Antimalarial Activity of Methoxylated Flavonoid Fraction (Cirsimaritin-Dominated) from Artemisia kopetdaghensis in Plasmodium berghei-Infected Mice with in silico Analysis.

BACKGROUND: Building on our previous investigation of the total semipolar extract (SPE) of Artemisia kopetdaghensis in Plasmodium berghei-infected mice, the present study focuses specifically on the methoxylated flavonoid fraction (MFF). Flavonoids are increasingly recognized for their ability to suppress parasite growth and modulate host immunity. To clarify their role, we isolated and characterized the major flavonoid constituents of this plant and evaluated their antimalarial potential, both alone and in combination with chloroquine, in a mouse model.

METHODS: Aerial parts of A. kopetdaghensis were extracted using a chloroform:acetone (2:1) solvent mixture. The extract was then fractionated by column chromatography. Based primarily on ^1H-NMR spectra, the MFF was selected and further purified by preparative HPLC. Isolated compounds were identified by 1D and 2D NMR and mass spectrometry. In the in vivo antimalarial study, thirty-six infected female Balb/c mice were treated with MFF and evaluated for key parameters. Docking and molecular interaction studies were conducted using AutoDock v4.2.6 software to examine the interactions of constituents with cytokine protein targets: 1D9C [interferon-gamma (IFN-γ)], 1B6C (TGF-β), 1BBN [(interleukin-4 (IL-4)], and 4HR9 (IL-17), separately.

RESULTS: Phytochemical analysis of the MFF by HPLC revealed three flavones: 6-methoxytricin (20%), cirsilineol (10%), and cirsimaritin (70%), identified for the first time in A. kopetdaghensis, with cirsimaritin as the dominant constituent. In vivo, MFF treatment significantly reduced parasitemia and enhanced parasite suppression in P. berghei-infected mice. Cytokine profiling demonstrated suppression of TGF-β and IL-4 followed by their recovery, together with marked elevations of IFN-γ and IL-17, indicating balanced modulation between pro-inflammatory and regulatory responses. These immunological findings were corroborated by molecular docking analyses, which confirmed binding interactions of cirsimaritin with cytokine receptor targets, providing mechanistic support for its immunomodulatory activity.

CONCLUSIONS: This work extends our previous study on the SPE by moving from extract-level observations to constituent-specific insights. By combining phytochemical isolation with in silico receptor interaction analysis, we demonstrate that methoxylated flavonoids, particularly cirsimaritin, are key modulators of host immunity and promising candidates for further development as adjuncts or leads in antimalarial therapy.