Validated HPLC Analysis of Puerarin in <i>Pueraria lobata</i> –Derived Exosome‐Like Nanoparticles With Intestinal Barrier Enhancement

A high-performance liquid chromatography with diode array detection (HPLC-DAD) method was developed and validated to quantify puerarin encapsulated in exosome-like nanoparticles derived from Pueraria lobata (ENsP). The method demonstrated high specificity, excellent linearity (r² = 0.9999), and low limits of detection (LOD: 0.60 µg/mL) and quantification (LOQ: 1.83 µg/mL). Matrix effects were negligible, and accuracy and precision tests revealed high recovery rates (95.8%-105.5%) with low relative standard deviations (0.58%-4.10%). Horwitz ratio values (0.15-0.66) and relative expanded uncertainties (13.8%-16.6%) complied with CODEX standards (≤22%). The puerarin content in ENsP was determined to be 1.08 ± 0.02 mg/g, and its stability was maintained over 10 days at 4°C. In a cyclophosphamide (CPA)-induced immunosuppressed BALB/c mouse model, oral administration of ENsP significantly improved small intestinal morphology, including restoration of intestinal length and the villus-to-crypt ratio, and enhanced expression of barrier-related genes and proteins ZO-1 and MUC2. These results highlight ENsP as a stable and biocompatible carrier with functional bioactivity, offering promising potential for pharmaceutical and nutraceutical applications targeting intestinal barrier integrity. PRACTICAL APPLICATIONS: This study developed and validated an HPLC-DAD method for the quantification of functional compounds in exosome-like nanoparticles derived from Pueraria lobata (ENsP). This advancement establishes a foundation for quality control and the further development of functional food ingredients.