Punicalagin rich pomegranate peel extract -loaded chitosan nanoparticles ameliorate experimental arthritis by downregulating TNF-α and CD20 gene expression

Rheumatoid arthritis (RA) is a chronic, symmetric, inflammatory autoimmune disease. Punicalagin is the most abundant compound in pomegranate peel, with antioxidant and anti-inflammatory properties, and can be used as an herbal agent for treating diseases. However, the effectiveness of polyphenols depends on maintaining their stability. Using encapsulated polyphenols instead of free compounds can effectively overcome these limitations. After extracting phenolic compounds such as punicalagin from pomegranate peel, they are prone to degradation under adverse environmental conditions. In this context, chitosan nanoparticles have the potential to encapsulate various plant extracts, owing to their non-toxicity and biocompatibility. The pomegranate peel extract was encapsulated in chitosan nanoparticles, and the particle size, structure, and zeta potential were evaluated. Subsequently, 20 female Wistar rats with induced rheumatoid arthritis with bovine collagen type II solution and complete Freund’s adjuvant CFA were divided into the following groups: RA Model, Control, Punicalagin treatment, and Punicalagin encapsulated with chitosan nanoparticles treatment group. Malondialdehyde level is measured as a biomarker of lipid peroxidation induced by oxidative stress. Rheumatoid factors (RF) served as markers of local inflammation, and TNF-α and CD20 gene expression were evaluated by real-time PCR. Field Emission Scanning Electron Microscopy (FESEM) imaging revealed that the blank nanoparticles were monodisperse and spherical, with an average diameter of 132 nm. The average diameter of the control nanoparticles was measured at 98 nm. The zeta potential of the nanoparticles was recorded at -28 mV. Based on the results, the punicalagin encapsulated in chitosan nanoparticles confirmed an anti-inflammatory effect (4.3), which was superior to the punicalagin (6.5). Treatment with punicalagin encapsulated in chitosan nanoparticles showed a decreasing trend in RF levels compared with punicalagin alone. Similarly, serum malondialdehyde levels were significantly reduced in the encapsulated punicalagin group. Additionally, the expression of the TNF-α and CD20 genes was significantly decreased in the encapsulated punicalagin group compared to the punicalagin-only group. In conclusion, although punicalagin alone reduced inflammatory markers and alleviated rheumatoid arthritis symptoms, encapsulating it in chitosan nanoparticles led to superior therapeutic outcomes.