## 4 Discussion  
Cardiovascular disease is the major cause of morbidity and mortality in chronic glycemia and hypertension [27; 28]. As such, diabetes and hypertension are closely linked by similar risk factors, such as endothelial dysfunction, oxidative stress, arterial atherosclerosis, obesity, and inflammation processes [27]. Diabetic and hypertensive pathways also overlap some cardiovascular complications related primarily to micro- and macrovascular disease [27]. Common mechanisms, such as upregulation of the renin-angiotensin-aldosterone system, oxidative stress, inflammation, and activation of the immune system likely contribute to the close relationship between diabetes and hypertension. Nutritional interventions were consistently proposed as a part of a comprehensive strategy to lower the incidence and severity of cardiovascular diseases and their risk factors [1]. In addition to the immense benefits of phenolic and non-peptic compounds, bioactive peptides are considered a viable treatment and prevention alternative, owing to their inhibitory nature of various disease-related enzymes [2]. Edible insects are undoubtedly an underexploited source of protein and bioactive peptides. Literature evidence shows that the most therapeutically effective edible insect preparations are in the form of peptides derived from protein hydrolyses, which showed inhibitory effects towards inflammation, hypertension, diabetes, and microbial growth [9]. In our previous work, we evaluated the potential antioxidant, ACE, and DPP-IV inhibiting capabilities of various cricket protein hydrolyses (CPH), using the commercial enzyme alcalase. CPH prepared with the highest enzyme concentration (3.0%) and incubated for 80 min, showed the best bioactivity among other hydrolysate preparations. The observed activity remained or improved after SGID, suggesting further liberation of active peptides or stability towards gastrointestinal enzymes [7]. This study highlighted the conditions necessary to produce cricket protein hydrolyses that might be biologically active. However, the peptide/peptide group responsible was not yet characterized. The main objective of this study was to isolate and characterize peptide groups present within cricket protein hydrolyses, primarily responsible for its potential bioactivity. In general, active enzymatic hydrolyses contain peptides with a wide range of molecular weights, amino acid sequences, hydrophobicity and hydrophilicity, charge, and bioactive efficacies; therefore, this work focused on assay-directed fractionation. The SGID digests of the protein hydrolyses (CPHD) were chosen as the starting material for fractionation, since peptides likely exhibit their properties after digestion and absorbance into the blood system. Following the data described in Figure 1 and Table 1, the CPHD were first separated by molecular weight size. There were four major groups of peptides (Fractions S1-S4) with distinguished molecular weights and retention time. Fraction S4 was identified as having the best inhibiting activity towards ACE, DPP-IV, and \(\alpha\)-glucosidase, and was further separated using RP-HPLC. From the chromatographic profile at 214 nm (Supplementary Materials Figure S1a), fraction S4 was still a complex mixture of peptides, which was then separated according to the hydrophobicity of the peptides (F1-F6). ACE and \(\alpha\)-glucosidase inhibition assays revealed that fraction F6 had better inhibition activity compared to the other fractions. After two stages of fractionation, S6 and F4 likely contained hydrophobic peptides below 10 amino acids, similar to known biologically relevant peptides [2]. Food-derived bioactive peptides usually do not have any specific consensus on amino acid sequences that are responsible for their biological activity, but most maintain certain common features, such as having a positive/neutral charge and relatively hydrophobic and amphipathic structure [29].