NPs-Chip-Ligand Fishing: A novel strategy for exploring targets and screening active ingredients in food-derived natural products

Natural products (NPs) from food sources represent valuable reservoirs of bioactive compounds with health-promoting properties, yet systematically elucidating the relationships between specific active ingredients, their molecular targets, and physiological effects remains a significant challenge. Here, we present an innovative NPs-Chip-Ligand Fishing Strategy to elucidate these complex interactions, using ginsenosides from Panax ginseng C. A. Mey., a functional food ingredient, as a proof-of-concept model. Utilizing this platform, we reproducibly identified 12 potential target proteins across independent experiments, with malate dehydrogenase 2 (MDH2) emerging as the top-ranking candidate. Given that MDH2 dysregulation is intrinsically linked to oxygen-glucose deprivation/reperfusion (OGD/R)-induced oxidative stress and cell injury, we validated that ginsenosides exert potent cytoprotection by enhancing cell viability, mitigating LDH release, and suppressing ROS generation. Furthermore, integrated SPR and magnetic bead-based ligand fishing with recombinant MDH2 screened four bioactive ginsenosides (Rb1, Rd, Rf, Rg3), with Rb1 exhibiting the highest binding affinity (KD=5.02×10 -5 M). Furthermore, molecular docking and enzyme activity assays confirmed that Rb1 directly binds to MDH2 and inhibits its catalytic activity in a dose-dependent manner. This strategy offers a robust platform to link complex extracts with their molecular targets, providing critical mechanistic insights to drive the evidence-based design of nutraceuticals and functional foods. • NPs-chip-ligand fishing strategy developed for food natural products • The strategy simultaneously identifies targets and active compounds • MDH2 identified as a validated target for ginsenosides against OGD/R injury • Ginsenosides Rb1, Rd, Rf, and Rg3 identified as potent MDH2 inhibitors • Ginsenoside Rb1 directly binds to and inhibits MDH2 (KD=5.02×10 -5 M)