Ultrasensitive spectrofluorimetric analysis of harmalacidine HCl: A potent underexplored MAO-A inhibitor from Peganum harmala L

Peganum harmala seeds are a rich source of β-carboline alkaloids with diverse pharmacological properties; however, their major constituent, harmalacidine HCl, remains underexplored. This work reports the extraction, isolation, spectrofluorimetric quantification, comparative monoamine oxidase A (MAO-A) inhibitory activity, and cytotoxic evaluation of harmalacidine HCl alongside other P. harmala alkaloids. Harmalacidine HCl was identified by NMR spectroscopy. A novel spectrofluorimetric method was developed and validated for the ultrasensitive quantification of harmine (H1), harmaline (H2), and harmalacidine HCl (H3), showing excellent sensitivity down to nanogram levels (LOQ: 0.88 ng/mL for harmalacidine HCl) and linearity (r ≥ 0.9994). The study highlights the critical role of pH adjustment in optimizing the extraction and fluorimetric detection of H3 (harmalacidine). In vitro MAO-A inhibition assay revealed that harmalacidine HCl inhibits MAO-A with an IC₅₀ of 457 ± 179 nM, surpassing harmine (IC₅₀ = 506 ± 163 nM) and comparable to the standard inhibitor, clorgyline. Molecular docking studies supported these findings, demonstrating that H3 forms additional interactions with amino acid residues in the active site of MAO-A, potentially accounting for its enhanced inhibitory activity. The cytotoxicity (MTT) assay revealed that H3 exhibited relatively low antiproliferative activity against A549 and HCT116 cancer cell lines, as well as normal HEK293T cells, with an EC₅₀ of approximately 10 μM. In vitro cytotoxicity testing indicated a preliminary therapeutic index of approximately 19, suggesting a substantial margin between the effective and cytotoxic concentrations of harmalacidine HCl, and indicating an acceptable initial safety profile. Given its abundance in P. harmala and promising MAO-A inhibitory activity, harmalacidine HCl could be a promising lead for further development as a reversible MAO-A inhibitor, with potential applications in treating neuropsychiatric disorders.