Integrative multi-omics analysis to decipher the mechanism by which the interaction between Danggui Buxue Tang and gut microbiota drives isoflavone metabolic transformation

• Gut microbiota transforms DBT isoflavones via three-step pathway. • DBT enriches Bifidobacterium (0.05%→25.5%), inhibits Porphyromonas . • Biotransformation cuts toxicity 90%, doubles drug clearance. • Metabolites strongly bind hematopoiesis targets like CDK2, GSK3B. • Multi-omics reveals microbiota-enzyme-metabolite-target network. Danggui Buxue Tang (DBT) is a classic herbal formula traditionally used to "tonify Qi and generate blood." Its efficacy is believed to depend on the biotransformation of its bioactive compounds by the gut microbiota; however, the specific metabolic profile and the key bacterial taxa involved remain unclear. An in vitro anaerobic fermentation model was established to simulate gut microbial biotransformation of DBT. This model allowed for a controlled and reproducible investigation of DBT-microbiota interactions. We integrated untargeted metabolomics (LC-MS) with 16S rRNA gene sequencing to analyze the concomitant changes in metabolites and the microbial community. PICRUSt2 was used for functional prediction. Additionally, network pharmacology and molecular docking were employed to construct a "microbiota-enzyme-metabolite-target" network. Untargeted metabolomics analysis identified 734 significantly altered metabolites following DBT fermentation, including notable increases in formononetin, biochanin A, and xenognosin B. 16S rRNA gene sequencing revealed that DBT markedly increased the relative abundance of Bifidobacterium (from 0.05% to 25.5%) while suppressing Porphyromonas. Functional prediction indicated significant enrichment in the "Drug metabolism - other enzymes" pathway. Correlation analysis revealed significant associations between specific gut microbiota and metabolites. A three-step metabolic cascade (glycoside hydrolysis → methylation → hydroxylation) was identified, which reduced toxicity by 90% and increased clearance twofold. The metabolites exhibited strong binding to targets such as CDK2 (binding energy ≤ -7.24 kcal/mol) and were enriched in hematopoiesis-related pathways, including the PI3K-Akt signaling pathway. This study systematically elucidates how the gut microbiota transforms DBT isoflavones into active aglycones that target core hematopoietic proteins, providing a novel perspective on the mechanism of orally administered, poorly absorbable traditional Chinese medicine formulas. Our results confirm that the gut microbiota is indispensable for the efficacy of DBT.