Identification and Comparative Evaluation of Analgesic Components in Toad Venom on Inflammatory Pain.

Objective: Although toad venom has been utilized in traditional Chinese medicine for centuries to manage pain, a systematic evaluation of its material basis against inflammatory pain remains lacking. This study sought to rigorously assess the analgesic efficacy of toad venom extracts and their active constituents, identify the principal bioactive components, and explore their potential underlying mechanisms through the integration of network pharmacology and experimental observation, thereby providing a scientific foundation for its potential therapeutic application. Materials and Methods: Activity-guided fractionation and LC-Q-TOF-MS were employed to identify the key active compounds of toad venom. Analgesic activities of the extracts, fractions, and selected monomers were evaluated using a carrageenan-induced mouse pain model. Network pharmacology was utilized to predict potential therapeutic targets. The predicted mechanisms were subsequently validated in vitro using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and in vivo across distinct murine inflammatory models. Finally, a preliminary toxicological evaluation was conducted to assess in vivo safety. Results: Ethyl acetate and methanol fractions were identified as the primary active extracts. Subsequent screening revealed that Cinobufagin and Resibufogenin exhibited the most prominent analgesic properties among the tested monomers. Focusing on an examined subset of the network pharmacology predictions, in vitro assays indicated that both compounds effectively downregulated the PI3K/AKT/NF-κB signaling axis. Notably, Resibufogenin demonstrated superior anti-inflammatory efficacy and significantly mitigated oxidative stress by restoring cellular antioxidant defenses (GSH and TEAC). In vivo evaluations further showed that Resibufogenin robustly attenuated carrageenan-induced paw edema, xylene-induced ear swelling, and LPS-induced systemic inflammation. Furthermore, therapeutic dosages of Resibufogenin (3 and 10 mg/kg) exhibited no detectable toxicity under the tested conditions without inducing observable pathological lesions in major organs. Conclusion: Through origin screening, bioassay-guided fractionation, and LC-Q-TOF-MS analysis, Cinobufagin and Resibufogenin were identified as the principal analgesic constituents of toad venom. Integrated bioinformatic and experimental analyses suggest that their efficacy is associated with anti-inflammatory mechanisms. Notably, Resibufogenin, exhibiting no detectable toxicity under the tested conditions and alleviated inflammatory pain, which may involve the suppression of the PI3K/AKT/NF-κB cascade and alleviating oxidative stress, highlighting its potential as a promising lead compound for novel analgesic therapeutics.