Polysaccharide-driven quality control of Gastrodia elata Blume: A multidimensional strategy integrating LC-MS/MS-based quantitative analysis, machine learning, and anti-neuroinflammatory bioactivity.

Gastrodia elata Blume (GE), a highly regarded traditional Chinese medicinal herb, is widely recognized for its diverse pharmacological activities. Current quality control primarily relies on small molecule biomarkers, yet critical research gaps persist in understanding the complex interactions of the key ingredient, Gastrodia elata polysaccharides (GEP), with bioactivity-related quality due to analytical challenges. To overcome the limitations of traditional non-specific degradation methods, this study established a novel multidimensional quality control strategy using optimized α-amylase-assisted Fenton's initiation toward defined oligosaccharide groups (FITDOG) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the simultaneous qualitative and quantitative characterization of specific GEP domains. Analysis of 205 batches across varying grades and origins identified α-1,4/1,6-glucans (0.3-40%) as robust, grade-dependent markers. Integration of these macromolecular profiles with small-molecule markers into an Extreme Gradient Boosting (XGBoost) model, superior cultivar discrimination was achieved (Area Under the Curve, AUC > 0.89), while SHapley Additive exPlanations (SHAP) analysis highlighting the predictive significance of the glucan content. Analytical findings were biologically validated in lipopolysaccharide (LPS)-stimulated human microglial clone 3 cells (HMC3), demonstrating that elevated levels of these α-1,4/1,6-glucans significantly correlated with enhanced cell viability (p < 0.05) and anti-neuroinflammatory regulation, characterized by suppression of IL6 and upregulation of TGFB1. This study proposes α-1,4/1,6-glucans as novel quality markers for GE and establishes a robust analytical framework that integrates chemical profiling and biological validation. This advancement enhances the precision of quality control in traditional Chinese medicine and underscores the potential of polysaccharide-driven strategies for the standardization of medicinal herbs.