Shahrekord University of Medical Sciences Journal of Herbmed Pharmacology 2345-5004 15 1 2026 01 01 Phytochemical characterization and biological evaluation of Carica papaya L. leaf alkaloids: In vitro and in silico insights into antioxidant, cytotoxicity, and anti-inflammatory activities 66 74 10.34172/jhp.2026.53250 EN Arya J https://orcid.org/0009-0003-7833-7375 Krishnakumar K https://orcid.org/0000-0001-6191-121X Alex George https://orcid.org/0000-0002-4962-3917 Remya Chandran https://orcid.org/0000-0002-2407-2442 Akash Prakash https://orcid.org/0009-0005-8878-4907 Mathew John https://orcid.org/0000-0002-8856-9984 Journal Article 10.34172/jhp.2026.53250 2025 07 05 2025 11 03 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 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric-reducing antioxidant power (FRAP) assays. Cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Anti-inflammatory potential was assessed by quantifying cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS) activities, and cellular nitrite levels in LPS-stimulated RAW 264.7 macrophage cells. In silico analysis was performed to assess the binding interactions of alkaloid compounds with COX-2 and iNOS. Results: Carpaine and carpamic acid were identified as potential alkaloid compounds. DPPH assay demonstrated concentration-dependent antioxidant activity (IC50 = 0.77 (0.06) mg/mL), and FRAP assay showed potential ferric-ion-reducing ability. COX-2 and iNOS activities were significantly decreased (P < 0.01) with IC50 values of 31.02 (4.53) μg/mL and 28.85 (3.27) μg/mL, respectively. Nitric oxide production decreased in a concentration-dependent manner (P < 0.01). In silico analysis revealed that carpamic acid had a stronger binding affinity for iNOS and COX-2 compared to carpaine. MTT assay demonstrated cytotoxicity with an LC50 of 49.69 (0.89) 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.