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  3. Integrating six-stage cascade focused strategy to reveal the potential mechanisms and effective components of Ershiwei Roudoukou pills in anti-atherosclerosis

Integrating six-stage cascade focused strategy to reveal the potential mechanisms and effective components of Ershiwei Roudoukou pills in anti-atherosclerosis

  • Phytomedicine. 2026 May:154:158052. doi: 10.1016/j.phymed.2026.158052.
Hongbin Zhang 1,  Li Qiao 2,  Fan Yang 1,  Le Li 1,  Yuhao Zhang 3,  Yi Wu 4,  Zhenhua Tian 5,  Haiqiang Jiang 6
Affiliations

Affiliations

  • 1 School of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan, 250355, China.
  • 2 Experimental Center, Shandong University of Traditional Chinese Medicine, Jinan, 250355, China.
  • 3 Innovative Institute of Chinese Medicine and Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan, 250355, China.
  • 4 College of Veterinary Medicine , Yunnan Agricultural University, Kunming, 650000, China. Electronic address: [email protected].
  • 5 Experimental Center, Shandong University of Traditional Chinese Medicine, Jinan, 250355, China. Electronic address: [email protected].
  • 6 School of Pharmacy, Shandong University of Traditional Chinese Medicine, Jinan, 250355, China; Key Laboratory of Traditional Chinese Medicine Classical Theory, Ministry of Education, Shandong University of Traditional Chinese Medicine, Jinan, 250355, China; Shandong Key Laboratory of Digital Traditional Chinese Medicine, Shandong University of Traditional Chinese Medicine Jinan, 250355, China. Electronic address: [email protected].
PMID: 41833102 DOI: 10.1016/j.phymed.2026.158052
Abstract

Background: Elucidating effective components and mechanisms of traditional Chinese medicine (TCM) formulas remains a critical challenge for modernization. ErShiWei RouDouKou Pills (ESWRDK), a Tibetan formula with cardiovascular potential, lacks systematic exploration of its anti-atherosclerotic (AS) material basis and mechanisms.

Purpose: A novel six-stage cascade focused strategy integrating three-dimensional filtering mode, qualitative characterization, multi-component quantification, anti-AS efficacy, multi-lipidomics and bioactive compounds evaluation was proposed, advancing TCM research by holistic and multi-layered approach.

Methods: UHPLC-MS combined with mass defect-ion intensity filtering (MD-ITF), DPIs, Nl and FBMN employed for profiling. Nine characteristic components were quantitated. A 12-week high-fat diet was fed to apoE⁻/⁻ mice for AS model and the efficacy was assessed by lipid level, pro-inflammatory cytokines, HE and Oil red O staining. MALDI-TOF-MS based lipidomics identified tissue specific biomarkers. WB probed target and metabolic signaling in liver. Candidate Binders were screened through IF-MALDI-TOF-MS, molecular docking, enzyme inhibition assays and fluorescence analysis.

Results: Firstly, the MD-ITF method and structural classification was established for complicated matrix. Secondly, 426 chemical components including 74 low-abundance were characterized. Thirdly, 9 characteristic components were quantified, and content distribution were profiled. Fourthly, ESWRDK reduced lipids, inflammation, and aortic plaques in AS mice. Fifthly, a total of 38, 23 and 48 differential biomarkers were identified predominantly linked to glycerophospholipids (GP) metabolism. WB confirmed ESWRDK downregulated hepatic PLA2, upregulated p-AMPK/AMPK and PPAR-α, and suppressed SREBP-1, orchestrating and mitigating lipid dysregulation. Finally, dehydrodiisoeugenol and agarotetrol bound PLA2, formed stable 1:1 static quenchingand inhibited PLA2 activity in vitro.

Conclusion: A novel six-stage cascade-focused strategy was successfully established to elucidate ESWRDK's anti-AS mechanisms, offering feasible paradigm for advancing modernization of TCM.

Keywords

Active components; Atherosclerosis; Ershiwei Roudoukou pills; Lipidomics; Six-stage cascade focused strategy.

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