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

Abstract

Background: Building on our previous investigation of the total semipolar extract (SPE) of Artemisiakopetdaghensis in Plasmodiumberghei-infected mice, thepresent study focuses specifically on the methoxylated flavonoid fraction (MFF). Flavonoids are increasingly recognized for their ability to suppress parasitegrowth and modulate host immunity. To clarify their role, we isolated and characterized the major flavonoid constituents of this plant and evaluated theirantimalarial 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 columnchromatography. Based primarily on <>1H-NMR spectra, the MFF was selected and further purified by preparative HPLC. Isolated compounds were identified by 1Dand 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 keyparameters. Docking and molecular interaction studies were conducted using AutoDock v4.2.6 software to examine the interactions of constituents withcytokine protein targets: 1D9C interferon-gamma (IFN-gamma), 1B6C (TGF-beta), 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 thefirst time in A.kopetdaghensis, with cirsimaritin as the dominant constituent. In vivo, MFF treatment significantly reduced parasitemia and enhanced parasitesuppression in P.berghei-infected mice. Cytokine profiling demonstrated suppression of TGF-beta and IL-4 followed by their recovery, together with markedelevations of IFN-gamma and IL-17, indicating balanced modulation between pro-inflammatory and regulatory responses. These immunological findings werecorroborated by molecular docking analyses, which confirmed binding interactions of cirsimaritin with cytokine receptor targets, providing mechanisticsupport 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 combiningphytochemical isolation with in silico receptor interaction analysis, we demonstrate that methoxylated flavonoids, particularly cirsimaritin, are key modulatorsof host immunity and promising candidates for further development as adjuncts or leads in antimalarial therapy.