Phytochemical characterization and biological evaluation of Carica papaya L. leaf alkaloids: In vitro and in silico insights into antioxidant, cytotoxicity, and anti-inflammatory activities

Introduction: Carica papaya L. leaf alkaloids have gained pharmacological interest for their anti-thrombocytopenic activity. Inflammation and oxidative stress exacerbate disease severity. This study evaluated the in vitro antioxidant, cytotoxicity, and anti-inflammatory activities of the alkaloid fraction of C. papaya leaves. Methods: The alkaloid fraction was isolated and analyzed using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) and Fourier transform infrared (FTIR) spectroscopy. Antioxidant potential was evaluated by the ۲,۲-diphenyl-۱-picrylhydrazyl (DPPH) and ferric-reducing antioxidant power (FRAP) assays. Cytotoxicity was determined by the ۳-(۴,۵-dimethylthiazol-۲-yl)-۲,۵-diphenyltetrazolium bromide (MTT) assay. Anti-inflammatory potential was assessed by quantifying cyclooxygenase-۲ (COX-۲), inducible nitric oxide synthase (iNOS) activities, and cellular nitrite levels in LPS-stimulated RAW ۲۶۴.۷ macrophage cells. In silico analysis was performed to assess the binding interactions of alkaloid compounds with COX-۲ and iNOS. Results: Carpaine and carpamic acid were identified as potential alkaloid compounds. DPPH assay demonstrated concentration-dependent antioxidant activity (IC۵۰ = ۰.۷۷ (۰.۰۶) mg/mL), and FRAP assay showed potential ferric-ion-reducing ability. COX-۲ and iNOS activities were significantly decreased (P < ۰.۰۱) with IC۵۰ values of ۳۱.۰۲ (۴.۵۳) μg/mL and ۲۸.۸۵ (۳.۲۷) μg/mL, respectively. Nitric oxide production decreased in a concentration-dependent manner (P < ۰.۰۱). In silico analysis revealed that carpamic acid had a stronger binding affinity for iNOS and COX-۲ compared to carpaine. MTT assay demonstrated cytotoxicity with an LC۵۰ of ۴۹.۶۹ (۰.۸۹) μg/mL and a favorable selectivity index. Conclusion: The alkaloid fraction of C. papaya leaves exhibits significant antioxidant, cytotoxicity, and anti-inflammatory activities at non-toxic concentrations. Further in vivo studies are needed to validate and expand on the current findings.