rich source of piperine, a bioactive alkaloid with pharmaceutical, cosmetic, nutritional supplement, and agricultural applications, yet efficient and sustainable extraction methods remain underexplored. Methods: This study compared ultrasonic bath extraction (UBE) and ultrasonic probe extraction (UPE) using natural deep eutectic solvents (NADES) for isolating piperine from PNL fruits. Six NADES formulations were screened, with NADES-5 (choline chloride:glycerin:urea, 1:1:1) showing superior performance. Response surface methodology with a Box–Behnken design optimized extraction parameters, including liquid-to-solid ratio, extraction time, temperature, and water content, for both UBE and UPE. Results: Optimized UPE consistently outperformed UBE, yielding 49.97 mg/g of piperine versus 25.67 mg/g under identical NADES conditions. Comprehensive characterization using TLC, HPTLC, UV, FTIR, Raman, HPLC, NMR, XRD, SEM, and EDX confirmed the successful isolation and structural integrity of piperine, with samples obtained via UPE exhibiting higher purity (98.7% vs. 95.2%) and enhanced crystallinity. In vitro cytotoxicity assays demonstrated that piperine extracted by UPE showed stronger activity against C2C12 myoblasts (IC50: 24.3 μg/mL vs. 40.6 μg/mL) and greater anticancer effects in MCF-7 and HT-29 cells compared to piperine extracted by UBE. Antioxidant evaluation via DPPH, ABTS, FRAP, and TAC assays, along with intracellular reactive oxygen and nitrogen species suppression in THP-1 and RAW 264.7 macrophages, further confirmed the superior biological potential of the UPE-derived piperine sample. Conclusions: These findings indicate that UPE using NADES is a sustainable approach for high-yield piperine extraction with enhanced purity and bioactivity, supporting its potential for pharmaceutical applications.