*3.1. Antihypertensive Peptides*

Cardiovascular disease (CVD) is one of the leading causes of mortality in the world today and hypertension is a significant risk factor for CVD. The regulation of blood pressure is mainly maintained by the renin angiotensin pathway. Briefly, the renin angiotensin system works by the secretion of renin into the blood system from the kidneys. Renin then binds the peptide angiotensinogen and forms angiotensin I. The angiotensin converting enzyme (ACE) binds and cleaves angiotensin I and transforms it into the highly potent vasoconstrictor angiotensin II, thus increasing blood pressure [65].

Fitzgerald et al. [66] extracted protein from the macroalgae *P. palmata* and performed an enzymatic hydrolysis with papain, identifying within the hydrolysate the peptide IRLIIVLMPILHA which potently inhibited the enzyme renin. Moreover, when this peptide sequence undergoes an in vitro digestion process, the gastrointestinal enzymes cleaved the peptide resulting in the production of the di-peptide IR with high anti-renin activity. In a follow up in vivo study using spontaneously hypertensive rats (SHR) and dosing with oral gavage, captopril reduced the blood pressure by 29 mm Hg, while the *P. palmata* hydrolysate reduced it by 34 mm Hg and IRLIIVLMPILHA peptide showed a reduction of 34 mm Hg [67].

ACE is a highly druggable target and several widely prescribed antihypertensive agents (e.g., captopril) are ACE inhibitors [68]. These inhibitors function by preventing the ACE-mediated conversion of angiotensin I into angiotensin II, preventing an increase in blood pressure. Captopril is a proline-based synthetic analog of a peptide present in snake venom that is a competitive inhibitor of ACE [69]. However, drugs like captopril, enalapril and lisinopril have serious adverse side effects that include dry cough, skin rashes, renal failure, and congenital malformations amongs<sup>t</sup> others [70,71]. Thus, there is a growing interest in isolating new peptides with ACE inhibitory activity from natural sources, including those in food [72].

Multiple peptides with ACE inhibitory properties have been isolated from protein extracts from the microalgae *C. vulgaris* and *A. platensis* followed by enzymatic hydrolysis with pepsin [73]. In vivo tests evaluating the efficiency of peptides in SHR revealed that the oral administration of the tetrapeptide IAPG—isolated from *A. platensis*—resulted in a decrease in systolic blood pressure by approximately 50 mm Hg within 1 h of its ingestion [73]. The tripeptide FAL—isolated from *Chlorella*—was less potent in the SHR model, leading to a decrease of approximately 40 mm Hg within 2 h of ingestion. Moreover, the physiological effects of both IAPG and FAL in the SHR were sustained for 4 h postingestion [73].

Using a similar approach, Sun et al. [74] prepared hydrolysates from the macroalga *Ulva intestinalis* protein using trypsin, pepsin, papain, α-chymotrypsin and alcalase, and determined the in vitro activity of these hydrolysates when inhibiting ACE. The authors determined that trypsin-derived hydrolysates had the greatest inhibitory effect and identified the peptides FGMPLDR and MELVLR as those responsible for this effect. The authors also performed molecular docking with AutoDock 4.2 to reveal that while both peptides were bound to the active site, the mode of binding was different [74]. FGMPLDR interacted with Glu123, Ala354, Ala356, Glu384, and Arg522 and in particular with Ala354 and

Glu384 which are both present in the S1 pocket of ACE, interacting with a well-known ACE inhibitor, lisinopril. In contrast, MELVLR was predicted to interact with Asn70, Glu143, Gln281, His383, and Lys511, with Gln281 and Lys511 of particular importance and located in the S2 pocket of the active site of ACE [74].

To our knowledge, there are not many studies with bioactive peptides from algae linking their structure to a proposed mechanism of action. Zarei et al. [75] studied the ACE inhibitory mechanism of action of the bioactive peptides YLLLK, WAFS and GVQE-GAGHYALL identified from palm kernel cake. The authors noted concentration-dependent effects on enzyme inhibition, consistent with the presence of more than one binding site for the peptides and potentially multiple modes of inhibition [75]. Moreover, differences were appreciated in the way that these peptides achieved their activity, as some peptides showed variable degrees of degradation upon pre-incubation with ACE. The authors concluded that the peptide YLLLK acted as a competitive inhibitor and exhibited a higher number of total interactions with ACE compared to the other two peptides [75]. The action of the peptide YLLLK at the ACE active site visualized using molecular docking is represented in Figure 1. Ni, Li, Liu and Hu [69] determined that the ACE inhibition of the yeas<sup>t</sup> peptide TPTQQS was caused by non-competitive interactions by displacing the Zn cofactor from the active site of the enzyme so the reaction cannot occur. The majority of the peptide is attached outside of the active site; however, the tail end of the peptide containing the serine 6 residue is what comes into contact and sequesters the zinc ion by forming a coordination bond with it [69].

**Figure 1.** Automated molecular docking of the peptide YLLLK at the ACE active site. ACE hydrophobic residues are represented in green, positively charged residues in blue, and negatively charged residues in red; hydrogen bonds are purple arrows, polar residues are in turquoise color, and other residues and the zinc atom are represented automatically. Image obtained from Zarei, Abidin, Auwal, Chay, Abdul Haiyee, Md Sikin and Saari [75] originally published by MDPI.
