Hempseed and hempseed protein extract: antioxidant potential, peptidomic analysis and muscle cell protection under heat stress conditions

The progressive intensification of climate change has led to a marked rise in global temperatures, raising critical concerns about heat stress (HS) and its detrimental effects on both human and animal health. Among the most affected tissues, skeletal muscle is particularly vulnerable due to its high metabolic demand, underscoring the need for strategies that enhance cellular resilience. Nutrition has emerged as a key area of investigation in this context. Hemp (Cannabis sativa L.), although still underexplored, has attracted scientific interest for its rich functional profile. This study investigated the functional properties of two hemp-based products, hempseed (HSD) and HSD protein extract, by assessing their total phenolic content (TPC), antioxidant activity (FRAP and ABTS assays), and angiotensin-converting enzyme inhibitory (ACE-I) potential following in vitro digestion. In parallel, peptide profiling was performed using nano-LC-MS/MS, with peptide annotation through the SATPdb and DFBP databases. The resulting digestates were then applied to murine C2C12 myoblasts under both standard culture conditions and HS conditions (41.5°C for 3 h). Cell viability was assessed using the Alamar Blue assay. Both HSD and its protein extract showed promising functional properties, as confirmed by peptidomic analysis, which identified 1273 peptides in HSD and over 1781 in the protein extract. Many of these peptides exhibited known antioxidant or ACE-I bioactivities. In cell-based assays, both digested matrices supported C2C12 cell viability under standard conditions at specific concentrations. However, under HS, only HSD at 0.69 and 0.35 mg/mL was able to preserve cell viability, significantly preventing the decline observed in untreated controls. This protective effect was not observed with the protein extract and is likely attributable to the lipid fraction of whole HSD—particularly omega-3 and omega-6 polyunsaturated fatty acids and tocopherols—which are known modulators of oxidative stress and inflammation. These results support its potential role as a functional dietary ingredient capable of enhancing muscle cell resilience to HS. This study underscores the value of sustainable, plant-based resources such as HSD in the development of nutritional strategies aimed at mitigating the physiological impacts of climate change.