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

Hempseed and hempseed protein extract: antioxidant potential, peptidomic analysis and muscle cell protection under heat stress conditions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Hempseed and hempseed protein extract: antioxidant potential, peptidomic analysis and muscle cell protection under heat stress conditions Davide Lanzoni, Elena Petrosillo, Francesca Grassi Scalvini, Joshua Grana, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6511890/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract 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. Biological sciences/Biotechnology/Proteomics Biological sciences/Biotechnology Biological sciences/Biochemistry/Peptides Biological sciences/Cell biology/Cell growth Antioxidant Activity Digestion Heat Stress Muscle Cell Hempseed Peptidomic Figures Figure 1 Figure 2 Figure 3 Introduction Climate change is causing global temperatures to rise rapidly, with significant consequences for human health. According to Climate Copernicus [ 1 ], 2023 was the warmest year recorded since 1850. In just twelve months, between February 2023 and January 2024, the average global temperature exceeded pre-industrial levels by 1.5°C, highlighting the gravity of the climate crisis [ 2 ]. This increase in temperatures not only affects the environment, but also has a direct impact on human physiological well-being, increasing the risk of heat stress (HS) [ 2 ]. Body temperature is a crucial physical property that influences the structure and function of biological tissues [ 3 ]. Humans, as homeothermic organisms, maintain a constant tissue temperature, generally between 36–38°C under resting conditions [ 3 ]. This balance is essential to ensure normal physiological functioning. However, as pointed out by Rhoads et al. [ 4 ] and Cramer et al. [ 3 ], exposure to high temperatures can cause significant alterations and damage at the cellular level. Intracellular molecular structures, stabilised by relatively weak interactions, are particularly sensitive to changes in the microenvironment, such as changes in temperature or pH. Heat can alter plasma membrane fluidity, transmembrane transport rates and the three-dimensional configuration of proteins, interfering with their synthesis to the point of causing cell death [ 3 – 5 ]. Of all biological tissues, muscle tissue, being highly metabolically active, is particularly vulnerable. Muscle tissue is not only susceptible to HS caused by external environmental factors that raise body temperature, but also to HS induced by physical exertion, specifically resulting from intense exercise [ 6 ]. This dual exposure makes muscles particularly susceptible to temperature-related damage, underlining the need for preventive measures under conditions of HS. One of the most effective strategies to mitigate the effects of HS is represented by nutrition [ 4 ]. In this context, hemp ( Cannabis sativa L.) emerges as a promising solution due to its dual role. On the one hand, its cultivation requires a reduced amount of water and pesticides and is actively involved in carbon sequestration, thus contributing to reducing the ecological footprint and potentially by mitigating the increase in global temperatures. On the other, it can offer products with a high nutritional and functional profile [ 7 , 8 ]. Of these, hempseed, previously recognised as a processing waste product, is the best known. It is characterised by a carbohydrate content of 20–30 g/100 g (mostly dietary fibre), 25–35 g/100 g lipids with a balanced fatty acid composition and 20–25 g/100 g protein easy to digest (85.2%), featured by an interesting peptidomic profile [ 7 , 9 ]. In parallel, from a functional point of view, hempseeds are distinguished by a high antioxidant profile [ 8 ]. This latter aspect, in the context of HS, represents a crucial point, as HS stimulates the generation of reactive oxygen species (ROS), known for their pro-oxidant effect, which lead to the chemical modification and destruction of molecules due to the development of hypoxia, which accompanies almost all tissue damage [ 10 ]. Although hemp-based products are known for their interesting functional profile, their potential role in mitigating HS on muscle tissue has not yet been fully explored in the scientific literature. This study aims to fill this gap by focusing on several key analyses. The objective was to evaluate the antioxidant profile of two hemp-based products, namely hempseed, which we will refer to in the case of the sample under investigation with HSD, and the HSD protein extract after in vitro digestion process. Following the measurement of total phenolic content (TPC), two established assays were used for this evaluation: the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and the ferric reducing antioxidant power (FRAP) assays. In parallel, the inhibitory activity of angiotensin-converting enzyme-I (ACE-I), a parameter closely linked to the regulation of oxidative stress and tissue protection, was investigated. Results obtained were simultaneously correlated with the presence of bioactive peptides released following in vitro digestion. Finally, the digests were tested on a murine muscle cell line (C2C12) exposed to experimental HS condition. This step is a critical component of the study, as it enables the evaluation of the potential of hemp-based products to maintain and safeguard muscle cell function when exposed to HS conditions. Results Functional profile evaluation of HSD and HSD protein extract after in vitro digestion The results related to the evaluation of functional activity after in vitro digestion are shown in Table 1. Table 1 . Functional activity evaluation (TPC, ABTS, FRAP and ACE-I assay). Values are represented as mean ± SEM. Different superscript letters indicate statistically significant ( p < 0.05 ) differences between the two samples for the same assay. TPC Total Phenolic Content; FRAP: Ferric Reducing Antioxidant Power; ACE-I: Angiotensin Converting Enzyme-I; TE: Trolox Equivalent; HSD: Hempseed. HSD HSD protein extract TPC (mg TAE/100 g) 231.86 ± 16.68 a 509.25 ± 18.68 b ABTS (mg TE/100 g) 2477.87 ± 406.09 2815.76 ± 450.52 FRAP (mg FeSO 4 /100 g) 11.71 ± 0.58 a 15.36 ± 0.18 b ACE-I (%) 11.06 ± 1.66 a 26.73 ± 2.09 b As shown in Table 1, the TPC exhibited statistically significant differences ( p < 0.05 ) between the HSD protein extract (509.25 ± 18.68 mg TAE/100 g) and HSD (231.68 ± 16.68 mg TAE/100 g). This result was corroborated by the FRAP assay, with significantly higher values observed for the HSP protein extract (15.37 ± 0.18 mg FeSO₄/g) compared to HSD (11.71 ± 0.58 mg FeSO₄/g). Similarly, although not statistically significant, a similar trend was observed for the ABTS assay. Finally, ACE-I activity was significantly higher ( p < 0.05 ) for the HSD protein extract (26.73 ± 2.09%) compared to HSD (11.06 ± 1.66%). Peptidomic profile of HSD and HSD protein extract after in vitro digestion The peptidomic profiles of HSD and HSD protein extract after digestion and filtration were obtained by means of a shotgun label free approach which allowed the identification of 1273 peptides in HSD and 1781 peptides in HSD protein extract, respectively. The lists of all the peptides identified with high confidence are reported in Supplementary Table 1 and 2, respectively. All the peptides were searched in DFBP, a database of bioactive peptides from food sources and in SATPdb, a database of structurally annotated therapeutic peptides. The peptides identified as bioactive according to these two data bases were classified by category as shown in Table 2 and Supplementary Table 3, that report the number of the bioactive peptides identified in HSD and HSD protein extract (Table 2) and the sequence of the bioactive peptides listed in SATPbd and DFBP databases identified in HSD and HSD protein extract data sets (Supplementary Table 3). Table 2. Number of bioactive peptides identified in HSD and HSD protein extract The Table reports the number of peptides identified as bioactive following the search in SATPbd and DFBP data bases of all the peptides identified in HSD and HSD protein extract. SATPdb DFBP BIOACTIVE PEPTIDES common peptides in HSD and HSD protein extract (165) peptides exclusively in HSD protein extract (1616) peptides exclusively in HSD (1108) common peptides in HSD and HSD protein extract (165) peptides exclusively in HSD protein extract (1616) peptides exclusively in HSD (1108) Antioxidant 2 19 18 3 28 24 ACE-inhibitor 1 36 12 11 64 37 Antibacterial 3 1 Antihypertensive 8 53 37 1 9 3 Anticancer 1 2 Antifungal 3 2 Antimicrobial 3 5 1 Antiparasitic 1 Antiviral 7 3 1 3 Cell-cell comunication 1 1 Drug delivery vehicle 4 Toxic 2 1 Drug deliv vehicle 1 2 DPP IV-inhibitory 2 11 10 Immunomodulatory 1 Opioid 1 PEP-inhibitory 3 1 Mineral-binding 1 Neuropeptides 2 Celiac disease 1 4 3 Renin inhibitory 1 Antithrombotic 5 6 TOTAL 12 110 72 19 128 93 Cell viability and evaluation of digests in HS mitigation As described in material and methods, for HSD and HSD protein extract, the freeze-dried samples were resuspended in 15 mL of standard medium, reaching final concentrations of 41.1 and 48.4 mg/mL, respectively. However, the assessment of cell viability with Alamar Blue, was conducted using concentrations of 10.28 mg/mL for HSD and 12.10 mg/mL for HSD protein extract, values characterised by a non-toxic pH for the cells. Figure 1 As illustrated in Figure 1(a), for HSDs, concentrations of 10.28 and 5.14 mg/mL resulted in a significant ( p < 0.05 ) reduction in cell viability (63.48 ± 7.38% and 74.91 ± 4.98%, respectively) compared to the positive control (10% FBS, 118.80 ± 5.79%). In contrast, the lower concentrations of 0.17, 0.09, 0.05 and 0.02 mg/mL showed no significant differences compared to the positive control. The best results were obtained with the concentrations of 2.77, 1.39, 0.69 and 0.35 mg/mL which produced a significant ( p < 0.05 ) increase in cell viability (180.05 ± 4.58%, 173.37 ± 6.60%, 149.77 ± 10.04% and 133.43 ± 6.40 % respectively) compared to 10% FBS. For HSD protein extracts (Figure 1b), the concentrations of 12.10, 6.05, 0.19, 0.09, 0.05 and 0.02 mg/mL showed no differences compared to 10% FBS. In parallel, the concentrations of 3.0 mg/mL (185.80 ± 11.79%), 1.50 mg/mL (177.80 ± 11.11%), 0.75 mg/mL (165.66 ± 9.01%) and 0.3 mg/mL (153.32 ± 7.20%) significantly ( p < 0.05) increased viability compared to the positive control (111.78 ± 4.83%). On the basis of the reported data, the following concentrations were selected for the evaluation of HS mitigation: 2.77, 1.39, 0.69 and 0.35 mg/mL for HSD, and 3.00, 1.50, 0.75 and 0.38 mg/mL for the HSD protein extract. As previously described and depicted in Figure 2, HS was evaluated for 3 h at 41.5 °C. Figure 2 Analysis of cell viability using the Alamar Blue test showed a significantly increasing reduction with a rising incubation time at 41.5 °C. In particular, the increase from 1 h (88.22 ± 1.99 %, p < 0.05 ) to 2 h (87.71 ± 1.20 %, p < 0.01 ) and then to 3 h (79.64 ± 1.92 %, p < 0.001 ) of exposure resulted in a significant decrease in viability compared to the optimal culture conditions observed in the control group at 37 °C. Figure 3 shows the effects of the selected concentrations on HS mitigation. For HSD protein extract (Fig. 3b), none of the tested concentrations were able to counteract the toxic effect of HS on cell viability, showing statistically significant differences between cells grown at 37 °C and those at 41.5 °C. In contrast, different and promising results were observed for HSD (Fig. 3a). Although higher concentrations, such as 2.77 and 1.39 mg/mL, did not ensure the maintenance of cell viability compared to the 37 °C condition, the concentrations of 0.69 mg/mL (130.75 ± 8.28%) and 0.35 mg/mL (132.14 ± 8. 09%) tested at 41.5 °C showed no statistically significant differences from the control condition at 37 °C (149.77 ± 10.04% and 133.43 ± 6.40%, respectively), highlighting their ability to preserve cell viability. Discussion Functional profile of HSD and HSD protein extract after in vitro digestion Interest in nutraceuticals continues to grow, fuelled by significant advances in research aimed at identifying the properties and potential applications of functional products, as well as by increasing consumer interest in safe and increasingly healthy food [ 11 ]. In this context, hemp could be a valid solution. For this reason, two hemp-based products were considered in this study, HSD and HSD protein extract, partially characterised in our previous work [ 8 ]. From a nutritional point of view, both matrices presented a very interesting chemical profile. In particular, they showed a protein content of 23.1 ± 0.57 and 45.1 ± 0.84 g/100 g, and a lipid content of 27.9 ± 0.75 and 9.2 ± 0.09 g/100 g for HSD and HSD protein extract, respectively, suggesting their application in food and feed sector. In parallel, also the TPC (Table 1 ) showed interesting levels. Surprisingly, but in agreement with our previous work [ 8 ], the HSD protein extract showed a significantly higher absolute phenolic value than HSD after the digestion process. This result can be explained by the strong interaction between the phenolic compounds and the fibre content [ 12 ]. Indeed, the fibre is able to trap phenols via hydrophobic interactions, hydrogen bonds (between the oxygen atoms of the glycosidic chains of the polysaccharides and the hydroxyl groups of the phenolic compounds) and covalent bonds, which are particularly strong in the acid-detergent fibre (ADF) and in the acid-detergent lignin (ADL), which represent the main localisation regions of the phenolic compounds. Although, as reported by Farinon et al. [ 7 ], phenolic compounds mainly localise at the hull level, it is plausible to assume that the lower content of ADF (20.2 ± 1.02 g/100 g) and ADL (9.5 ± 0.62 g/100 g) present in HSD protein extract compared to HSD (33.2 ± 0.31, 14.4 ± 0.32 g/100, respectively for ADF and ADL) may favour a higher bioavailability of phenolic compounds in the HSD protein extract during the digestive process, resulting in higher values (Table 1 ). This trend is highly correlated with antioxidant activity. ABTS method reported high values for both samples. The values observed for HSD (2386.57 ± 117.90 mg TE/100 g) and HSD protein extract (2815.7 6 ± 450.52 mg TE/100 g) are overlapping (2477.87 ± 406.09; 3936.79 ± 59.29 mg TE/100 g, respectively) with what was observed in our previous work [ 8 ], despite the application of different digestion models. This decrease in recorded antioxidant activity, observed also for TPC, is attributable to two main causes. The first factor concerns differences in in vitro digestion protocols. As pointed out by Giromini et al. [ 13 ], the use of different enzymes, with varying concentrations and specificities, can significantly affect the extent of protein hydrolysis and, consequently, the amount of bioactive peptides released. At the same time, it is conceivable that the filtration process (≤ 10 kDa) may have excluded larger peptides or bioactive compounds that are actively involved in modulating the antioxidant profile. The above is also applicable for the values obtained in the FRAP assay. Despite this, HSD and the HSD protein extract proved to be matrices characterised by a high antioxidant value. This is related to both the presence of phenolic compounds and the interesting peptidomic profile shown in Table 1 . More precisely, several works in the literature have demonstrated a high correlation between TPC and antioxidant activity, not only in hemp-based products, but also in different plant matrices, where the increase in the concentration of phenolic compounds at different stages of digestion showed a correlation with increased antioxidant activity, in line with the results obtained in this study [ 14 – 16 ]. At the same time, although as reported by Tang et al. [ 17 ], Wang et al. [ 18 ] and Malomo et al. [ 19 ], the native structure of hempseed proteins is characterised by low functionality, the hydrolysis process results in the formation of bioactive peptides, generally with a molecular weight of less than 10 kDa (molecular weight chosen in this study to study bioactivity), which increases the functional value of hemp seed proteins. More specifically, as reported by Tang et al. [ 17 ], Wang et al. [ 18 ] and Malomo et al. [ 19 ], the peptides resulting from hydrolysis are characterised by a high antioxidant value, in particular Fe scavenging and chelation, mechanisms of action of the ABTS and FRAP assays, respectively; confirming the peptidomic profile of Table 2 and the results of Table 1 [ 17 – 19 ]. However, it is important to emphasise, that the concentration at which these hydrolysates exert their functional effects strongly depends on the specific hydrolysis conditions employed. This underlines how different hydrolysis methods produce peptides with distinct structural and functional properties, thus influencing their biological activity [ 7 ]. The functional evaluation, as illustrated in Table 1 , included the analysis of ACE-I activity, a crucial parameter in determining the functionality of a food-derived product. As reported by Pfeffer [ 20 ], ACE-I plays a key role in the regulation of blood pressure and the pathogenesis of hypertension. ACE-I catalyses the conversion of angiotensin-I, an inactive precursor, into angiotensin-II, a potent vasoconstrictor agent, while inactivating bradykinin, a peptide with vasodilatory action [ 21 ]. For these reasons, ACE-I are frequently used in the treatment of myocardial infarction [ 22 ], hypertension [ 23 ] and other related cardiovascular diseases, which to date, as reported by Li et al. [ 24 ], afflict around 20% of the world's population. Consequently, scientific research is actively investigating food matrices with such functional characteristics, given their potential benefits and applications [ 25 – 27 ]. Among these, HSD and HDS protein extract showed promising results, with ACE-I values comparable to those of other hemp-based products, in particular leaves and roots (18%) [ 28 ]. As shown by Vermeirssen et al. [ 29 ] and Lanzoni et al. [ 30 ], ACE-I activity tends to increase significantly following the digestive process. This rise is attributable to the disruption of the proteins' native structure, which leads to the release of bioactive peptides capable of modulating this functional activity. Nevertheless, comparing data with those found in the literature is complex and sometimes speculative due to the many variables that influence the results. Among these, the chemical composition of the sample, the degree of purity, the hydrolysis methods adopted and the exposure time to enzymes play a crucial role. As reported by Segura Campos et al. [ 31 ], low-molecular-weight peptides derived from enzymatic hydrolysis show a higher inhibitory activity on ACE-I than those with a high molecular weight, highlighting how peptide size is a determining factor. This relationship was further confirmed by Teh et al. [ 32 ], who showed that the type of extraction applied to hempseed protein hydrolysates, acidic or alkaline, can alter the protein conformation and, consequently, the inhibitory efficacy on ACE-I. In particular, alkaline hydrolysis produced significantly better results, reaching a maximum inhibition of 70.61 ± 0.06%. Nevertheless, as shown in Table 2 , the samples revealed a high ACE-I peptidomic profile, particularly for the HSD protein extract, confirming the results in Table 1 . These data highlight the considerable potential of hemp-based products as functional matrices with ACE-I properties, reinforcing their role in the prevention and management of hypertension. As reported by Israili et al. [ 33 ], despite significant advances in the prevention, diagnosis and treatment of hypertension, this condition remains a major public health challenge. Indeed, hypertension is associated with an increased risk of mortality and morbidity from stroke, coronary artery disease, congestive heart failure and end-stage renal disease. For this reason, the high presence of anti-hypertensive bioactive peptides (Table 2 ) from HSD and HSD protein extract, also confirmed by Teh et al. [ 32 ] and Malomo et al. [ 19 ], is an important solution for the treatment of this disorder. Effect of digested HSD and HSD protein extract on C2C12 cell viability The above data effectively illustrate the functional aspect of the two hemp-based products. After the digestive process, the gastrointestinal tract is certainly the first target organ of HSD and HSD protein extract. As pointed out by Leonard et al. [ 34 ], certain hempseed fractions showed positive effects on intestinal tissue. Similar benefits have also been observed in the cardiovascular and neurological systems [ 35 , 36 ]. Although the fundamental link between nutrition and muscle tissue is widely documented [ 37 – 39 ], to our knowledge there are no studies exploring the effects of digested hemp-based products on muscle tissue. Therefore, we decided to investigate this further using murine C2C12 muscle cells, a widely used model recognised for its ability to reflect human physiology [ 40 ]. As illustrated in Fig. 1 , both HSD and HSD protein extract showed a similar trend. The lower concentrations did not produce significant effects compared to the control, probably due to an insufficient amount to promote an increase in cell viability. On the other hand, although the higher concentrations of HSD protein extract showed no difference to the control, in the case of HSD, the concentrations of 10.28 and 5.14 mg/mL resulted in a significant reduction in viability, as previously observed. This difference is most probably attributable to their composition. Whereas HSD protein extract is a processed, pure product, and intended for direct human consumption, HSD represents a raw, unpurified product containing all its natural components, including anti-nutritional factors (AF), known to be present in high amounts in hempseeds [ 7 ]. Therefore, it is reasonable to assume that high AF concentrations negatively affected cell viability. This effect would not occur directly, but would act by limiting the uptake by cells of essential nutrients, such as nitrogen compounds and minerals, that are crucial for maintaining cell viability [ 7 , 41 – 43 ]. Among the AFs within hempseeds, those most present, although highly comparable with those of other oil-seeds, are the condensed tannins, trypsin inhibitors and phytic acid [ 7 ]. In particular, condensed tannins are phenolic compounds that can form insoluble complexes with proteins and minerals, negatively affecting their availability [ 44 ]. Trypsin inhibitors impair the digestibility of dietary proteins and the availability of essential amino acids. These compounds show remarkable stability in the gastric environment, resisting both the action of pepsin and acidic pH conditions. In addition, they inhibit the activity of intestinal digestive enzymes, thus limiting the absorption of nitrogen compounds essential for cell viability [ 45 ]. Similarly, phytic acid, due to its peculiar molecular structure, is able to bind to multivalent cations and positively charged proteins, forming insoluble complexes known as phytates. These complexes, including phytate-minerals and phytate-proteins, cannot be digested and adversely affect the bioavailability of dietary minerals and proteins, reducing nutritional efficiency [ 7 ]. As shown in Fig. 1 , and as previously described, successive concentrations up to 0.35 and 0.38 mg/mL for HSD and HSD protein extract, respectively, supported cell viability. Of the factors involved, it is presumed that cell viability was supported by fundamental factors such as nitrogen source, glucose supply (essential for energy metabolism) and micro-nutrients. The nitrogen source is a key element for cell metabolism and protein synthesis [ 46 ]. As reported by Wang and Zheng [ 47 ], cells need a readily available nitrogen source to effectively support anabolic processes. Due to their high digestibility, hemp-derived proteins are particularly suitable for this purpose. Indeed, the digestive process generates peptides and amino acids that are easily assimilated by cells. This aspect was confirmed by Mamone et al. [ 48 ], who highlighted the presence of a reduced number of complex peptides in the digestion products of hemp-based proteins. Consequently, it can be hypothesised that this highly bioavailable nitrogen source contributes to stimulating cell growth by providing the necessary substrates for protein synthesis and supporting the metabolic processes essential for cell proliferation. At the muscular level, in addition to availability, it is crucial to consider the quality of the nitrogen source [ 49 ]. In particular, essential amino acids are the most effective nutrients for stimulating muscle protein synthesis, although the molecular mechanisms responsible for this effect are still being investigated. In addition to acting as essential precursors for protein synthesis, essential amino acids appear to exert a direct effect in promoting synthesis processes [ 49 ]. Among these, a crucial role is played branched-chain amino acids, which act not only as major transporters of amine nitrogen between the viscera and peripheral tissues, including skeletal muscle, but also as direct stimulators of muscle protein synthesis. Among the branched-chain amino acids, leucine emerges as the main bioactive amino acid, distinguished by its ability to activate anabolic muscle processes, making it a crucial element in maintaining and promoting muscle mass [ 49 ]. In this context, HSD and HSD protein extract may have contributed to the supply of essential amino acids. As reported by Callaway [ 50 ], House et al. [ 9 ], Mattila et al. [ 51 ], and Oseyko et al. [ 52 ], both raw hempseed and purified hempseed proteins are notable for containing all essential amino acids, with leucine being particularly abundant, ranking just after arginine, asparagine, and glutamine. This compositional profile supports the hypothesis that hemp-derived protein can provide a nutritionally valuable source of essential amino acids, particularly leucine, to stimulate muscle protein synthesis and support anabolic processes. In parallel, cell viability may have been supported by an energy supply in the form of free monosaccharides. The importance of glucose in muscle tissue was highlighted by Carbone et al. [ 46 ], who showed that glucose plays a crucial role in supporting the metabolic functions of muscle cells. Furthermore, as described by Nedachi et al. [ 53 ], the growth and differentiation of C2C12 muscle cells are significantly compromised when they are cultured under low-glucose conditions, suggesting that an adequate amount of glucose is necessary for the proper development and maintenance of cell viability. In this context, hempseeds could represent also a valuable source of carbohydrates, as reported by Wei et al. [ 54 ], who showed a particularly interesting carbohydrate profile, characterised by high concentrations of glucose and arabinose. Therefore, it is plausible to assume that the monosaccharides released during the digestive process were utilised by the cells as an energy source, contributing to their metabolism and, consequently, to improve cell viability. In the assessment of muscle cell viability, minerals such as magnesium (Mg), phosphorus (P), zinc (Zn) and iron (Fe) play essential roles. Mg constitutes approximately 27% of the mineral content in muscles and is the predominant cation within cells, preceded only by potassium [ 55 ]. This mineral is crucial for the functioning of numerous enzymes, neuromuscular transmission and support of muscle contraction, also aiding post-exercise recovery. Furthermore, as reported by the National Research Council [ 56 ], Mg has a stabilising function on DNA and facilitates protein synthesis, thus contributing to the maintenance of muscle mass. Zn, involved in protein synthesis and tissue repair, is crucial for the proliferation of muscle cells, which are responsible for muscle growth and recovery [ 57 ]. P, as a key component of ATP, is essential for energy metabolism and the synthesis of crucial molecules, promoting muscle function [ 58 ]. Fe, essential for oxygen transport and mitochondrial activity, is involved in the production of energy required for muscle contractions, and its deficiency impairs energy efficiency and muscle capacity [ 59 ]. In this context, the minerals mentioned above are crucial for muscle health, facilitating tissue growth, and optimising overall muscle function. An aspect of fundamental importance to this study concerns the mineral content of hempseeds. Considering EFSA's Dietary Reference Value [ 60 ], hempseed is confirmed as an excellent natural source of Mg, P, Zn and Fe. In particular, the content of P, the most abundant mineral in hempseeds, is higher than flax (Linum usitatissimum) seeds, which are widely used in the human diet [ 7 ]. In light of these considerations, it is reasonable to speculate that the release of essential minerals during the digestive process may have promoted their uptake by muscle cells, thereby optimising their functionality and contributing to improved cellular vitality. Minerals may have been utilised by muscle cells to support fundamental processes such as protein synthesis, tissue repair, energy metabolism and muscle satellite cell proliferation. Heat Stress evaluation on C2C12 cell viability As previously discussed, the main objective of the present study was to evaluate the potential protective effect of HSD and HSD protein extract against HS. Understanding the physiological and cellular impacts of HS is a critical area of research in today's scientific landscape, given that global warming poses one of the most pressing and multifaceted environmental threats worldwide [ 61 ]. Human-induced climate changes have a profound influence on the physiological and perceptual responses of living organisms, affecting them in both direct and indirect ways, including alterations in the regulation of core body temperature, heart rate, skin temperature, and thermal comfort [ 62 ]. Such changes can significantly compromise the maintenance of homeostasis, exacerbating physiological vulnerabilities and reducing the ability of organisms to adapt to changing environmental conditions [ 3 – 5 ]. Exposure to 41.5°C, as shown in Fig. 2 , resulted in a significant reduction in cell viability, with the effect being directly proportional to exposure time. These results are consistent with those reported by Park et al. [ 63 ] and Summers and Valentine [ 64 ]. The decrease in cell viability can be traced to several molecular mechanisms. In particular, HS is known to induce a marked apoptotic response, as highlighted by Gao et al. [ 65 ]. Furthermore, as described by Mylostyva et al. [ 10 ], HS promotes the generation of ROS, which cause oxidative damage to essential biomolecules, including proteins, lipids and nucleic acids. This effect is particularly damaging to mitochondrial proteins. In particular, as noted by Belhadj-Slimen et al. [ 66 ], oxidation-induced damage to mitochondrial proteins includes the subunits of the pyruvate decarboxylase complex, ATP synthase and tricarboxylic acid cycle enzymes, all of which are essential components in the production of cellular energy, thus representing an important factor in the reduction of cell viability. However, the main mechanism behind this impairment seems to be related to the effects of high temperatures on protein folding. Under these conditions, misfolding, denaturation and aggregation of proteins occur, severely affecting cellular homeostasis [ 67 ]. In response to HS, cells activate an adaptation strategy characterised by the rapid synthesis of heat shock proteins (HSPs). These proteins, which act as molecular chaperones, play a central role in cellular protection. Heat shock proteins participate in critical processes such as repair of damaged proteins, proper protein folding and prevention of stress-induced protein aggregation. In skeletal muscle cells, HSPs are critical for maintaining homeostasis and mitigating damage caused by environmental stresses, helping to preserve the structural and functional integrity of tissues [ 67 ]. However, although HSPs are crucial for restoring cellular homeostasis, it is important to emphasise, as pointed out by Pechan [ 68 ], that under stress conditions, cells prioritises the production of stress-related proteins over those necessary for cell proliferation, which in turn results in a reduction in cell viability and overall cellular health. In parallel, molecular chaperones influence various cellular components, including the cytoskeleton [ 69 ]. This phenomenon has been extensively described by Coss and Linnemans [ 70 ], who reported how hyperthermia can destabilise the cytoskeleton, impairing its form and function, with negative effects on cell adhesion and proliferation. The above could provide an explanation for the reduction in cell viability observed in this study, attributable to the effects of the HS. Evaluation of HSD and HSP protein extract digest mitigation on HS Given the significant implications for human health, there is growing interest in strategic diet modification in an attempt to improve the HS response [ 4 ]. Surprisingly, no concentration evaluated for HSD protein extract allowed viability to be sustained at 41.5°C (Fig. 3 ). In contrast, for HSD, only the lower concentrations (0.69 and 0.35 mg/mL) were able to contain the negative effects on viability by HS, in contrast to the higher concentrations (2.77 and 1.39 mg/mL). This result could be related to the phenomenon of hormesis . The term hormesis is used to describe a biphasic dose-effect response, characterised by a stimulation or beneficial effect at low doses and an inhibitory or toxic effect at high doses [ 71 ]. This principle is extensively documented in the dose-response assessment of food-derived bioactive compounds [ 72 ]. Therefore, in this study, it is plausible to hypothesise that higher concentrations may act as pro-oxidants, favouring the accumulation of ROS and consequently leading to a reduction in cell viability. This protective effect could be attributed to several factors. However, considering that no protective effect was observed in HSD protein extract, it is reasonable to assume that, in the case of HSD, this protection stems neither from the peptide fraction nor from the phenolic component. In our opinion, these results can mainly be attributed to the higher lipid content of HSD compared to HSD protein extract (27.9 ± 0.75; 9.2 ± 0.09 g/100 g, respectively) [ 8 ]. Notably, as reported in the literature, hempseeds are characterised by a lipid profile rich in Omega-3 and Omega-6 polyunsaturated fatty acids (PUFAs), which are widely recognised for their beneficial effects on general health [ 7 ]. Among the predominant PUFAs in hempseeds, linoleic acid (18:2, n-6, LA) and α-linolenic acid (18:3, n-3, ALA) are the most abundant. These fatty acids are classified as essential, as they must be obtained exclusively through the diet, and serve as precursors for biologically active long-chain PUFAs in animals and humans. Specifically, LA is converted to arachidonic acid (20:4, n-6, AA), while ALA serves as a precursor to eicosapentaenoic acid (20:5, n-3, EPA) and docosahexaenoic acid (22:6, n-3, DHA). 7 In particular, EPA and DHA are actively involved in multiple physiological processes, including the preservation of cell membrane integrity, modulation of anti-inflammatory and anti-oxidant activity [ 7 , 73 , 74 ]. This was also confirmed by Mickleborough [ 75 ]. More specifically, the author observed how the administration of EPA and DHA mitigated the pro-inflammatory effects and ROS production at the muscle level during physical activity; one of the factors involved in HS, as previously reported. At the same time, the lipid fraction of hempseed is characterised by a high concentration of fat-soluble vitamins, including tocopherols, which are the main component [ 76 ]. As highlighted by Montserrat-De La Paz and colleagues [ 76 ], tocopherols are characterised by marked antioxidant activity, mainly attributable to their ability to neutralise free radicals (scavenging) and to counteract ROS, the main mediators of oxidative damage associated with HT syndrome [ 10 ]. The antioxidant efficacy of tocopherols against HS is widely documented in the literature. This effect has been observed both in human health studies, under conditions of oxidative stress caused by intense exercise [ 77 , 78 ] and in studies on animal models exposed to HS [ 79 – 81 ]. Therefore, in light of the above evidence, it is reasonable to assume that the lipid component of HSD contributed significantly to the observed positive effects against HS, compared to the HSD protein extract. In conclusion, both HSD and HSD protein extract, after in vitro digestion, showed a highly promising functional profile, with particular reference to antioxidant and ACE-I activity, results further confirmed by the presence of bioactive peptides identified through peptidomic characterisation. Although both HSD and HSD protein extract showed a good ability to support cell viability under optimal growth conditions, only the lowest concentrations of HSD revealed a protective effect against HS. This could be due to the intrinsic complexity of the matrix considered.’ These results suggest that HSD, beyond their recognised nutritional value, may serve as a functional ingredient capable of increasing cellular resilience under HS conditions. However, further studies are needed to confirm these effects and explore their applicability in dietary strategies aimed at mitigating the impact of HS on muscle tissue and overall physiological health. Materials and methods HSD and HSD protein extract HSDs ( Cannabis sativa L., variety Futura) were purchased from a company in Chrastice (Czech Republic). They were sown in April/May 2021 and harvested when they reached 70% maturity. After harvest, the HSDs were dried at 40°C to reduce the moisture content to 7%, which is essential for proper storage. Cannabinoid content analysis, performed at the Institute of Animal Science of the Czech Republic, reported cannabidiol levels of 30 µg/g and a Δ9-tetrahydrocannabinol (THC) content of 0 µg/g, respecting the legal limits imposed by the European regulation (1370/2013) [ 82 , 83 ]. The protein extract of HSDs was purchased from a commercial supplier. In vitro digestion of HSD and HSD protein extract In vitro digestion was performed following the protocols of Minekus et al. [ 84 ] and Brodkorb et al. [ 85 ]. The reagents and enzymes used were purchased from Sigma Chemical Co. ( St. Louis, MO, USA ). Simulated digestion fluids for oral (SOF), gastric (SGF) and intestinal (SIF) phases were prepared in agreement with Brodkorb and colleagues [ 85 ]. Precisely, 1.0 ± 0.05 g of each sample, previously ground, was diluted in SOF and incubated for 2 min at 37°C with alpha-amylase (75 U mL-1, pH 7.0), under stirring, to simulate the oral phase. At the end, the oral bolus was diluted with SGF and pepsin (2000 U mL-1, pH 3.0), and incubated at 37 ºC for 2 h, under stirring. At the end, the gastric chyme was diluted with SIF and incubated with bile salts (10 mM, pH 7.0) and the pancreatic enzymes (100 U mL -1 , pH 7.0) for 2 h at 37 ºC, under stirring. Once digestion was completed, the digested fraction was separated as reported by Regmi et al. [ 86 ]. Specifically, the undigested fraction was collected in a filtration unit using a porcelain funnel covered with filter paper (Whatman 54 Florham Park, NJ). Subsequently, the filtered component, corresponding to the digested fraction, was ultra-filtered using centrifuge filters (10 kDa) (Pierce™, Protein Concentrator PES, Thermo Fisher Scientific, UK), for high weight protein depletion [ 30 ]. After filtration, the samples were aliquoted and immediately stored at -20 ºC for analysis, as reported below. Evaluation of functional activity of HSD and HSD protein extract Total Phenolic Content of HSD and HSD protein extract after in vitro digestion The TPC was quantified according to the protocol described by Attard [ 87 ]. Reagents, including tannic acid, methanol, Folin–Ciocalteu (FC) reagent, and sodium carbonate (Na₂CO₃), were procured from Sigma Chemical Co. (St. Louis, MO, USA). A standard curve was generated by preparing a series of 1:2 serial dilutions of tannic acid in distilled water, ranging from 480 to 0 µg/mL. The FC reagent was diluted 1:10 with distilled water, and a 1 M solution of Na₂CO₃ was prepared. For the assay, 100 µL of each sample was combined with 500 µL of the diluted FC reagent and 400 µL of the Na₂CO₃ solution. The reaction mixtures were incubated in the dark at room temperature (RT) for 20 min. Following incubation, absorbance was measured at a wavelength of 630 nm using a spectrophotometer. Solvent blanks were included as controls in each assay to account for background absorbance. The TPC was determined based on the standard curve and expressed as tannic acid equivalents (mg TAE/100 g). ABTS Assay of HSD and HSD protein extract after in vitro digestion Reagents including ABTS, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and potassium persulfate (K₂S₂O₈) were procured from Sigma Chemical Co. (St. Louis, MO, USA). The ABTS assay was conducted following the protocol described by Re et al [ 88 ]. Trolox, prepared as a 2.5 mM solution and used as the antioxidant standard, was diluted into six concentrations ranging from 0 to 2000 µM/mL. To generate the ABTS radical cation (ABTS⁺), 88 µL of 2.45 mM K₂S₂O₈ were added to 5 mL of 7 mM ABTS solution. This mixture was incubated in the dark at RT for 16 h before use. For antioxidant activity measurements, the ABTS⁺ solution was diluted with ethanol to achieve an absorbance of 0.70 ± 0.02 at 734 nm. Subsequently, 10 µL of each sample was combined with 1 mL of the diluted ABTS⁺ solution and incubated in the dark at RT for 6 min. After incubation, the absorbance of the reaction mixture was recorded at 734 nm using a spectrophotometer. Solvent blanks were included for accuracy in all analyses. Antioxidant activity was expressed as Trolox Equivalents (mg TE/100 g). FRAP Assay of HSD and HSD protein extract after in vitro digestion Reagents used for the FRAP assay, including ferric chloride hexahydrate [FeCl₃·6H₂O], ferrous sulfate (FeSO₄), sodium acetate trihydrate, glacial acetic acid, and 2,4,6-tripyridyl-s-triazine (TPTZ), were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The assay was conducted based on the method described by Abdelalem and Elbassiony [ 89 ] with minor modifications [ 30 ]. The reagents were prepared as follows: a) Acetate buffer (300 mM, pH 3.6): 2.69 g of sodium acetate trihydrate were dissolved in 16 mL of glacial acetic acid and diluted to 1 L with distilled water. b) TPTZ solution (10 mM): 31.2 mg of TPTZ were dissolved in 10 mL of 40 mM HCl. c) Ferric chloride solution (20 mM): 54 mg of FeCl₃·6H₂O was dissolved in 10 mL of distilled water. Ferrous sulfate (FeSO₄) was used as the antioxidant standard, prepared in six dilutions ranging from 0 to 1500 µM/L. The FRAP working reagent was prepared by mixing 2.5 mL of TPTZ solution, 2.5 mL of ferric chloride solution and 25 mL of acetate buffer. For the analysis, 10 µL of each sample were added to 300 µL of the FRAP reagent, incubated at RT for 20 min in the dark, and the absorbance was measured at 595 nm. Solvent blanks were included in each analysis to ensure accuracy. Antioxidant activity was expressed as mg FeSO₄/100 g. ACE-I Assay of HSD and HSD protein extract after in vitro digestion The ACE-I assay was conducted as described by Giromini et al. [ 90 ] with minor modifications as outlined by Shalabi et al [ 91 ]. The synthetic substrate furanacroloyl-Phe-Glu-Glu (FAPGG) was used for the ACE-I enzyme. Briefly, 150 µL of FAPGG were pre-incubated at 37°C for 1 min. After this incubation, 10 µL of each sample and 10 µL of ACE-I enzyme (15 mU) were added to the substrate to initiate the enzymatic reaction. The reaction kinetics were monitored with a Synergy KTX microplate reader at a wavelength of 340 nm, with measurements taken every minute for a period of 30 min. Captopril served as a positive control. Hydrolysis of FAPGG by ACE-I resulted in a reduction in absorbance at 340 nm. Complete inhibition of ACE-I activity corresponded to 100% inhibition. The % of ACE-I inhibition was calculated using the formula: %ACE-I = ((Abs no sample − Abs sample )/Abs no sample )×100. More precisely, Abs no sample​ refers to the absorbance of the enzyme-substrate mixture without the addition of any test sample, whereas Abs sample​ denotes the absorbance of the mixture containing the matrices under study. MS/MS peptidomic methods of HSD and HSD protein extract after in vitro digestion Endogenous peptides were identified using a peptidomic approach based on LC-nano tandem ESI mass spectrometry. This label-free shotgun method was performed without enzymatic digestion prior to MS/MS analysis. As part of the sample preparation, the permeate samples were first filtered through Whatman 54 paper filters (Florham Park, NJ), following the protocol described by Regmi et al. [ 86 ]. The filtered component, representing the digested fraction, was further treated by ultrafiltration using 10 kDa centrifuge filters (Pierce™, Protein Concentrator PES, Thermo Fisher Scientific, UK) to remove high molecular weight proteins [ 92 ]. The resulting fraction was then lyophilised to prepare it for mass spectrometric (MS) analysis. Prior to MS, the lyophilised flow through- of HSD and HSD protein extract, containing peptides and low molecular weight proteins, was reconstituted in 0.3% (v/v) formic acid and subjected to desalting using Zip-Tip C18 columns (Millipore, Billerica, MA, USA) [ 92 , 93 ]. Nano-HPLC coupled with MS/MS analysis was conducted using a Dionex Ultimate 3000 HPLC system equipped with an EASY-Spray™ capillary column (15 cm × 150 µm) packed with 2 µm C18 particles (100 Å). The system was interfaced with a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). The analysis utilized mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in 20/80 acetonitrile/water, v/v) at a flow rate of 0.300 µL/min, with the column temperature maintained at 35°C. The acquired raw files were subjected to data analysis using Proteome Discoverer software (version 1.4). The searches were performed against the NCBI reference Cannabis sativa (updated on November 2023; 33846 sequences) for HSD and HSD protein extract. The enzyme specificity was set as unspecific and methionine oxidation, and asparagine/glutamine deamidation were set as variable modifications [ 94 ]. Only peptides with high confidence were included for positive identification (X correlation ≥ 2.5). All peptides were searched in SATPdb, a database of structurally annotated therapeutic peptides, and in DFBP, a database of food-derived bioactive peptides [ 95 , 96 ]. To consider possible further proteolysis, the search was performed keeping a minimum sequence length of four amino acids and applying an “IF” nested function to a matrix which compared the sequence of each peptide found with the ones of the database (Microsoft Excel 2023, version 16.80) [ 30 ]. Cell viability studies Samples (HSD and HSD protein extract) obtained following the digestive process were immediately frozen at -20°C to preserve their integrity. To assess their effect on C2C12 cells, the samples were subsequently subjected to freeze-drying. After this, the freeze-dried samples were accurately weighed and resuspended in 15 mL of cell growth medium (no-complete medium) [Dulbecco's Modified Eagle's Medium (DMEM) with high glucose content (Sigma-Aldrich, Milan, Italy)] and immediately filtered under vacuum at 0.22 µm. This procedure resulted in final concentrations of 41.1 mg/mL for HSDs and 48.4 mg/mL for HSDs protein extract. However, the initial concentration tested on the cells was 10.28 mg/mL for HSDs and 12.1 mg/mL for HSDs protein extract; values characterised by a non-toxic pH for the cells. The murine skeletal muscle cell line C2C12 (IZSLER, Brescia, Italy) was grown in complete growth medium for 15–16 passages in 75 cm 2 culture flasks. The complete growth medium consisted of DMEM high in glucose (Sigma-Aldrich, Milan, Italy) supplemented with 10% foetal bovine serum (FBS, Immunological Sciences, Rome, Italy), 2 mM L-glutamine (Sigma-Aldrich, Milan, Italy) and 1% penicillin/streptomycin (Immunological Sciences, Rome, Italy). Cells were detached at 60–70% confluence with 5 mL of trypsin-EDTA 1X (Immuno-logical Sciences, Rome, Italy) and cell counts were estimated using the trypan blue staining method (Sigma-Aldrich, Milan, Italy) with a haemocytometer. After trypsinization, the cells were seeded into 96-well plates at a density of 1.0×10⁵ cells/mL, using complete medium as previously described. Following 24 h of incubation (time required for cell attachment and proliferation) at 37°C with 5% CO₂, the growth medium was removed and replaced with the specific treatments, as reported in Table 3 . The cells were then incubated for an additional 24 h under the same conditions. Table 3 Table 3 Concentration of cellular treatments with HSDs and HSDS protein extracts . Values are expressed in mg/mL. The negative control consists of medium without Foetal Bovine Serum (FBS), while the positive control is complete growth medium. HSDs: Hempseeds. TREATMENTS HSDs HSDs protein extract 10.28 mg/mL 0.17 mg/mL 12.1 mg/mL 0.19 mg/mL 5.14 mg/mL 0.09 mg/mL 6.05 mg/mL 0.09 mg/mL 2.77 mg/mL 0.05 mg/mL 3.00 mg/mL 0.05 mg/mL 1.39 mg/mL 0.02 mg/mL 1.50 mg/mL 0.02 mg/mL 0.69 mg/mL Negative control (0% FBS) 0.75 mg/mL Negative control (0% FBS) 0.35 mg/mL Positive control (10% FBS) 0.38 mg/mL Positive control (10% FBS) Cell viability was assessed using the Alamar Blue colorimetric kit (Immunological Sciences, Società Italiana Chimici, Rome, Italy). For the assay, 15 µL of ready-to-use Alamar Blue reagent was added to each well containing 150 µL of medium. Each plate was shaken for 30 ss and incubated for 3 h at 37°C with 5% CO₂. At the end of the incubation, the plates were read with a spectrophotometer at a wavelength of 570 nm. To account for background noise, the growth media for each treatment (without cells) were read at 600 nm. Cell viability was calculated by subtracting the optical density (OD) values of the media from the OD values measured at 570 nm for the cells. Each value was then standardised on the negative control (cells grown in DMEM without FBS) and statistically compared to the positive control (cells grown in complete growth medium). For the evaluation of HS, a temperature of 41.5°C was selected based on the study by Tang et al. [ 97 ]. Specifically, C2C12 cells were seeded into 96-well plates at a concentration of 1.0×10⁵ cells/mL in complete growth medium and incubated for 24 h at 37°C with 5% CO₂. After the initial 24 h incubation, the supernatant was removed and replaced with no-complete growth medium. Immediately the plates were incubated at 37°C (control, physiological condition) or exposed to 41.5°C for 1, 2, or 3 h. Viability was tested with the Alamar Blue kit, as previously described. Based on the data obtained and shown in results section, the 3 h incubation period was chosen for the evaluation of digest against HS. To evaluate the effects of digestates in mitigating HS, concentrations of 2.77, 1.39, 0.69, and 0.35 mg/mL for HSD and 3.00, 1.50, 0.75 and 0.38 mg/mL for HSD protein extract were chosen; i.e. concentrations that demonstrated greater efficacy in supporting cell viability. Specifically, C2C12 cells were seeded in 96-well plates at a density of 1.0 × 10⁵ cells/mL in complete growth medium and incubated for 24 h at 37°C with 5% CO₂. Following the incubation, the growth medium was removed and replaced with the above treatments, which were incubated at the same conditions. After treatment, all media were removed and the cells were incubated in DMEM for 3 h at 37°C or 41.5°C. At the end of the HS period, cell viability was assessed with the Alamar Blue assay, as previously described. Statistical analysis All the data were analysed using GraphPad Prism 9 9.3.1 (GraphPad Software Inc., San Diego, CA, USA). The functional activities (TPC, ABTS, FRAP and ACE-I) were analysed using an independent samples t-test, assuming equal variances between the groups. Data on the effects of digestates on cell viability and HS determination at 1 h, 2 h and 3 h at 41.5°C were analysed using one-way ANOVA followed by Tukey's multiple comparison test. The analysis of the effect of digestates on HS was conducted using two-way ANOVA ( Time x Treatment ) followed by Tukey's multiple comparison test. All data are reported as mean ± standard error of the mean (SEM) of at least three independent experiments. Values are considered statistically significant for a 95% confidence intervale ( p-value = 0.05 ). Abbreviations ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) ACE-I angiotensin-converting enzyme-I ADF Acid Detergent Fibre ADL Acid Detergent Lignin DMEM Dulbecco's Modified Eagle's Medium FAPGG furanacroloyl-Phe-Glu-Glu FBS Foetal Bovine Serum FC Folin-Ciocalteu FRAP Ferric Reducing Antioxidant Power HS Heat Stress HSD hempseed (when referred to the sample object of study) HSPS Heat-shock proteins MS Mass Spectrometric NDF Neutral Detergent Fibre OD Optical Density ROS Reactive Oxygen Species RT Room Temperature SEM standard error of the mean SGF Simulated Gastric Fluids SIF Simulated Intestinal Fluids SOF Simulated Oral Fluids TE Trolox Equivalent TPC Total Phenolic Content TPTZ 2,4,6-tripyridyl-s-triazine. Declarations Acknowledgements This work was carried out in the frame of the JRC Visiting Scientist agreement No. 12/JRC.F.2/2023 (Directorate F - Health and Food JRC.F.2 Health Technologies). Funding sources This research received no specific grant from any funding agency, commercial or not-for-profit section. Data availability statement The datasets that were generated for this study are available on request to the corresponding author. Author Contributions D.L. and C.G. conceived and designed the study. Methodology was developed by D.L., E.P., F.G.S., G.T., and C.G. Software implementation and data validation were performed by D.L., E.P., F.G.S., G.T., and C.G. Data curation was carried out by D.L., E.P., F.G.S., G.T., and C.G. The original draft of the manuscript was written by D.L., S.G., I.G., and C.G. All authors contributed to the review and editing of the final manuscript and approved its submission. ORCID Davide Lanzoni: 0000-0002-8233-659X. Elena Petrosillo: 0009-0000-8222-6430. Francesca Grassi Scalvini: 0000-0002-2995-1112. Joshua Grana: 0009-0007-9141-8726. Gabriella Tedeschi: 0000-0003-2082-6443. Sabrina Gioria: 0000-0001-7150-9523. Eva Pěchoučková: 0000-0001-9336-5436. Ian Givens: 0000-0002-6754-6935. Carlotta Giromini: 0000-0002-3717-5336. Competing interests The authors declare that they have no competing interests. References Climate Copernicus. 2023 is the hottest year on record, with global temperatures close to the 1.5°C limit. Climate Copernicus. (2024). https://www.climate.copernicus.eu Kovats, R. S. & Hajat, S. Heat stress and public health: a critical review. Annu. Rev. Public. Health . 29 , 41–55 (2008). Cramer, M. N., Gagnon, D., Laitano, O. & Crandall, C. G. Human temperature regulation under heat stress in health, disease, and injury. Physiol. Rev. 102 , 1907–1989 (2022). Rhoads, R. P., Baumgard, L. H., Suagee, J. K. & Sanders, S. R. Nutritional interventions to alleviate the negative consequences of heat stress. Adv. Nutr. 4 , 267–276 (2013). Ganesan, S. et al. Short-term heat stress causes altered intracellular signaling in oxidative skeletal muscle. J. Anim. Sci. 95 , 2438–2451 (2017). Zhao, L. et al. Heat stress decreases metabolic flexibility in skeletal muscle of growing pigs. AJP-Regul Integr. Comp. Physiol. 315 , R1096–R1106 (2018). Farinon, B., Molinari, R., Costantini, L. & Merendino, N. The seed of industrial hemp (Cannabis sativa L.): Nutritional quality and potential functionality for human health and nutrition. Nutrients 12 , 1935 (2020). Lanzoni, D. et al. Total phenolic content and antioxidant activity of in vitro digested hemp-based products. Foods 12 , 601 (2023). House, J. D., Neufeld, J. & Leson, G. Evaluating the quality of protein from hemp seed (Cannabis sativa L.) products through the use of the protein digestibility-corrected amino acid score method. J. Agric. Food Chem. 58 , 11801–11807 (2010). Mylostуva, D. et al. Biochemical changes during heat stress in productive animals with an emphasis on the antioxidant defense system. J. Anim. Behav. Biometeorol. 10 , 0–0 (2021). Keservani, R. K. et al. Nutraceutical and functional food as future food: A review. Der Pharm. Lett. 2 , 106–116 (2010). Leonard, W., Zhang, P., Ying, D. & Fang, Z. Hempseed in food industry: Nutritional value, health benefits, and industrial applications. Compr. Rev. Food Sci. Food Saf. 19 , 282–308 (2020). Giromini, C., Lanzoni, D., Rebucci, R. & Baldi, A. Bioactive peptides from milk and dairy proteins: models of digestion and intestinal barrier. In: Protein Digestion-Derived Peptides, Academic, 145–173. (2024). Lanzoni, D. et al. Phenolic profile and antioxidant activity of hemp co-products following green chemical extraction and ex vivo digestion. Ital. J. Anim. Sci. 23 , 651–663 (2024). Lucas-González, R., Viuda-Martos, M., Álvarez, J. A. P. & Fernández-López, J. Changes in bioaccessibility, polyphenol profile and antioxidant potential of flours obtained from persimmon fruit (Diospyros kaki) co-products during in vitro gastrointestinal digestion. Food Chem. 256 , 252–258 (2018). Ma, Y. et al. Phenolics and antioxidant activity of bamboo leaves soup as affected by in vitro digestion. Food Chem. Toxicol. 135 , 110941 (2020). Tang, C. H., Wang, X. S. & Yang, X. Q. Enzymatic hydrolysis of hemp (Cannabis sativa L.) protein isolate by various proteases and antioxidant properties of the resulting hydrolysates. Food Chem. 114 , 1484–1490 (2009). Wang, X. S., Tang, C. H., Chen, L. & Yang, X. Q. Characterization and antioxidant properties of hemp protein hydrolysates obtained with Neutrase®. FTB 47 , 428–434 (2009). Malomo, S. A., Onuh, J. O., Girgih, A. T. & Aluko, R. E. Structural and antihypertensive properties of enzymatic hemp seed protein hydrolysates. Nutrients 7 (2015). Pfeffer, M. A. Angiotensin-converting enzyme inhibition in congestive heart failure: benefit and perspective. Am. Heart J. 126 , 789–793 (1993). Raghavan, S. & Kristinsson, H. G. ACE-inhibitory activity of tilapia protein hydrolysates. Food Chem. 117 , 582–588 (2009). Flather, M. D. et al. Long-term ACE-inhibitor therapy in patients with heart failure or left-ventricular dysfunction: a systematic overview of data from individual patients. Lancet 355 , 1575–1581 (2000). Pahor, M. et al. Therapeutic benefits of ACE inhibitors and other antihypertensive drugs in patients with type 2 diabetes. Diabetes Care . 23 , 888–892 (2000). Li, Y., Zhou, J., Huang, K., Sun, Y. & Zeng, X. Purification of a novel angiotensin I-converting enzyme (ACE) inhibitory peptide with an antihypertensive effect from loach (Misgurnus anguillicaudatus). J. Agric. Food Chem. 60 , 1320–1325 (2012). Vermeirssen, V., Van Camp, J. & Verstraete, W. Fractionation of angiotensin I converting enzyme inhibitory activity from pea and whey protein in vitro gastrointestinal digests. J. Sci. Food Agric. 85 , 399–405 (2005). Miguel, M., Manso, M. A., López-Fandiño, R., Alonso, M. J. & Salaices, M. Vascular effects and antihypertensive properties of κ-casein macropeptide. Int. Dairy. J. 17 , 1473–1477 (2007). Bougatef, A. et al. Angiotensin I-converting enzyme (ACE) inhibitory activities of sardinelle (Sardinella aurita) by-products protein hydrolysates obtained by treatment with microbial and visceral fish serine proteases. Food Chem. 111 , 350–356 (2008). Ahmad, I., Yanuar, A., Mulia, K. & Mun’im, A. Review of angiotensin-converting enzyme inhibitory assay: Rapid method in drug discovery of herbal plants. Phcog Rev. 11 , 1 (2017). Vermeirssen, V., Van Camp, J., Devos, L. & Verstraete, W. Release of angiotensin I converting enzyme (ACE) inhibitory activity during in vitro gastrointestinal digestion: From batch experiment to semicontinuous model. J. Agric. Food Chem. 51 , 5680–5687 (2003). Lanzoni, D. et al. Antioxidant capacity and peptidomic analysis of in vitro digested Camelina sativa L. Crantz and Cynara cardunculus co-products. Sci. Rep. 14 , 14456 (2024). Segura Campos, M. R., Peralta González, F., Chel Guerrero, L. & Betancur Ancona, D. Angiotensin I-converting enzyme inhibitory peptides of chia (Salvia hispanica) produced by enzymatic hydrolysis. Int. J. Food Sci. 1 , 158482 (2013). Teh, S. S., Bekhit, A. E. D. A., Carne, A. & Birch, J. Antioxidant and ACE-inhibitory activities of hemp (Cannabis sativa L.) protein hydrolysates produced by the proteases AFP, HT, Pro-G, actinidin and zingibain. Food Chem. 203 , 199–206 (2016). Israili, Z. H., Hernández-Hernández, R. & Valasco, M. The future of antihypertensive treatment. Am. J. Ther. 14 , 121–134 (2017). Leonard, W., Xiong, Y., Zhang, P., Ying, D. & Fang, Z. Enhanced lignanamide absorption and antioxidative effect of extruded hempseed (Cannabis sativa L.) hull in Caco-2 intestinal cell culture. J. Agric. Food Chem. 69 , 11259–11271 (2021). Schwab, U. S. et al. Effects of hempseed and flaxseed oils on the profile of serum lipids, serum total and lipoprotein lipid concentrations and haemostatic factors. Eur. J. Nutr. 45 , 470–477 (2006). Rezapour-Firouzi, S. et al. Immunomodulatory and therapeutic effects of Hot-nature diet and co-supplemented hemp seed, evening primrose oils intervention in multiple sclerosis patients. CAM 21 , 473–480 (2013). Weinert, D. J. Nutrition and muscle protein synthesis: A descriptive review. JCCA 53 , 186 (2009). Mithal, A. et al. Impact of nutrition on muscle mass, strength, and performance in older adults. Osteoporos. Int. 24 , 1555–1566 (2013). McGlory, C., van Vliet, S., Stokes, T., Mittendorfer, B. & Phillips, S. M. The impact of exercise and nutrition on the regulation of skeletal muscle mass. J. Physiol. 597 , 1251–1258 (2019). Manabe et al. Characterization of an acute muscle contraction model using cultured C2C12 myotubes. PloS One . 7 , e52592 (2012). Miller, D. D. Minerals. in: Fennema’s Food Chemistry, CRC, 627–679 (2017). Roeseler, D. A. et al. Muscle protein signaling in C2C12 cells is stimulated to similar degrees by diverse commercial food protein sources and experimental soy protein hydrolysates. J. Agric. Food Chem. 65 , 2956–2964 (2017). Kamei, Y., Hatazawa, Y., Uchitomi, R., Yoshimura, R. & Miura, S. Regulation of skeletal muscle function by amino acids. Nutrients 12 , 261 (2020). Russo, R. & Reggiani, R. Evaluation of protein concentration, amino acid profile and antinutritional compounds in hempseed meal from dioecious and monoecious varieties. Am. J. Plant. Sci. 6 , 14–22 (2015). Weder, J. K. & Kahleyss, R. Reaction of Lentil Trypsin – Chymotrypsin Inhibitors with Human and Bovine Proteinases. J. Agric. Food Chem. 51 , 8045–8050 (2003). Carbone, J. W., McClung, J. P. & Pasiakos, S. M. Skeletal muscle responses to negative energy balance: effects of dietary protein. Adv. Nutr. 3 , 119–126 (2012). Wang, Z. A. & Zheng, J. S. Advances in peptide synthesis. In: Peptide and Peptidomimetic Therapeutics. Academic, 73–97. (2022). Mamone, G., Picariello, G., Ramondo, A., Nicolai, M. A. & Ferranti, P. Production, digestibility and allergenicity of hemp (Cannabis sativa L.) protein isolates. Food Res. Int. 115 , 562–571 (2019). Fujita, S. & Volpi, E. Amino acids and muscle loss with aging. J. Nutr. 136 , 277S–280S (2006). Callaway, J. C. Hempseed as a nutritional resource: An overview. Euphytica 140 , 65–72 (2004). Mattila, P. et al. Nutritional value of commercial protein-rich plant products. Plant. Foods Hum. Nutr. 73 , 108–115 (2018). Oseyko, M., Sova, N., Lutsenko, M. & Kalyna, V. Chemical aspects of the composition of industrial hemp seed products. Ukr. Food J. 8 , 544–559 (2019). Nedachi, T., Kadotani, A., Ariga, M., Katagiri, H. & Kanzaki, M. Ambient glucose levels qualify the potency of insulin myogenic actions by regulating SIRT1 and FoxO3a in C2C12 myocytes. Am. J. Physiol. Endocrinol. Metab. 294 , E668–E678 (2008). Wei, P., Tang, Y., Zhou, K., Wei, Z. & Liu, G. Characteristics of polysaccharides from industrial hemp (Cannabis sativa L.) kernels. Foods 13 , 3429 (2024). Shils, M. E. Magnesium. In: (eds Shils, M. E. & Young, V. R.) Modern Nutrition in Health and Disease, 7th ed., Lea & Febiger, Philadelphia, 159–192. (1988). National Research Council. Minerals. In: Diet and Health: Implications for Reducing Chronic Disease Risk, National Academies Press (US), (1989). Bhowmik, D., Chiranjib, K. & Kumar, S. A potential medicinal importance of zinc in human health and chronic. Int. J. Pharm. 1 , 5–11 (2010). Fuller, T. J., Carter, N. W., Barcenas, C. A. & Knochel, J. P. Reversible changes of the muscle cell in experimental phosphorus deficiency. J. Clin. Invest. 57 , 1019–1024 (1976). Gupta, C. P. Role of iron (Fe) in body. IOSR J. Appl. Chem. 7 , 38–46 (2014). EFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies). Overview on dietary reference values for the EU population as derived by the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), European Food Safety Authority, 2017. Available online: https://www.efsa.europa.eu/sites/default/files/assets/DRV_Summary_tables_jan_17.pdf Brenkert, A. L. & Malone, E. L. Modeling vulnerability and resilience to climate change: a case study of India and Indian states. Clim. Change . 72 , 57–102 (2005). Habibi, P. et al. Climate change and heat stress resilient outdoor workers: findings from systematic literature review. BMC Public. Health . 24 , 1711 (2024). Park, H. G., Han, S. I., Oh, S. Y. & Kang, H. S. Cellular responses to mild heat stress. CMLS 62 , 10–23 (2005). Summers, C. M. & Valentine, R. J. Two hours of heat stress induces MAP-kinase signaling and autophagasome accumulation in C2C12 myotubes. Cell. Biochem. Biophys. 80 , 367–373 (2022). Gao et al. Heat stress inhibits proliferation, promotes growth, and induces apoptosis in cultured Lantang swine skeletal muscle satellite cells. J. Zhejiang Univ. Sci. B . 16 , 549 (2015). Belhadj et al. Reactive oxygen species, heat stress and oxidative-induced mitochondrial damage: a review. Int. J. Hyperth. 30 , 513–523 (2014). Locke, M. & Celotti, C. The effect of heat stress on skeletal muscle contractile properties. Cell. Stress Chaperones . 19 , 519–527 (2014). Pechan, P. M. Heat shock proteins and cell proliferation. FEBS Lett. 280 , 1–4 (1991). Liang, P. & MacRae, T. H. Molecular chaperones and the cytoskeleton. J. Cell. Sci. 110 , 1431–1440 (1997). Coss, R. A. & Linnemans, W. A. M. The effects of hyperthermia on the cytoskeleton: a review. Int. J. Hyperth. 12 , 173–196 (1996). Mattson, M. P. Hormesis defined. Ageing Res. Rev. 7 , 1–7 (2008). Hayes, D. P. Nutritional hormesis. Eur. J. Clin. Nutr. 61 , 147–159 (2007). Li, G. et al. Antioxidant activity of docosahexaenoic acid (DHA) and its regulatory roles in mitochondria. J. Agric. Food Chem. 69 , 1647–1655 (2021). Xiao, B. et al. Eicosapentaenoic acid (EPA) exhibits antioxidant activity via mitochondrial modulation. Food Chem. 373 , 131389 (2022). Mickleborough, T. D. Omega-3 polyunsaturated fatty acids in physical performance optimization. IJSNEM 23 , 83–96 (2013). Montserrat-de la Paz, S., Marín-Aguilar, F., García-Gimenez, M. D. & Fernández-Arche, M. A. Hemp (Cannabis sativa L.) seed oil: analytical and phytochemical characterization of the unsaponifiable fraction. J. Agric. Food Chem. 62 , 1105–1110 (2014). Fischer, C. P. et al. Vitamin E isoform-specific inhibition of the exercise-induced heat shock protein 72 expression in humans. J. Appl. Physiol. 100 , 1679–1687 (2006). Cumming, K. T. et al. Effects of vitamin C and E supplementation on endogenous antioxidant systems and heat shock proteins in response to endurance training. Physiol. Rep. 2 , e12142 (2014). Sahin, K., Sahin, N., Onderci, M., Yaralioglu, S. & Kucuk, O. Protective role of supplemental vitamin E on lipid peroxidation, vitamins E, A and some mineral concentrations of broilers reared under heat stress. Vet. Med-CZECH . 46 , 140 (2001). Sivakumar, A. V. N., Singh, G. & Varshney, V. P. Antioxidants supplementation on acid-base balance during heat stress in goats. Asian-Australasian J. Anim. Sci. 23 , 1462–1468 (2010). Ajakaiye, J. J., Perez-Bello, A. & Mollineda-Trujillo, A. Impact of heat stress on egg quality in layer hens supplemented with l-ascorbic acid and dl-tocopherol acetate. Vet. Arh . 81 , 119–132 (2010). European Union. Council Regulation (EU) 1370/2013 of 16 December 2013 determining measures on fixing certain aids and refunds related to the common organisation of the markets in agricultural products. (2013). https://eur-lex.europa.eu . Sorrentino, G. Introduction to emerging industrial applications of cannabis (Cannabis sativa L). Rend. Lin Sci. Fis. 32 , 233–243 (2021). Minekus, M. et al. A standardised static in vitro digestion method suitable for food—an international consensus. Food Funct. 5 , 1113–1124 (2014). Brodkorb et al. INFOGEST static in vitro simulation of gastrointestinal food digestion. Nat. Protoc. 14 , 991–1014 (2019). Regmi, P. R., Ferguson, N. S. & Zijlstra, R. T. In vitro digestibility techniques to predict apparent total tract energy digestibility of wheat in grower pigs. J. Anim. Sci. 87 , 3620–3629 (2009). Attard, E. A rapid microtitre plate Folin-Ciocalteu method for the assessment of polyphenols. Open. Life Sci. 8 , 48–53 (2013). Re, R. et al. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol. Med. 26 , 1231–1237 (1999). Abdelaleem, M. A. & Elbassiony, K. R. A. Evaluation of phytochemicals and antioxidant activity of gamma-irradiated quinoa (Chenopodium quinoa). Braz J. Biol. 81 , 806–813 (2020). Giromini, C., Fekete, Á. A., Givens, D. I., Baldi, A. & Lovegrove, J. A. Short-communication: a comparison of the in vitro angiotensin-1-converting enzyme inhibitory capacity of dairy and plant protein supplements. Nutrients 9 , 1352 (2017). Shalaby, S. M., Zakora, M. & Otte, J. Performance of two commonly used angiotensin-converting enzyme inhibition assays using FA-PGG and HHL as substrates. J. Dairy. Res. 73 , 178–186 (2006). Tavares, T. G. et al. Optimisation, by response surface methodology, of degree of hydrolysis and antioxidant and ACE-inhibitory activities of whey protein hydrolysates obtained with cardoon extract. Int. Dairy. J. 21 , 926–933 (2011). Nonnis, S. et al. Acute environmental temperature variation affects brain protein expression, anxiety and explorative behaviour in adult zebrafish. Sci. Rep. 11 , 2521 (2021). Capraro, J. et al. Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells. Biochem. Biophys. Res. Commun. 437 , 648–652 (2013). Singh et al. SATPdb: a database of structurally annotated therapeutic peptides. Nucleic Acids Res. 44 , D1119–D1126 (2016). Qin, D. et al. DFBP: a comprehensive database of food-derived bioactive peptides for peptidomics research. Bioinformatics 38 , 3275–3280 (2022). Tang, J. et al. Damage to the myogenic differentiation of C2C12 cells by heat stress is associated with up-regulation of several selenoproteins. Sci. Rep. 8 , 1–9 (2018). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6511890","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":461825884,"identity":"736e0852-c251-433a-83f0-c15114d1c53c","order_by":0,"name":"Davide Lanzoni","email":"","orcid":"","institution":"Università degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"Davide","middleName":"","lastName":"Lanzoni","suffix":""},{"id":461825886,"identity":"75812679-141b-4581-89f3-ff20ea9e66b3","order_by":1,"name":"Elena Petrosillo","email":"","orcid":"","institution":"Università degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"Elena","middleName":"","lastName":"Petrosillo","suffix":""},{"id":461825888,"identity":"24609a5d-b182-452a-832f-0ca82d3934b3","order_by":2,"name":"Francesca Grassi Scalvini","email":"","orcid":"","institution":"Università degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"Francesca","middleName":"Grassi","lastName":"Scalvini","suffix":""},{"id":461825889,"identity":"51723cc5-a7bd-40d7-be8e-07be592cff03","order_by":3,"name":"Joshua Grana","email":"","orcid":"","institution":"Università degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"Joshua","middleName":"","lastName":"Grana","suffix":""},{"id":461825890,"identity":"048fbee7-70df-49d4-9cbf-d16a33cb22b0","order_by":4,"name":"Gabriella Tedeschi","email":"","orcid":"","institution":"Università degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"Gabriella","middleName":"","lastName":"Tedeschi","suffix":""},{"id":461825891,"identity":"99497a76-e075-41e7-830c-b46809068cfb","order_by":5,"name":"Sabrina Gioria","email":"data:image/png;base64,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","orcid":"","institution":"European Commission, Joint Research Centre (JRC)","correspondingAuthor":true,"prefix":"","firstName":"Sabrina","middleName":"","lastName":"Gioria","suffix":""},{"id":461825892,"identity":"ddac6719-e110-41cb-95ab-e5e6125a3986","order_by":6,"name":"Eva Pěchoučková","email":"","orcid":"","institution":"Czech University of Life Sciences Prague","correspondingAuthor":false,"prefix":"","firstName":"Eva","middleName":"","lastName":"Pěchoučková","suffix":""},{"id":461825893,"identity":"43cb5d7d-47bb-4277-b85c-dbfa8506e0dc","order_by":7,"name":"Ian Givens","email":"","orcid":"","institution":"University of Reading","correspondingAuthor":false,"prefix":"","firstName":"Ian","middleName":"","lastName":"Givens","suffix":""},{"id":461825894,"identity":"6a2ebb74-6cbf-461f-9e55-26ea79aa1fdf","order_by":8,"name":"Carlotta Giromini","email":"","orcid":"","institution":"Università degli Studi di Milano","correspondingAuthor":false,"prefix":"","firstName":"Carlotta","middleName":"","lastName":"Giromini","suffix":""}],"badges":[],"createdAt":"2025-04-23 10:53:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6511890/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6511890/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83538281,"identity":"6ffbfd09-d3d0-48b4-ac1b-ea1952da3d57","added_by":"auto","created_at":"2025-05-28 07:19:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":9676,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell viability (%) assessed with the Alamar Blue assay\u003c/strong\u003e. a) HSD; b) HSD protein extract. Values, expressed in %, are represented as mean ± SEM. Values are standardised on growth medium (0% FBS) and expressed in mg/mL. * indicates statistically significant differences (\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e) compared to positive control (10% FBS).\u003c/p\u003e","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6511890/v1/aebb82b3fc0586bb5ccab807.png"},{"id":83538149,"identity":"2e719d54-cb81-4602-bea3-ee9cc091bd24","added_by":"auto","created_at":"2025-05-28 07:11:12","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHeat stress evaluation at 41.5 °C for 1h, 2h and 3h\u003c/strong\u003e. Values, expressed in %, are represented as mean ± SEM. Values were standardised to the control (37 °C). * \u0026lt; 0.05; ** \u0026lt; 0.01; *** \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6511890/v1/c4bffa05513a9ca0546c43da.png"},{"id":83538158,"identity":"bae1c3f2-9295-420d-9065-54d98bb6934c","added_by":"auto","created_at":"2025-05-28 07:11:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10704,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of HSD (a) and HSD protein extract (b) on cell viability under HS conditions\u003c/strong\u003e. Values, expressed in %, are represented as mean ± SEM. * indicates statistically significant differences (\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e) between treatments at 37 °C and 41.5 °C.\u003c/p\u003e","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6511890/v1/f2811d7bcac92585f0ab95e6.png"},{"id":98306393,"identity":"2788f73c-e0b8-4177-bbe6-e08791316f07","added_by":"auto","created_at":"2025-12-16 11:10:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1510017,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6511890/v1/15695b26-cde6-40d8-90d1-76dcc4bcf13d.pdf"},{"id":83538152,"identity":"3dddca2f-be30-4629-b597-85979d12c763","added_by":"auto","created_at":"2025-05-28 07:11:12","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":60796,"visible":true,"origin":"","legend":"","description":"","filename":"LegendofSupplementarymaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6511890/v1/2cd3ba395a157bcb822af6d8.pdf"},{"id":83538157,"identity":"e53f310a-3a09-4899-9b25-04511c3b757b","added_by":"auto","created_at":"2025-05-28 07:11:14","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":85032,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6511890/v1/1d86a7df80c9555b2d677285.xlsx"},{"id":83538153,"identity":"34f72c13-39c8-4731-812a-23b702a118ed","added_by":"auto","created_at":"2025-05-28 07:11:12","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":114086,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6511890/v1/61d41cc7ed57e4f5a1308ee0.xlsx"},{"id":83538154,"identity":"9c450e0a-2e3a-4375-8627-62672b79027b","added_by":"auto","created_at":"2025-05-28 07:11:12","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":100911,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6511890/v1/98679de45db0a4fb70e8cd88.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Hempseed and hempseed protein extract: antioxidant potential, peptidomic analysis and muscle cell protection under heat stress conditions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eClimate change is causing global temperatures to rise rapidly, with significant consequences for human health. According to Climate Copernicus [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], 2023 was the warmest year recorded since 1850. In just twelve months, between February 2023 and January 2024, the average global temperature exceeded pre-industrial levels by 1.5\u0026deg;C, highlighting the gravity of the climate crisis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This increase in temperatures not only affects the environment, but also has a direct impact on human physiological well-being, increasing the risk of heat stress (HS) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Body temperature is a crucial physical property that influences the structure and function of biological tissues [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Humans, as homeothermic organisms, maintain a constant tissue temperature, generally between 36\u0026ndash;38\u0026deg;C under resting conditions [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. This balance is essential to ensure normal physiological functioning. However, as pointed out by Rhoads et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and Cramer et al. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], exposure to high temperatures can cause significant alterations and damage at the cellular level. Intracellular molecular structures, stabilised by relatively weak interactions, are particularly sensitive to changes in the microenvironment, such as changes in temperature or pH. Heat can alter plasma membrane fluidity, transmembrane transport rates and the three-dimensional configuration of proteins, interfering with their synthesis to the point of causing cell death [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Of all biological tissues, muscle tissue, being highly metabolically active, is particularly vulnerable. Muscle tissue is not only susceptible to HS caused by external environmental factors that raise body temperature, but also to HS induced by physical exertion, specifically resulting from intense exercise [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. This dual exposure makes muscles particularly susceptible to temperature-related damage, underlining the need for preventive measures under conditions of HS. One of the most effective strategies to mitigate the effects of HS is represented by nutrition [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In this context, hemp (\u003cem\u003eCannabis sativa\u003c/em\u003e L.) emerges as a promising solution due to its dual role. On the one hand, its cultivation requires a reduced amount of water and pesticides and is actively involved in carbon sequestration, thus contributing to reducing the ecological footprint and potentially by mitigating the increase in global temperatures. On the other, it can offer products with a high nutritional and functional profile [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Of these, hempseed, previously recognised as a processing waste product, is the best known. It is characterised by a carbohydrate content of 20\u0026ndash;30 g/100 g (mostly dietary fibre), 25\u0026ndash;35 g/100 g lipids with a balanced fatty acid composition and 20\u0026ndash;25 g/100 g protein easy to digest (85.2%), featured by an interesting peptidomic profile [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In parallel, from a functional point of view, hempseeds are distinguished by a high antioxidant profile [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This latter aspect, in the context of HS, represents a crucial point, as HS stimulates the generation of reactive oxygen species (ROS), known for their pro-oxidant effect, which lead to the chemical modification and destruction of molecules due to the development of hypoxia, which accompanies almost all tissue damage [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Although hemp-based products are known for their interesting functional profile, their potential role in mitigating HS on muscle tissue has not yet been fully explored in the scientific literature. This study aims to fill this gap by focusing on several key analyses. The objective was to evaluate the antioxidant profile of two hemp-based products, namely hempseed, which we will refer to in the case of the sample under investigation with HSD, and the HSD protein extract after \u003cem\u003ein vitro\u003c/em\u003e digestion process. Following the measurement of total phenolic content (TPC), two established assays were used for this evaluation: the 2,2\u0026prime;-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and the ferric reducing antioxidant power (FRAP) assays. In parallel, the inhibitory activity of angiotensin-converting enzyme-I (ACE-I), a parameter closely linked to the regulation of oxidative stress and tissue protection, was investigated. Results obtained were simultaneously correlated with the presence of bioactive peptides released following \u003cem\u003ein vitro\u003c/em\u003e digestion. Finally, the digests were tested on a murine muscle cell line (C2C12) exposed to experimental HS condition. This step is a critical component of the study, as it enables the evaluation of the potential of hemp-based products to maintain and safeguard muscle cell function when exposed to HS conditions.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eFunctional profile evaluation of HSD and HSD protein extract after in vitro digestion\u003c/p\u003e\n\u003cp\u003eThe results related to the evaluation of functional activity after \u003cem\u003ein vitro\u003c/em\u003e digestion are shown in Table 1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e. \u003cstrong\u003eFunctional activity evaluation (TPC, ABTS, FRAP and ACE-I assay).\u003c/strong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eValues are represented as mean \u0026plusmn; SEM. Different superscript letters indicate statistically significant (\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e) differences between the two samples for the same assay. TPC Total Phenolic Content; FRAP: Ferric Reducing Antioxidant Power; ACE-I: Angiotensin Converting Enzyme-I; TE: Trolox Equivalent; HSD: Hempseed.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"656\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 219px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 219px;\"\u003e\n \u003cp\u003eHSD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 219px;\"\u003e\n \u003cp\u003eHSD protein extract\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 219px;\"\u003e\n \u003cp\u003eTPC (mg TAE/100 g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 219px;\"\u003e\n \u003cp\u003e231.86 \u0026plusmn; 16.68\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 219px;\"\u003e\n \u003cp\u003e509.25 \u0026plusmn; 18.68\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 219px;\"\u003e\n \u003cp\u003eABTS (mg TE/100 g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 219px;\"\u003e\n \u003cp\u003e2477.87 \u0026plusmn; 406.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 219px;\"\u003e\n \u003cp\u003e2815.76 \u0026plusmn; 450.52\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 219px;\"\u003e\n \u003cp\u003eFRAP (mg FeSO\u003csub\u003e4\u003c/sub\u003e/100 g)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 219px;\"\u003e\n \u003cp\u003e11.71 \u0026plusmn; 0.58\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 219px;\"\u003e\n \u003cp\u003e15.36 \u0026plusmn; 0.18\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 219px;\"\u003e\n \u003cp\u003eACE-I (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 219px;\"\u003e\n \u003cp\u003e11.06 \u0026plusmn; 1.66\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 219px;\"\u003e\n \u003cp\u003e26.73 \u0026plusmn; 2.09\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;As shown in Table 1, the TPC exhibited statistically significant differences (\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e) between the HSD protein extract (509.25 \u0026plusmn; 18.68 mg TAE/100 g) and HSD (231.68 \u0026plusmn; 16.68 mg TAE/100 g). This result was corroborated by the FRAP assay, with significantly higher values observed for the HSP protein extract (15.37 \u0026plusmn; 0.18 mg FeSO₄/g) compared to HSD (11.71 \u0026plusmn; 0.58 mg FeSO₄/g). Similarly, although not statistically significant, a similar trend was observed for the ABTS assay. Finally, ACE-I activity was significantly higher (\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e) for the HSD protein extract (26.73 \u0026plusmn; 2.09%) compared to HSD (11.06 \u0026plusmn; 1.66%).\u003c/p\u003e\n\u003cp\u003ePeptidomic profile of HSD and HSD protein extract after in vitro digestion\u003c/p\u003e\n\u003cp\u003eThe peptidomic profiles of HSD and HSD protein extract after digestion and filtration were obtained by means of a shotgun label free approach which allowed the identification of 1273 peptides in HSD and 1781 peptides in HSD protein extract, respectively. The lists of all the peptides identified with high confidence are reported in Supplementary Table 1 and 2, respectively. All the peptides were searched in DFBP, a database of bioactive peptides from food sources and in SATPdb, a database of structurally annotated therapeutic peptides. The peptides identified as bioactive according to these two data bases were classified by category as shown in Table 2 and Supplementary Table 3, that report the number of the bioactive peptides identified in HSD and HSD protein extract (Table 2) and \u0026nbsp;the sequence of the bioactive peptides listed in SATPbd and DFBP databases identified in HSD and HSD protein extract data sets (Supplementary Table 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2. Number of bioactive peptides identified in HSD and HSD protein extract\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Table reports the number of peptides identified as bioactive following the search in SATPbd and DFBP data bases of all the peptides identified in HSD and HSD protein extract.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"738\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"bottom\" style=\"width: 308px;\"\u003e\n \u003cp\u003eSATPdb\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"bottom\" style=\"width: 304px;\"\u003e\n \u003cp\u003eDFBP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eBIOACTIVE PEPTIDES\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003ecommon peptides in HSD and \u0026nbsp;HSD protein extract \u0026nbsp; (165)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003epeptides exclusively in HSD protein extract \u0026nbsp; (1616)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003epeptides exclusively in HSD \u0026nbsp; \u0026nbsp; \u0026nbsp; (1108)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003ecommon peptides in HSD and \u0026nbsp;HSD protein extract \u0026nbsp; \u0026nbsp; (165)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003epeptides exclusively in HSD protein extract \u0026nbsp; (1616)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003epeptides exclusively in HSD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; (1108)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eAntioxidant\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e24\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eACE-inhibitor\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e64\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eAntibacterial\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eAntihypertensive\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eAnticancer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eAntifungal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eAntimicrobial\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eAntiparasitic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eAntiviral\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eCell-cell comunication\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eDrug delivery vehicle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eToxic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eDrug deliv vehicle\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eDPP IV-inhibitory\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eImmunomodulatory\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eOpioid\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003ePEP-inhibitory\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eMineral-binding\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eNeuropeptides\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eCeliac disease\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eRenin inhibitory\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 127px;\"\u003e\n \u003cp\u003eAntithrombotic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 116px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 102px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 91px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 111px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 96px;\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 97px;\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 127px;\"\u003e\n \u003cp\u003eTOTAL\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 116px;\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 102px;\"\u003e\n \u003cp\u003e110\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 91px;\"\u003e\n \u003cp\u003e72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 111px;\"\u003e\n \u003cp\u003e19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 96px;\"\u003e\n \u003cp\u003e128\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 97px;\"\u003e\n \u003cp\u003e93\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eCell viability and evaluation of digests in HS mitigation\u003c/p\u003e\n\u003cp\u003eAs described in material and methods, for HSD and HSD protein extract, the freeze-dried samples were resuspended in 15 mL of standard medium, reaching final concentrations of 41.1 and 48.4 mg/mL, respectively. However, the assessment of cell viability with Alamar Blue, was conducted using concentrations of 10.28 mg/mL for HSD and 12.10 mg/mL for HSD protein extract, values characterised by a non-toxic pH for the cells.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs illustrated in Figure 1(a), for HSDs, concentrations of 10.28 and 5.14 mg/mL resulted in a significant (\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e) reduction in cell viability (63.48 \u0026plusmn; 7.38% and 74.91 \u0026plusmn; 4.98%, respectively) compared to the positive control (10% FBS, 118.80 \u0026plusmn; 5.79%). In contrast, the lower concentrations of 0.17, 0.09, 0.05 and 0.02 mg/mL showed no significant differences compared to the positive control. The best results were obtained with the concentrations of 2.77, 1.39, 0.69 and 0.35 mg/mL which produced a significant (\u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e) increase in cell viability (180.05 \u0026plusmn; 4.58%, 173.37 \u0026plusmn; 6.60%, 149.77 \u0026plusmn; 10.04% and 133.43 \u0026plusmn; 6.40 % respectively) compared to 10% FBS.\u003c/p\u003e\n\u003cp\u003eFor HSD protein extracts (Figure 1b), the concentrations of 12.10, 6.05, 0.19, 0.09, 0.05 and 0.02 mg/mL showed no differences compared to 10% FBS. In parallel, the concentrations of 3.0 mg/mL (185.80 \u0026plusmn; 11.79%), 1.50 mg/mL (177.80 \u0026plusmn; 11.11%), 0.75 mg/mL (165.66 \u0026plusmn; 9.01%) and 0.3 mg/mL (153.32 \u0026plusmn; 7.20%) significantly (\u003cem\u003ep \u0026lt; 0.05)\u003c/em\u003e increased viability compared to the positive control (111.78 \u0026plusmn; 4.83%).\u003c/p\u003e\n\u003cp\u003eOn the basis of the reported data, the following concentrations were selected for the evaluation of HS mitigation: 2.77, 1.39, 0.69 and 0.35 mg/mL for HSD, and 3.00, 1.50, 0.75 and 0.38 mg/mL for the HSD protein extract.\u003c/p\u003e\n\u003cp\u003eAs previously described and depicted in Figure 2, HS was evaluated for 3 h at 41.5 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis of cell viability using the Alamar Blue test showed a significantly increasing reduction with a rising incubation time at 41.5 \u0026deg;C. In particular, the increase from 1 h (88.22 \u0026plusmn; 1.99 %, \u003cem\u003ep \u0026lt; 0.05\u003c/em\u003e) to 2 h (87.71 \u0026plusmn; 1.20 %, \u003cem\u003ep \u0026lt; 0.01\u003c/em\u003e) and then to 3 h (79.64 \u0026plusmn; 1.92 %, \u003cem\u003ep \u0026lt; 0.001\u003c/em\u003e) of exposure resulted in a significant decrease in viability compared to the optimal culture conditions observed in the control group at 37 \u0026deg;C.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFigure 3 shows the effects of the selected concentrations on HS mitigation. For HSD protein extract (Fig. 3b), none of the tested concentrations were able to counteract the toxic effect of HS on cell viability, showing statistically significant differences between cells grown at 37 \u0026deg;C and those at 41.5 \u0026deg;C. In contrast, different and promising results were observed for HSD (Fig. 3a). Although higher concentrations, such as 2.77 and 1.39 mg/mL, did not ensure the maintenance of cell viability compared to the 37 \u0026deg;C condition, the concentrations of 0.69 mg/mL (130.75 \u0026plusmn; 8.28%) and 0.35 mg/mL (132.14 \u0026plusmn; 8. 09%) tested at 41.5 \u0026deg;C showed no statistically significant differences from the control condition at 37 \u0026deg;C (149.77 \u0026plusmn; 10.04% and 133.43 \u0026plusmn; 6.40%, respectively), highlighting their ability to preserve cell viability.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eFunctional profile of HSD and HSD protein extract after in vitro digestion\u003c/h2\u003e \u003cp\u003eInterest in nutraceuticals continues to grow, fuelled by significant advances in research aimed at identifying the properties and potential applications of functional products, as well as by increasing consumer interest in safe and increasingly healthy food [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In this context, hemp could be a valid solution. For this reason, two hemp-based products were considered in this study, HSD and HSD protein extract, partially characterised in our previous work [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. From a nutritional point of view, both matrices presented a very interesting chemical profile. In particular, they showed a protein content of 23.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57 and 45.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84 g/100 g, and a lipid content of 27.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75 and 9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 g/100 g for HSD and HSD protein extract, respectively, suggesting their application in food and feed sector. In parallel, also the TPC (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) showed interesting levels. Surprisingly, but in agreement with our previous work [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], the HSD protein extract showed a significantly higher absolute phenolic value than HSD after the digestion process. This result can be explained by the strong interaction between the phenolic compounds and the fibre content [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Indeed, the fibre is able to trap phenols via hydrophobic interactions, hydrogen bonds (between the oxygen atoms of the glycosidic chains of the polysaccharides and the hydroxyl groups of the phenolic compounds) and covalent bonds, which are particularly strong in the acid-detergent fibre (ADF) and in the acid-detergent lignin (ADL), which represent the main localisation regions of the phenolic compounds. Although, as reported by Farinon et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], phenolic compounds mainly localise at the hull level, it is plausible to assume that the lower content of ADF (20.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02 g/100 g) and ADL (9.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62 g/100 g) present in HSD protein extract compared to HSD (33.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31, 14.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 g/100, respectively for ADF and ADL) may favour a higher bioavailability of phenolic compounds in the HSD protein extract during the digestive process, resulting in higher values (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis trend is highly correlated with antioxidant activity. ABTS method reported high values for both samples. The values observed for HSD (2386.57\u0026thinsp;\u0026plusmn;\u0026thinsp;117.90 mg TE/100 g) and HSD protein extract (2815.7 6\u0026thinsp;\u0026plusmn;\u0026thinsp;450.52 mg TE/100 g) are overlapping (2477.87\u0026thinsp;\u0026plusmn;\u0026thinsp;406.09; 3936.79\u0026thinsp;\u0026plusmn;\u0026thinsp;59.29 mg TE/100 g, respectively) with what was observed in our previous work [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], despite the application of different digestion models. This decrease in recorded antioxidant activity, observed also for TPC, is attributable to two main causes. The first factor concerns differences in \u003cem\u003ein vitro\u003c/em\u003e digestion protocols. As pointed out by Giromini et al. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], the use of different enzymes, with varying concentrations and specificities, can significantly affect the extent of protein hydrolysis and, consequently, the amount of bioactive peptides released. At the same time, it is conceivable that the filtration process (\u0026le;\u0026thinsp;10 kDa) may have excluded larger peptides or bioactive compounds that are actively involved in modulating the antioxidant profile. The above is also applicable for the values obtained in the FRAP assay.\u003c/p\u003e \u003cp\u003eDespite this, HSD and the HSD protein extract proved to be matrices characterised by a high antioxidant value.\u003c/p\u003e \u003cp\u003eThis is related to both the presence of phenolic compounds and the interesting peptidomic profile shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. More precisely, several works in the literature have demonstrated a high correlation between TPC and antioxidant activity, not only in hemp-based products, but also in different plant matrices, where the increase in the concentration of phenolic compounds at different stages of digestion showed a correlation with increased antioxidant activity, in line with the results obtained in this study [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. At the same time, although as reported by Tang et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], Wang et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and Malomo et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], the native structure of hempseed proteins is characterised by low functionality, the hydrolysis process results in the formation of bioactive peptides, generally with a molecular weight of less than 10 kDa (molecular weight chosen in this study to study bioactivity), which increases the functional value of hemp seed proteins. More specifically, as reported by Tang et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], Wang et al. [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] and Malomo et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], the peptides resulting from hydrolysis are characterised by a high antioxidant value, in particular Fe scavenging and chelation, mechanisms of action of the ABTS and FRAP assays, respectively; confirming the peptidomic profile of Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and the results of Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, it is important to emphasise, that the concentration at which these hydrolysates exert their functional effects strongly depends on the specific hydrolysis conditions employed. This underlines how different hydrolysis methods produce peptides with distinct structural and functional properties, thus influencing their biological activity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe functional evaluation, as illustrated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, included the analysis of ACE-I activity, a crucial parameter in determining the functionality of a food-derived product. As reported by Pfeffer [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], ACE-I plays a key role in the regulation of blood pressure and the pathogenesis of hypertension. ACE-I catalyses the conversion of angiotensin-I, an inactive precursor, into angiotensin-II, a potent vasoconstrictor agent, while inactivating bradykinin, a peptide with vasodilatory action [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. For these reasons, ACE-I are frequently used in the treatment of myocardial infarction [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], hypertension [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and other related cardiovascular diseases, which to date, as reported by Li et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], afflict around 20% of the world's population.\u003c/p\u003e \u003cp\u003eConsequently, scientific research is actively investigating food matrices with such functional characteristics, given their potential benefits and applications [\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Among these, HSD and HDS protein extract showed promising results, with ACE-I values comparable to those of other hemp-based products, in particular leaves and roots (18%) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. As shown by Vermeirssen et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and Lanzoni et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], ACE-I activity tends to increase significantly following the digestive process. This rise is attributable to the disruption of the proteins' native structure, which leads to the release of bioactive peptides capable of modulating this functional activity. Nevertheless, comparing data with those found in the literature is complex and sometimes speculative due to the many variables that influence the results. Among these, the chemical composition of the sample, the degree of purity, the hydrolysis methods adopted and the exposure time to enzymes play a crucial role. As reported by Segura Campos et al. [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], low-molecular-weight peptides derived from enzymatic hydrolysis show a higher inhibitory activity on ACE-I than those with a high molecular weight, highlighting how peptide size is a determining factor. This relationship was further confirmed by Teh et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], who showed that the type of extraction applied to hempseed protein hydrolysates, acidic or alkaline, can alter the protein conformation and, consequently, the inhibitory efficacy on ACE-I. In particular, alkaline hydrolysis produced significantly better results, reaching a maximum inhibition of 70.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06%. Nevertheless, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the samples revealed a high ACE-I peptidomic profile, particularly for the HSD protein extract, confirming the results in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. These data highlight the considerable potential of hemp-based products as functional matrices with ACE-I properties, reinforcing their role in the prevention and management of hypertension. As reported by Israili et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], despite significant advances in the prevention, diagnosis and treatment of hypertension, this condition remains a major public health challenge. Indeed, hypertension is associated with an increased risk of mortality and morbidity from stroke, coronary artery disease, congestive heart failure and end-stage renal disease. For this reason, the high presence of anti-hypertensive bioactive peptides (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) from HSD and HSD protein extract, also confirmed by Teh et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] and Malomo et al. [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], is an important solution for the treatment of this disorder.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eEffect of digested HSD and HSD protein extract on C2C12 cell viability\u003c/h2\u003e \u003cp\u003eThe above data effectively illustrate the functional aspect of the two hemp-based products. After the digestive process, the gastrointestinal tract is certainly the first target organ of HSD and HSD protein extract. As pointed out by Leonard et al. [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], certain hempseed fractions showed positive effects on intestinal tissue. Similar benefits have also been observed in the cardiovascular and neurological systems [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Although the fundamental link between nutrition and muscle tissue is widely documented [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], to our knowledge there are no studies exploring the effects of digested hemp-based products on muscle tissue. Therefore, we decided to investigate this further using murine C2C12 muscle cells, a widely used model recognised for its ability to reflect human physiology [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, both HSD and HSD protein extract showed a similar trend. The lower concentrations did not produce significant effects compared to the control, probably due to an insufficient amount to promote an increase in cell viability. On the other hand, although the higher concentrations of HSD protein extract showed no difference to the control, in the case of HSD, the concentrations of 10.28 and 5.14 mg/mL resulted in a significant reduction in viability, as previously observed. This difference is most probably attributable to their composition. Whereas HSD protein extract is a processed, pure product, and intended for direct human consumption, HSD represents a raw, unpurified product containing all its natural components, including anti-nutritional factors (AF), known to be present in high amounts in hempseeds [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, it is reasonable to assume that high AF concentrations negatively affected cell viability. This effect would not occur directly, but would act by limiting the uptake by cells of essential nutrients, such as nitrogen compounds and minerals, that are crucial for maintaining cell viability [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Among the AFs within hempseeds, those most present, although highly comparable with those of other oil-seeds, are the condensed tannins, trypsin inhibitors and phytic acid [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In particular, condensed tannins are phenolic compounds that can form insoluble complexes with proteins and minerals, negatively affecting their availability [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Trypsin inhibitors impair the digestibility of dietary proteins and the availability of essential amino acids. These compounds show remarkable stability in the gastric environment, resisting both the action of pepsin and acidic pH conditions. In addition, they inhibit the activity of intestinal digestive enzymes, thus limiting the absorption of nitrogen compounds essential for cell viability [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Similarly, phytic acid, due to its peculiar molecular structure, is able to bind to multivalent cations and positively charged proteins, forming insoluble complexes known as phytates. These complexes, including phytate-minerals and phytate-proteins, cannot be digested and adversely affect the bioavailability of dietary minerals and proteins, reducing nutritional efficiency [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and as previously described, successive concentrations up to 0.35 and 0.38 mg/mL for HSD and HSD protein extract, respectively, supported cell viability. Of the factors involved, it is presumed that cell viability was supported by fundamental factors such as nitrogen source, glucose supply (essential for energy metabolism) and micro-nutrients. The nitrogen source is a key element for cell metabolism and protein synthesis [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. As reported by Wang and Zheng [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], cells need a readily available nitrogen source to effectively support anabolic processes. Due to their high digestibility, hemp-derived proteins are particularly suitable for this purpose. Indeed, the digestive process generates peptides and amino acids that are easily assimilated by cells. This aspect was confirmed by Mamone et al. [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], who highlighted the presence of a reduced number of complex peptides in the digestion products of hemp-based proteins. Consequently, it can be hypothesised that this highly bioavailable nitrogen source contributes to stimulating cell growth by providing the necessary substrates for protein synthesis and supporting the metabolic processes essential for cell proliferation. At the muscular level, in addition to availability, it is crucial to consider the quality of the nitrogen source [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In particular, essential amino acids are the most effective nutrients for stimulating muscle protein synthesis, although the molecular mechanisms responsible for this effect are still being investigated. In addition to acting as essential precursors for protein synthesis, essential amino acids appear to exert a direct effect in promoting synthesis processes [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Among these, a crucial role is played branched-chain amino acids, which act not only as major transporters of amine nitrogen between the viscera and peripheral tissues, including skeletal muscle, but also as direct stimulators of muscle protein synthesis. Among the branched-chain amino acids, leucine emerges as the main bioactive amino acid, distinguished by its ability to activate anabolic muscle processes, making it a crucial element in maintaining and promoting muscle mass [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In this context, HSD and HSD protein extract may have contributed to the supply of essential amino acids. As reported by Callaway [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e], House et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], Mattila et al. [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], and Oseyko et al. [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e], both raw hempseed and purified hempseed proteins are notable for containing all essential amino acids, with leucine being particularly abundant, ranking just after arginine, asparagine, and glutamine. This compositional profile supports the hypothesis that hemp-derived protein can provide a nutritionally valuable source of essential amino acids, particularly leucine, to stimulate muscle protein synthesis and support anabolic processes.\u003c/p\u003e \u003cp\u003eIn parallel, cell viability may have been supported by an energy supply in the form of free monosaccharides. The importance of glucose in muscle tissue was highlighted by Carbone et al. [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], who showed that glucose plays a crucial role in supporting the metabolic functions of muscle cells. Furthermore, as described by Nedachi et al. [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], the growth and differentiation of C2C12 muscle cells are significantly compromised when they are cultured under low-glucose conditions, suggesting that an adequate amount of glucose is necessary for the proper development and maintenance of cell viability. In this context, hempseeds could represent also a valuable source of carbohydrates, as reported by Wei et al. [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e], who showed a particularly interesting carbohydrate profile, characterised by high concentrations of glucose and arabinose. Therefore, it is plausible to assume that the monosaccharides released during the digestive process were utilised by the cells as an energy source, contributing to their metabolism and, consequently, to improve cell viability.\u003c/p\u003e \u003cp\u003eIn the assessment of muscle cell viability, minerals such as magnesium (Mg), phosphorus (P), zinc (Zn) and iron (Fe) play essential roles. Mg constitutes approximately 27% of the mineral content in muscles and is the predominant cation within cells, preceded only by potassium [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. This mineral is crucial for the functioning of numerous enzymes, neuromuscular transmission and support of muscle contraction, also aiding post-exercise recovery. Furthermore, as reported by the National Research Council [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], Mg has a stabilising function on DNA and facilitates protein synthesis, thus contributing to the maintenance of muscle mass. Zn, involved in protein synthesis and tissue repair, is crucial for the proliferation of muscle cells, which are responsible for muscle growth and recovery [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. P, as a key component of ATP, is essential for energy metabolism and the synthesis of crucial molecules, promoting muscle function [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Fe, essential for oxygen transport and mitochondrial activity, is involved in the production of energy required for muscle contractions, and its deficiency impairs energy efficiency and muscle capacity [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. In this context, the minerals mentioned above are crucial for muscle health, facilitating tissue growth, and optimising overall muscle function. An aspect of fundamental importance to this study concerns the mineral content of hempseeds. Considering EFSA's Dietary Reference Value [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], hempseed is confirmed as an excellent natural source of Mg, P, Zn and Fe. In particular, the content of P, the most abundant mineral in hempseeds, is higher than flax (Linum usitatissimum) seeds, which are widely used in the human diet [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In light of these considerations, it is reasonable to speculate that the release of essential minerals during the digestive process may have promoted their uptake by muscle cells, thereby optimising their functionality and contributing to improved cellular vitality. Minerals may have been utilised by muscle cells to support fundamental processes such as protein synthesis, tissue repair, energy metabolism and muscle satellite cell proliferation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHeat Stress evaluation on C2C12 cell viability\u003c/h3\u003e\n\u003cp\u003eAs previously discussed, the main objective of the present study was to evaluate the potential protective effect of HSD and HSD protein extract against HS. Understanding the physiological and cellular impacts of HS is a critical area of research in today's scientific landscape, given that global warming poses one of the most pressing and multifaceted environmental threats worldwide [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Human-induced climate changes have a profound influence on the physiological and perceptual responses of living organisms, affecting them in both direct and indirect ways, including alterations in the regulation of core body temperature, heart rate, skin temperature, and thermal comfort [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Such changes can significantly compromise the maintenance of homeostasis, exacerbating physiological vulnerabilities and reducing the ability of organisms to adapt to changing environmental conditions [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Exposure to 41.5\u0026deg;C, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, resulted in a significant reduction in cell viability, with the effect being directly proportional to exposure time. These results are consistent with those reported by Park et al. [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] and Summers and Valentine [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. The decrease in cell viability can be traced to several molecular mechanisms. In particular, HS is known to induce a marked apoptotic response, as highlighted by Gao et al. [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Furthermore, as described by Mylostyva et al. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], HS promotes the generation of ROS, which cause oxidative damage to essential biomolecules, including proteins, lipids and nucleic acids. This effect is particularly damaging to mitochondrial proteins. In particular, as noted by Belhadj-Slimen et al. [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], oxidation-induced damage to mitochondrial proteins includes the subunits of the pyruvate decarboxylase complex, ATP synthase and tricarboxylic acid cycle enzymes, all of which are essential components in the production of cellular energy, thus representing an important factor in the reduction of cell viability. However, the main mechanism behind this impairment seems to be related to the effects of high temperatures on protein folding. Under these conditions, misfolding, denaturation and aggregation of proteins occur, severely affecting cellular homeostasis [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. In response to HS, cells activate an adaptation strategy characterised by the rapid synthesis of heat shock proteins (HSPs). These proteins, which act as molecular chaperones, play a central role in cellular protection. Heat shock proteins participate in critical processes such as repair of damaged proteins, proper protein folding and prevention of stress-induced protein aggregation. In skeletal muscle cells, HSPs are critical for maintaining homeostasis and mitigating damage caused by environmental stresses, helping to preserve the structural and functional integrity of tissues [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. However, although HSPs are crucial for restoring cellular homeostasis, it is important to emphasise, as pointed out by Pechan [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e], that under stress conditions, cells prioritises the production of stress-related proteins over those necessary for cell proliferation, which in turn results in a reduction in cell viability and overall cellular health. In parallel, molecular chaperones influence various cellular components, including the cytoskeleton [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. This phenomenon has been extensively described by Coss and Linnemans [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e], who reported how hyperthermia can destabilise the cytoskeleton, impairing its form and function, with negative effects on cell adhesion and proliferation. The above could provide an explanation for the reduction in cell viability observed in this study, attributable to the effects of the HS.\u003c/p\u003e\n\u003ch3\u003eEvaluation of HSD and HSP protein extract digest mitigation on HS\u003c/h3\u003e\n\u003cp\u003eGiven the significant implications for human health, there is growing interest in strategic diet modification in an attempt to improve the HS response [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Surprisingly, no concentration evaluated for HSD protein extract allowed viability to be sustained at 41.5\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, for HSD, only the lower concentrations (0.69 and 0.35 mg/mL) were able to contain the negative effects on viability by HS, in contrast to the higher concentrations (2.77 and 1.39 mg/mL). This result could be related to the phenomenon of \u003cem\u003ehormesis\u003c/em\u003e. The term \u003cem\u003ehormesis\u003c/em\u003e is used to describe a biphasic dose-effect response, characterised by a stimulation or beneficial effect at low doses and an inhibitory or toxic effect at high doses [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. This principle is extensively documented in the dose-response assessment of food-derived bioactive compounds [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. Therefore, in this study, it is plausible to hypothesise that higher concentrations may act as pro-oxidants, favouring the accumulation of ROS and consequently leading to a reduction in cell viability.\u003c/p\u003e \u003cp\u003eThis protective effect could be attributed to several factors. However, considering that no protective effect was observed in HSD protein extract, it is reasonable to assume that, in the case of HSD, this protection stems neither from the peptide fraction nor from the phenolic component. In our opinion, these results can mainly be attributed to the higher lipid content of HSD compared to HSD protein extract (27.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75; 9.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 g/100 g, respectively) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Notably, as reported in the literature, hempseeds are characterised by a lipid profile rich in Omega-3 and Omega-6 polyunsaturated fatty acids (PUFAs), which are widely recognised for their beneficial effects on general health [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Among the predominant PUFAs in hempseeds, linoleic acid (18:2, n-6, LA) and α-linolenic acid (18:3, n-3, ALA) are the most abundant. These fatty acids are classified as essential, as they must be obtained exclusively through the diet, and serve as precursors for biologically active long-chain PUFAs in animals and humans. Specifically, LA is converted to arachidonic acid (20:4, n-6, AA), while ALA serves as a precursor to eicosapentaenoic acid (20:5, n-3, EPA) and docosahexaenoic acid (22:6, n-3, DHA).\u003csup\u003e\u003cem\u003e7\u003c/em\u003e\u003c/sup\u003e In particular, EPA and DHA are actively involved in multiple physiological processes, including the preservation of cell membrane integrity, modulation of anti-inflammatory and anti-oxidant activity [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. This was also confirmed by Mickleborough [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. More specifically, the author observed how the administration of EPA and DHA mitigated the pro-inflammatory effects and ROS production at the muscle level during physical activity; one of the factors involved in HS, as previously reported.\u003c/p\u003e \u003cp\u003eAt the same time, the lipid fraction of hempseed is characterised by a high concentration of fat-soluble vitamins, including tocopherols, which are the main component [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. As highlighted by Montserrat-De La Paz and colleagues [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e], tocopherols are characterised by marked antioxidant activity, mainly attributable to their ability to neutralise free radicals (scavenging) and to counteract ROS, the main mediators of oxidative damage associated with HT syndrome [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The antioxidant efficacy of tocopherols against HS is widely documented in the literature. This effect has been observed both in human health studies, under conditions of oxidative stress caused by intense exercise [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e] and in studies on animal models exposed to HS [\u003cspan additionalcitationids=\"CR80\" citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTherefore, in light of the above evidence, it is reasonable to assume that the lipid component of HSD contributed significantly to the observed positive effects against HS, compared to the HSD protein extract.\u003c/p\u003e \u003cp\u003eIn conclusion, both HSD and HSD protein extract, after \u003cem\u003ein vitro\u003c/em\u003e digestion, showed a highly promising functional profile, with particular reference to antioxidant and ACE-I activity, results further confirmed by the presence of bioactive peptides identified through peptidomic characterisation. Although both HSD and HSD protein extract showed a good ability to support cell viability under optimal growth conditions, only the lowest concentrations of HSD revealed a protective effect against HS. This could be due to the intrinsic complexity of the matrix considered.\u0026rsquo; These results suggest that HSD, beyond their recognised nutritional value, may serve as a functional ingredient capable of increasing cellular resilience under HS conditions. However, further studies are needed to confirm these effects and explore their applicability in dietary strategies aimed at mitigating the impact of HS on muscle tissue and overall physiological health.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHSD and HSD protein extract\u003c/h2\u003e \u003cp\u003eHSDs (\u003cem\u003eCannabis sativa\u003c/em\u003e L., variety Futura) were purchased from a company in Chrastice (Czech Republic). They were sown in April/May 2021 and harvested when they reached 70% maturity. After harvest, the HSDs were dried at 40\u0026deg;C to reduce the moisture content to 7%, which is essential for proper storage. Cannabinoid content analysis, performed at the Institute of Animal Science of the Czech Republic, reported cannabidiol levels of 30 \u0026micro;g/g and a Δ9-tetrahydrocannabinol (THC) content of 0 \u0026micro;g/g, respecting the legal limits imposed by the European regulation (1370/2013) [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e, \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. The protein extract of HSDs was purchased from a commercial supplier.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro digestion of HSD and HSD protein extract\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e digestion was performed following the protocols of Minekus et al. [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e] and Brodkorb et al. [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. The reagents and enzymes used were purchased from Sigma Chemical Co. (\u003cem\u003eSt. Louis, MO, USA\u003c/em\u003e). Simulated digestion fluids for oral (SOF), gastric (SGF) and intestinal (SIF) phases were prepared in agreement with Brodkorb and colleagues [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]. Precisely, 1.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 g of each sample, previously ground, was diluted in SOF and incubated for 2 min at 37\u0026deg;C with alpha-amylase (75 U mL-1, pH 7.0), under stirring, to simulate the oral phase. At the end, the oral bolus was diluted with SGF and pepsin (2000 U mL-1, pH 3.0), and incubated at 37 \u0026ordm;C for 2 h, under stirring. At the end, the gastric chyme was diluted with SIF and incubated with bile salts (10 mM, pH 7.0) and the pancreatic enzymes (100 U mL\u003csup\u003e-1\u003c/sup\u003e, pH 7.0) for 2 h at 37 \u0026ordm;C, under stirring. Once digestion was completed, the digested fraction was separated as reported by Regmi et al. [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. Specifically, the undigested fraction was collected in a filtration unit using a porcelain funnel covered with filter paper (Whatman 54 Florham Park, NJ). Subsequently, the filtered component, corresponding to the digested fraction, was ultra-filtered using centrifuge filters (10 kDa) (Pierce\u0026trade;, Protein Concentrator PES, Thermo Fisher Scientific, UK), for high weight protein depletion [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. After filtration, the samples were aliquoted and immediately stored at -20 \u0026ordm;C for analysis, as reported below.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEvaluation of functional activity of HSD and HSD protein extract\u003c/h2\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003eTotal Phenolic Content of HSD and HSD protein extract after in vitro digestion\u003c/h2\u003e \u003cp\u003eThe TPC was quantified according to the protocol described by Attard [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. Reagents, including tannic acid, methanol, Folin\u0026ndash;Ciocalteu (FC) reagent, and sodium carbonate (Na₂CO₃), were procured from Sigma Chemical Co. (St. Louis, MO, USA). A standard curve was generated by preparing a series of 1:2 serial dilutions of tannic acid in distilled water, ranging from 480 to 0 \u0026micro;g/mL. The FC reagent was diluted 1:10 with distilled water, and a 1 M solution of Na₂CO₃ was prepared. For the assay, 100 \u0026micro;L of each sample was combined with 500 \u0026micro;L of the diluted FC reagent and 400 \u0026micro;L of the Na₂CO₃ solution. The reaction mixtures were incubated in the dark at room temperature (RT) for 20 min. Following incubation, absorbance was measured at a wavelength of 630 nm using a spectrophotometer. Solvent blanks were included as controls in each assay to account for background absorbance. The TPC was determined based on the standard curve and expressed as tannic acid equivalents (mg TAE/100 g).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eABTS Assay of HSD and HSD protein extract after in vitro digestion\u003c/h2\u003e \u003cp\u003eReagents including ABTS, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), and potassium persulfate (K₂S₂O₈) were procured from Sigma Chemical Co. (St. Louis, MO, USA). The ABTS assay was conducted following the protocol described by Re et al [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. Trolox, prepared as a 2.5 mM solution and used as the antioxidant standard, was diluted into six concentrations ranging from 0 to 2000 \u0026micro;M/mL. To generate the ABTS radical cation (ABTS⁺), 88 \u0026micro;L of 2.45 mM K₂S₂O₈ were added to 5 mL of 7 mM ABTS solution. This mixture was incubated in the dark at RT for 16 h before use. For antioxidant activity measurements, the ABTS⁺ solution was diluted with ethanol to achieve an absorbance of 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 at 734 nm. Subsequently, 10 \u0026micro;L of each sample was combined with 1 mL of the diluted ABTS⁺ solution and incubated in the dark at RT for 6 min. After incubation, the absorbance of the reaction mixture was recorded at 734 nm using a spectrophotometer. Solvent blanks were included for accuracy in all analyses. Antioxidant activity was expressed as Trolox Equivalents (mg TE/100 g).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFRAP Assay of HSD and HSD protein extract after in vitro digestion\u003c/h2\u003e \u003cp\u003eReagents used for the FRAP assay, including ferric chloride hexahydrate [FeCl₃\u0026middot;6H₂O], ferrous sulfate (FeSO₄), sodium acetate trihydrate, glacial acetic acid, and 2,4,6-tripyridyl-s-triazine (TPTZ), were obtained from Sigma Chemical Co. (St. Louis, MO, USA). The assay was conducted based on the method described by Abdelalem and Elbassiony [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e] with minor modifications [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The reagents were prepared as follows: a) Acetate buffer (300 mM, pH 3.6): 2.69 g of sodium acetate trihydrate were dissolved in 16 mL of glacial acetic acid and diluted to 1 L with distilled water. b) TPTZ solution (10 mM): 31.2 mg of TPTZ were dissolved in 10 mL of 40 mM HCl. c) Ferric chloride solution (20 mM): 54 mg of FeCl₃\u0026middot;6H₂O was dissolved in 10 mL of distilled water. Ferrous sulfate (FeSO₄) was used as the antioxidant standard, prepared in six dilutions ranging from 0 to 1500 \u0026micro;M/L. The FRAP working reagent was prepared by mixing 2.5 mL of TPTZ solution, 2.5 mL of ferric chloride solution and 25 mL of acetate buffer. For the analysis, 10 \u0026micro;L of each sample were added to 300 \u0026micro;L of the FRAP reagent, incubated at RT for 20 min in the dark, and the absorbance was measured at 595 nm. Solvent blanks were included in each analysis to ensure accuracy. Antioxidant activity was expressed as mg FeSO₄/100 g.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eACE-I Assay of HSD and HSD protein extract after in vitro digestion\u003c/h2\u003e \u003cp\u003eThe ACE-I assay was conducted as described by Giromini et al. [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e] with minor modifications as outlined by Shalabi et al [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. The synthetic substrate furanacroloyl-Phe-Glu-Glu (FAPGG) was used for the ACE-I enzyme. Briefly, 150 \u0026micro;L of FAPGG were pre-incubated at 37\u0026deg;C for 1 min. After this incubation, 10 \u0026micro;L of each sample and 10 \u0026micro;L of ACE-I enzyme (15 mU) were added to the substrate to initiate the enzymatic reaction. The reaction kinetics were monitored with a Synergy KTX microplate reader at a wavelength of 340 nm, with measurements taken every minute for a period of 30 min. Captopril served as a positive control. Hydrolysis of FAPGG by ACE-I resulted in a reduction in absorbance at 340 nm. Complete inhibition of ACE-I activity corresponded to 100% inhibition. The % of ACE-I inhibition was calculated using the formula:\u003c/p\u003e \u003cp\u003e%ACE-I = ((Abs \u003csub\u003eno sample\u003c/sub\u003e \u0026minus; Abs \u003csub\u003esample\u003c/sub\u003e)/Abs \u003csub\u003eno sample\u003c/sub\u003e)\u0026times;100.\u003c/p\u003e \u003cp\u003eMore precisely, Abs no sample​ refers to the absorbance of the enzyme-substrate mixture without the addition of any test sample, whereas Abs sample​ denotes the absorbance of the mixture containing the matrices under study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMS/MS peptidomic methods of HSD and HSD protein extract after in vitro digestion\u003c/h2\u003e \u003cp\u003eEndogenous peptides were identified using a peptidomic approach based on LC-nano tandem ESI mass spectrometry. This label-free shotgun method was performed without enzymatic digestion prior to MS/MS analysis. As part of the sample preparation, the permeate samples were first filtered through Whatman 54 paper filters (Florham Park, NJ), following the protocol described by Regmi et al. [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. The filtered component, representing the digested fraction, was further treated by ultrafiltration using 10 kDa centrifuge filters (Pierce\u0026trade;, Protein Concentrator PES, Thermo Fisher Scientific, UK) to remove high molecular weight proteins [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. The resulting fraction was then lyophilised to prepare it for mass spectrometric (MS) analysis.\u003c/p\u003e \u003cp\u003ePrior to MS, the lyophilised flow through- of HSD and HSD protein extract, containing peptides and low molecular weight proteins, was reconstituted in 0.3% (v/v) formic acid and subjected to desalting using Zip-Tip C18 columns (Millipore, Billerica, MA, USA) [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e, \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e]. Nano-HPLC coupled with MS/MS analysis was conducted using a Dionex Ultimate 3000 HPLC system equipped with an EASY-Spray\u0026trade; capillary column (15 cm \u0026times; 150 \u0026micro;m) packed with 2 \u0026micro;m C18 particles (100 \u0026Aring;). The system was interfaced with a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). The analysis utilized mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in 20/80 acetonitrile/water, v/v) at a flow rate of 0.300 \u0026micro;L/min, with the column temperature maintained at 35\u0026deg;C. The acquired raw files were subjected to data analysis using Proteome Discoverer software (version 1.4). The searches were performed against the NCBI reference Cannabis sativa (updated on November 2023; 33846 sequences) for HSD and HSD protein extract. The enzyme specificity was set as unspecific and methionine oxidation, and asparagine/glutamine deamidation were set as variable modifications [\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e]. Only peptides with high confidence were included for positive identification (X correlation\u0026thinsp;\u0026ge;\u0026thinsp;2.5). All peptides were searched in SATPdb, a database of structurally annotated therapeutic peptides, and in DFBP, a database of food-derived bioactive peptides [\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e, \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e]. To consider possible further proteolysis, the search was performed keeping a minimum sequence length of four amino acids and applying an \u0026ldquo;IF\u0026rdquo; nested function to a matrix which compared the sequence of each peptide found with the ones of the database (Microsoft Excel 2023, version 16.80) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eCell viability studies\u003c/h2\u003e \u003cp\u003eSamples (HSD and HSD protein extract) obtained following the digestive process were immediately frozen at -20\u0026deg;C to preserve their integrity. To assess their effect on C2C12 cells, the samples were subsequently subjected to freeze-drying. After this, the freeze-dried samples were accurately weighed and resuspended in 15 mL of cell growth medium (no-complete medium) [Dulbecco's Modified Eagle's Medium (DMEM) with high glucose content (Sigma-Aldrich, Milan, Italy)] and immediately filtered under vacuum at 0.22 \u0026micro;m. This procedure resulted in final concentrations of 41.1 mg/mL for HSDs and 48.4 mg/mL for HSDs protein extract. However, the initial concentration tested on the cells was 10.28 mg/mL for HSDs and 12.1 mg/mL for HSDs protein extract; values characterised by a non-toxic pH for the cells.\u003c/p\u003e \u003cp\u003eThe murine skeletal muscle cell line C2C12 (IZSLER, Brescia, Italy) was grown in complete growth medium for 15\u0026ndash;16 passages in 75 cm\u003csup\u003e2\u003c/sup\u003e culture flasks. The complete growth medium consisted of DMEM high in glucose (Sigma-Aldrich, Milan, Italy) supplemented with 10% foetal bovine serum (FBS, Immunological Sciences, Rome, Italy), 2 mM L-glutamine (Sigma-Aldrich, Milan, Italy) and 1% penicillin/streptomycin (Immunological Sciences, Rome, Italy). Cells were detached at 60\u0026ndash;70% confluence with 5 mL of trypsin-EDTA 1X (Immuno-logical Sciences, Rome, Italy) and cell counts were estimated using the trypan blue staining method (Sigma-Aldrich, Milan, Italy) with a haemocytometer. After trypsinization, the cells were seeded into 96-well plates at a density of 1.0\u0026times;10⁵ cells/mL, using complete medium as previously described. Following 24 h of incubation (time required for cell attachment and proliferation) at 37\u0026deg;C with 5% CO₂, the growth medium was removed and replaced with the specific treatments, as reported in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The cells were then incubated for an additional 24 h under the same conditions.\u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eConcentration of cellular treatments with HSDs and HSDS protein extracts\u003c/b\u003e. Values are expressed in mg/mL. The negative control consists of medium without Foetal Bovine Serum (FBS), while the positive control is complete growth medium. HSDs: Hempseeds.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eTREATMENTS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003eHSDs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eHSDs protein extract\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e10.28 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.17 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.1 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.19 mg/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5.14 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.09 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.05 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.09 mg/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2.77 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.05 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.00 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.05 mg/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1.39 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.02 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.50 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.02 mg/mL\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.69 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNegative control (0% FBS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.75 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNegative control (0% FBS)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e0.35 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePositive control (10% FBS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.38 mg/mL\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ePositive control (10% FBS)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eCell viability was assessed using the Alamar Blue colorimetric kit (Immunological Sciences, Societ\u0026agrave; Italiana Chimici, Rome, Italy). For the assay, 15 \u0026micro;L of ready-to-use Alamar Blue reagent was added to each well containing 150 \u0026micro;L of medium. Each plate was shaken for 30 ss and incubated for 3 h at 37\u0026deg;C with 5% CO₂. At the end of the incubation, the plates were read with a spectrophotometer at a wavelength of 570 nm. To account for background noise, the growth media for each treatment (without cells) were read at 600 nm. Cell viability was calculated by subtracting the optical density (OD) values of the media from the OD values measured at 570 nm for the cells. Each value was then standardised on the negative control (cells grown in DMEM without FBS) and statistically compared to the positive control (cells grown in complete growth medium).\u003c/p\u003e \u003cp\u003eFor the evaluation of HS, a temperature of 41.5\u0026deg;C was selected based on the study by Tang et al. [\u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e]. Specifically, C2C12 cells were seeded into 96-well plates at a concentration of 1.0\u0026times;10⁵ cells/mL in complete growth medium and incubated for 24 h at 37\u0026deg;C with 5% CO₂. After the initial 24 h incubation, the supernatant was removed and replaced with no-complete growth medium. Immediately the plates were incubated at 37\u0026deg;C (control, physiological condition) or exposed to 41.5\u0026deg;C for 1, 2, or 3 h. Viability was tested with the Alamar Blue kit, as previously described. Based on the data obtained and shown in results section, the 3 h incubation period was chosen for the evaluation of digest against HS.\u003c/p\u003e \u003cp\u003eTo evaluate the effects of digestates in mitigating HS, concentrations of 2.77, 1.39, 0.69, and 0.35 mg/mL for HSD and 3.00, 1.50, 0.75 and 0.38 mg/mL for HSD protein extract were chosen; i.e. concentrations that demonstrated greater efficacy in supporting cell viability. Specifically, C2C12 cells were seeded in 96-well plates at a density of 1.0 \u0026times; 10⁵ cells/mL in complete growth medium and incubated for 24 h at 37\u0026deg;C with 5% CO₂. Following the incubation, the growth medium was removed and replaced with the above treatments, which were incubated at the same conditions. After treatment, all media were removed and the cells were incubated in DMEM for 3 h at 37\u0026deg;C or 41.5\u0026deg;C. At the end of the HS period, cell viability was assessed with the Alamar Blue assay, as previously described.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll the data were analysed using GraphPad Prism 9 9.3.1 (GraphPad Software Inc., San Diego, CA, USA). The functional activities (TPC, ABTS, FRAP and ACE-I) were analysed using an independent samples t-test, assuming equal variances between the groups. Data on the effects of digestates on cell viability and HS determination at 1 h, 2 h and 3 h at 41.5\u0026deg;C were analysed using one-way ANOVA followed by Tukey's multiple comparison test. The analysis of the effect of digestates on HS was conducted using two-way ANOVA (\u003cem\u003eTime x Treatment\u003c/em\u003e) followed by Tukey's multiple comparison test. All data are reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM) of at least three independent experiments. Values are considered statistically significant for a 95% confidence intervale (\u003cem\u003ep-value\u0026thinsp;=\u0026thinsp;0.05\u003c/em\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eABTS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e(2,2\u0026prime;-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eACE-I\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eangiotensin-converting enzyme-I\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eADF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAcid Detergent Fibre\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eADL\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAcid Detergent Lignin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDMEM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDulbecco's Modified Eagle's Medium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eFAPGG\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003efuranacroloyl-Phe-Glu-Glu\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eFBS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFoetal Bovine Serum\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eFC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFolin-Ciocalteu\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eFRAP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFerric Reducing Antioxidant Power\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHeat Stress\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHSD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehempseed (when referred to the sample object of study)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHSPS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHeat-shock proteins\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMass Spectrometric\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNDF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eNeutral Detergent Fibre\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eOD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOptical Density\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eROS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReactive Oxygen Species\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eRT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRoom Temperature\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSEM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estandard error of the mean\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSGF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSimulated Gastric Fluids\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSIF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSimulated Intestinal Fluids\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSOF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eSimulated Oral Fluids\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTE\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTrolox Equivalent\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTPC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTotal Phenolic Content\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTPTZ\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2,4,6-tripyridyl-s-triazine.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was carried out in the frame of the JRC Visiting Scientist agreement No. 12/JRC.F.2/2023 (Directorate F - Health and Food JRC.F.2 Health Technologies).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding sources \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research received no specific grant from any funding agency, commercial or not-for-profit section.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets that were generated for this study are available on request to the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eD.L. and C.G. conceived and designed the study. Methodology was developed by D.L., E.P., F.G.S., G.T., and C.G. Software implementation and data validation were performed by D.L., E.P., F.G.S., G.T., and C.G. Data curation was carried out by D.L., E.P., F.G.S., G.T., and C.G. The original draft of the manuscript was written by D.L., S.G., I.G., and C.G. All authors contributed to the review and editing of the final manuscript and approved its submission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eORCID\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDavide Lanzoni: 0000-0002-8233-659X.\u003c/p\u003e\n\u003cp\u003eElena Petrosillo: 0009-0000-8222-6430.\u003c/p\u003e\n\u003cp\u003eFrancesca Grassi Scalvini: 0000-0002-2995-1112.\u003c/p\u003e\n\u003cp\u003eJoshua Grana: 0009-0007-9141-8726.\u003c/p\u003e\n\u003cp\u003eGabriella Tedeschi: 0000-0003-2082-6443.\u003c/p\u003e\n\u003cp\u003eSabrina Gioria: 0000-0001-7150-9523.\u003c/p\u003e\n\u003cp\u003eEva Pěchoučkov\u0026aacute;: 0000-0001-9336-5436.\u003c/p\u003e\n\u003cp\u003eIan Givens: 0000-0002-6754-6935.\u003c/p\u003e\n\u003cp\u003eCarlotta Giromini: 0000-0002-3717-5336.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eClimate Copernicus. 2023 is the hottest year on record, with global temperatures close to the 1.5\u0026deg;C limit. Climate Copernicus. (2024). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.climate.copernicus.eu\u003c/span\u003e\u003cspan address=\"https://www.climate.copernicus.eu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKovats, R. S. \u0026amp; Hajat, S. Heat stress and public health: a critical review. \u003cem\u003eAnnu. Rev. Public. Health\u003c/em\u003e. \u003cb\u003e29\u003c/b\u003e, 41\u0026ndash;55 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCramer, M. N., Gagnon, D., Laitano, O. \u0026amp; Crandall, C. G. Human temperature regulation under heat stress in health, disease, and injury. \u003cem\u003ePhysiol. Rev.\u003c/em\u003e \u003cb\u003e102\u003c/b\u003e, 1907\u0026ndash;1989 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRhoads, R. P., Baumgard, L. H., Suagee, J. K. \u0026amp; Sanders, S. R. Nutritional interventions to alleviate the negative consequences of heat stress. \u003cem\u003eAdv. Nutr.\u003c/em\u003e \u003cb\u003e4\u003c/b\u003e, 267\u0026ndash;276 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGanesan, S. et al. Short-term heat stress causes altered intracellular signaling in oxidative skeletal muscle. \u003cem\u003eJ. Anim. Sci.\u003c/em\u003e \u003cb\u003e95\u003c/b\u003e, 2438\u0026ndash;2451 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao, L. et al. Heat stress decreases metabolic flexibility in skeletal muscle of growing pigs. \u003cem\u003eAJP-Regul Integr. Comp. Physiol.\u003c/em\u003e \u003cb\u003e315\u003c/b\u003e, R1096\u0026ndash;R1106 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarinon, B., Molinari, R., Costantini, L. \u0026amp; Merendino, N. The seed of industrial hemp (Cannabis sativa L.): Nutritional quality and potential functionality for human health and nutrition. \u003cem\u003eNutrients\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 1935 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLanzoni, D. et al. Total phenolic content and antioxidant activity of in vitro digested hemp-based products. \u003cem\u003eFoods\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 601 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHouse, J. D., Neufeld, J. \u0026amp; Leson, G. Evaluating the quality of protein from hemp seed (Cannabis sativa L.) products through the use of the protein digestibility-corrected amino acid score method. \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cb\u003e58\u003c/b\u003e, 11801\u0026ndash;11807 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMylostуva, D. et al. Biochemical changes during heat stress in productive animals with an emphasis on the antioxidant defense system. \u003cem\u003eJ. Anim. Behav. Biometeorol.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 0\u0026ndash;0 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKeservani, R. K. et al. Nutraceutical and functional food as future food: A review. \u003cem\u003eDer Pharm. Lett.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, 106\u0026ndash;116 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeonard, W., Zhang, P., Ying, D. \u0026amp; Fang, Z. Hempseed in food industry: Nutritional value, health benefits, and industrial applications. \u003cem\u003eCompr. Rev. Food Sci. Food Saf.\u003c/em\u003e \u003cb\u003e19\u003c/b\u003e, 282\u0026ndash;308 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiromini, C., Lanzoni, D., Rebucci, R. \u0026amp; Baldi, A. Bioactive peptides from milk and dairy proteins: models of digestion and intestinal barrier. In: Protein Digestion-Derived Peptides, Academic, 145\u0026ndash;173. (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLanzoni, D. et al. Phenolic profile and antioxidant activity of hemp co-products following green chemical extraction and ex vivo digestion. \u003cem\u003eItal. J. Anim. Sci.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 651\u0026ndash;663 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLucas-Gonz\u0026aacute;lez, R., Viuda-Martos, M., \u0026Aacute;lvarez, J. A. P. \u0026amp; Fern\u0026aacute;ndez-L\u0026oacute;pez, J. Changes in bioaccessibility, polyphenol profile and antioxidant potential of flours obtained from persimmon fruit (Diospyros kaki) co-products during in vitro gastrointestinal digestion. \u003cem\u003eFood Chem.\u003c/em\u003e \u003cb\u003e256\u003c/b\u003e, 252\u0026ndash;258 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa, Y. et al. Phenolics and antioxidant activity of bamboo leaves soup as affected by in vitro digestion. \u003cem\u003eFood Chem. Toxicol.\u003c/em\u003e \u003cb\u003e135\u003c/b\u003e, 110941 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang, C. H., Wang, X. S. \u0026amp; Yang, X. Q. Enzymatic hydrolysis of hemp (Cannabis sativa L.) protein isolate by various proteases and antioxidant properties of the resulting hydrolysates. \u003cem\u003eFood Chem.\u003c/em\u003e \u003cb\u003e114\u003c/b\u003e, 1484\u0026ndash;1490 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, X. S., Tang, C. H., Chen, L. \u0026amp; Yang, X. Q. Characterization and antioxidant properties of hemp protein hydrolysates obtained with Neutrase\u0026reg;. \u003cem\u003eFTB\u003c/em\u003e \u003cb\u003e47\u003c/b\u003e, 428\u0026ndash;434 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalomo, S. A., Onuh, J. O., Girgih, A. T. \u0026amp; Aluko, R. E. Structural and antihypertensive properties of enzymatic hemp seed protein hydrolysates. \u003cem\u003eNutrients\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePfeffer, M. A. Angiotensin-converting enzyme inhibition in congestive heart failure: benefit and perspective. \u003cem\u003eAm. Heart J.\u003c/em\u003e \u003cb\u003e126\u003c/b\u003e, 789\u0026ndash;793 (1993).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRaghavan, S. \u0026amp; Kristinsson, H. G. ACE-inhibitory activity of tilapia protein hydrolysates. \u003cem\u003eFood Chem.\u003c/em\u003e \u003cb\u003e117\u003c/b\u003e, 582\u0026ndash;588 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFlather, M. D. et al. Long-term ACE-inhibitor therapy in patients with heart failure or left-ventricular dysfunction: a systematic overview of data from individual patients. \u003cem\u003eLancet\u003c/em\u003e \u003cb\u003e355\u003c/b\u003e, 1575\u0026ndash;1581 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePahor, M. et al. Therapeutic benefits of ACE inhibitors and other antihypertensive drugs in patients with type 2 diabetes. \u003cem\u003eDiabetes Care\u003c/em\u003e. \u003cb\u003e23\u003c/b\u003e, 888\u0026ndash;892 (2000).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, Y., Zhou, J., Huang, K., Sun, Y. \u0026amp; Zeng, X. Purification of a novel angiotensin I-converting enzyme (ACE) inhibitory peptide with an antihypertensive effect from loach (Misgurnus anguillicaudatus). \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cb\u003e60\u003c/b\u003e, 1320\u0026ndash;1325 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVermeirssen, V., Van Camp, J. \u0026amp; Verstraete, W. Fractionation of angiotensin I converting enzyme inhibitory activity from pea and whey protein in vitro gastrointestinal digests. \u003cem\u003eJ. Sci. Food Agric.\u003c/em\u003e \u003cb\u003e85\u003c/b\u003e, 399\u0026ndash;405 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiguel, M., Manso, M. A., L\u0026oacute;pez-Fandi\u0026ntilde;o, R., Alonso, M. J. \u0026amp; Salaices, M. Vascular effects and antihypertensive properties of κ-casein macropeptide. \u003cem\u003eInt. Dairy. J.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 1473\u0026ndash;1477 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBougatef, A. et al. Angiotensin I-converting enzyme (ACE) inhibitory activities of sardinelle (Sardinella aurita) by-products protein hydrolysates obtained by treatment with microbial and visceral fish serine proteases. \u003cem\u003eFood Chem.\u003c/em\u003e \u003cb\u003e111\u003c/b\u003e, 350\u0026ndash;356 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmad, I., Yanuar, A., Mulia, K. \u0026amp; Mun\u0026rsquo;im, A. Review of angiotensin-converting enzyme inhibitory assay: Rapid method in drug discovery of herbal plants. \u003cem\u003ePhcog Rev.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 1 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVermeirssen, V., Van Camp, J., Devos, L. \u0026amp; Verstraete, W. Release of angiotensin I converting enzyme (ACE) inhibitory activity during in vitro gastrointestinal digestion: From batch experiment to semicontinuous model. \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cb\u003e51\u003c/b\u003e, 5680\u0026ndash;5687 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLanzoni, D. et al. Antioxidant capacity and peptidomic analysis of in vitro digested Camelina sativa L. Crantz and Cynara cardunculus co-products. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 14456 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSegura Campos, M. R., Peralta Gonz\u0026aacute;lez, F., Chel Guerrero, L. \u0026amp; Betancur Ancona, D. Angiotensin I-converting enzyme inhibitory peptides of chia (Salvia hispanica) produced by enzymatic hydrolysis. \u003cem\u003eInt. J. Food Sci.\u003c/em\u003e \u003cb\u003e1\u003c/b\u003e, 158482 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTeh, S. S., Bekhit, A. E. D. A., Carne, A. \u0026amp; Birch, J. Antioxidant and ACE-inhibitory activities of hemp (Cannabis sativa L.) protein hydrolysates produced by the proteases AFP, HT, Pro-G, actinidin and zingibain. \u003cem\u003eFood Chem.\u003c/em\u003e \u003cb\u003e203\u003c/b\u003e, 199\u0026ndash;206 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIsraili, Z. H., Hern\u0026aacute;ndez-Hern\u0026aacute;ndez, R. \u0026amp; Valasco, M. The future of antihypertensive treatment. \u003cem\u003eAm. J. Ther.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 121\u0026ndash;134 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeonard, W., Xiong, Y., Zhang, P., Ying, D. \u0026amp; Fang, Z. Enhanced lignanamide absorption and antioxidative effect of extruded hempseed (Cannabis sativa L.) hull in Caco-2 intestinal cell culture. \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cb\u003e69\u003c/b\u003e, 11259\u0026ndash;11271 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchwab, U. S. et al. Effects of hempseed and flaxseed oils on the profile of serum lipids, serum total and lipoprotein lipid concentrations and haemostatic factors. \u003cem\u003eEur. J. Nutr.\u003c/em\u003e \u003cb\u003e45\u003c/b\u003e, 470\u0026ndash;477 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRezapour-Firouzi, S. et al. Immunomodulatory and therapeutic effects of Hot-nature diet and co-supplemented hemp seed, evening primrose oils intervention in multiple sclerosis patients. \u003cem\u003eCAM\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, 473\u0026ndash;480 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeinert, D. J. Nutrition and muscle protein synthesis: A descriptive review. \u003cem\u003eJCCA\u003c/em\u003e \u003cb\u003e53\u003c/b\u003e, 186 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMithal, A. et al. Impact of nutrition on muscle mass, strength, and performance in older adults. \u003cem\u003eOsteoporos. Int.\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e, 1555\u0026ndash;1566 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMcGlory, C., van Vliet, S., Stokes, T., Mittendorfer, B. \u0026amp; Phillips, S. M. The impact of exercise and nutrition on the regulation of skeletal muscle mass. \u003cem\u003eJ. Physiol.\u003c/em\u003e \u003cb\u003e597\u003c/b\u003e, 1251\u0026ndash;1258 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eManabe et al. Characterization of an acute muscle contraction model using cultured C2C12 myotubes. \u003cem\u003ePloS One\u003c/em\u003e. \u003cb\u003e7\u003c/b\u003e, e52592 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiller, D. D. Minerals. in: Fennema\u0026rsquo;s Food Chemistry, CRC, 627\u0026ndash;679 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoeseler, D. A. et al. Muscle protein signaling in C2C12 cells is stimulated to similar degrees by diverse commercial food protein sources and experimental soy protein hydrolysates. \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cb\u003e65\u003c/b\u003e, 2956\u0026ndash;2964 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKamei, Y., Hatazawa, Y., Uchitomi, R., Yoshimura, R. \u0026amp; Miura, S. Regulation of skeletal muscle function by amino acids. \u003cem\u003eNutrients\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 261 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRusso, R. \u0026amp; Reggiani, R. Evaluation of protein concentration, amino acid profile and antinutritional compounds in hempseed meal from dioecious and monoecious varieties. \u003cem\u003eAm. J. Plant. Sci.\u003c/em\u003e \u003cb\u003e6\u003c/b\u003e, 14\u0026ndash;22 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeder, J. K. \u0026amp; Kahleyss, R. Reaction of Lentil Trypsin\u0026thinsp;\u0026ndash;\u0026thinsp;Chymotrypsin Inhibitors with Human and Bovine Proteinases. \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cb\u003e51\u003c/b\u003e, 8045\u0026ndash;8050 (2003).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarbone, J. W., McClung, J. P. \u0026amp; Pasiakos, S. M. Skeletal muscle responses to negative energy balance: effects of dietary protein. \u003cem\u003eAdv. Nutr.\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e, 119\u0026ndash;126 (2012).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, Z. A. \u0026amp; Zheng, J. S. Advances in peptide synthesis. In: Peptide and Peptidomimetic Therapeutics. Academic, 73\u0026ndash;97. (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMamone, G., Picariello, G., Ramondo, A., Nicolai, M. A. \u0026amp; Ferranti, P. Production, digestibility and allergenicity of hemp (Cannabis sativa L.) protein isolates. \u003cem\u003eFood Res. Int.\u003c/em\u003e \u003cb\u003e115\u003c/b\u003e, 562\u0026ndash;571 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFujita, S. \u0026amp; Volpi, E. Amino acids and muscle loss with aging. \u003cem\u003eJ. Nutr.\u003c/em\u003e \u003cb\u003e136\u003c/b\u003e, 277S\u0026ndash;280S (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCallaway, J. C. Hempseed as a nutritional resource: An overview. \u003cem\u003eEuphytica\u003c/em\u003e \u003cb\u003e140\u003c/b\u003e, 65\u0026ndash;72 (2004).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMattila, P. et al. Nutritional value of commercial protein-rich plant products. \u003cem\u003ePlant. Foods Hum. Nutr.\u003c/em\u003e \u003cb\u003e73\u003c/b\u003e, 108\u0026ndash;115 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOseyko, M., Sova, N., Lutsenko, M. \u0026amp; Kalyna, V. Chemical aspects of the composition of industrial hemp seed products. \u003cem\u003eUkr. Food J.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 544\u0026ndash;559 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNedachi, T., Kadotani, A., Ariga, M., Katagiri, H. \u0026amp; Kanzaki, M. Ambient glucose levels qualify the potency of insulin myogenic actions by regulating SIRT1 and FoxO3a in C2C12 myocytes. \u003cem\u003eAm. J. Physiol. Endocrinol. Metab.\u003c/em\u003e \u003cb\u003e294\u003c/b\u003e, E668\u0026ndash;E678 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei, P., Tang, Y., Zhou, K., Wei, Z. \u0026amp; Liu, G. Characteristics of polysaccharides from industrial hemp (Cannabis sativa L.) kernels. \u003cem\u003eFoods\u003c/em\u003e \u003cb\u003e13\u003c/b\u003e, 3429 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShils, M. E. Magnesium. In: (eds Shils, M. E. \u0026amp; Young, V. R.) Modern Nutrition in Health and Disease, 7th ed., Lea \u0026amp; Febiger, Philadelphia, 159\u0026ndash;192. (1988).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNational Research Council. Minerals. In: Diet and Health: Implications for Reducing Chronic Disease Risk, National Academies Press (US), (1989).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBhowmik, D., Chiranjib, K. \u0026amp; Kumar, S. A potential medicinal importance of zinc in human health and chronic. \u003cem\u003eInt. J. Pharm.\u003c/em\u003e \u003cb\u003e1\u003c/b\u003e, 5\u0026ndash;11 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFuller, T. J., Carter, N. W., Barcenas, C. A. \u0026amp; Knochel, J. P. Reversible changes of the muscle cell in experimental phosphorus deficiency. \u003cem\u003eJ. Clin. Invest.\u003c/em\u003e \u003cb\u003e57\u003c/b\u003e, 1019\u0026ndash;1024 (1976).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta, C. P. Role of iron (Fe) in body. \u003cem\u003eIOSR J. Appl. Chem.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 38\u0026ndash;46 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEFSA NDA Panel (EFSA Panel on Dietetic Products, Nutrition and Allergies). Overview on dietary reference values for the EU population as derived by the EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), European Food Safety Authority, 2017. Available online: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.efsa.europa.eu/sites/default/files/assets/DRV_Summary_tables_jan_17.pdf\u003c/span\u003e\u003cspan address=\"https://www.efsa.europa.eu/sites/default/files/assets/DRV_Summary_tables_jan_17.pdf\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrenkert, A. L. \u0026amp; Malone, E. L. Modeling vulnerability and resilience to climate change: a case study of India and Indian states. \u003cem\u003eClim. Change\u003c/em\u003e. \u003cb\u003e72\u003c/b\u003e, 57\u0026ndash;102 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHabibi, P. et al. Climate change and heat stress resilient outdoor workers: findings from systematic literature review. \u003cem\u003eBMC Public. Health\u003c/em\u003e. \u003cb\u003e24\u003c/b\u003e, 1711 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePark, H. G., Han, S. I., Oh, S. Y. \u0026amp; Kang, H. S. Cellular responses to mild heat stress. \u003cem\u003eCMLS\u003c/em\u003e \u003cb\u003e62\u003c/b\u003e, 10\u0026ndash;23 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSummers, C. M. \u0026amp; Valentine, R. J. Two hours of heat stress induces MAP-kinase signaling and autophagasome accumulation in C2C12 myotubes. \u003cem\u003eCell. Biochem. Biophys.\u003c/em\u003e \u003cb\u003e80\u003c/b\u003e, 367\u0026ndash;373 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao et al. Heat stress inhibits proliferation, promotes growth, and induces apoptosis in cultured Lantang swine skeletal muscle satellite cells. \u003cem\u003eJ. Zhejiang Univ. Sci. B\u003c/em\u003e. \u003cb\u003e16\u003c/b\u003e, 549 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelhadj et al. Reactive oxygen species, heat stress and oxidative-induced mitochondrial damage: a review. \u003cem\u003eInt. J. Hyperth.\u003c/em\u003e \u003cb\u003e30\u003c/b\u003e, 513\u0026ndash;523 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLocke, M. \u0026amp; Celotti, C. The effect of heat stress on skeletal muscle contractile properties. \u003cem\u003eCell. Stress Chaperones\u003c/em\u003e. \u003cb\u003e19\u003c/b\u003e, 519\u0026ndash;527 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePechan, P. M. Heat shock proteins and cell proliferation. \u003cem\u003eFEBS Lett.\u003c/em\u003e \u003cb\u003e280\u003c/b\u003e, 1\u0026ndash;4 (1991).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang, P. \u0026amp; MacRae, T. H. Molecular chaperones and the cytoskeleton. \u003cem\u003eJ. Cell. Sci.\u003c/em\u003e \u003cb\u003e110\u003c/b\u003e, 1431\u0026ndash;1440 (1997).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoss, R. A. \u0026amp; Linnemans, W. A. M. The effects of hyperthermia on the cytoskeleton: a review. \u003cem\u003eInt. J. Hyperth.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 173\u0026ndash;196 (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMattson, M. P. Hormesis defined. \u003cem\u003eAgeing Res. Rev.\u003c/em\u003e \u003cb\u003e7\u003c/b\u003e, 1\u0026ndash;7 (2008).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHayes, D. P. Nutritional hormesis. \u003cem\u003eEur. J. Clin. Nutr.\u003c/em\u003e \u003cb\u003e61\u003c/b\u003e, 147\u0026ndash;159 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi, G. et al. Antioxidant activity of docosahexaenoic acid (DHA) and its regulatory roles in mitochondria. \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cb\u003e69\u003c/b\u003e, 1647\u0026ndash;1655 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiao, B. et al. Eicosapentaenoic acid (EPA) exhibits antioxidant activity via mitochondrial modulation. \u003cem\u003eFood Chem.\u003c/em\u003e \u003cb\u003e373\u003c/b\u003e, 131389 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMickleborough, T. D. Omega-3 polyunsaturated fatty acids in physical performance optimization. \u003cem\u003eIJSNEM\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 83\u0026ndash;96 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMontserrat-de la Paz, S., Mar\u0026iacute;n-Aguilar, F., Garc\u0026iacute;a-Gimenez, M. D. \u0026amp; Fern\u0026aacute;ndez-Arche, M. A. Hemp (Cannabis sativa L.) seed oil: analytical and phytochemical characterization of the unsaponifiable fraction. \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cb\u003e62\u003c/b\u003e, 1105\u0026ndash;1110 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFischer, C. P. et al. Vitamin E isoform-specific inhibition of the exercise-induced heat shock protein 72 expression in humans. \u003cem\u003eJ. Appl. Physiol.\u003c/em\u003e \u003cb\u003e100\u003c/b\u003e, 1679\u0026ndash;1687 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCumming, K. T. et al. Effects of vitamin C and E supplementation on endogenous antioxidant systems and heat shock proteins in response to endurance training. \u003cem\u003ePhysiol. Rep.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e, e12142 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSahin, K., Sahin, N., Onderci, M., Yaralioglu, S. \u0026amp; Kucuk, O. Protective role of supplemental vitamin E on lipid peroxidation, vitamins E, A and some mineral concentrations of broilers reared under heat stress. \u003cem\u003eVet. Med-CZECH\u003c/em\u003e. \u003cb\u003e46\u003c/b\u003e, 140 (2001).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSivakumar, A. V. N., Singh, G. \u0026amp; Varshney, V. P. Antioxidants supplementation on acid-base balance during heat stress in goats. \u003cem\u003eAsian-Australasian J. Anim. Sci.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e, 1462\u0026ndash;1468 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAjakaiye, J. J., Perez-Bello, A. \u0026amp; Mollineda-Trujillo, A. Impact of heat stress on egg quality in layer hens supplemented with l-ascorbic acid and dl-tocopherol acetate. \u003cem\u003eVet. Arh\u003c/em\u003e. \u003cb\u003e81\u003c/b\u003e, 119\u0026ndash;132 (2010).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEuropean Union. Council Regulation (EU) 1370/2013 of 16 December 2013 determining measures on fixing certain aids and refunds related to the common organisation of the markets in agricultural products. (2013). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://eur-lex.europa.eu\u003c/span\u003e\u003cspan address=\"https://eur-lex.europa.eu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSorrentino, G. Introduction to emerging industrial applications of cannabis (Cannabis sativa L). \u003cem\u003eRend. Lin Sci. Fis.\u003c/em\u003e \u003cb\u003e32\u003c/b\u003e, 233\u0026ndash;243 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMinekus, M. et al. A standardised static in vitro digestion method suitable for food\u0026mdash;an international consensus. \u003cem\u003eFood Funct.\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e, 1113\u0026ndash;1124 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrodkorb et al. INFOGEST static in vitro simulation of gastrointestinal food digestion. \u003cem\u003eNat. Protoc.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 991\u0026ndash;1014 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRegmi, P. R., Ferguson, N. S. \u0026amp; Zijlstra, R. T. In vitro digestibility techniques to predict apparent total tract energy digestibility of wheat in grower pigs. \u003cem\u003eJ. Anim. Sci.\u003c/em\u003e \u003cb\u003e87\u003c/b\u003e, 3620\u0026ndash;3629 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAttard, E. A rapid microtitre plate Folin-Ciocalteu method for the assessment of polyphenols. \u003cem\u003eOpen. Life Sci.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 48\u0026ndash;53 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRe, R. et al. Antioxidant activity applying an improved ABTS radical cation decolorization assay. \u003cem\u003eFree Radic Biol. Med.\u003c/em\u003e \u003cb\u003e26\u003c/b\u003e, 1231\u0026ndash;1237 (1999).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdelaleem, M. A. \u0026amp; Elbassiony, K. R. A. Evaluation of phytochemicals and antioxidant activity of gamma-irradiated quinoa (Chenopodium quinoa). \u003cem\u003eBraz J. Biol.\u003c/em\u003e \u003cb\u003e81\u003c/b\u003e, 806\u0026ndash;813 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiromini, C., Fekete, \u0026Aacute;. A., Givens, D. I., Baldi, A. \u0026amp; Lovegrove, J. A. Short-communication: a comparison of the in vitro angiotensin-1-converting enzyme inhibitory capacity of dairy and plant protein supplements. \u003cem\u003eNutrients\u003c/em\u003e \u003cb\u003e9\u003c/b\u003e, 1352 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShalaby, S. M., Zakora, M. \u0026amp; Otte, J. Performance of two commonly used angiotensin-converting enzyme inhibition assays using FA-PGG and HHL as substrates. \u003cem\u003eJ. Dairy. Res.\u003c/em\u003e \u003cb\u003e73\u003c/b\u003e, 178\u0026ndash;186 (2006).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTavares, T. G. et al. Optimisation, by response surface methodology, of degree of hydrolysis and antioxidant and ACE-inhibitory activities of whey protein hydrolysates obtained with cardoon extract. \u003cem\u003eInt. Dairy. J.\u003c/em\u003e \u003cb\u003e21\u003c/b\u003e, 926\u0026ndash;933 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNonnis, S. et al. Acute environmental temperature variation affects brain protein expression, anxiety and explorative behaviour in adult zebrafish. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e11\u003c/b\u003e, 2521 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCapraro, J. et al. Internalisation and multiple phosphorylation of γ-Conglutin, the lupin seed glycaemia-lowering protein, in HepG2 cells. \u003cem\u003eBiochem. Biophys. Res. Commun.\u003c/em\u003e \u003cb\u003e437\u003c/b\u003e, 648\u0026ndash;652 (2013).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh et al. SATPdb: a database of structurally annotated therapeutic peptides. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cb\u003e44\u003c/b\u003e, D1119\u0026ndash;D1126 (2016).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQin, D. et al. DFBP: a comprehensive database of food-derived bioactive peptides for peptidomics research. \u003cem\u003eBioinformatics\u003c/em\u003e \u003cb\u003e38\u003c/b\u003e, 3275\u0026ndash;3280 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang, J. et al. Damage to the myogenic differentiation of C2C12 cells by heat stress is associated with up-regulation of several selenoproteins. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 1\u0026ndash;9 (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Antioxidant Activity, Digestion, Heat Stress, Muscle Cell, Hempseed, Peptidomic","lastPublishedDoi":"10.21203/rs.3.rs-6511890/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6511890/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe 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 (\u003cem\u003eCannabis sativa\u003c/em\u003e 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 \u003cem\u003ein vitro\u003c/em\u003e 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\u0026deg;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\u0026mdash;particularly omega-3 and omega-6 polyunsaturated fatty acids and tocopherols\u0026mdash;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.\u003c/p\u003e","manuscriptTitle":"Hempseed and hempseed protein extract: antioxidant potential, peptidomic analysis and muscle cell protection under heat stress conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-28 07:11:07","doi":"10.21203/rs.3.rs-6511890/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4bd3f232-a05d-449f-a6b0-322275f9d0b1","owner":[],"postedDate":"May 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49040477,"name":"Biological sciences/Biotechnology/Proteomics"},{"id":49040478,"name":"Biological sciences/Biotechnology"},{"id":49040479,"name":"Biological sciences/Biochemistry/Peptides"},{"id":49040480,"name":"Biological sciences/Cell biology/Cell growth"}],"tags":[],"updatedAt":"2025-12-16T11:10:07+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-28 07:11:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6511890","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6511890","identity":"rs-6511890","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}