Trace component fishing strategy based on offline two-dimensional liquid chromatography combined with PRDX3-surface plasmon resonance for Uncaria alkaloids.
10.1016/j.jpha.2025.101244
- Author:
Hui NI
1
;
Zijia ZHANG
2
;
Ye LU
3
;
Yaowen LIU
1
;
Yang ZHOU
2
;
Wenyong WU
2
;
Xinqin KONG
2
;
Liling SHEN
2
;
Sihan CHEN
2
;
Huali LONG
2
;
Cheng LUO
4
;
Hao ZHANG
3
;
Jinjun HOU
2
;
Wanying WU
1
Author Information
1. School of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, 210029, China.
2. 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.
3. Institute of Interdisciplinary Integrative Medicine Research, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China.
4. University of Chinese Academy of Sciences, Beijing, 100049, China.
- Publication Type:Journal Article
- Keywords:
Offline two-dimensional liquid chromatography;
Peroxiredoxin 3;
Surface plasmon resonance;
Trace component fishing strategy;
Uncaria alkaloids
- From:
Journal of Pharmaceutical Analysis
2025;15(9):101244-101244
- CountryChina
- Language:English
-
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.
