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, as an effective antioxidant, exists in walnut leaves and is supposed to maintain the structural integrity of the band 3 protein of red blood cell (rbcs). in this context, an attempt was made to explore in-silico and in-vitro hemostatic effects of chitosan/gelatin hydrogels containing hydro-alcoholic extract of iranian walnut leaves (juglans regia l.), rich in quercetin-3-o-rhamnoside.methods: in-silico molecular docking predicted the potential interaction between quercetin-3-o-rhamnoside and band 3 protein. hydroalcoholic extraction (1:1 water: 70%ethanol) of walnut leaves was performed in a soxhlet extractor. the hemostatic activity from various extract concentrations (0%, 2.5%, 5%, 10%, and 20% v/v) was assayed using prothrombin time test on citrated blood samples. gelatin/chitosan hydrogels (3:1 and 2:1) loaded with the hemostatic concentrations were prepared by the casting method, and their adhesion ability to rbcs was tested by measuring the concentration of free hemoglobin in comparison with controls such as sterile gauze and free-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 4ku with -7.11 kcal/mol. the nine amino acids found to be important in this interaction include thr728, ser465, val729, arg730, ile531, lys851, ile528, phe532, and phe792. all hydrogels incorporating with 2.5% v/v of extract could significantly enhance rbc adhesion controls (p < 0.05).conclusion: the hydroalcoholic extract from juglans regia l. may be of value as an antioxidant and hemostatic agent. quercetin-3-o-rhamnoside is suggested to be extracted from walnut leaves, and directly incorporated into the hydrogel, and its hemostatic and antioxidant properties can be investigated at both in vitro and in vivo levels.