Trace component fishing strategy based on offline two-dimensional liquid chromatography combined with PRDX3-surface plasmon resonance for <i>Uncaria</i> alkaloids

Hui Ni; Zijia Zhang; Ye Lu; Yaowen Liu; Yang Zhou; Wenyong Wu; Xinqin Kong; Liling Shen; Sihan Chen; Huali Long; Cheng Luo; Hao Zhang; Jinjun Hou; Wanying Wu

doi:10.1016/j.jpha.2025.101244

Volume 15 Issue 9

Sep. 2025

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Article Navigation > Journal of Pharmaceutical Analysis > 2025 > 15(9): 101244

Hui Ni, Zijia Zhang, Ye Lu, Yaowen Liu, Yang Zhou, Wenyong Wu, Xinqin Kong, Liling Shen, Sihan Chen, Huali Long, Cheng Luo, Hao Zhang, Jinjun Hou, Wanying Wu. Trace component fishing strategy based on offline two-dimensional liquid chromatography combined with PRDX3-surface plasmon resonance for Uncaria alkaloids[J]. Journal of Pharmaceutical Analysis, 2025, 15(9): 101244. doi: 10.1016/j.jpha.2025.101244

Citation:

Hui Ni, Zijia Zhang, Ye Lu, Yaowen Liu, Yang Zhou, Wenyong Wu, Xinqin Kong, Liling Shen, Sihan Chen, Huali Long, Cheng Luo, Hao Zhang, Jinjun Hou, Wanying Wu. Trace component fishing strategy based on offline two-dimensional liquid chromatography combined with PRDX3-surface plasmon resonance for Uncaria alkaloids[J]. Journal of Pharmaceutical Analysis, 2025, 15(9): 101244. doi: 10.1016/j.jpha.2025.101244

Citation:

Hui Ni, Zijia Zhang, Ye Lu, Yaowen Liu, Yang Zhou, Wenyong Wu, Xinqin Kong, Liling Shen, Sihan Chen, Huali Long, Cheng Luo, Hao Zhang, Jinjun Hou, Wanying Wu. Trace component fishing strategy based on offline two-dimensional liquid chromatography combined with PRDX3-surface plasmon resonance for Uncaria alkaloids[J]. Journal of Pharmaceutical Analysis, 2025, 15(9): 101244. doi: 10.1016/j.jpha.2025.101244

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Trace component fishing strategy based on offline two-dimensional liquid chromatography combined with PRDX3-surface plasmon resonance for Uncaria alkaloids

doi: 10.1016/j.jpha.2025.101244

Hui Ni^a,b,
Zijia Zhang^b,
Ye Lu^c,
Yaowen Liu^a,b,
Yang Zhou^b,
Wenyong Wu^b,
Xinqin Kong^b,d,
Liling Shen^b,d,
Sihan Chen^b,d,
Huali Long^b,
Cheng Luo^d,e,
Hao Zhang^{c
,
,},
Jinjun Hou^{b
,
,},
Wanying Wu^{a,b,d
,
,}

a. School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, 210029, China;
b. Shanghai Research Center for Modernization of Traditional Chinese Medicine, National Engineering Research Center of TCM Standardization Technology, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China;
c. Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China;
d. University of Chinese Academy of Sciences, Beijing, 100049, China;
e. Chemical Biology Research Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China

Funds:

This work was supported by Shanghai Institute of Materia Medica - Shanghai University of Traditional Chinese Medicine (SIMM-SHUTCM) Traditional Chinese Medicine Innovation Joint Research Program, China (Grant No.: 2022, E2G808H096), the National Key Research and Development Program of China (Grant Nos.: 2022YFC3501704 and 2023YFC3504205), and Sanming Project of Medicine in Shenzhen, China (Grant No.: ZZYSM202106004).

Received Date: Sep. 02, 2024
Accepted Date: Feb. 18, 2025
Rev Recd Date: Jan. 25, 2025
Publish Date: Sep. 30, 2025

Abstract

Abstract

The rapid screening of bioactive constituents within traditional Chinese medicine (TCM) presents a significant challenge to researchers. Prevailing strategies for the screening of active components in TCM often overlook trace components owing to their concealment by more abundant constituents. To address this limitation, a fishing strategy based on offline two-dimensional liquid chromatography (2D-LC) combined with surface plasmon resonance (SPR) was utilized to screen bioactive trace components targeting peroxiredoxin 3 (PRDX3), using Uncaria alkaloids (UAs) as a case study. Initially, an orthogonal preparative offline 2D-LC system combining a positively charged C₁₈ column and a conventional C₁₈ column under disparate mobile phase conditions was constructed. To fully reveal the trace alkaloids, 13 2D fractions of UAs were prepared, and their components were characterized using mass spectrometry (MS). Subsequently, employing PRDX3 as the targeting protein, a SPR-based screening approach was established and rigorously validated with geissoschizine methyl ether (GSM) serving as a positive control for binding. Employing this refined strategy, 29 candidate binding alkaloids were fished from the 13 2D fractions. Notably, combining offline 2D-LC with SPR increased the yield of candidate binding components from 10 to 29 when compared to SPR-based screening alone. Subsequent binding affinity assays confirmed that PRDX3 was a direct binding target for the 12 fished alkaloids, with isovallesiachotamine (IV), corynoxeine N-oxide (CO-N), and cadambine (CAD) demonstrating the highest affinity for PRDX3. Their interactions were further validated through molecular docking analysis. Subsequent intracellular H₂O₂ measurement assays and transfection experiments confirmed that these three trace alkaloids enhanced PRDX3-mediated H₂O₂ clearance. In conclusion, this study introduced an innovative strategy for the identification of active trace components in TCM. This approach holds promise for accelerating research on medicinal components within this field.
- Offline two-dimensional liquid chromatography,
- Peroxiredoxin 3,
- Surface plasmon resonance,
- Trace component fishing strategy,
- Uncaria alkaloids

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References(55)

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