The Potential Role of 6-gingerol and 6-shogaol as ACE Inhibitors in Silico Study

Hypertension has become the third highest cause of death in Indonesia. The condition is correlated with angiotensin-converting enzyme (ACE), and possibly managed with the use of drugs. In addition, some natural compounds, including 6-shogaol and 6-gingerol from ginger, are used to decrease blood pressure. However, the mechanism and binding site of these compounds to ACE protein is currently unclear. This study, therefore, aims to investigate the potential role of these compounds as an angiotensinconverting enzyme inhibitor. The ACE protein was downloaded from Protein Data Bank (PDB) database with the ID: 3bkk, while the 6-shogaol (CID: 5281794) and 6-gingerol (CID: 44559528) ligands were obtained from the PubChem database. Meanwhile, molecular docking was established using HEX 8.0.0 software. The analysis examined the amino acid residues and the bonds formed from these interactions. According to the results, 14 amino acid residues were formed by the interaction between 6-shogaol and ACE, while the interaction between 6-gingerol and ACE formed eight amino acids. Also, 13 amino acid residues in the novelty binding site of ACE were discovered to be blocked by the ligands from ginger. Therefore, the compounds have potential roles as inhibitors, and this possibly helps to prevent regulation of the renin-angiotensin system. These interactions also formed hydrogen bonds, as well as electrostatic, unfavorable, and hydrophobic sites, making the binding stronger than others.


INTRODUCTION
Type 2 Diabetes Mellitus (T2DM) is a metabolic disease characterized by insulin resistance, hyperglycemia, and pancreatic βcell dysfunction (Ashcroft & Rorsman, 2012;Hameed et al., 2015;Bare et al., 2018). The World Health Organization (WHO) predicts T2DM prevalence to have increased in Indonesia by 2030. According to Tarigan et al. (2018), the condition is closely related to hypertension, the third highest cause of death within the country. Hypertension is related to the Angiotensin-converting enzyme (ACE), and this protein hydrolyzes and converts inactive angiotensin-I to active angiotensin-II (Ouwerkerk et al., 2017;Messerli et al., 2018). Synthetic drugs are able to maintain hypertension by reducing blood pressure. However, these drugs tend to have side effects. Therefore, the accuracy of drugs ought to be evaluated in order to achieve the therapeutic goals, as redactors of cardiovascular morbidity and mortality. The administration of inappropriate antihypertensive medicine tends to increase blood pressure and lead to heart attack, stroke, kidney disease, as well as other ailments. Traditional treatment using natural herbs has provided an effective alternative for reducing blood pressure. The bioactive compounds in herbs are less toxic compared to synthetic drugs. Furthermore, natural medicine helps to exploit biological resources (Chiou et al., 2017;Kesuma et al., 2018). Various natural compounds have been reported as ACE inhibitors. These include quercetin (Larson et al., 2010;Muhammad & Fatima, 2015;Liu et al., 2020), chlorogenic acid (Agunloye & Oboh, 2018;Bare et al., 2019a), hyperin (Huang et al., 2017, tannic acid, flavonoid, and phenolic compounds (Nileeka- Balasuriya & Vasantha-Rupasinghe, 2011;Al Shukor et al., 2013).
Ginger (Zingiber officinale) is one of the natural herbs to reduce blood pressure. In Sikka, ginger is used to treat skin disease, strokes, hypertension, and diabetes mellitus. The herb also contains 6-gingerol, 6-shogaol, 8gingerol, 8-shogaol, 10-gingerol, and 10shogaol and other polyphenols (Mao et al., 2019) with anti-diabetic (Shivanna et al., 2013), anti-hypertensive property (Akinyemi et al., 2013;Bare et al., 2019b). According to the in-vitro study, ginger extract prevents hypertension and manages blood pressure (Akinyemi et al., 2013). Furthermore, the herb contains bioactive compounds with antagonistic effects on ACE-1 (Liu et al., 2013;Zhao et al., 2018;Bare et al., 2019b). However, the inhibition mechanism of these compounds on ACE is not currently clear. Therefore, this study analyzed the bioactive compounds in ginger as appropriate nutrients for treating genomic hypertension through ACE inhibition.

MATERIALS AND METHODS
Ligand and Protein Preparation. The model ACE (ID: 3bkk) was obtained from the Protein Data Bank (PDB) database http://www.rcsb.org/pdb/home/home.do, while the 6-shogaol ligand (CID: 5281794) and 6gingerol (CID: 44559528) were acquired from the PubChem database. Subsequently, the energy of these ligands were minimized using the PyRx virtual screening program Open Babel tool and converted from SDF into PDB files. The protein obtained were then cleaned to extract water molecules and binding ligands, using Discovery Studio Client 4.1 ( Bare et al., 2019a). Molecular Interaction. HEX 8.0.0 software was used to dock the prepared protein and ligand. The docking parameters utilized were Shape, electro and DARS. Subsequently, the protein-ligand's 3D model, ACE binding sites, interaction types, and Ramachandran plot were determined and visualized using Discovery Studio Client 4.1 software (Bare et al., 2019b).

Fig. 2.
The physicochemical complex of ACE-6-gingerol: a. hydrophobicity, and ACE-6 gingerol hydrophobicity plot; b. hydrogen bond and hydrogen bond plot ACE-6 gingerol; c. active side load of ACE-6 gingerol; d. ionization; e. solvent accessible surface (SAS). Based on Fig. 2a, the interaction between 6-gingerol and ACE indicates low hydrophobicity on the surface of the ligand. Fig. 2b and Table 1 show the 6-gingerol compound also functions as a donor and acceptor of ACE in GLU376 and ASN167, while Fig. 2c and Fig. 2d indicate the 6-gingerol compound tends to be neutral, thus the ligand is zero charged. According to Fig. 2e, the value of the solvent-accessible surface (SAS) for 6gingerol surface is relatively high. SAS values are related to the van der Waals forces between ligands and ACE proteins. The 6-shogaol-ACE complex exhibited a potential mechanism to inactivate the ACE conformation. Fig. 3c and Fig. 3d show the complex's active sites performing functional inhibition of ACE. These are GLU376 (hydrogen Bond type Carbon hydrogen), LYS454, LYS511, ZN701 (electrostatic tractive charge), HIS383, PHE457, TYR523, HIS383 (electrostatic Pi-Anion), VAL379 (hydrophobic Pi-Sigma), GLN281 (unfavorable bump), GLU376, ASP145, ASP453 (unfavorable negative-negative), LYS454 (unfavorable donor-donor). Meanwhile, Table 1 shows the binding energy of 6-shogaol-ACE was -256.6 cal/mol. This is typically quite strong to interact with the protein.  Fig. 4a shows the low hydrophobicity level of 6-shogaol-ACE, denoted by blue color. Meanwhile Fig. 4b and Table 1 show a potential residue, GLU376 capable of functioning as an ACE donor or acceptor of ACE. Fig. 4c and Fig.  4d show the 6-shogaol-ACE complex's zero charge, denoted by brown color. Based on Fig.  4e, the complex has a high solvent-accessible surface (SAS) value, depicted by green color.
The interaction between 6-gingerol, 6shogaol, and ACE indicated positive results. According to Table 1, twenty amino acid residues performed as ACE active sites, leading ACE inactivation. A study by Ouwerkerk et al. (2017) stated several bioactive compounds significant for reducing the pathological impact on metabolism are currently being studied, while Al Shukor et al. (2013) reported molecular interactions between ACE inhibitors and the ACE binding site from plant phenolic compound in twelve amino acid residues. Similarly, 6 of these 12 amino acid residues were found to interact with the ligand in this study. These are TYR523, ALA354, LYS511, GLN281, ASP453, and ASP415. Meanwhile, Muhammad & Fatimah (2015) reported other amino acid residues capable of binding with quercetin glycosides. These are ARG124, TYR135, ILE204, ALA208, GLU216, TYR215, and GLU96. This study also discovered novel binding sites from amino acid residues interacting with 6-gingerol and 6shogaol. These are TYR253, HIS383, HIS387, ASP377, GLU162, ASN167, GLU376, ZN701, HIS383, PHE457, VAL379, ASP145, and LYS454, and these are different from the quercetin and phenolic compound binding sites. In addition, the presence of several hydrogen bonds in the ligand-protein complex suggested strong binding (Bare et al., 2019b) Candrakirana, 2019; Kataria & Khatkar, 2019). These bonds also promoted ligand binding affinities and stabilized the ligand-protein interaction (Zhou et al., 2012). The lower binding distance between ALA354 and 6gingerol leads to the formation of a stronger and Vol 8(2), December 2020 Biogenesis: Jurnal Ilmiah Biologi 217 tighter hydrogen bond, compared to the others (Santoso et al., 2016).
ACE inhibition is a critical stage of hypertension treatment because ACE has a crucial role in regulating the renin-angiotensin system (Guang et al., 2012;Liao et al., 2019). This method has a positive effect on therapy and has been linked to improved health, outside of blood pressure regulation (Bhullar et al., 2014). Liu et al. (2013) reported 6-gingerol to be a molecular target drug for curving hypertension. This study also proposed the molecular interaction this compound and another amino acid residue. Meanwhile, Akinyemi et al. (2013) and Zhao et al. (2018) stated ginger varieties inhibited ACE and also protected the heart from Fe 2+ -and SNP-induced lipid peroxidation. Recent research also showed ginger extract influences blood pressure and lipid level in hypertensive and hyperlipidemic Wistar rats model (Sanghal et al., 2011, Sahardi & Makpol, 2019. Similarly, a study by Akinyemi et al. (2014) reported ginger extract to exhibit ACE inhibitory activity. The interaction between ligan and protein has a positive impact, as ACE inhibitor was produced by renin to convert angiotensin I into angiotensin II. Also, Sahardi & Makpol (2019), disclosed ginger is able to reduce the blood pressure of hypertension patients, as well as lipid peroxidation in the heart.

CONCLUSION
This study confirmed the binding of 6shogaol and 6-gingerol to ACE protein is tight, and the complex formed possibly has potential ACE inhibitory activity within ACE active sites. This interaction was observed to occur in twenty amino acid residues. The interaction also formed hydrogen bonds, electrostatic, unfavorable, and hydrophobic bonds, making binding stronger, compared to others.