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 ...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 C_(18)column and a conventional C_(18)column under disparate mobile phase conditions was constructed.To fully reveal the trace alkaloids,132D 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 132D 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 H_(2)O_(2)measurement assays and transfection experiments confirmed that these three trace alkaloids enhanced PRDX3-mediated H_(2)O_(2)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.展开更多
基金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)Sanming Project of Medicine in Shenzhen,China(Grant No.:ZZYSM202106004).
文摘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 C_(18)column and a conventional C_(18)column under disparate mobile phase conditions was constructed.To fully reveal the trace alkaloids,132D 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 132D 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 H_(2)O_(2)measurement assays and transfection experiments confirmed that these three trace alkaloids enhanced PRDX3-mediated H_(2)O_(2)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.
