Anti-inflammatory effects of water-soluble polysaccharides from Annona squamosa L. pulp in LPS-induced zebrafish embryos: Network pharmacology-guided analysis and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Annona squamosa L. (custard apple) is widely used in traditional folk medicine across tropical regions for treating inflammatory conditions and metabolic disorders. However, the antioxidant and anti-inflammatory potential of water-soluble polysaccharides from the edible pulp (WPAS) has not been previously investigated.

AIM OF THE STUDY: To evaluate the antioxidant and anti-inflammatory activities of WPAS and to explore its potential mechanisms using integrated computational approaches and zebrafish models.

MATERIALS AND

METHODS

The in vitro antioxidant capacity of WPAS was evaluated using DPPH radical- and hydroxyl radical-scavenging assays. DFT, network pharmacology, molecular docking, molecular dynamics simulation, QSAR, and PASS prediction were employed to characterize representative monosaccharide motifs that served as simplified structural proxies for WPAS and to generate exploratory hypotheses regarding potential interaction networks and inflammation-associated molecular markers. The in vivo anti-inflammatory effects were assessed in an LPS-induced zebrafish embryo inflammation model by evaluating ROS levels, neutrophil recruitment, inflammatory protein expression, and cytokine gene expression.

RESULTS

WPAS exhibited concentration-dependent radical scavenging activity, with IC50 values of 71.84 μg/mL for DPPH radicals and 804.6 μg/mL for hydroxyl radicals, indicating appreciable in vitro antioxidant potential, particularly against DPPH radicals. Monosaccharide-based computational analyses suggested MMP9-related interaction patterns within the in silico framework and indicated possible associations with pathway-related processes, including NF-κB, JAK-STAT, and PI3K-Akt-related processes. In LPS-challenged zebrafish embryos, WPAS exhibited favorable biosafety and significantly attenuated inflammatory responses in a dose-dependent manner, as evidenced by reduced intracellular ROS, decreased neutrophil infiltration, lowered protein levels of TNF-α, NF-κB p65, and MMP9, and downregulated mRNA expression of TNF-α, IL-1β, and IL-6.

CONCLUSION

This study provides the first evidence that WPAS exhibits antioxidant and anti-inflammatory activities. The protective effects observed in zebrafish were associated with reduced ROS production, decreased neutrophil recruitment, and changes in inflammatory marker levels, including NF-κB p65, TNF-α, and MMP9. These findings support the traditional use of Annona squamosa L. and highlight WPAS as a promising natural polysaccharide candidate for further investigation of its antioxidant and anti-inflammatory properties.