Structural features of α-Glucans from Gymnadenia orchidis Lindl. and ability to inhibit the LPS-induced inflammatory response and osteoclast differentiation in RAW264.7 macrophage via the TLR4/RANKL/NF-κB signaling pathway

Gymnadenia orchidis Lindl. — a traditional Orchidaceae Gymnadenia medicinal plant—exhibits validated pharmacological activities, including potent anti-inflammatory and antioxidant capacities. Nonetheless, current research on its resource development, utilization, and underlying pharmacological mechanisms remains limited. Its polysaccharides’ potential in periodontitis management aligns with market demands for high-quality natural oral healthcare products. This study aimed to characterize Gymnadenia orchidis polysaccharides (GOP), assess anti-inflammatory/anti-osteoclastogenic potencies on Lipopolysaccharide (LPS)-induced RAW264.7 macrophage, and clarify the mechanisms. GOP was purified via ultrasonic-assisted aqueous extract isolation and refined via DEAE Sepharose Fast Flow chromatography. structural features were analyzed by Methylation analysis, Fourier-transform infrared (FT-IR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, and microscopic morphological analysis In an LPS-elicited inflammatory model established in RAW264.7 macrophage, cell viability, inflammatory cytokine production, and the underlying mechanisms were evaluated using the Reverse Transcription-quantitative PCR (RT-qPCR), Enzyme-Linked Immunosorbent Assay (ELISA), Cell Counting Kit-8 (CCK-8), and Western blot. Two fractions (GOP-N: 2.52 kDa; GOP-A: 27.56 kDa), mainly composed of α-1,4- D -Glc p (GOP-A additionally containing α-1,4,6- D -Glc p ), exhibited lamellar morphologies. In vitro anti-inflammatory bioassessments demonstrated that in an LPS-elicited RAW264.7 cell inflammation models, GOP-N and GOP-A exert dual regulatory potencies by impeding the TLR4/RANKL/NF-κB pathway: they inhibit osteoclast differentiation, downregulate proinflammatory mediator secretion, and upregulate IL-10 expression, exerting potent anti-inflammatory activity. Collectively, GOP possess significant immunomodulatory activity and are potential periodontitis agents, providing a theoretical foundation for polysaccharide-based oral products and supporting rational G. orchidis resource development. • Novel α-1,4/α-1,4,6-D-Glcp from Gymnadenia orchidis Lindl.: sheet-like, non-starch and multi-technique characterized. • Inhibits LPS-induced TLR4/RANKL/NF-κB and osteoclast differentiation, revealing a new anti-periodontitis mechanism. • Turns discarded Gymnadenia orchidis Lindl. residues into high-value polysaccharide for crop sustainability and oral care.