Exploring the mechanism of Buxue Yimu Pill on hemorrhagic anemia through molecular docking, network pharmacology and experimental validation

Buxue Yimu Pill (BYP) is a classic gynecological medicine in China, which is composed of Angelica sinensis (Oliv.) Diels, Leonurus japonicus Houtt, Astragalus membranaceus (Fisch.) Bunge, Colla corii asini and Citrus reticulata Blanco. It has been widely used in the clinical therapy with the function of enriching Blood, nourishing Qi, and removing blood stasis. The main aim of this study was to determine the bioactive molecules and therapeutic mechanism of BYP against hemorrhagic anemia. In the present study, GC-MS and UPLC/Q-TOF-MS/MS were employed to identify the chemical compounds from BYP. The genecards database (https: //www.genecards.org/)was used to obtain the potential target proteins related to hemorrhagic anemia. And autodock/Vina was adopted to evaluate the binding ability of the protein receptors and chemical ligands. Gene ontology and KEGG pathway enrichment analysis were conducted using the ClusterProfiler. As a result, a total of 62 candidate molecules were identified and 152 targets related to hemorrhagic anemia were obtained. 34 active molecules and 140 targets were obtained through the virtual screening experiment. Furthermore, the data of molecular-target (M-T), target-pathway (T-P), and molecular-target-pathway (M-T-P) network suggested that 32 active molecules could enhance hematopoiesis and activate the immune system by regulating 57 important targets. Pharmacological experiments showed that BYP could significantly increase the RBC, HGB, and HCT contents, and significantly down-regulate the EPO, IL-6, CSF3, NOS2, VEGFA, PDGFRB, TGFB1 expression. The results also showed that leonurine, leonuriside B, leosibiricin, ononin, rutin, astragaloside I, riligustilide and levistolide A, were the active molecules closely related to enriching Blood. In conclusion, based on the molecular docking, network pharmacology and validation experimental results, the enriching blood effect of BYP on hemorrhagic anemia might be associated with hematopoiesis produce, anti-inflammatory, and immunity enhancement.