Bioactive peptides are formed by linking two or more proteinogenic amino acids via peptide bonds, typically ranging in length from 2 to 20 amino acids [1]. These peptides are inert in their primary protein structure and require complete hydrolysis to exhibit their functional properties [2]. They can be released through hydrolysis by proteolytic enzymes, fermentation, or gastrointestinal digestion [3]. Due to their high activity, selectivity and minimal side effects, bioactive peptides have attracted considerable attention. Various functional peptides have been documented, encompassing antihypertensive [4], antidiabetic [5], anti-aging [6], antibacterial [7], antioxidant [8], anti-inflammatory [9], anticancer [10], and immunomodulatory properties [11]. The activity or function of a peptide is primarily dictated by its structural characteristics, including molecular weight, length, sequence (especially the type of C- and N-terminal residues), hydrophobicity, charge characteristics, and spatial conformation [12]. Exploring the structure-activity relationship reveals the connection between a compound's molecular structure and its biological activity, providing essential guidance for drug design and optimization [13].

With the growing interest in bioactive peptides, numerous angiotensin converting enzyme (ACE) inhibitory peptides have been identified and characterized. Additionally, computational models, such as artificial neural networks and Quantitative Structure-Activity Relationship (QSAR) modeling, have emerged to elucidate the structure-activity relationship [14,15]. ACE is a Zn2+ dependent carboxypeptidase with an active site comprising three subsites: S1, S2, and S1’ [16]. Peptides with shorter chains can reach the active pocket of ACE more easily, so potent ACE inhibitory peptides typically consist of 2–12 amino acid residues [17]. The binding affinity of peptides to ACE is primarily influenced by their hydrophobic amino acid content, which strongly correlates with their inhibitory potency. Statistical analysis by Ding et al. revealed a high prevalence of hydrophobic amino acids at the N-terminus (65.70 %) and C-terminus (57.46 %) of ACE inhibitory peptides [18]. These findings underscore the critical role of terminal hydrophobic amino acids in ACE inhibition. Moreover, previous QSAR studies had highlighted the C-terminus of the peptides play more significant role on ACE inhibition than the N-terminal residue [[19], [20], [21]], which strongly hinted the hydrophobic C-terminus favoring the ACE inhibition. For instance, studies indicated that the existence of Ile, Leu, Phe, Pro, Trp, or Val in the C-terminal region enhanced ACE inhibitory activity [22]. This enhancement is likely due to the strong interaction between hydrophobic residues at the C-terminus and the ACE active site, which prefer nonpolar amino acids. Hydrophobic interactions facilitate peptide binding, improving stability within the active site and increasing inhibitory potency [18,23,24].

The gut microbiota and its metabolites play a critical role in multiple physiological processes, including digestion, immune function, metabolic regulation, maintenance of the intestinal barrier, and modulation of the nervous system [25]. The association between gut microbiota and blood pressure has been firmly established in recent years [26]. For instance, fecal microbiota transplantation (FMT) from hypertensive patients to germ-free mice has been shown to significantly elevates systolic and diastolic blood pressure, directly demonstrating the impact of gut microbiota on host blood pressure [27]. Furthermore, medical interventions like FMT or supplementation with specific microbial strains effectively modulate blood pressure in animal models [28]. Short-chain fatty acids (SCFAs), the primary metabolites produced by gut microbiota, play a significant role in blood pressure regulation after absorption in the gut [25]. Maintaining gut microbiota homeostasis is essential for effective blood pressure management, highlighting its potential as a novel therapeutic target for hypertension. Recent studies indicate that ACE inhibitory peptides can simultaneously lower blood pressure and modulate gut microbiota composition. For example, the walnut-derived peptide FDWLR and the α-lactalbumin-derived peptide VGINYW exhibit potent antihypertensive effects by regulating the renin-angiotensin-aldosterone system (RAAS), gut microbiota, and SCFA levels [29,30].

Pistachio is a nutritious food, rich in proteins, lipids, vitamins, and essential minerals [31]. With a protein content of 19.80 ± 0.49 g/100 g in the edible portion and a hydrophobic amino acid percentage of 40.7 %, pistachio stands out as a promising source of ACE inhibitory peptides [32]. Our previous work had demonstrated that hydrolysates of pistachio kernel proteins, prepared using gastrointestinal digestive enzymes, exhibited antihypertensive effects in rats. Moreover, a peptide (ACKEP) pivotal for ACE binding was isolated from the hydrolysate, indicating the potential of pistachio kernel proteins as a source of bioactive peptides with antihypertensive properties [33].

Therefore, the objective of this work is to discover novel and potent antihypertensive peptides derived from the tryptic hydrolysate of pistachio proteins and explore the correlation between peptide structure and activity. Pistachio kernel powder was hydrolyzed using trypsin, followed by the isolation and purification of the hydrolysate via ultrafiltration, gel chromatography, and reversed-phase high performance liquid chromatography (RP-HPLC) techniques. Subsequently, the peptide exhibiting the highest ACE inhibitory activity was utilized as a model molecule to elucidate the key role of the C-terminal amino acids in ACE inhibition. Furthermore, the acute and subacute antihypertensive effects of the peptide were evaluated in vivo, as well as the mechanism illustration based on serum ACE activity and angiotensin Ⅱ (Ang Ⅱ) levels, fecal SCFA levels, and gut microbiota analysis.