Anti-Inflammatory Activity of Mandragora autumnalis Ethanolic Extract: In Vitro and Cellular Mechanistic Insights

Plant-based remedies have been employed since ancient times to manage and prevent a wide range of diseases. Background: Their bioactive constituents provide natural alternatives to synthetic anti-inflammatory drugs, often with reduced toxicity and multiple biological benefits. Mandragora autumnalis, a medicinal plant rich in secondary metabolites, has not been extensively investigated for its anti-inflammatory properties. Methods: The anti-inflammatory activity of Mandragora autumnalis ethanolic extract (MAE) was evaluated using in vitro biochemical assays, including heat-induced protein denaturation (casein and bovine serum albumin) and red blood cell membrane stabilization. Cellular anti-inflammatory effects were assessed in LPS-stimulated RAW 264.7 macrophages by measuring nitric oxide production, pro-inflammatory cytokine levels, macrophage migration, gene and protein expression of inflammatory mediators, and the activation status of NF-κB, STAT3, and MAPK signaling pathways. Results: Preliminary screening revealed that MAE effectively inhibited heat-induced protein denaturation (casein and bovine serum albumin) and heat-induced red blood cell (RBC) hemolysis, indicating strong protein- and membrane-stabilizing properties associated with anti-inflammatory activity. In lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, MAE markedly suppressed the inflammatory response by downregulating the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), leading to a significant reduction in nitric oxide (NO) production and pro-inflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Furthermore, MAE inhibited macrophage migration and attenuated key inflammatory signaling pathways, notably nuclear factor kappa B (NF-κB), signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinases (MAPKs). Molecular docking analysis was conducted to explore the potential interactions between the most abundant chemical compounds and key proteins involved in inflammatory signaling pathways, including ERK and iNOS. Conclusions: Overall, these results indicate that MAE exhibits strong anti-inflammatory activity by stabilizing proteins, protecting cellular membranes, and inhibiting key inflammatory mediators and signaling pathways. These findings highlight its potential as a natural therapeutic candidate for the prevention and management of chronic inflammation-related disorders, such as arthritis, cardiovascular diseases, and cancer. However, further mechanistic investigations and in vivo studies are required to confirm its therapeutic potential and clinical relevance.