Objective  To study the active ingredients and potential molecular mechanism of Notopterygii rhizoma et radix in treating Alzheimer's disease (AD), establish an high performance liquid chromatographytandem mass spectrometry (HPLC-MS/MS) method which can simultaneously determine the content of five components in Notopterygii rhizoma et radix, and provide a basis for experimental research on its pharmacological substance basis and molecular mechanism.

Methods  The active ingredients and targets of Notopterygii rhizoma et radix were obtained from the Traditional Chinese Medicine Systems Pharmacology (TCMSP) database, and AD related targets were predicted from GeneCards, Online Mendelian Inheritance in Man (OMIM), and National Center for Biotechnology Information (NCBI) databases. The Cytoscape software was used to construct a drug-ingredient-target-disease visualization network, the STRING database online platform was used to construct a protein-protein interaction network, and GO function and KEGG pathway enrichment analysis were performed. The main active ingredients and key targets were molecularly linked using Maestro software to validate the network pharmacology results. HPLC-MS/MS method was used to simultaneously determine the content of five components, namely, nodakenin, bergaptin, diosmetin, notopterol, and isoimperatorin. XTerra MS C₁₈ column (100 mm × 2.1 mm, 3.5 µm) was used. The methanol-water was used as the mobile phase with the gradient elution, and the flow rate was 0.3 mL/min. The column temperature was 30 ℃, and the sample amount was 5 µL. The electric spray ion source and multi reaction monitoring positive ion mode were adopted.

Results  A total of 13 active ingredients in Notopterygii rhizoma et radix and 36 anti AD targets were obtained from network pharmacology. Gene enrichment analysis showed that there were 165 GO functional items and 89 pathways (P<0.05). The molecular docking results indicated that the main active ingredients of Notopterygii rhizoma et radix had the best binding activity with the key target retinoic acid X receptor. There was a good linear relationship between the peak area and the concentrations of nodakenin, bergaptin, diosmetin, notopterol, and isoimperatorin in the ranges of 69.74-8 472.85, 8.51-1  033.89, 0.35-37.00, 15.42-1 873.27, and 58.85-7 150.49 ng/mL, respectively (r ﹥0.990 0).

Conclusion  The potential mechanism of multi-component, multi-target, and multi pathway anti-AD effects of Notopterygii rhizoma et radix has been preliminarily explored. The established HPLC-MS/MS method is easy to operate, reliable, highly specific, and reproducible, and can be used for quality control of Notopterygii rhizoma et radix. This study will provide a basis for the research on the pharmacological substance basis, molecular mechanism, and quality control of related preparations of Notopterygii rhizoma et radix against AD.

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