Integrated NMR, HPLC-Chemometrics, and Network Pharmacology Strategy for Quality Assessment and Mechanism Elucidation of Gelatinous Chinese Medicines

Gelatinous Chinese medicines (GCMs), such as Ejiao, are animal-derived solid preparations traditionally used for blood and qi deficiency. With growing market demand, adulteration has become a critical quality concern. This study established a multimodal quality assessment system for GCMs. Using 1H NMR, we characterized the acid hydrolysates of GCMs and identified 17 components, including 16 total hydrolysable amino acids and taurine, revealing compositional similarity across seven GCMs. Stability studies under acid hydrolysis were conducted on 19 amino acids, all of which remained stable except methionin, cystine, and tryptophan, leading to the selection of the remaining 16 amino acids as target analytes. A pre-column derivatization–HPLC method was validated for the quantification of 16 total hydrolysable amino acids, showing excellent linearity (r2 > 0.999), high precision (RSD < 2.71%), and satisfactory recoveries (90.74–109.3%). Content determination of 65 batches revealed batch-to-batch variations in total hydrolysable amino acid profiles. Combined with chemometrics, the method successfully discriminated dermal, horn, bone, and shell glues, and further enabled subcategory differentiation. Network pharmacology analysis suggested that these amino acid components may interact with key targets (e.g., GRM5, GABRA1, GABBR2) and pathways, primarily neuroactive ligand–receptor interaction. Collectively, this integrated analytical and network-based strategy not only supports chemical authentication but also provides insights into the potential molecular basis of the traditionally described restorative effects of GCMs, offering a scientific foundation for their modernization and international recognition.