Hemostatic Properties of Green Hydrogel Synthesized from Chitosan/Gelatin Polymers and Hydroalcoholic Extract of Juglans Regia L: An In-Silico and In-Vitro Study

Objectives: Quercetin-3-O-rhamnoside, an effective antioxidant, exists in walnut leavesand is supposed to maintain the structural integrity of the Band 3 protein of red bloodcells (RBCs). In this context, an attempt was made to explore the in-silico and in-vitrohemostatic effects of chitosan/gelatin hydrogels containing hydro-alcoholic extract ofIranian walnut leaves (Juglans regia L.), rich in quercetin-3-O-rhamnoside. Methods: In-silico molecular docking predicted the potential interaction betweenquercetin-3-O-rhamnoside and Band 3 protein. Hydroalcoholic extraction (1:1 water:70% ethanol) of walnut leaves was performed in a Soxhlet extractor. The hemostaticactivity from various extract concentrations (0%, 2.5%, 5%, 10%, and 20% v/v) wasassayed using the prothrombin time test on citrated blood samples. Gelatin/Chitosanhydrogels (3:1 and 2:1) loaded with the hemostatic concentrations were prepared bythe casting method, and their adhesion ability to RBCs was tested by measuring theconcentration of free hemoglobin in comparison with controls such as sterile gauze andfree-hydrogel positive controls. Results: Quercetin-3-O-rhamnoside exhibited a high affinity for Band 3 protein,presenting a binding energy of -8.39 kcal/mol compared to the standard ligand 4KUwith -7.11 kcal/mol. The nine amino acids found to be important in this interactioninclude THR728, SER465, VAL729, ARG730, ILE531, LYS851, ILE528, PHE532, andPHE792. All hydrogels incorporating 2.5% v/v of extract could significantly enhanceRBC adhesion controls (p < 0.05). Conclusion: The hydroalcoholic extract from Juglans regia L. may be of value as anantioxidant and hemostatic agent. Quercetin-3-O-rhamnoside is suggested to be extractedfrom walnut leaves and directly incorporated into the hydrogel, and its hemostatic andantioxidant properties can be investigated at both in vitro and in vivo levels