Targeting Inflammatory Pathways via Molecular Docking and In vivo Assessment of Prunus amygdalus Stem Bark-Derived Flavonoids and Rutin

Almond (Prunus amygdalus) steam bark, an agro-industrial by-product rich in polyphenolic compounds, represents a sustainable source of bioactive flavonoids, particularly rutin, with potential pharmacological applications. In this study, total flavonoids were extracted using ethanol-based maceration combined with ultrasonic treatment and vacuum concentration, yielding a high-purity fraction (98.0 ± 0.8%), while rutin was isolated through semi-preparative HPLC. Acute oral toxicity assessment in male mice at a limit dose of 5,000 mg/kg revealed no mortality, no significant changes in body weight (p > 0.05), and only mild transient behavioral alterations, indicating an LD50 > 5,000 mg/kg and classification under GHS Category 5 (low toxicity). In the carrageenan-induced paw edema model, the flavonoid fraction at 100 mg/kg and rutin at 25 mg/kg exhibited the highest anti-inflammatory activity, with 61.6% and 55.6% inhibition of edema formation at the third hour, respectively, while higher doses showed moderate but sustained effects, suggesting a non-linear dose-response relationship. Molecular docking studies demonstrated strong binding affinities of rutin to inflammation-related protein targets, including TNF-α (–7.8 kcal/mol, Ki = 1.92 µM), AKT1 (–8.8 kcal/mol, Ki = 0.36 µM), and ESR1 (–8.5 kcal/mol, Ki = 0.61 µM), as well as high-affinity binding to serum albumin (–10.4 kcal/mol, Ki = 23.2 nM). These findings indicate that almond steam bark-derived flavonoids and rutin are safe at high oral doses, exert potent anti-inflammatory effects, and act via multi-target modulation of inflammatory pathways, supporting their potential as natural therapeutic or nutraceutical agents. HIGHLIGHTS Almond (Prunus amygdalus) steam bark is a rich source of bioactive flavonoids and rutin Optimized ethanol-ultrasound extraction yielded 98% pure total flavonoid fraction Semi-preparative HPLC enabled efficient isolation of high-purity rutin Both compounds showed LD50 > 5,000 mg/kg in acute oral toxicity tests (GHS Category 5) Strong anti-inflammatory effects observed in carrageenan-induced paw edema model Molecular docking revealed multi-target modulation of inflammatory pathways GRAPHICAL ABSTRACT

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