An integrated strategy for deciphering the action mechanism of emplastrum: Prescription analysis- component identification- virtual screening and affinity testing in the case of Yaoshen Gao.

ETHNOPHARMACOLOGICAL RELEVANCE

Emplastrum has a long history of use in the clinical practice of traditional Chinese medicine (TCM), valued for its convenient external application and pronounced therapeutic effects. Traditionally, the emplastrum was composed of numerous herbal medicines. The elucidation of their mechanisms of action are of great importance. YaoShen Gao (YSG), as a traditional example of emplastrum, was composed of more than 20 medicinal herbs. Clinically, YSG has been used to treat benign prostatic hyperplasia (BPH). However, the active components and therapeutic targets of YSG remain unclear, requiring further investigation.

AIM OF THE STUDY

To establish an integrated strategy to uncover the mechanisms underlying the potential active ingredients and therapeutic targets in complex TCM emplastra, using YSG for BPH treatment as a case study.

MATERIALS AND METHODS

A BPH rat model was established via castration and testosterone propionate injections. The therapeutic efficacy of YSG was evaluated comprehensively through phenotypic, pathological, physiological, and biochemical analyses. Prescription analysis was conducted based on the principles of "monarch, minister, assistant, and courier," as well as clinical dosage and efficacy. Based on the strategy of representative compounds-single herbal medicine -YSG formulation, the chemical profile of YSG was performed using UPLC-Q Exactive Orbitrap- MS. Network pharmacology identified preliminary targets, while molecular docking and literature mining further narrowed these down. Finally, molecular dynamics simulations and Bio-Layer Interferometry (BLI) were used to validate binding affinities of active components to targets.

RESULTS

The efficacy indicators demonstrated that treatment of YSG significantly reduced prostate wet weight and prostate index in BPH rats, with notable improvements observed in glandular structure. Additionally, YSG inhibited the expression of inflammatory factors (TNF- α, IL- 8) and fibrosis-related proteins (VEGF, TGF-β). Twelve key herbal medicines were identified by prescription analysis from the 20 herbs in YSG, such as Cistanche deserticola, Epimedium sagittatum and so on. High-resolution mass spectrometry (HR-MS) characterized 125 chemical components, and Venn analysis identified 409 common targets between YSG components and BPH. Subsequently, GO and KEGG analyses revealed that these targets are predominantly involved in protein phosphorylation, cellular components, and key signaling pathways. Protein-protein interaction (PPI) analysis identified 10 key targets, suggesting that the therapeutic effects of YSG on BPH are mediated through 39 active compounds and 12 relevant signaling pathways. Molecular docking analysis identified 14 target-compound pairs, and literature supported their relevance in PI3K/AKT, VEGF, TNF, and TGF-β pathways. Molecular dynamics simulations and BLI further validated the strong interactions between representative target-small molecule pairs like AKT1 with bavachalcone (KD = 46.8 μM) and PIK3R1 with apigenin (KD = 47.9 μM).

CONCLUSIONS

A systematic strategy for identifying active ingredients and therapeutic targets in complex TCM emplastra was developed. YSG contains active components, including bavachalcone, apigenin, schisandrin C, liquiritigenin, 8-prenyldaidzein, estrone, isopimpinellin, 8-prenylkaempferol, which act on six key targets, such as AKT1 and PIK3R1, to regulate the AKT/PI3K, AGE-RAGE, AR, VEGF, TGF-β, TNF and others signaling pathways. These pathways further modulate cellular proliferation, fibrosis, inflammation, and angiogenesis, thereby effectively contributing to the treatment of BPH.