Objective To establish an analytical method for fingerprinting and multi-component quantification by HPLC, and to predict the spleen-tonifying active components in Atractylodis macrocephalae Rhizoma. combined with network pharmacology.

Methods The HPLC fingerprints of Atractylodis macrocephalae Rhizomea. were established, and the similarity of 10 batches of samples from different origins was evaluated using the Similarity Evaluation System for Chromatographic Fingerprints of Traditional Chinese Medicine (2012.1 version). The differential components were determined by hierarchical cluster analysis (HCA) and orthogonal partial least squares discriminant analysis (OPLS-DA). A "traditional Chinese medicine-active component-target-disease" network was constructed via network pharmacology. Combined with the detectability, specificity and effectiveness of components, the core spleen-tonifying active components of Atractylodis macrocephalae Rhizoma. were predicted, and their contents were determined.

Results The similarities between the established HPLC fingerprint and the reference fingerprint of Atractylodis macrocephalae Rhizoma. ranged from 0.949 to 0.992. A total of 27 common peaks were identified, three of which were assigned. HCA clustered the 10 batches of samples into two categories. The differential components screened by OPLS-DA were peak 21, atractylenolide Ⅲ, atractylenolide Ⅱ, atractylenolide Ⅰ, peak 7, peak 19, and peak 24. Network pharmacology analysis showed that the spleen-tonifying active components of Atractylodis macrocephalae Rhizoma. exerted spleen-tonifying effects by acting on core targets including EGFR, SRC, TNF, PIK3CA, JAK2, ESR1, IL1B, ERBB2, PPARG, APP, AR, and ALK, and regulating signaling pathways such as pathways in cancer, proteoglycans in cancer, and pancreatic cancer. Atractylenolide Ⅰ, Ⅱ and Ⅲ were predicted as the core spleen-tonifying active components. The contents of atractylenolide Ⅰ, Ⅱ and Ⅲ in 10 batches were 0.0587-0.1652, 0.0201-0.2288, and 0.0448-0.3961 mg/g, respectively.

Conclusion The established HPLC fingerprint and multi-component quantitative analysis method for Atractylodis macrocephalae Rhizoma. is accurate and easy to operate. Combined with network pharmacology, it scientifically and reasonably predicts the spleen-tonifying active components, which can provide a scientific reference for the quality evaluation of Atractylodis macrocephalae Rhizoma. and further research on its spleen-tonifying mechanism.

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