¹Division of Biochemistry, Chemistry Department, Aleppo University, Syrian Arab Republic ²Biochemical Technology Program, Faulty of Applied Sciences, Dhamar University, Yemen

^*Address for Correspondence: Al-Madhagi HA, Biochemical Technology Program, Faulty of Applied Sciences, Dhamar University, Yemen, E-mail: bio.haitham@gmail.com

Submission: 21 September, 2022 Accepted: 25 October, 2022 Published: 29 October, 2022

Copyright: © 2022 Kanawati A, et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Hypertension (HTN) still constitutes a worldwide problem to the healthcare section and individual’s life. The current angiotensin converting enzyme (ACE) inhibitors are first-choice option but pose deleterious side effects upon prolonged administration. The goal of the present in silico study is to evaluate the Allium sativum and Solanum lycopersicum proteins as source for anti-hypertensive natural peptides. The corresponding protein sequences were obtained from the UniProt database and then inputted to antihypertensive peptides predictor online tool. Using the same tool, 3 digestive enzymes (pepsin, trypsin and elastase 1) were chosen for the digestion of proteins into small peptides and subsequently assess their hypotensive activity. Some biochemical characteristics of positive bioactive peptides were calculated via Pepstats web interface while HAPPENN tool examined hemotoxicity. Afterwards, the secondary structure of positive bioactive peptides was de novo predicted through PEPstrMOD server prior to docking against human ACE (PDB ID: 1o86) using HPEPDOCK and ClusPro platforms. A.sativum peptides are more potent than S.lycopersicum ones in terms of IC50 as well as docking score albeit neither of which showed hemotoxicity. The present in silico work suggests the hypotensive activity of A.sativum and S.lycopersicum as natural treatment option of HTN.

Hypertension; Anti-hypertensive peptide; Peptide-protein docking; Allium sativum; Solanum lycopersicum

Hypertension (HTN) continues to be the second main risk factor of death cases from cardio- and cerebrovascular accidents as well as end-organs damage globally [1]. It is estimated in the United States (US) that one in every three persons suffers from HTN [2]. The recent redefinition of HTN is blood pressure (BP) levels ≥ 130/80 mm Hg or having HTN medicines. Accordingly, the percentage of prevalence of HTN in US rises to 50% [3]. Moreover, about half of COVID-19- hospitalized patients have HTN which increases the risk of mortality even more [4].

Various risk factors are traced to drive BP up that can be categorized into modifiable and non-modifiable factors (Figure 1). Modifiable factors include, but not limited to, diet (particularly Narich fast foods), sedentary lifestyle and obese people. Non-modifiable factors involve aging and the presence of chronic diseases besides such as type 2 diabetes and kidney diseases [5,6]. In addition, genetic inheritance and, more recently, epigenetics programming especially in prenatal stage increases the incidence of HTN later in adulthood [7].

Once patient’s BP got elevated, therapeutic intervention should be introduced to restore the levels of BP to the accepted values. One of the mainstay pathways regulating BP is renin-angiotensinaldosterone system (RAAS) (Figure 2). As the name implies, there are 3 cooperating hormones to control BP, renin (also known as angiotensinogenase), angiotensin and aldosterone. Driven by the HTN, juxtaglomerular cells of kidney sense this rise in BP and respond by secreting renin to the bloodstream. Therein, it proteolytically activates angiotensinogen produced from liver forming angiotensin I and the latter catalyzed by the enzyme angiotensin-converting enzyme 1 (ACE1) is cleaved further to angiotensin II. The produced angiotensin II acts as powerful vasoconstrictor by activating its corresponding receptors ATR (angiotensin II receptors) on smooth muscle cells. With the assistance of ATR binding by angiotensin II, aldosterone is secreted from adrenal gland to enhance Na reabsorption contributing further to the establishment of HTN [8]. Therefore, interrupting ACE1 action cancels the vasoconstriction as well as Na reabsorption actions reducing BP to the normal set. ACE1 inhibitors are considered first-choice treatment of HTN [9].

Not only small organic molecules can achieve ACE1 inhibition. Many food proteins upon enzymic digestion in the gut releases bioactive peptides exhibiting inhibitory action toward different targets of RAAS including ACE1 [10,11]. So, we purposed to explore the anti-hypertensive activity of peptides digested from some A.sativum proteins in silico. The present study provides a probable means to nutraceutically treat HTN through the food taken on a regular daily routine.

The Repeated workflow strategy employed in this study is illustrated in Figure 3. However, the details of each step are explained below.

The primary structure (amino acid sequence) of the examined proteins was retrieved from UniProt database (https://www. uniprot.org/) [12]. Three proteins were chosen randomly: Alliin lyase 1 (Uniprot ID#Q01594), mannose-specific lectin (Uniprot ID#P83886) and peroxidase (Uniprot ID#H2CLW6) limiting the organism to Allium sativum only. Similarly, three proteins from Solanum lycopersicum were selected for further analysis (Uniprot ID#P27260, G1JUH1 & C1K5M2).

The 3 mentioned proteins were subjected to three digestive enzymes (randomly chosen) namely: Pepsin, Trypsin and Elastase 1 in combination. Afterwards, the anti-hypertensive activity of the generated peptides were evaluated using anti-hypertensive peptide predictor (AHPP) server [13]. This server enables enzymatic digestion and activity assessment simultaneously. Only the peptide showed positive anti-hypertensive activity and having ≥ 7 AA were selected for further analysis.

Despite of posing biological activity, some peptides have a coinciding side effects including hemotoxicity and many neglect such aspect [14]. HAPPENN web server was utilized to examine the hemotxicity of the positive peptides [15]. Results are expressed as probability score between 0 (non-hemolytic) to 1 (highly hemolytic).

The peptides succeeded in achieving positive antihypertensive activity and had at least seven AA were uploaded to pepstats web server (https://www.ebi.ac.uk/Tools/seqstats/emboss_pepstats/) for detecting some characteristics such as molecular weight, isoelectric point and charge, among others. The obtained results were collected and tubulated.

The secondary structure of the best 3 peptides were predicted de novo via PEPstrMOD online tool [16] (https://webs.iiitd.edu. in/raghava/pepstrmod/) in provision to be docked against human ACE1(PDB ID: 1O86) after removal of water molecules, chloride and zinc ions and the inhibitor lisinopril by UCSF Chimera v1.16 [17]. Then, peptides and receptor PDB files to uploaded to and HPEPDOCK protein-protein docking platform [18].

Table 1 Obtained biochemical features of the fragmented peptides from A.sativum proteins along with hemotoxicity values.

The 3 examined proteins of A.sativum, only 8 peptides found positive (antihypertensive). As we chose 3 digestive enzymes (pepsin, trypsin and elastase 1) the residues were short (<10 AA). Most of the fragmented peptides had neutral charge and, hence, pI around 5. Furthermore, the digested peptides posed high probability of being expressed in inclusion bodies which facilitates the large-scale production and isolation of the examined peptides. Moreover, none of the peptides showed hemolytic activity as reflected by values near zero (indicative of non-hemolytic) (Table 1). Overall, the digested peptides from A.sativum are both anti-hypertensive as well as nonhemolytic peptides particularly 3^rd peptide (FTGHSGSR). This outcome is good for provision to molecular docking against human ACE1 to further confirm the findings.

Only 6 positive peptides were obtained from precursor proteins of Solanum lycopersicum. The fragmented peptides were significantly potent in terms of anti-hypertensive activity as mirrored by the IC₅₀ values. The digested peptides are composed of a combination of neutral, acidic and basic amino acids with net charge between -2 to 1 and pI in the range 3.55 to 8.9. In addition, none of the obtained peptides exhibited hemolytic activity (Table 2).

Table 3 Docking output of A.sativum peptides to ACE1 using HPEPDOCK and ClusPro servers

Apparently from Table 3, the fragmented peptides from allium sativum exhibited good inhibition profile toward ACE1, confirming thus their anti-hypertensive activity predicted via AHPP server. The docking scores in HPEPDOCK ranged from -185 to -240 kcal/mole. Similarly, from -561 up to -805 kcal/mole predicted against ACE1 via ClusPro platform. The ranking of peptides in the two servers are almost the same which confirm the results of each other with the 3^rd peptide were the best in the two servers.

To further reveal the detailed peptide-protein interactions between 3rd peptide and ACE1, the 3D surface view and indepthrepresentation have been performed (Figure 4). It has been figured out that peptides lie well within the active center of ACE1 as shown in surface view (Figure 4A).

The docking profile of the digested peptides from S.lycopersicum showed slightly weaker binding energy to ACE1 explored by HPEPDOCK server in comparison to A.sativum peptides. While the highest docking score of S.lycopersicum peptides were peptide 6 which gave inhibitory value of -225 kcal/mole, 3rd peptide of A.sativum had a docking score of -240 kcal/mole, suggesting better potency. Nonetheless, peptide 4 of S.lycopersicum showed best docking energy compared to the 6th peptide in HPEPDOCK (-862 vs -711 kcal/mole) (Table 4). This docking energy is even stronger than best docking energy of 3rd peptide (best one) of A. sativum peptides.

Developing HTN worsen individual’s health life since it is accompanied by complications progression such as cardiovascular diseases, stroke and dementia. Beyond 1970s, BP levels have been declined in high-income nations whilst raised substantially in Middle-east and Southeast regions [19]. In 2016, about third of adult population worldwide are diagnosed with HTN [20]. Men on common have age-standardized systolic BP higher than women counterparts in several countries [21]. This data necessitates should urge to find more potent therapeutics with minimized side effects because the first-choice treatments (ACE1 inhibitors) exhibit deleterious side effects involving headache, visual disturbances, cough, dizziness and insomnia on prolonged use [22]. In recent years, the direction of HTN treatment has been transformed into food peptides including milk, egg, meat and marine meats. The hypotensive peptides are released upon hydrolysis through gastrointestinal tract enzymes [23]. Jogi et al. reviewed long list of hypotensive peptides derived from food sources [24]. However, garlic and tomato-hypotensive peptides were not mentioned. The present in silico study was designed to simulate the in vitro and in vivo works for assessment of hypotensive peptides generated from food sources and predict their ACE1 inhibition. The results of the current study suggest the hypotensive activity of both garlic (A.sativum) and tomato (S.lycopersicum) as evidenced by IC₅₀ values and validating molecular docking against ACE1 target albeit garlic peptides are more potent. Moreover, the fragmented peptides are non-hemolytic and had a high probability of being expressed in inclusion bodies for large-scale production. Overall, the present theoretical exploration provided a notable suggestion that should be validated by in-vitro and in-vivo experiments